NPR - California Center for Algae Biotechnology · 2020. 2. 27. · PPant arms for biosynthetic...

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The phosphopan

aDepartment of Chemistry and Biochemistry

Gilman Drive, La Jolla, CA 92093-0358, USA

434 1360bHoward Hughes Medical Institute, The S

H. Skirball Center for Chemical Biology

Road, La Jolla, CA 92037, USA

† Electronic supplementary informa10.1039/c3np70054b

‡ Authors contributed equally.

§ Current address: Department of Syste

Denmark, Søltos Plads 221, 2800 Kgs. Ly

Cite this: Nat. Prod. Rep., 2014, 31, 61

Received 11th June 2013

DOI: 10.1039/c3np70054b

www.rsc.org/npr

This journal is © The Royal Society of C

tetheinyl transferases: catalysis ofa post-translational modification crucial for life†

Joris Beld,‡a Eva C. Sonnenschein,‡§a Christopher R. Vickery,‡ab Joseph P. Noelb

and Michael D. Burkart*a

Covering: up to 2013

Although holo-acyl carrier protein synthase, AcpS, a phosphopantetheinyl transferase (PPTase), was

characterized in the 1960s, it was not until the publication of the landmark paper by Lambalot et al. in

1996 that PPTases garnered wide-spread attention being classified as a distinct enzyme superfamily. In

the past two decades an increasing number of papers have been published on PPTases ranging from

identification, characterization, structure determination, mutagenesis, inhibition, and engineering in

synthetic biology. In this review, we comprehensively discuss all current knowledge on this class of

enzymes that post-translationally install a 40-phosphopantetheine arm on various carrier proteins.

1 Introduction2 Types of PPTases2.1 Family I: holo-acyl carrier protein synthase (AcpS-type

PPTases)2.2 Family II: Sfp-type PPTases2.3 Family III: type I integrated PPTases3 Importance in primary and secondary metabolism3.1 Bacteria3.2 Archaea3.3 Cyanobacteria3.4 Protista3.5 Fungi3.6 Type I integrated PPTases3.7 Plants and algae3.8 Animals3.9 Homo sapiens3.10 Evolution and phylogeny4 Carrier protein(s)4.1 Specicity and activity for carrier proteins

, University of California-San Diego, 9500

. E-mail: [email protected]; Tel: +1 858

alk Institute for Biological Studies, Jack

and Proteomics, 10010 N. Torrey Pines

tion (ESI) available. See DOI:

ms Biology, Technical University of

ngby, Denmark.

hemistry 2014

4.2 The other phosphopantetheinylated proteins and theirPPTases

4.3 Carrier protein recognition by PPTases4.4 Peptide mimics of carrier proteins as substrate of PPTases4.5 Regulation by 40-phosphopantetheinylation5 Structures5.1 Structural features of AcpS-type PPTases5.2 Structural features of Sfp-type PPTases5.3 Structural features of type I integrated PPTases5.4 Identication of residues important for PPTase activity5.5 pH and metal effects on PPTase activity5.6 Activity and specicity for CoA donor5.7 Measuring the activity of PPTases6 Biotechnological use of PPTases6.1 In vitro labeling6.2 In vivo tagging and labeling6.3 Other applications6.4 Production of natural products in heterologous hosts6.5 Drug discovery7 Outlook8 Acknowledgements9 References

1 Introduction

Phosphopantetheinyl transferases (PPTases)1 are essential for cellviability across all three domains of life: bacteria, archaea andeukaryota. PPTases post-translationally modify modular anditerative synthases acting in a processive fashion, namely fattyacid synthases (FASs), polyketide synthases (PKSs), and non-ribosomal peptide synthetases (NRPSs). The central component of

Nat. Prod. Rep., 2014, 31, 61–108 | 61

http://dx.doi.org/10.1039/c3np70054b

http://pubs.rsc.org/en/journals/journal/NP

http://pubs.rsc.org/en/journals/journal/NP?issueid=NP031001

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these chain-elongating synthases is non-catalytic and is either atranslationally linked domain of a larger polypeptide chain or anindependently translated protein. Regardless, this proteincomponent is referred to as a carrier protein (CP), or alternativelyas a thiolation domain.2 A CP is responsible for timing and effi-ciency in shuttling the rapidly changing chemical intermediates

Joris Beld received his MSc inchemical engineering from theUniversity of Twente, Nether-lands, under the tutelage of Prof.David Reinhoudt. He obtainedhis PhD in biochemistry from theETH Zurich, Switzerland,working on selenium and oxida-tive protein folding in the lab ofProf. Donald Hilvert. He iscurrently a postdoctoral fellow atUC San Diego involved in a rangeof projects investigating micro-algal and bacterial biochemistryand fatty acid biosynthesis.

Eva Sonnenschein graduatedfrom Kiel University, Germany, in2007 and received her PhD inMarine Microbiology from JacobsUniversity Bremen and the MaxPlanck Institute for MarineMicrobiology in 2011. She hasbeen working as a postdoctoralresearcher at UC San Diegoexploring the details of secondarymetabolism in microalgae andaiming towards heterologousexpression of natural-product

synthases in these organisms. Coming from an aquatic ecologybackground, Eva wants to understand the molecular basics ofmicrobial interactions with the goal to predict community adaptionto environmental changes in the future.

Christopher Vickery graduatedfrom the University of Maryland,College Park in 2009 with a B.S.in Biochemistry. He has been agraduate student for Prof.Michael Burkart and Prof. JosephNoel in the Department ofChemistry and Biochemistry atthe University of California, SanDiego, and the Salk Institute,since 2009. His current work isfocused on the structural andfunctional characteristics of

phosphopantetheinyl transferases and their role in both known anduncharacterized biosynthetic pathways in different organisms.

62 | Nat. Prod. Rep., 2014, 31, 61–108

due to chain elongation between the structurally diverse multi-enzyme complexes of these pathways. The CP tethers the growingintermediates on a 40-phosphopantetheine (PPant) arm of 20 Athrough a reactive thioester linkage. PPant is thought of as a“prosthetic arm” on which all substrates and intermediates ofthese pathways are covalently yet transiently held during theorderly progression of enzymatic modications to the extendingchain. PPTases mediate the transfer and covalent attachment ofPPant arms from coenzyme A (CoA) to conserved serine residuesof the CP domain through phosphoester bonds. These essentialpost-translation protein modications convert inactive apo-syn-thases to active holo-synthases (Fig. 1). Recently, mechanisticallydistinct classes of enzymes have been identied that requirePPant arms for biosynthetic catalysis. These include enzymesinvolved in the biosynthesis of lipid A, D-alanyl-lipoteichoic acid,

Joseph Noel obtained a Bachelorof Science degree in Chemistryfrom the University of Pittsburghat Johnstown in 1985. Hereceived his Ph.D. in chemistryfrom the Ohio State University in1990, working on the enzymologyof phospholipases with ProfessorMing-DawTsai. As a postdoctoralfellow with the late Paul B. Siglerin the Department of MolecularBiophysics and Biochemistry atYale, Joe elucidated the structure

of heterotrimeric G-proteins. Joe is currently director of the Jack H.Skirball Center for Chemical Biology and Proteomics at the SalkInstitute, professor at the Salk Institute and investigator with theHoward Hughes Medical Institute.

A native Texan, Michael Burkartreceived a BA in chemistry fromRice University in 1994. Hereceived a PhD in OrganicChemistry from the ScrippsResearch Institute in 1999 underthe mentorship of Prof. Chi-HueyWong, aer which time hepursued postdoctoral studies atHarvard Medical School withProf. Christopher Walsh. Since2002 he has taught and per-formed research at the University

of California, San Diego, where he is a Professor of Chemistry andBiochemistry and Associate Director of the San Diego Center forAlgae Biotechnology. His research interests include natural-productsynthesis and biosynthesis, enzyme mechanism, and metabolicengineering. Prof. Burkart has been a fellow of the Alfred P. SloanFoundation and Ellison Medical Foundation and was the RSCOrganic and Biomolecular Chemistry Lecturer for 2010.

This journal is © The Royal Society of Chemistry 2014


Fig. 1 General reaction scheme of post-translational phospho-pantetheinylation by a PPTase. The PPTase transfers the PPant moietyfrom CoA to a conserved serine residue on the apo-CP to produceholo-CP, here showcased by a typical NRPS module containing the C(condensation), A (adenylation), and CP (carrier protein) domains. 30,50-PAP is 30,50-phosphoadenosine phosphate.

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lipo-chitin nodulation factor, b-alanine–dopamine conjugates,carboxylic acid reductions, dehydrogenation of a-aminoadipatesemialdehyde and reduction of 10-formyltetrahydrofolate. Inshort PPTases are essential for the biosynthesis of fatty acids andlysine, many bioactive secondary metabolites, and a variety ofother central biosynthetic pathways in both primary and special-ized metabolism.

The essential enzymatic role of PPTases in general fatty acidbiosynthesis was recognized in the groundbreaking work ofVagelos and Elovson.3 Since then, many other PPTases have beendiscovered to play the same role in a wide variety of secondarymetabolic pathways.1,4 Many of these PPTases have beendescribed on the gene and protein levels providing for intensebiochemical characterization. With the mapping of their activesites, their interactions and catalytic mechanisms accompanyingCoA and CP recognitions have provided quantitative clarity, insome cases delineating predictable strategies for their molecularengineering for an assortment of basic and applied applications.

Due to their key metabolic positions in metabolism, PPTasesare considered a prime drug and antibiotic target in medicineas well as agriculture. Being essential for the biosynthesis ofnatural products with antibiotic, immunosuppressive, andcytotoxic activities, as well as valuable hydrocarbons, efficientand selective PPTases serve pivotal roles for metabolic engi-neering efforts in the pharmaceutical and biofuel industries.

This is the rst comprehensive review of PPTases. We havestructured this overviewrst onpublished results over the last twodecades with goals of understanding the identity, activity, andfunctions of PPTases across the three domains of life. Whereappropriate, we also identify knowledge gaps for futureinvestigations.

2 Types of PPTases

FASs, PKSs or NRPSs are either assembled from one or moremulti-domain polypeptides (type I, possibly originating fromancient gene fusion events) or from separate protein partners


(type II). In general, prokaryotes utilize type II FASs andeukaryotes utilize type I FASs. CPs of FASs and PKSs are calledacyl carrier proteins (ACPs), whereas the CPs of NRPSs arereferred to as peptidyl carrier proteins (PCPs). In order for thesesynthases to function in primary and secondary metabolism of(iteratively) produced intermediates and products, conservedserine residues of the CPs must be functionalized by PPTasecatalysis with a PPant arm. Given the diversity of CP sequencesand structures, the PPTase superfamily can be divided intothree related yet distinguishable families, based on amino acidsequence conservation and alignments, three-dimensionalstructures, and their target-elongating synthases.

Holo-ACP synthase (AcpS) is the archetypical enzyme of therst family of PPTases recognized (Fig. 2). It encompasses 120 aa,forms a homo-trimeric quaternary structure with active sitesshared across each homotypic interface, and acts on type II FASACP (AcpP). Surfactin phosphopantetheinyl transferase (Sfp)represents the second family of PPTases. Sfp is the PPTasenecessary for installing PPant on the PCP of surfactin synthase. Incontrast to AcpS, Sfp exists as a pseudo-homodimer of �240 aa,resembling two AcpS monomers with one active site at thepseudo-dimer interface, and possesses a much broader substrateacceptance. The third family of PPTases are translationally fusedC-terminal transferases residing in the megasynthases as one ofseveral catalytic domains in type I yeast and fungal FAS mega-synthases. This third family of PPTases post-translationallymodify apo-ACPs prior to assembly of the megasynthases. In thissection, we review these three families, rst focusing on theirdiscovery, divergent primary sequences and their biochemistry(note: structures will be discussed in Section 5).

2.1 Family I: holo-acyl carrier protein synthase (AcpS-typePPTases)

The discovery of AcpS in the late 1960s was instigated by studieson ACP and CoA in Escherichia coli.7 Feeding radioactivepantothenate to a pantothenate-requiring auxotroph revealedthat 90% of the radioactivity was associated with the proteinfractions, and 90% of that total amount was associated with FASACP (AcpP). Radioactive CoA was also observed early, corre-lating with the amount of pantothenate fed, but then dropped.However, the concentration of radioactive ACP was stable undera broad range of conditions, suggesting that CoA formationpreceded radioactive incorporation into ACP.7 Indeed, Elovsonand Vagelos partially puried AcpS from E. coli and showed thatAcpS catalyzed the incorporation of radioactivity into ACP usingCoA and apo-ACP as substrates to form holo-ACP and 30,50-PAP.The authors also demonstrated that the reaction requires eitherMg2+ or Mn2+, and described AcpS as being an unstableprotein.3 Years later, Polacco and Cronan mutagenized thepantothenate auxotroph used earlier by Elovson and Vagelos,then selected strains that only grew on very high levels ofexogenous pantothenate. One strain in particular had nomeasurable AcpS activity aer cell lysis and the site of thegenetic lesion was mapped to the genomic region later shown toinclude the gene encoding AcpS (acpS).8

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Fig. 2 PPTases. Overview of the three families of PPTases,1 typified by AcpS, Sfp and the integrated PPTase domain of Saccharomyces cerevisiaeFAS2, and a sequence alignment of archetypical PPTases using T-Coffee and ESPript5,6 (Bs, Bacillus subtilis; Ec, Escherichia coli; Hs, Homosapiens; and Sc, S. cerevisiae).

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Later work on plants, which synthesize their fatty acidsprimarily in the chloroplast using a type II FAS similar to thebacterial FAS, afforded AcpS isolations from a second domain oflife, the eukaryota. AcpS was isolated from spinach leaves anddeveloping castor bean endosperm. Both AcpS fractionsexhibited similar biochemical characteristics as the enzymeisolated earlier from E. coli.9 Moreover, in plants, AcpS activityappeared to reside in the cytosol, and the PPant attached to apo-ACP before holo-ACP translocated into the chloroplast.9 In 1995,Lambalot and Walsh puried AcpS from E. coli 70 000-fold.Curiously, although they achieved a 70 000-fold purication,the protein was still not homogeneous, suggesting that it isexpressed at very low levels. Later heterologous overexpressionin E. coli of the dpj gene provided pure AcpS, which appeared tospan 125 aa, and purify as a 28 kDa dimer. The original dpj genewas renamed acpS.10 Subsequent work in the Walsh labdescribed AcpS as a trimer not a dimer and that it accepted notonly bacterial AcpP but a variety of CPs from type II elongatingsystems including Lactobacillus casei D-alanyl carrier protein,Rhizobium protein NodF and Streptomyces ACPs involved infrenolicin, granaticin, oxytetracycline and tetracenomycinpolyketide biosynthesis.11 Ironically, CPs from type I elongatingsystems were not substrates for AcpS, as shown by the inabilityof E. coli AcpS to install a PPant arm on apo-EntF, the E. colienterobactin synthase, or apo-TycA, the Bacillus brevis tyroci-dine synthase. Although AcpSs exhibit a moderate level of CPsubstrate permissiveness in type II elongating systems, AcpSsare primarily used for post-translational modication andactivation of the CPs of FASs (primary metabolism) across adiversity of organisms making them the most commonly foundPPTase.

2.2 Family II: Sfp-type PPTases

In the early nineties, Grossman et al. identied genes involvedin the biosynthesis of the siderophores enterobactin and sur-factin from E. coli and B. subtilis, respectively.12 These genes,called entD and sfp, were soon grouped with the gsp gene from

64 | Nat. Prod. Rep., 2014, 31, 61–108

B. brevis, which plays a role in gramicidin S biosynthesis.13 In1996, a landmark paper co-authored by ve different labs waspublished. The work comprehensively described the identi-cation of many PPTases using bioinformatics, the character-ization of family II proteins by heterologous expression,purication and biochemical characterization and the clari-cation of the link between Sfp, EntD and Gsp in natural-productbiosynthesis.1 This paper and previously published workmarked the wide-spread discovery of many acpS and sfp genesacross all three domains of life.14

Sfp expresses well in E. coli and other heterologous hosts andshows highly permissive catalytic activity towards CPs using notonly CoA but CoA-like substrates. These properties now affor-ded many labs with the ability to delve deeply into the biosyn-thesis of many natural products. Until recently, Sfp was also theonly family II PPTase for which the three-dimensional structurewas reported affording an atomic resolution understanding ofsubstrate recognition and CP maturation.15 Sfp quickly becamethe go-to PPTase used in many in vitro assays requiring PPTaseactivity for holo-CP synthesis. Also, its utility in metabolicengineering was widely recognized because it relieved amajor bottleneck associated with the concentration of bioactiveholo-CPs needed for in vivo production of targeted metabolites.

The workhorse bacterium E. coli K12 afforded the identi-cation of an even greater variety of PPTases from a singleorganism. As described earlier, the PPTase AcpS (family I) wasrst identied in E. coli as responsible for phosphopantethei-nylating AcpP; however, feeding radioactive pantothenate toE. coli revealed two other proteins that incorporated radioac-tivity. One was later identied as EntF,16 the CP of the enter-obactin synthase complex. This discovery then showed thatEntF is phosphopantetheinylated by the family II PPTase EntD.The other protein is EntB, a small isochorismate lyase-carrierprotein fusion involved in the initiation of enterobactinsynthesis. Bioinformatic tools and the newly uncovered E. coligenome revealed another PPTase-like sequence. Originallynamed o195 (aer ORF195), it was later renamed AcpT. AcpTshowed sequence similarity to EntD and Sfp. AcpT exhibited



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poor in vitro PPTase activity when using either ACP or EntF asCP acceptors.1 Surprisingly, AcpT rescued an E. coli strain withdefects in YejM, a membrane protein with unknown function.Evenmore baffling, AcpT did not need to be a catalytically activeenzyme to rescue the yejM-defective E. coli strain (seeSection 3.1).17

More broadly, in family II PPTases, the genes encoding thePPTase oen reside in close proximity to, or part of, a synthaseoperon. Nevertheless, there are also many cases where thePPTase genes are found far removed from their CP substrategenes and the other synthase operon genes. With improvedbioinformatic tools, family II PPTases could be grouped basedon phylogenic distributions and sequence alignments.1,14,18 Forexample, sequence alignments of PPTases identied two highlyconserved regions, called ppt-1 and ppt-3, now generalized asthe bipartite sequence, (I/V/L)G(I/V/L/T)D(I/V/L/A/)(x)n(F/W)(A/S/T/C)xKE(S/A)h(h/S)K(A/G), where ‘x’ are chemically disparateamino acids, n is 42–48 aa for AcpS (family I) and 38–41 aa forSfp-type (family II) PPTases, and ‘h’ is an amino acid with ahydrophobic side chain. Moreover, the sub-motifs WxxKEA orFxxKES are linear ngerprints for at least two differentsubclasses of Sfp-type PPTases (family II) discussed phyloge-netically in Section 3.9 and structurally in Section 5.

2.3 Family III: type I integrated PPTases

In the eukaryote Saccharomyces cerevisiae (yeast), three differentPPTases have been identied: PPT2 that activates a recentlydiscovered type II mitochondrial FAS ACP, a PPTase dedicatedto a-aminoadipate semialdehyde dehydrogenase (AASDH)activity, and a translationally fused PPTase domain sitting at theC-terminal end of a cytosolic type I FAS. Curiously, this trans-lational fusion in yeast and other fungi differs from anothereukaryotic system, the cytosolic type I mammalian FAS. In theselatter FASs, their translationally fused CPs are phosphopante-theinylated by independently translated family II PPTases.

The mammalian and fungal FASs arrange as strikinglydifferent oligomeric structures. Type I mammalian synthasesare multidomain single polypeptide monomers that organizephysiologically as 540 kDa dimers. In contrast, type I fungalsynthases contain one (or sometimes two) multidomain poly-peptides that assemble into large, 2.6 MDa hexameric or het-erododecameric complexes.19 In the yeast S. cerevisiae, its FASmegasynthase consists of two related proteins, FAS1 and FAS2,that form a hexameric a6b6 oligomer. It is thought that thePPTase of one a-subunit installs the PPant arm on ACP of thesecond a-subunit, suggesting that an a2 dimer, formed betweenthe terminal PPTase domains, needs to be assembled beforepost-translational modication. This is supported by theprevious observation that activation of apo-FAS was more effi-cient aer in vitro re-association.20 However, the crystal struc-ture of the fungal FAS revealed that the PPTase domain does notassociate with other protein modules, sitting 60 A away from thenearest ACP. Moreover, the PPTase module is located on theoutside of the barrel-like a6b6 oligomer, whereas fatty acidbiosynthesis takes place inside the barrel.21 This static struc-tural arrangement suggests that the formation of a transient


dimer of two PPTase modules forms rst to allow for post-translational modication of ACP modules which is then fol-lowed by full barrel assembly.22

When the integrated PPTase domain of S. cerevisiae FAS wassubcloned and independently expressed from the entire mega-synthase, the PPTase formed a trimeric complex similar to AcpS(family I), with three active sites formed by the inter-subunitinterfaces. Unexpectedly, both the full-length FAS retaining thePPTase domain and the excised PPTase trimer were equallyactive in vitro. Surprisingly, the fully assembled a6b6 FAS wasstill able to phosphopantetheinylate free apo-ACP in vitro, sug-gesting that dynamics of the a6b6 oligomer occur and playcritical roles in ACP maturation and FAS function.22 Recently,cryo-electron microscopy of the FAS particle again showed thatthe PPTase domain resided on the outside of the barrel, butsubstantial exibility in the wall of the barrel was also inferredfrom multiple particle reconstructions.23 Nevertheless, how thePPTase domain of this family forms an active enzyme, whendoes this catalytic activity arise during assembly of the mega-synthases, and how the mature FAS particle retains phospho-pantetheinyl transfer activity remain major unansweredquestions.

3 Importance in primary andsecondary metabolism

Since the discovery of the large superfamily of PPTases,1 manyhomologs have been discovered in various organisms, and thepast 25 years has seen a surge of literature on both the char-acterization of PPTases and identication of PPTase genes in(newly uncovered) biosynthetic clusters. Here we summarizethe discovery, and the identication and the target(s) of nearlyall PPTases that have been found to date across diverse species.Bacteria and cyanobacteria commonly produce secondarymetabolites, thereby representing a rich spectrum of PPTases(Table 1). Since AcpS-type (family I) PPTases are found in manyspecies, and in all cases responsible for FAS activation, thisclass of PPTases is only briey mentioned in this section.PPTases activate secondary metabolite biosynthetic clusters,and these clusters along with their products are discussed indetail. Further, in some cases, the family II PPTases are nottranslated due to genetic inactivation. This mutational eventleads to altered secondary metabolism and interesting pheno-types likely under selective pressure. Finally, we assembledlarge sets of PPTase sequences and constructed phylogenetictrees, showcasing fascinating evolutionary groupings of PPTasenatural diversity. This section aims to emphasize the ubiquity ofPPTases, their broad function and diverse catalytic specicities,and their critical role in many secondary metabolism pathways.

3.1 Bacteria

Bacteria are notable producers of CP-mediated secondarymetabolites that include pigments, siderophores and antibi-otics. Presumably, this arsenal of molecules are produced toscavenge nutrients (e.g. siderophores) and/or to provide thehost organisms with enhanced tness in the face of challenging

Nat. Prod. Rep., 2014, 31, 61–108 | 65


Tab

le1

Functionally

describedPPTases

PPTase

Proteinaccessionno.

Section

Phylum

Species

Function,s

pecicity

Citation

CELT

O4G

9NP_

5081

53Anim

alNem

atod

aCaeno

rhab

ditiselegan

sn.d.

1DmPP

TNP_

7297

88Anim

alArthropo

daDrosoph

ilamelan

ogaster

FAS,

Ebo

ny

24AASH

DPP

TNP_

0562

38.2

H.sap

iens

Chorda

taH.sap

iens

FAS,

mitoF

AS,

AASD

H,T

HF,

mito-THF

25,26

Gsp

CAA53

988

Bacteria

Firm

icutes

B.b

revis

Gramicidin

13Ps

f-1

P558

10Bacteria

Firm

icutes

Bacilluspu

milus

Surfactin

1,27

Sfp,

Lpa-8

P391

35,B

AA09

125

Bacteria

Firm

icutes

B.sub

tilis

Surfactin,p

lipa

statin

B1

28,29

Lpa-14

,Lpa

-B3

2113

333A

,P39

144

Bacteria

Firm

icutes

B.sub

tilis

Iturin

A,s

urfactin,fen

gycin

1,30

,31

Bli

AAO74

604

Bacteria

Firm

icutes

Bacilluslicheniformis

Bacitracin

1,32

–34

Acp

SP2

4224

Bacteria

Proteo

bacteria

E.coli

FAS

1EntD

P199

25Bacteria

Proteo

bacteria

E.coli

Enteroba

ctin

1Acp

T,o

195

NP_

2900

41Bacteria

Proteo

bacteria

E.coli

n.d.

35ClbA

CAJ763

00Bacteria

Proteo

bacteria

E.coli

Colibactin

103,

104

HIO

152

P439

54Bacteria

Proteo

bacteria

Haemop

hilusinuenzae

FAS

1Pp

tTNP_

2173

10Bacteria

Actinob

acteria

Mycob

acterium

tubercolosis

Seconda

rymetab

olism

36–38

MuP

ptYP_

9060

28Bacteria

Actinob

acteria

Mycob

acterium

ulcerans

Mycolactone

Npt

ABI836

56Bacteria

Actinob

acteria

Nocardiasp

.NRRL56

54AcidRed

uction

39PP

1183

AAN66

807

Bacteria

Proteo

bacteria

Pseudo

mon

aspu

tida

KT24

40n.d.

40Pc

pSBAK88

897

Bacteria

Proteo

bacteria

Pseudo

mon

asaerugino

saPA

O1

FAS,

side

roph

oremetab

olism

40–42

Mup

NAAM12

928

Bacteria

Proteo

bacteria

Pseudo

mon

asuo

rescens

Mup

irocin

43SePP

T1

Q6T

710

Bacteria

Actinob

acteria

Saccha

ropo

lysporaerythraea

Erythromycin

44SePP

T2

A4F

C68

Bacteria

Actinob

acteria

S.erythraea

Erythromycin

44PigL

Q5W

260

Bacteria

Proteo

bacteria

Serratia

marcescens

Althiomycin

(NRPS

-PKS)

45Ps

wP

Q75

PZ2

Bacteria

Proteo

bacteria

S.marcescens

Althiomycin

(NRPS

-PKS)

45MtaA

AAF1

9809

Bacteria

Proteo

bacteria

Stigmatella

aurantiaca

Myxothiazol

46,47

Nsh

CNsh

-ORFC

Bacteria

Actinob

acteria

Streptom

yces

actuosus

Nosihep

tide

1EntD

-typ

eQ53

636

Bacteria

Proteo

bacteria

Salm

onella

austin

Enteroba

ctin

1Sim10

Q93

FA6

Bacteria

Actinob

acteria

Streptom

yces

antibioticus

Simocyclinon

e48

,49

ScAcp

SO86

785

Bacteria

Actinob

acteria

Streptom

yces

coelicolor

PKSan

dFA

S50

Red

UNP_

6300

04Bacteria

Actinob

acteria

S.coelicolor

PKSan

dFA

S51

SCO66

73NP_

6307

48Bacteria

Actinob

acteria

S.coelicolor

PKSan

dFA

S50

KirP

CAN89

630

Bacteria

Actinob

acteria

Streptom

yces

collinus

Kirromycin

52EntD

-typ

eP0

A3C

0Bacteria

Proteo

bacteria

Shigella

exneri

Enteroba

ctin

1Fd

mW

AAQ08

936

Bacteria

Actinob

acteria

Streptom

yces

griseus

Fred

ericam

ycin

53NysF

Q9L

4X7

Bacteria

Actinob

acteria

Streptom

yces

noursei

Nystatin

54EntD

-typ

eQ56

064

Bacteria

Actinob

acteria

Salm

onella

typh

imurium

Enteroba

ctin

1JadM

AAF3

4678

Bacteria

Actinob

acteria

Streptom

yces

venezuelae

Jado

mycin

55Svp

AAG43

513

Bacteria

Actinob

acteria

Streptom

yces

verticillus

Bleom

ycin

56Vab

DABG82

032

Bacteria

Proteo

bacteria

Vibrio

anguillarum

Van

chroba

ctin

57,58

AngD

YP_

0045

6669

8Bacteria

Proteo

bacteria

V.an

guillarum

Angu

ibactin

58Xab

AAAG28

384

Bacteria

Proteo

bacteria

Xan

thom

onas

albilinean

sAlbicidin

59NsP

PT,P

ptNs,

NhcS,H

etI

AAY42

632,

AAW67

221

Cyanob

acteria

Cyanob

acteria

Nod

ularia

spum

igenaNSO

R10

Glycolipid,

nod

ularin

14,60

NgcS

YP_

0018

6378

2Cyanob

acteria

Cyanob

acteria

Nostocpu

nctiform

ePC

C73

102/

ATCC29

133

Glycolipid

61

HetI

AAA22

003,

BAB77

058

Cyanob

acteria

Cyanob

acteria

Anab

aena

(Nostoc)

sp.P

CC71

20Glycolipid

61–64

OsP

PTZP

_071

0928

1Cyanob

acteria

Cyanob

acteria

OscillatoriaPC

C65

06Anatoxin-a,H

omoa

natoxin-a

65Sp

pT,H

etI

BAA10

326,

NP_

4422

56Cyanob

acteria

Cyanob

acteria

Synechocystissp

.PCC68

03FA

S66

66 | Nat. Prod. Rep., 2014, 31, 61–108 This journal is © The Royal Society of Chemistry 2014

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Tab

le1

(Contd.)

PPTase

Proteinaccessionno.

Section

Phylum

Species

Function,s

pecicity

Citation

Npg

A,C

fwA

XP_

6637

44Fu

ngi

Ascom

ycota

Aspergillusnidu

lans

NRPS

,PKS,

lysine

67–69

PptA

CAK46

165

Fungi

Ascom

ycota

Aspergillusniger

PKSan

dNRPS

70Pp

tAAY60

7103

Fungi

Ascom

ycota

Aspergillusfumigatus

PKSan

dNRPS

71,72

PptB

XP_

7465

91Fu

ngi

Ascom

ycota

A.fumigatus

mitoF

AS

73Lys5

AAO26

020

Fungi

Ascom

ycota

Can

dida

albicans

Lysine

74Pp

t1AER36

018

Fungi

Ascom

ycota

Cochliobo

lussativus

Lysine,

NRPS

,PKS

75Pp

t1DQ02

8305

Fungi

Ascom

ycota

Colletotrichu

mgram

inicola

Lysine,

NRPS

,PKS

232

Ppt1,F

fPpt1

HE61

4113

Fungi

Ascom

ycota

Fusarium

fujik

uroi

Lysine,

NRPS

,PKS

76Integrated

PPTases

Fungi

Ascom

ycota

Part

ofPK

Stype

Ior

FAStype

IFA

S20

PptasePc

hr,Pc

13g0

4050

XP_

0025

5884

1Fu

ngi

Ascom

ycota

Penicillium

chrysogenu

mLysine,

NRPS

,PKS

77Lys5

CAA96

866

Fungi

Ascom

ycota

S.cerevisiae

Lysine

1,78

,79

PPT2

Q12

036

Fungi

Ascom

ycota

S.cerevisiae

mitoF

AS

80New

8G2T

RL9

Fungi

Ascom

ycota

Schizosaccha

romyces

pombe

mitoF

AS(putatively)

8113

1415

4/Lys7

Q10

474

Fungi

Ascom

ycota

S.po

mbe

Lysine

1,82

Ppt1

EHK16

960

Fungi

Ascom

ycota

Trichod

ermavirens

Lysine,

NRPS

,PKS

83CpP

PTAAW50

594

Protista

Apicomplexa

Cryptospo

ridium

parvum

FAS

84DiAcp

SEAL6

9712

Protista

Mycetozoa

Dictyostelium

discoideum

mit-FAS

85DiSfp

EAL6

4498

Protista

Mycetozoa

D.d

iscoideum

PKSan

dFA

S85

SupC

A4U

8R1

Bacteria

n.d.

Aplysina

aeroph

obasymbion

tPK

S86

,87

LubD

F8S2

77Bacteria

n.d.

A.aeroph

obasymbion

tNRPS

87,88


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biotic and abiotic environments. In bacteria, pigments aregenerally not produced for color-mediated recognition, butinstead serve as UV protectants, antioxidants, siderophores orantibiotics. Soil-dwelling bacteria, like Streptomyces and Bacilli,produce many diverse secondary metabolites biosynthesized byelongating synthases, and therefore encode a variety of PPTasesthat activate synthase-mediated pathways. Even the mostcommon laboratory bacterium, E. coli K12, hosts more than onePPTase gene.

Escherichia coli. The genome of E. coli K12 contains threegenetically encoded PPTases: AcpS, EntD and AcpT. AcpS wasidentied by Elovson and Vagelos in 1968,3 shortly aer thediscovery of the FAS ACP (AcpP).89 Overexpression of AcpSallowed for detailed characterization of this enzyme.90

EntD is the PPTase of the enterobactin biosynthetic cluster(Fig. 3). Enterobactin91 is a siderophore that is secreted toscavenge iron from the environment for bacterial viability. Aerbiosynthesis, enterobactin is exported from the cytosol to theperiplasm, from the periplasm to the outside of the cell, andaer binding of iron, imported back into the bacterium.92 Thebiosynthetic Ent cluster and the natural product, then calledenterochelin, were isolated and studied in the early 1970s.93 Atthat time, it was known that the proteins encoded in the genecluster associate and require ATP and Mg2+ for activity. Thebiosynthesis starts with the conversion of chorismate to 2,3-dihydroxybenzoate catalyzed by EntA, B and C. Serine and 2,3-dihydroxybenzoate are then assembled into enterobactin byEntD-EntG, which encompass an NRPS cluster. Later, EntD wasoverexpressed by two laboratories independently and found tobe membrane localized, but no function could be assigned tothe protein.94–96 EntD was identied as a PPTase by Lambalotet al.1 and shown tomodify the PCP EntF. The C-terminal carrierprotein domain of EntB, a bifunctional isochorismate lyase,also served as a substrate for EntD.97 The assembly line enzy-mology of enterobactin and other bacterial siderophores hasbeen reviewed by Crosa and Walsh.98

The gene entD has been knocked out in E. coli (e.g. strainAN90-60).99 The resulting strain does not produce enterobactin,based upon visualization using low-iron CAS indicator plates,on which wild-type strains show a yellow halo and siderophore-decient strains do not. Overproduction of AcpS cannotcompensate the absence of EntD.100 Conversely, overexpressingentD on an inducible plasmid could not complement theabsence of acpS.100,101 However, in vitro EntD seems to modifyapo-AcpP from E. coli, albeit at a very slow rate.1 It is noteworthythat in acpS knockout B. subtilis strains, fatty acid biosynthesisis maintained, presumably due to the latent activity of Sfp infatty acid biosynthesis.102 Surprisingly, the specicity of EntDfor carrier proteins or CoA analogs has not been examined indetail. Chalut et al.36 observed that when expressing PKSmodules from mycobacteria in E. coli, some synthases werephosphopantetheinylated. To discover the protein responsiblefor modifying mycobacterial synthases in E. coli, an entDknockout mutant was constructed, which did not show 40-phosphopantetheinylation of PKS modules. So, although EntDis part of an NRPS (activating PCPs), the enzyme also seems tobe active on other elongating synthases carrying ACPs affording

Nat. Prod. Rep., 2014, 31, 61–108 | 67


Fig. 3 Biosynthesis of enterobactin and the corresponding gene cluster including the PPTase entD, labelled in red (GenBank acc. no.NP_415115.2) (data extracted from the E. coli genome with the GenBank acc. no. NC_000913.2). ICL represents isochorismate lyase; PCP,peptidyl carrier protein; C, condensation domain; A, adenylation domain; and TE, thioesterase domain.

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complementation of synthase activity in the absence of theircognate PPTases.

The third PPTase from E. coli, AcpT, was identied by Lam-balot et al.,1 but has been difficult to characterize.35 In E. coliO157, AcpT lies in the O-island 138 gene cluster which containsfatty acid biosynthesis-like genes, including two putative carrierproteins Z4853 and Z4854. When these ACPs were expressed inE. coli K12 (which lacks the O-island 138 gene cluster), 40-phosphopantetheinylation was independent of the presence ofAcpS but dependent upon the presence of AcpT. In AcpSknockout strains, AcpT is only able to restore very slow growth,suggesting a latent but low-level permissiveness to other non-cognate substrates. The gene cluster present in E. coli O157between yhht and acpT, which reside next to each other in E. coliK12 (thus lacking the O-island 138), consists of a set of inter-esting FAS-related enzymes (Fig. 4). Sequence alignment of theACPs Z4853 (or Ecs4328) and Z4854 (or Ecs4329) reveals theconserved serine for PPant modication, but with a motif thatcontains “DSI” instead of the typical AcpP DSL motif. This motifis also found in PUFA (poly-unsaturated fatty acid) synthases aswell as in type I PKS synthases.2

Fig. 4 Comparison of E. coli O157 (top) and E. coli K12 (bottom) in the

68 | Nat. Prod. Rep., 2014, 31, 61–108

Another pathogenic E. coli strain of the phylogenetic groupB2 produces a compound that induces DNA double-strandbreakage in eukaryotic cells.103 The biosynthetic origin of thistoxin was found in a hybrid PKS-NRPS cluster (also known as“PKS island”) producing colibactin (Fig. 5). Within this cluster,clbA was identied as a PPTase.103,104 Colibactin has never beenisolated and its structure remains unknown. However, recently,advances have been made in the elucidation of this natural-product structure. ClbP (Fig. 5) was identied as a member of aunique group of D-asparagine peptidases involved in thematuration of non-ribosomal peptides.105,106 ClbP was shown tobe membrane-bound and its enzymatic activity located in theperiplasm, suggesting biosynthesis of pre-colibactin in thecytosol, export to the periplasm and subsequent activation(thereby preventing toxicity to E. coli itself).107 The NRPS ClbNand the NRPS portion of ClbB were recently characterized invitro.108 ClbN synthesizes myristoyl-D/L-asparagine andClbB(NRPS) condenses L-Ala or L-Val onto this scaffold. ClbPcleaves offmyristoyl-D-Asn. Recently, in vivo, the clb gene clusterwas knocked-out and the metabolites analyzed. The onlynatural product found in the wild-type strain, which was not

region of the PPTase acpT (in red).



Fig. 5 Colibactin biosynthetic gene cluster with the PPTase gene clbA (GenBank acc. no. AM229678.1). NRPS genes: yellow, PKS genes: blue,PKS-NRPS hybrid genes: green, PPTase gene: red.

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present in the knockout strain, was myristoyl-D-Asn; the struc-ture of the full-length natural product remains elusive.109

Other E. coli strains harbor this cluster, including the non-pathogenic probiotic E. coli Nissle 1917.110,111 Screening 1565isolates by PCR revealed that the PKS island is also present inKlebsiella pneumoniae, Enterobacter aerogenes, and Citrobacterkoseri.112 There is considerable interest in colibactin, since itmight be that its biosynthesis is associated with colon cancer.113

E. coli Nissle 1917 is used as a probiotic and improves chronicinammatory bowel disease, but its mode of action is unknown.Interestingly, deletion of the PPTase clbA gene causes abolish-ment of its DNA damage activity, but at the same time also lossof its probiotic activity.114

The PPTase ClbA was recently characterized in moredetail.115 Besides enterobactin and colibactin, some E. colistrains also produce yersiniabactin. Yersiniabactin is encodedby the high-pathogenicity island and in contrast to Yersiniapestis (in Y. pestis YbtD is the dedicated PPTase)116 no PPTase isfound in the E. coli genome that seems to activate this synthase.In vitro and in vivo, EntD and ClbA can activate yersiniabactinand enterobactin synthases, but the colibactin synthase cannotbe activated by EntD. In vitro, YbtD, Sfp and PptT can activatecolibactin synthase. Interestingly, the PKS island and the high-pathogenicity island are strongly associated and it is hypothe-sized that this feature is selected in virulent E. coli speciesbecause the PPTase ClbA can activate both siderophore andgenotoxin biosynthesis (Table 2).115

Burkholderia. Burkholderia are prolic Gram-negativebacteria that are animal, human and plant pathogens.Burkholderia cenocepacia produces the siderophores ornibactinand pyochelin by NRPSs. The pobA gene was identied as aPPTase and shown to efficiently complement an E. coli entDmutant.18

Table 2 Overview of the E. coli synthases and PPTases. Phylogeneticallysequence of the PPTases and complete synthases, we usedMultiGeneBlain”). The designations ‘+++’, ‘++’ and ‘+’ correspond to decreasing activitto unknown

Synthase Present in

PPTases

AcpSA, B1, B2, C, D, E, S

FAS A, B1, B2, C, D, E, S +++3

Enterobactin A, B1, B2, C, D, E, S —Yersiniabactin B1, B2, D (A, E) —Colibactin B2 —O157 D, E —


Burkholderia pseudomallei is the causative agent of melioi-dosis, a serious infectious disease in humans, and is resistant tomany antibiotics. Su et al. identied bacterial antigens that areimmunogenic in the human host, with the hope that thesebacterial proteins were upregulated during infection. Interest-ingly, one of the 109 proteins upregulated during infection wasthe PPTase BPSS2266.119

Burkholderia rhizoxinica is an intracellular symbiont of theplant pathogen Rhizopus microsporus and produces the antimi-totic polyketide rhizoxin for its fungal host. Rhizoxin is bio-synthesized by a PKS-NRPS hybrid and is also made byPseudomonas uorescens Pf-5, which uses a similar gene cluster.In B. rhizoxinica, the rhi synthase is encoded on the chromo-some and not on one of its several megaplasmids, as previouslythought. However, it seems remnants of the synthase cluster arepresent on one of the megaplasmids, including the PPTase Brp(GenBank: RBRH_02776).120

Burkholderia thailandensis produces a series of quorum-sensing quinolones bearing an unsaturated medium chain fattyacid tail. The hmq gene cluster has been shown to mediate thebiosynthesis of these 4-hydroxy-3-methyl-2-alkylquinolones,showing close homology to the pqs gene cluster in Pseudomonasaeruginosa, which is responsible for 4-hydroxy-2-alkylquinolineproduction. Recently, HmqF was identied as the source of theunsaturated fatty acid, and contains adenylation and dehydro-genase domains along with an ACP.121 The PPTase that post-translationally modies this ACP is so far unknown, but at leastfour PPTases (apart from AcpS) have been annotated in thegenome of B. thailandensis.

In Burkholderia K481-B101,122 glidobactin is synthesized by aPKS-NRPS hybrid.123 Glidobactin is an N-acylated depsipeptidewith a 12-membered macrolactam ring that inhibits the pro-teasome. The unsaturated fatty acid chain of glidobactin is

E. coli is divided into several reference groups.117 Using the nucleotidest118 to identify the presence of these genes in E. coli genomes (“Presenty of PPTase on synthase, whereas ‘—’ corresponds to no activity and ‘?’

EntD AcpT ClbAA, B1, B2, C, D, E, S A, B1, B2, C, D, E, S B2

+1 +1 ?+++97 ? ++115

+115 ? ++115

— ? +++115

? +35 ?

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installed by a part of the synthase called GlbF consisting of acondensation-, adenylation-, and PCP-domain, requiring 40-phosphopantetheinylation.122 Here again, the PPTase that isnecessary for this post-translational modication is currentlyunknown.

Streptomyces. Streptomyces, the largest genus of actino-bacteria, are famous for their large variety of natural productsproduced ribosomally, by PKS, NRPS or other pathways.124 Forexample, Streptomyces coelicolor is a Gram-positive bacteriumthat contains 30 biosynthetic secondary metabolite clusters thatproduce natural products, including actinorhodin, coelichelin,coelibactin, TW95a, EPA, methylenomycin (encoded on a giantlinear plasmid), prodiginines, germicidins and the antibioticCDA (Fig. 6).124 All of these natural products are produced bysynthases that require phosphopantetheinylation. Cox et al.identied an AcpS-type PPTase in S. coelicolor, which shows asurprising level of permissiveness for CoA substrates as well ascarrier proteins.50 For example, type I mammalian FAS, type Ifungal PKS, and several type II bacterial ACPs were efficientlyphosphopantetheinylated. The genome of S. coelicolor containstwo other PPTases: RedU, which is a dedicated PPTase forundecylprodigiosin biosynthesis; and SCO6673, required forCDA biosynthesis.125 Further in vivo studies reveal that bothRedU and SCO6673 have specic carrier protein targets,whereas ScAcpS shows broad activity. Recently, the crystalstructure of ScAcpS was determined, showcasing the basis forrelaxed substrate selection (discussed in Section 5).126

The existence of many biosynthetic pathways in Strepto-myces species led to some controversy over whether certain

Fig. 6 Secondary metabolites of S. coelicolor that depend on PPTase aNRPS natural product (nrp-cys) and another type I PKS product.124,127

70 | Nat. Prod. Rep., 2014, 31, 61–108

peptidic natural products were ribosomally produced (lanti-biotics) or non-ribosomally synthesized. For example, Strepto-myces actuosus produces the antibiotic nosiheptide,128 part of alarge family of cyclic thiopeptides,129,130 thought to be non-ribosomally synthesized. However, it was recently shown thatthese macrocylic peptides are ribosomally synthesized andpost-translationally modied by a nosiheptide biosyntheticcluster (Nos A to P).131 In 1990, Li et al. discovered a proteincalled NshORFC that sits upstream from the Nos cluster,132

which Lambalot et al. computationally identied as a PPTase,renaming it NshC.1 Since these thiopeptides are made ribo-somally, their biosynthesis does not require a carrier protein orPPTase; so why does this Streptomyces species have an EntD/Sfp-like PPTase in its genome closely located to nosiheptideproduction? Closer evaluation of the Nos biosynthetic clusterreveals the presence of unassigned protein NosJ with a pre-dicted length of 79 aa. It contains the highly conserved DSLmotif of carrier proteins, which suggests that the ribosomallyproduced pre-nosiheptide is loaded onto a Nos carrier proteinbefore it is post-translationally modied. NosI, directlyupstream of NosJ, is indeed annotated by Yu et al.131 as anAMP-dependent acyl-CoA ligase, which may be capable ofloading the pre-peptide onto the carrier protein in an adeny-lation type of reaction.

The Nos gene cluster shows similarity to nocathiacin (Noc),thiostrepton (Tsr) and siomycin (Sio). In these three, no NosJhomologs have been annotated as of yet. However, PSI-BLASTanalysis of NosJ reveals homologous hits in the large putativehydrolase/transferase proteins NocK, TsrI and SioP (see

ctivity. antiSMASH identifies additionally butyrolactone (type I PKS), an



Fig. 7 Comparison of the nosiheptide and nocathiacin gene clusters. The adenylation domain is shown in green, the putative carrier protein inblue and the ribosomal pre-peptide in yellow. Sequence alignment of E. coli AcpP and nosJ (ACR48339.1), sioP (ADR01086.1), nocK(ACN52299.1) and tsrP (ACN80653.1) show the conserved (D/T)SL motif, characteristic of a carrier protein. Sequence alignment was made usingT-Coffee and ESPript.5,6

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sequence alignment in Fig. 7), which all contain a similarsequence to NosJ and are directly neighboring the putativeadenylation domain NosI (and its homologs). Although specu-lative, it seems that these biosynthetic clusters have both ribo-somally encoded and non-ribosomally encoded synthasecharacteristics. Future studies will tell how PPTases, in partic-ular Nsh-ORFC, t into this story.

Streptomyces vertilicus produces bleomycin, and no PPTase isassociated with its biosynthetic gene cluster. By similarity toother Streptomyces PPTases, the PPTase Svp was identied,cloned and overproduced in E. coli. Svp is a catalyticallypromiscuous PPTase, active on both type I and type II ACPs aswell as some PCPs.56

Streptomyces noursei produces nystatin using a type Imodular polyketide synthase. At the 50 border of the biosyn-thetic cluster, a putative PPTase gene, nysF, was discovered.However, upon deletion of this gene, an increase in nystatinproduction was observed, suggesting a regulatory role for thisprotein.54 Since nysF has not been characterized in more detail,it is unclear whether this protein is a bona de PPTase, a regu-latory gene product, or both.

Streptomyces collinus Tue 365 produces kirromycin, a potentantibiotic that blocks translation in bacteria by interfering withthe elongation factor Ef-Tu.52 Kirromycin was discovered in1974 by Wolf et al.,133 and >200 publications have discussed itsbiosynthesis and mechanisms of action. In 2008, Weber et al.characterized the PKS-NRPS hybrid in detail and identied thePPTase KirP, essential for phosphopantetheinylating the largenumber of acyl- and peptidyl-carrier proteins.134 Interestingly,inactivation of KirP does not result in the total abolishment ofkirromycin biosynthesis.52


Streptomyces griseus produces fredericamycin, and fdmW hasbeen identied as a PPTase, within this PKS gene cluster.53

Inactivation of FdmW resulted in a 93% reduction of freder-icamycin production. Streptomyces antibioticus produces theamino-coumarin antibiotic simocyclinone, using 38 ORFs.48,49

Interestingly, two different PPTases (simA11 and simC8) areassociated with the biosynthesis of this natural product.SimA11 is similar to JadM and simC8 is similar to NysF(Table 1), but so far the carrier protein targets of these PPTasesare unknown.

The genome of Streptomyces avermitilis contains a stunning85 separate carrier proteins, from which some are involved in>70 PKS/NRPS synthase biosynthetic systems.135,136 The S. aver-mitilis genome also reveals four distinct PPTases, which havelow sequence similarity and seem to represent differentsubclasses of the Sfp-type family of PPTases.

Recently, dedicated PPTases have also been found associatedwith pactamycin biosynthesis (PctR) in Streptomyces pactum,137

A74528 biosynthesis (SanW) in Streptomyces sp. SANK61196,138

tirandamycin biosynthesis (TrdM) in Streptomyces sp.SCSIO1666,139 jadomycin biosynthesis (JadM) in S. venezuelae,55

oviedomycin biosynthesis (OvmF) in S. antibioticus ATCC11891,140 griseorhodin biosynthesis (GrhF) in Streptomyces liv-idans,141 and natamycin biosynthesis in Streptomyceschattanoogensis L10.142

Bacilli. Bacilli are Gram-positive bacteria that are usedextensively in agriculture for crop protection, due to their abilityto produce natural products that protect germinating seeds.This group of protective molecules are generally made byNRPSs, which require 40-phosphopantetheinylation. ThePPTase Sfp was rst identied serendipitously by Nakano

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et al.143 in attempts to transform the non-surfactin producingstrain B. subtilis JH642 into a surfactin producer. Interestingly,although B. subtilis JH642 does not produce surfactin, the sur-factin gene cluster is transcribed and expressed. It was shownthat a frameshi mutation led to expression of a truncated,inactive, Sfp protein of 165 aa.144 In 1994, Borchert et al.13

identied a gene from B. brevis, gsp, located directly upstreamfrom the gramicidin synthase gene, with 34% identity and 54%homology to Sfp. Gsp complements a Sfp deciency in a B.subtilismutant, but until 1996 its function remained unknown.1

Bacilli species produce several non-ribosomal peptides,including surfactin and iturin A.145 Before the identication andcharacterization of PPTases, Huang et al.30 discovered an openreading frame in B. subtilis RB14 that regulated the productionof iturin A. This ORF named lpa-14 showed homology to Sfp.Another B. subtilis strain, YB8, produces the lipopeptides sur-factin and plipastatin B1, both biosynthesized by their respec-tive NRPSs, and phosphopantetheinylated by lpa-8, which isbetter known as Sfp.29 B. subtilis strain B3 produces fengycinusing another NRPS, which is activated by lpa-B3 (lpa-14).31 TheB. subtilis strain RP24, isolated from the rhizoplane of a eld ofpigeon pea, possesses antifungal activity. Iturin A, surfactin andfengycin were identied in this agricultural isolate, which alsocontains a homolog to lpa-14 found to be the PPTase.146 Bacilluslichenformis produces bacitracin by an NRPS, and Bli is thePPTase that phosphopantetheinylates the PCP domain of thiselongating synthase.33,34 Bli was later expressed in E. coli andused to phosphopantetheinylate a domain from tyrocidinesynthase in vitro.32 Finally, the B. pumilus A-1 gene psf-1 wasfound to regulate the production of surfactin and later shown toencode an active PPTase.27

Many bacilli species, including Bacillus anthracis and somemarine bacteria, produce the siderophore petrobactin, which isbiosynthesized by a hybrid NRPS/non-NRPS siderophore syn-thase.147 The synthase-encoding cluster contains a stand-alonePCP domain, AsbD, which is phosphopantetheinylated by anunknown PPTase. There is no PPTase present in the genecluster itself and it has been suggested that BA2375, an EntDhomolog present in the enterobactin gene cluster, serves as thePPTase that installs the 40-phosphopantetheine arm on AsbD.148

Holo-AsbD is loaded with 3,4-dihydroxybenzoic acid by AsbCand this AsbD conjugate functions as the substrate for AsbE.AsbE, together with the stand-alone synthases AsbA and AsbB,catalyze the formation of petrobactin.

Mycobacteria. Like many bacteria, mycobacteria secretesiderophores to harvest iron from their hosts. These side-rophores are peptide-based and fall into two categories: thewater-soluble exochelins and the membrane-associated myco-bactins. These molecules have very similar core structures, butdiffer in the length of their fatty acid side chain (the water-soluble siderophores have a short C5-C9 chain and themembrane-associated siderophores a long C18-C21 chain). Bothof these siderophores are found in numerous bacteria, and inall cases are produced by the expression of NRPS gene clusters.During the characterization of the biosynthetic cluster ofmycobactin from Mycobacterium tuberculosis, PptT was identi-ed as the PPTase for the mycobactin synthase.38

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In addition to the mycobactin synthase, more than 18 type Ipolyketide synthases and two FAS genes have been annotated inthe genome ofM. tuberculosis, enabling this bacterial species togenetically encode a large variety of unusual membrane lipidsfor protection.149 For example, mycolic acid production relies ontwo FASs and one PKS. The presence of two FASs in oneorganism is not common across all three domains of life, but ismore prevalent in eukaryotes and protista. For M. tuberculosis,the second FAS (dedicated to mycolic acid biosynthesis) is dis-cussed in more detail in Section 4. The large number of carrierproteins in these synthases are post-translationally activated byPPTases. MtAcpS is responsible for phosphopantetheinylationof the two FASs and PptT for the phosphopantetheinylation ofthe NRPS and PKSs.36 Interestingly, a PptT knockout strain inMycobacterium bovis BCG is not viable, suggesting that thisPPTase is essential for organismal viability (although AcpS ispresent).

X-ray crystal structures have been published of MtAcpS,150

showing that the protein undergoes conformational changes atpHs >6.5, with resultant decreases in AcpS activity.151 Theintracellular pH of mycobacteria is between 6.1 and 7.2, evenwhen exposed to acid or base,152 and although purely specula-tive, it might be that siderophore or other natural-productproduction is regulated by the activity of the PPTase.

Recently, Leblanc and co-workers delved deeper into therequirement of mycobacteria to express PptT.37 Two conditionalpptT mutants in M. bovis BCG and M. tuberculosis H37Rvshowed retarded growth and persistence. Mutants in which thePPTase gene was controlled by a tetracycline promoter wereconstructed, allowing for conditional regulation of the PptTexpression. A 95% depletion of PptT was required to inhibitgrowth of M. bovis. Although the constructs in M. bovis and M.tuberculosis were identical, much higher concentrations oftetracycline were required for growth of M. tuberculosis, sug-gesting that either different tetracycline uptake rates, differentregulation of expression or higher levels of PptT are required forgrowth of M. tuberculosis. Nevertheless, PptT is necessary for invitro growth of mycobacteria, but the authors point out thatalthough essential in vitro, mycobacteria have been shown torequire enzymes in vitro that are not required in vivo. Thus, PptTknock-down strains were also tested for viability in macrophageand mice infections. Both knock-down mutant M. bovis and M.tuberculosis strains fail to multiply in vivo. Although PptTappears to be an excellent anti-mycobacterial target in vitro andin vivo, the cumulative effects of PPTase depletion are stillunknown, since mycolic acid, mycobactin, polyketide-derivedlipids, fatty acids, siderophores and some yet to be discoverednatural products all depend on PPTase activity for biosynthesis.

Recently, we identied a PPTase inM. ulcerans (unpublisheddata), MuPpt, presumably responsible for phosphopantethei-nylating mycolactone synthase. M. ulcerans is a human path-ogen and the causative agent of Buruli ulcer. The core ofmycolactone,153 a cytotoxic and immunosuppressive naturalproduct, is biosynthesized by two large PKSs (MLSA1, 1.8 MDaand MLSA2, 0.26 MDa) both of which are encoded on a largeplasmid (174 kb), pMUM001.154 The plasmid does not contain aPPTase, thus the synthases must be modied by MuPpt.



Fig. 8 Prodigiosin biosynthesis requires both PPTases PswP and PigL.156 Prodigiosin biosynthetic gene cluster (GenBank acc. no. AJ833002.1)including the PPTase gene pigL, labelled in red.

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Serratia. The biosyntheses of the red pigment prodigiosin andthe surfactant serrawettin W1 in S. marcescens depend on thepresence of the PPTase PswP.155 Mutants of PswP are unable toproduce pigment or surfactant but are still able to produce 2-methyl-3-n-amyl-pyrrole (MAP) and condense it with supplied 4-methoxy-2,20-bipyrrole-5-carbaldehyde (MBC) to form prodigiosin(Fig. 8). Serratia species appear to employ two PPTases, one toactivate the PCP of PigG and one to activate the ACPs of PigH.Similarly, in S. coelicolor, which also produces prodiginine, thePPTase RedU is responsible for 40-phosphopantetheinylation ofRedO but not for the other PCP and ACP domains.51 BesidesPswP, PigL has also been characterized as a PPTase.156

The insect pathogen S. marcescens Db10 also producesanother antibiotic, althiomycin, that inhibits growth of both B.subtilis and Staphylococcus aureus.45 Althiomycin is synthesizedby a PKS-NRPS hybrid and additional tailoring enzymes. TwoSfp/EntD-type PPTases were identied in S. marcescens, and aknockout mutant for each was constructed. Althiomycinproduction was eliminated upon deletion of one PPTase gene,SMA2452, whereas the other PPTase gene mutation, SMA4147,had no effect. Sequence alignments and PSI-BLASTing of pswPand pigL, previously identied as PPTases, shows that SMA2452is PswP. The althiomycin biosynthetic cluster has previouslybeen found in actinomycetes and Myxococcus, evolutionarilyunrelated bacteria, suggesting that S. marcescens, which isclosely related to E. coli, likely obtained this gene by horizontalgene transfer in either direction.


Recently, three broad-spectrum antibiotics were isolatedfrom Serratia plymuthica RVH1, called the zeamines. The genecluster responsible for their biosyntheses contains FAS, PKSand NRPS characteristics and resembles the pfa gene cluster,responsible for PUFA biosynthesis in marine bacteria. Apotential PPTase, Zmn5, was identied in the zeamine genecluster, which does not show homology with the dedicatedPUFA synthase PPTase PfaE, but instead shows a clearhomology with EntD and Sfp.157

Vibrio. Some Vibrio anguillarum strains produce the side-rophore vanchrobactin utilizing the NRPS VabF. VabD is thededicated PPTase used for 40-phosphopantetheinylation ofVabF.58,158 A VabD knockout strain shows no retarded growthunder iron-rich conditions, but reduced growth under iron-depletion.57 V. anguillarum strains also contain a second, moredominant, siderophore anguibactin, located on the pJM1plasmid. Anguibactin is also synthesized by an NRPS andrequires the PPTase AngD. Both VabD and AngD can complementone another, but since anguibactin is the more potent side-rophore, some strains have lost the vanchrobactin biosynthesispathway.159 Vibrio cholerae is a bacterial pathogen that bio-synthesizes a unique siderophore, vibriobactin, using biosyn-thetic machinery similar to enterobactin synthase. Both VibB andVibF are phosphopantetheinylated by the PPTase VibD.158,160

Pseudomonas. The genome of P. aeruginosa contains one Sfp-type PPTase, PaPcpS (previously named “PcpS”), with broadsubstrate specicity. Like Haemophilus inuenzae and

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cyanobacteria, no AcpS-type PPTase can be identied in thegenome.41 P. aeruginosa is a Gram-negative human pathogen thatis resistant to antibiotic treatment. Two siderophores, pyoverdinand pyochelin, are produced by NRPSs and associated with thevirulence of these bacteria. Pyoverdine biosynthesis requiresphosphopantetheinylation of PvdD, PvdI and PvdJ and pyochelinbiosynthesis requires activation of PchE and PchF. PaPcpS wasidentied by similarity to EntD, AcpS and Sfp, although the 242 aaprotein shows only 13% similarity to Sfp, and in contrast to Sfp isnot located in close proximity to a synthase. PaPcpS acts on FAS,PKS and NRPS acyl carrier proteins, but the PPTase is optimizedfor primarymetabolism, since its activity drops 30-fold for ACPs orPCPs from secondary metabolism in vitro.41 Construction of aPaPcpS knockout was unsuccessful, suggesting that the protein isessential. PaPcpS can genetically complement mutations in AcpSand EntD in E. coli, and it can also complement an Sfp mutant inB. subtilis.100 A PaPcpS knockout P. aeruginosa strain could only beconstructed in an E. coli AcpS (EcAcpS)-carrying strain, suggestingthat the essentiality of PaPcpS originates from its function inprimary metabolism. This mutant strain also shows no side-rophore production and does not grow in the presence of ironchelators.

Since Pseudomonas species are possibly good heterologoushosts for the production of natural products, Gross et al.screened six different carrier proteins from large synthases forefficient 40-phosphopantetheinylation by the endogenousPPTases. Indeed, the broad specicity of the single PPTasepresent in Pseudomonas sp. could be used to 40-phosphopante-theinylate various carrier proteins.42

Pseudomonas syringae produces the toxin coronatine, madeby a synthase requiring PPTase activity on two individual ACPs,two ACP domains and one PCP. The single PPTase fromP. syringaewas identied (PspT) and shown to have 62% identitywith PaPcpS. Interestingly, this PPTase, although having broadactivity, shows preference for secondary metabolism carrierproteins, in contrast to PaPcpS.161

Genome analysis suggests that other Pseudomonas speciesalso utilize one PPTase, although substrate specicity mightvary.43 P. uorescens produces the antibiotic mupirocin (pseu-domonic acid), which is thought to be biosynthesized by fourlarge type I PKS/FASs and a number of modifying enzymes.Eleven ACPs in the type I synthases and ve putative type IIACPs require 40-phosphopantetheinylation, and a putativePPTase in the gene cluster, MupN, has been identied.162

Indeed, when mupN was deleted, mupirocin production wasabolished, and it was shown in vitro that both type I and type IIACPs were modied by this PPTase.43 In contrast to P. aerugi-nosa, P. uorescens has two PPTases, PfPcpS and MupN, at itsdisposal, and it remains a question whether PaPcpS and PfPcpShave different substrate specicity.

Myxobacteria. Myxobacteria are natural-product factories.163

Myxothiazol was found in Stigmatella aurantiaca, epothilone inSorangium cellulosum and Myxococcus xanthus produces severalpolyketides and PKS-NRPS hybrid natural products.164 The Mtagene cluster is responsible for myxothiazol production and thePPTase MtaA for the 40-phosphopantetheinylation of this PKS-NRPS hybrid. Mutation of MtaA results in abolishment of the

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production of many metabolites, including myxothiazol.46 MtaAhas a relatively broad carrier protein substrate scope.47 Inter-estingly, M. xanthus DK1622 expresses two functionally redun-dant PPTases (MxPpt1, having�60% sequence identity to MtaA,and MxPpt2), both with relatively broad substrate specicity,supported by the fact that active myxothiazol-synthase andepothilone-synthase could be expressed in M. xanthus withoutthe need of coexpression of another PPTase.164

Xanthomonas albilineans. The plant pathogenic bacteriumX. albilineans is the causative agent of sugar cane leaf scald, andimportant factors in this disease are the albicidins produced bythe bacteria.165,166 Albicidins inhibit DNA replication in bacteriaand plastids. Albicidin is partially characterized, but due to theextremely low isolation yield, the biosynthesis is better under-stood than its chemistry.167 In 2000, a PPTase gene xabA wasidentied in the genome of X. albilineans and shown to beessential for production of the antibiotic, suggesting that thecompound is made by a synthase.59 A xabA knockout did notproduce albicidin, but when EntD was engineered into themutant strain, production was restored to the same levels as thewild-type strain. Albicidin is indeed produced by a large syn-thase, XabB, which is a 500 kDa PKS-NRPS hybrid synthase.168

The gene cluster was characterized in detail, and a theoreticalbackbone structure of the natural product was proposed.169

Although the PPTase XabA is essential for albicidin production,it is currently unknown whether this PPTase acts on all vecarrier proteins. To increase the production of albicidin, theXab gene cluster was expressed on two plasmids in Xanthomo-nas axonopodis pv. vesicatoria, and showed a 6-fold increasedyield of the antibiotic.170 Since non-producing albicidinmutantsare still plant pathogenic, Gabriel and Royer’s groups havesystematically studied the other factors that inuence patho-genicity.165,166 PPTase knockout mutations indeed showed noalbicidin production, but did not eliminate sugar cane leafscald. More dramatic effects were seen when transport proteins,OmpA family proteins or other (hypothetical proteins) weremutagenized. Recently, another three large NRPS clusters havebeen identied in X. albilineans genome, all with unknownfunction.171 Since there are multiple PPTases found in thegenome of X. albilineans, it is possible that, although hamperedby the XabA knockout, other PPTases still phosphopantethei-nylate carrier proteins in these NRPSs, contributing topathogenicity.

Other bacteria. The Gram-negative enterobacteria Salmo-nella produces enterobactin, which is a cyclic peptide side-rophore secreted under iron-deprevation.172 Lambalot et al.,1

identied three PPTases from Salmonella species as EntD-typeenzymes, but no actual Salmonella PPTases have been expressedor characterized.

The antibiotic erythromycin is made by the bacterium Sac-charopolyspora erythraea, using a modular polyketide synthasecontaining seven ACPs. Three PPTases have been identied inthe genome of S. erythraea, namely SeAcpS, SePptI and SePptII.In vitro characterization of these PPTases showed that SeAcpS isresponsible for FAS activation, SePptI is an integrated part of amodular PKS unit and SePptII is a stand-alone enzyme acti-vating an ACP-TE didomain of erythromycin synthase.44 The



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function of SePptI remains unknown but the genome ofS. erythraea contains four NRPSs and three PKSs, from whichthe products are unknown.

The causative bacterium of the plague is Y. pestis. Nine geneshave been identied in the high-pathogenicity island withNRPS/PKS character and yersiniabactin transport. Within thepgm locus no PPTases were found but by similarity to EntD, ybtDwas identied as a PPTase, and deletion of the ybtD generesulted in a strain decient in siderophore production.116

Poly-D-3-hydroxyalkanoates (PHAs) are biopolymers (poly-oxoesters) synthesized from CoA thioesters by the pha genecluster, found for example in the bacterium Ralstonia eutropha.The pha gene cluster consists of PhaA, a b-ketothiolase, PhaB,an acetoacetyl-CoA reductase, and PhaC, the synthase/poly-merase. Heterologous expression of the PHA gene cluster in E.coli resulted in the production of PHA.173 Feeding [3H]-b-alanineto a PHA-expressing and b-alanine auxotroph E. coli strain gavefour radioactive bands, corresponding to EntF, EntB, ACP andthe PHA synthase, suggesting that PhaC is phosphopantethei-nylated.173 However, construction of b-alanine auxotrophR. eutropha strains and feeding of [14C]-b-alanine did not showany labeled protein, except ACP.174 Two site-directed mutants ofconserved serine residues in PhaC had no in vivo or in vitrosynthase activity.174 However, when these serines were mutatedto alanine, [3H]-b-alanine was still incorporated in PhaC.175

Incubation of puried PhaC with [3H]-CoA and either of thePPTases ACPS, EntD, AcpT or Sfp did not result in labeledprotein.175 It remains thus the question whether this synthaserequires PPTase mediated activation.

Recently, more PPTases have been found (but not described indetail) in other bacteria, including those involved in factumycinbiosynthesis (FacP) in Acinetobacter baumannii,176 guadinominebiosynthesis (GdnS) in Streptomyces sp. K01-0509,177 pelgipeptinbiosynthesis (PlpC) in Paenibacillus elgii,178 aureusimine biosyn-thesis (AusB) in S. aureus,179–181 emetic toxin production (CesP) inBacillus cereus,182 yersiniabactin-related siderophore biosynthesis(NrpG) in Proteus mirabilis,183 quorum-sensing-related metaboliteproduction in Dickeya dadantii (VfmJ),184 siderophore cupriache-lin production in Cupriavidus necator H16 (CucB),185 siderophoretaiwanchelin production in Cupriavidus taiwanensis LMG19424(TaiQ),186 and the production of a metabolite involved in tobaccohypersensitive response and grape necrosis by Agrobacterium vitisF2/5 (F-avi5813).187

Nocardia. Some bacterial species can reduce aromaticcarboxylic acids to their corresponding alcohols (Fig. 9). A(promiscuous) carboxylic reductase (CAR) was isolated and

Fig. 9 Carboxylic acid reductase (CAR). CAR is a promiscuous enzymealdehyde. Carboxylic acids are first activated by an adenylation domainfashion reduced from the carrier protein by the reductase domain (R).alkanes.189


puried from Nocardia sp. NRRL 5646 and appeared to be a 128kDa protein that requires Mg2+, ATP and NADPH.188 When thecarboxylic reductase was expressed in E. coli, a 50-fold decreasein activity was observed. Incubation of the enzyme with CoA andcell-free extract resulted in an increase in activity. Combinedwith the presence of a “DSL” sequence-containing proteinbetween the adenylation domain and reductase domain, it wasthought that lack of 40-phosphopantetheinylation of the carrierprotein was the reason for the reduced activity. Indeed, in theNocardia strain a putative Sfp-type PPTase was identied, npt,cloned and expressed and shown to activate the reductase.Doubly-transformed E. coli with Npt and the reductase CARshows efficient transformation of vanillic acid to vanillin, incontrast to the non-transformed strain.39 Recently, this enzymehas received considerable interest in the realm of biofuels and itwas shown that overexpression of Sfp, reductase CAR (fromMycobacterium marinum) and an aldehyde reductase in E. coliresulted in the production of fatty alcohols.189

Photorhabdus luminescens. P. luminescens is a bacteriumsymbiotic with the nematode Heterorhabditis bacteriophora.Together, they infect insect larvae. The nematode needs thebacteria for pathogenicity, growth and reproduction, and thebacterium needs the nematode for spreading between insectprey. Only active bacteria can provide the factors necessary forthe nematode and dead cells or supernatants do not. Muta-genesis of the bacteria and subsequent screening for non-complementing behavior of the nematode revealed a mutantthat consistently failed to support the nematode. This mutantwas defective in siderophore and antibiotic biosynthesis andcontained a mutation in an EntD-like protein.190 Since thebacteria were still viable, this ngrA gene is not essential, butmost likely involved in the biosynthesis of some form ofhormonal, regulatory or signaling molecules made by an NRPSor PKS. Ciche et al. later showed that the inability of the bacteriato produce siderophores (including the newly identied side-rophore photobactin) did not hamper the nematode’s growthbut a mutation in ngrA did, suggesting that the biosynthesis ofsome other compound(s) requires the PPTase.191 P. luminescensalso produces bacteriocins and an antibiotic stilbenecompound that is biosynthesized via a unique ACP-dependentbiosynthetic pathway, unlike the canonical stilbene pathway inplants that utilizes type III PKSs.192 Although type II PKS clustersare rare in Gram-negative bacteria, Photorhabdus also producesan anthraquinone formed from a heptaketide precursor usingthis route.193 Within this cluster a dedicated PPTase, AntB, isfound, which cannot be replaced by NgrA.194 In the genome of P.

that reduces several (R0¼) acyl and aromatic carboxylic acids to an(A), loaded onto the carrier protein (CP) and in a NADPH-dependentThe aldehyde product can further be processed to yield alcohols or

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luminescens, ngrA sits in between two biosynthetic clusters: phfand hpa. The phf genes show close similarity to mbrialproteins, which are involved in adhesion. The hpa cluster isinvolved in hydroxyphenylacetate metabolism.195

Recently, Photorhabdus asymbiotica has been found inhuman infections in North America and Australia. Detailedgenomic comparison shows that insect virulence factors dis-appeared from its genome, but new human virulence factorsappeared from other human pathogenic bacteria. A closehomolog of ngrA is present in P. asymbiotica, making this aninteresting antibiotic target.196

Marine bacteria associated with sponges. Many naturalproducts are isolated frommarine sponges. Oen, these naturalproduct are not produced by the sponge, but by a bacterialecosystem living in the sponge. These bacteria are notoriouslydifficult to culture in a laboratory environment. In some cases,however, these bacteria have been studied in more detail. Forexample, when the metagenome of the sponges Theonellaswinhoei and Aplysina aerophoba were studied, PKSs were foundthat belong to the bacterium phylum Deinococcus thermus andproduces methyl-branched fatty acids.87 The gene cluster supwas identied and supC was annotated as a PPTase similar toPPTases from a sponge symbiont and the PPTase from cyano-bacterium Synechocystis sp. strain PCC6803. Later, SupC wascloned and expressed and shown to activate SupA-ACP.86 SupA-ACP is also activated by Sfp and Svp, but not by EcAcpS.Recently, two NRPSs were uncovered by genomic mining ofmarine sponges, one of which is abundant inmany sponges andshows affiliation with actinomycetes. The other NRPS wasfound in A. aerophoba and could be attributed to Chloroexisponge bacterial symbionts. Adjacent to the NRPS, a PPTase wasidentied (lubD), showing similarity to a PPTase from Methyl-obacterium nodulans.88

Marine bacteria and algae. Some marine bacteria and algaeproduce long-chain polyunsaturated fatty acids (PUFAs) likeeicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) by aunique pathway, different from the traditional FAS or elongase/desaturase pathways. These PUFAs are synthesized de novo by aPKS. Genetically, the bacterial and algal PKSs are organizeddifferently, but both require a PPTase. In the marine bacteriaShewanella sp. strain SCRC2738, ve open reading frames wereidentied that were sufficient to produce EPA in E. coli.197 In alandmark paper, Metz et al. characterize these ve open readingframes as PKS genes.198 Schizochytrium is a marine algae thatproduces large amounts of DHA, and genome analysis alsorevealed similar PKS genes in this eukaryote.198 The ve ORFsresponsible for PUFA production in Shewanella were laterrenamed as pfaA–E (Fig. 10), from which pfaE is a PPTase.199

Fig. 10 PUFA-PKS biosynthetic gene cluster (GenBank acc. no. EU7196

76 | Nat. Prod. Rep., 2014, 31, 61–108

Moritella marina produces DHA, and the pfaE gene cloned out ofthis organism could complement a PfaA-D-expressing E. colistrain.199 Recently it was shown that an EntD overexpressing E. colistrain can complement a PfaE-decient PUFA-synthase cluster.200

3.2 Archaea

Archaea form a third domain of life. The cell membranes ofarchaea are very different compared to bacteria and eukaryotes.Instead of using glycerol-ester lipids, archaea use glycerol-etherlipids; the stereochemistry of the glycerol moiety is reversed,and the hydrophobic tails are isoprenoids (containing cyclo-propane and cyclohexane rings). The total amount of fatty acidsisolated from archaea is thus minimal, and archaea seem tolack a traditional FAS.201 However, recently, an ACP-indepen-dent FAS was found, encompassing all of the bacterial FASenzymes except the ACP.202,203 The only published data on thePPTases of archaea are by Copp and Neilan, who identiedthree PPTases in archaeal species, namely Methanosarcina ace-tivorans, M. barkeri and M. mazei, clustering closely with Sfp.14

For this review, we PSI-BLASTed the PPTases EcAcpS, Sfp,Lys5, EntD, HetI and AASDHPPT against all archaeal genomesdeposited to date and found several convincing hits (Fig. 11). Toour surprise, we found both Sfp-type and AcpS-type PPTases. Wecombined these data with identication of PPant binding sitesusing ArchSchema204 and antiSMASH127 analyses of archaealgenomes for natural-product synthases. The archaea that encodeAcpS homologs do not seem to encode a traditional elongatingsynthase, but both Methanoregula boonei and Methanospirillumhungatei encode a rather unique –AcpS-ACP–‘AMP-dependent-ligase’–‘acyl-protein-synthetase/LuxE’–‘acyl-CoA-reductase’–DH–

cluster, all encoded on separate genes. Two of the Sfp-type PPTaseencoding archaea, Methanobrevibacter ruminantium and Meth-anocella paludicola, encode, large and rather unusual, NRPSclusters. Interestingly,Methanosarcina species contain an Sfp-typePPTase but no identiable synthases. Expression, characteriza-tion and identication of target carrier proteins is needed tounderstand the extent of PPTase diversity in archaea.

3.3 Cyanobacteria

Cyanobacteria are photosynthetic bacteria that, along witheukaryotic microalgae, are responsible for up to 50% of theworldwide carbon xation by photosynthesis (transversion ofatmospheric carbon dioxide into organic compounds).205 Somecyanobacterial species are a rich source of functionally andstructurally diverse natural products with various pharmaceu-tical applications.206 In the environment, these strains are alsoof global concern due to their ability to form “harmful algae

04.1) including the PPTase gene pfaE, labelled in red.



Fig. 11 Sequence alignments of archaeabacterial PPTases. Only the active-sites are shown. Bs is B. subtilis, Ec is E. coli. Sequence alignment wasmade using T-Coffee and ESPript.5,6

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blooms”. Cyanobacteria produce potent toxins such as saxitoxinand paralytic shellsh poisoning toxins that have a negativeeffect on the environment. Further, many cyanobacteria areknown for their unique ability to x atmospheric nitrogen. Thisprocess is located in a dedicated cell type, called heterocysts.Heterocysts are embedded in a glycolipid layer providing theanoxic environment essential for the activity of nitrogenase.207

Both processes – toxicity and nitrogen xation – are potentiallycontrolled by PPTases.

The majority of PPTases discovered in cyanobacteria are ofthe Sfp-type.14 These Sfp-like PPTases may be solely responsiblefor primary and secondary metabolism in cyanobacteria sinceAcpS-type PPTases are completely absent.14,60,66 A large-scalephylogenetic analysis of cyanobacterial PPTases showed that allcurrently described cyanobacterial PPTases fell within the W/KEA subfamily of the Sfp-type PPTases.14 A distinct clade ofcyanobacterial PPTases are involved in heterocyst differentia-tion. These include NsPPT in Nodularia spumigena NSOR10,60

HetI in Nostoc sp. PCC 712061–63 and NgcS in Nostoc punctiformeATCC 29133.61 These PPTases from heterocyst-forming cyano-bacteria were described as phylotype A, in contrast tophylotype B, which includes PPTases found in Prochlorococcus,Synechococcus and Gloeobacter. Besides understanding thenative function of cyanobacterial PPTases, they were furtherevaluated for their ability to activate non-cognate carrierproteins.

Unaware of its function, Wolk and Black reported the rstidentication of the cyanobacterial PPTase, HetI of Anabaena(also known as Nostoc) sp. PCC 7120.63 Knockout attempts werenot successful, leading to the hypothesis that HetI may berequired for maintaining vegetative growth. Further, the hetIgene is associated with a PKS gene cluster necessary forheterocyst glycolipid production.61,64,208 A genome-wide expres-sion study documented increased HetI levels under nitrogenstarvation,209 although overexpression of HetI had no inuenceon heterocyst formation.62

The genome of the closely related species N. punctiformePCC 73102 (ATCC 29133) contains three putative PPTase genes,each embedded within a unique gene cluster (Fig. 12).61 This


expansion of PPTases may be associated with the large genome(9.1 MB, http://www.jgi.doe.gov/), as well as the biosyntheticpotential of N. punctiforme for natural products. With 21 puta-tive PKSs, NRPSs and hybrid gene clusters, this strain’sbiosynthetic capabilities exceed those of any other describedcyanobacterium.210 Thus, the three PPTases are the potentialcore activators of an extensive natural-product biosyntheticmachinery.

The rst PPTase, NgcS (GenBank acc. no. YP_001863782),shows high sequence similarity to HetI of other cyanobacteria(Fig. 13) and is associated with the glycolipid-related HetMNIgene locus (Fig. 12A). It appears dedicated to its native glyco-lipid biosynthetic pathway, since it showed little activitytowards other cyanobacterial CPs.61 Despite high sequencesimilarity to other HetI-type PPTases (Fig. 13), the other twoPPTase genes (GenBank acc. no. YP_001865721, YP_001865651)identied in N. punctiforme are located within a putative PKSand a FAS gene cluster (Fig. 12B, C). All three proteins arephylogenetically distinct, demonstrating that PPTase phylogenyis not necessarily concordant with organismal phylogeny.61

In N. spumigena NSOR10, NsPPT, a homolog to HetI, is theonly PPTase present and displays a broad CP substrate accep-tance.60 It is involved in heterocyst glycoplipid and nodularintoxin synthesis. NsPPT further is able to activate the glycolipidsynthase NpArCP and nostopeptolide PKS NpACP of N. puncti-forme, the microcystin NRPS MPCP of Microcystis aeruginosa60

and SACP, the AcpP of Synechocystis sp. PCC6803 in vitro.66

In comparison to the previously described cyanobacteria,Synechocystis sp. PCC 6803 is not known to synthesize NRPS- orPKS-derived natural products.211 Its lone PPTase Sppt activatedthe cognate FAS carrier protein SACP in vitro, but activity waslow for non-cognate ACPs from secondary metabolism or theglycolipid biosynthetic pathway.66 Although its primarysequence aligns with Sfp, its activity resembles that of AcpS-typePPTases.

Like Synechocystis, the well-studied and environmentallyomnipresent species Prochlorococcus and Synechococcus are notprominent for their bioactive compounds, unlike Nostoc, Ana-baena or Nodularia.211 Further, their PPTases seem to be

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Fig. 12 Biosynthetic gene clusters surrounding the three PPTase genes of N. punctiforme PCC 73102 (ATCC 29133) (GenBank acc. no.NC_010628). (A) NgcS (GenBank acc. no. YP_001863782) with glycolipid PKS locus; (B) PPTase (GenBank acc. no. YP_001865721) with PKS-NRPS locus; (C) PPTase (GenBank acc. no. YP_001865651) with PKS locus. Domains identified with antiSMASH127 in bold. NRPS genes: yellow,PKS genes: blue, NRPS-PKS hybrid genes: green, PPTase gene: red. A respresents adenylation domain; ACP, acyl carrier protein; AT, acyltransferase; C, condensation domain; DH, dehydratase; DS, desaturase; E, epimerization domain; ER, enoylreductase; HP, hypothetical protein;KR, ketoreductase; KS, b-ketoacyl synthase; mCAT, malonyl CoA-ACP transacylase; MT, methyl transferase; Ox, NADH:flavin oxidoreductase;P5CR, pyrroline-5-carboxylate reductase; PCP, peptide carrier protein; PKS, polyketide synthase; SDR, short-chain dehydrogenase/reductase;TD, thioester reductase; TE, thioesterase; TP, thiamine pyrophosphate-binding domain containing protein.

Fig. 13 Alignment of HetI-like PPTases from Anabaena sp. PCC7120 (A7_HetI), N. punctiforme PCC 73102 (Np_NgcS) and N. spumigenaNSOR10 (Ns_NsPPT) against Sfp (Bs_Sfp) (GenBank acc. no. AAA22003, ACC78839, AAW67221, CAA44858). Sequence alignment was madeusing T-Coffee and ESPript.5,6

78 | Nat. Prod. Rep., 2014, 31, 61–108 This journal is © The Royal Society of Chemistry 2014

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phylogenetically distinct from those associated with hetero-cysts.14 Within this group of non-heterocyst-associatedenzymes, another PPTase was identied from Gloeobacter.14

Interestingly, however, a PPTase with 40% similarity to that ofGloeobacter violaceus was identied using environmental in vivoscreening, by activating the CP EntF of the E. coli enterobactinsynthase, thus demonstrating NRPS labeling abilities.212

Studying the anatoxin biosynthesis in Oscillatoria PPC 6506,the native PPTase OsPPT was functionally described to act onthe native PKS-CP AnaD.65 However, its ability to activate non-cognate CPs has so far not been tested.

Finally, two integrated PPTases have so far been identied inthe cyanobacteria G. violaceus (GenBank acc. no. BAC92166) andAzotobacter vinelandii (GenBank acc. no. ZP_00089517) with Sfp-like domain at the C-terminus of a PKS. However these arelacking further description.14

3.4 Protista

Apicomplexan parasites show an interesting diversity in fattyacid biosynthesis. Plasmodium falciparum contains a type II FASin its apicoplast, Cryptosporidium parvum contains a type I FAS,and Toxoplasma gondii uses both. The presence of PPTasescorresponds to their respective synthases.84 In the genome ofthe malaria parasite P. falciparum, only one PPTase (PfAcpS) canbe identied. PfAcpS shows close homology to EcAcpS, isputatively apicoplast targeted, and is fused to a hydrolasedomain of unknown function. In contrast, C. parvum has a 900kDa type I FAS and a large polyketide synthase (CpPKS1). Thetype I FAS has been reconstituted in vitro, aer expression offragments in E. coli, but appeared to be in its inactive apo-form.213 The native PPTase of C. parvum was later identied asan Sfp-type PPTase, shown to activate CpFASI84 and CpPKS1214

in vitro and in vivo. T. gondii uses both an AcpS-hydrolase fusionand an Sfp-type PPTase for its type I and type II FAS.

Trypanosoma have adopted an alternative fatty acid biosyn-thetic route that utilizes dedicated elongases instead of a type Ior type II cytosolic FAS.215 However, Trypanosoma also appear toproduce fatty acids in their mitochondria using a type II FAS,requiring PPTase activity.216 So far, no PPTases have beenidentied in Trypanosoma. We conducted detailed BLAST (BasicLocal Alignment Search Tool) analysis to identify putativePPTases in trypanosomal genomes, revealing PaPcpS homologsin Trypanosoma (e.g. CCD12699.1) and Sfp homologs in Leish-mania (e.g. XP_003860424.1) species.

Another interesting case is the amoeba Dictyostelium dis-coideum. The genome contains more than 45 type I polyketidesynthases, ranging in size between 1000 and 3000 aa. Two prod-ucts of these synthases have been characterized: differentiationinducing factor217 and 4-methyl-5-pentylbenzene-1,3-diol.218 Bothsynthases consist of six catalytic domains homologous to type IFASs, but instead of a thioesterase, an iterative PKSIII is used tooffload fatty acids. Interestingly, in the case of the PKS thatproduces 4-methyl-5-pentylbenzene-1,3-diol, the iterative PKSIIIdomain alone produces acyl pyrones, but in the presence of theinteracting ACP, an alkyl resorcinol scaffold is produced. These>45 PKS and FAS require 40-phosphopantetheinylation, and


recently two PPTases from D. discoideum were identied andcharacterized.85 One appears to be an AcpS-type and only workson a type II ACP that seems to be targeted to the mitochondria;whereas the other PPTase is an Sfp-type, which was shown toactivate type I synthases. Genetic knockouts of diAcpS or diSfpresulted in 50% and 20% survival, respectively.

3.5 Fungi

The kingdom of fungi is essential in global nutrient cycling dueto their ability to decompose organic matter. The ease of culti-vation and genetic modication has led to the use of fungi,especially yeasts, in metabolic engineering. As eukaryotes, fungiproduce a highly diverse range of proteins compared tobacteria. Furthermore, fungi have immense capabilities toproduce a wide variety of natural products. Fungal antibioticshave found various applications from pharmaceutics to agri-culture. However, this metabolic potential has also made fungione of the most difficult-to-combat pathogens of economicallyrelevant crops. Like bacteria, fungi produce bioactivecompounds via PKS and NRPS pathways and thus require aPPTase. Additionally, fungi have implemented an NRPS-likebiosynthetic mechanism into their lysine metabolism leading toa completely novel group of PPTases, the Lys5-like enzymes.

Yeasts. The rst indications of similarities between lysinemetabolism and NRPS systems were given by Morris and Jinks-Robertson in 1991.78 They identied a high sequence similarityof the protein Lys2 from the baker’s yeast S. cerevisiae to tyro-cidine synthetase of B. brevis.78 The proteins Lys2 and Lys5 werepreviously implicated in the reduction of a-aminoadipate toaminoadipate semialdehyde,219 but were described as oneprotein complex. Later, a PPant attachment site was identiedin Lys2, leading to the hypothesis that Lys5 activates Lys2 via 40-phosphopantetheinylation.1 This mechanism was conrmed bydetailed radiolabeling studies.79

Mootz and co-workers performed a functional characteriza-tion study in S. cerevisiae using a lys5 knockout strain.220 ThePPTases Sfp and Gsp from Bacillus spp., which are both involvedin non-ribosomal peptide synthesis, YdcB (AcpS) from B. sub-tilis, involved in fatty acid biosynthesis, and the then unchar-acterized PPTases q10474 (Lys7) from Schizosaccharomycespombe and NpgA from Aspergillus nidulans were evaluated.While Sfp, Gsp, Lys7 and NpgA were able to complement PPTaseactivity, YdcB could not. We now know that Lys7 corresponds toLys5 in S. cerevisiae, and thus is involved in lysine biosyn-thesis,82 while NpgA interacts with NRPSs.67,68 This led to thehypothesis that the activation of lysine metabolism and NRPSsare related to each other, but not to the FAS system.

Besides Lys5, S. cerevisiae contains an integrated PPTase(family III) and Ppt2, which is associated with the mitochon-drial FAS complex.221 The integrated PPTase was rst identiedvia sequence similarity.1,80 It is located within the FAS itself (onthe gene and protein level) providing autoactivation of theenzyme20 (see Section 3.6).

The third PPTase of S. cerevisiae, Ppt2, interacts with themitochondrial FAS.80 Gene disruption led to abolishment ofrespiration, and Ppt2 could label the native mitochondrial AcpP

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Fig. 14 Targets of PPTase activity in fungi, showcased by activities ofthe PPTase NpgA from A. nidulans.

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in vitro. This ACP was further labeled by EcAcpS, but not by thetype I FAS-specic PPTase Ppt1 from Brevibacterium ammonia-genes that was isolated shortly before this study. AcpS is knownfor its inability to interact with type I FAS enzymes, while Ppt1can. These results support the hypothesis that Ppt2 acts only onthe mitochondrial type II FAS, but not the cytoplasmic type IFAS of yeast.

Lys7 from the ssion yeast S. pombe interacts with Lys1, whichcorresponds to Lys5 and Lys2 in S. cerevisiae, respectively.82

However, evaluating heterologous vanillin biosynthesis in bothorganisms for biotechnological application revealed an endoge-nous activity mediating 40-phosphopantetheinylation in S. pombethat was absent in S. cerevisiae. Vanillin production in S. cerevisiaerequired co-expression of a heterologous PPTase.222 Additionally,mutational analysis showed that the amino acid residues neces-sary for the S. pombe Lys7 PPTase function were quite different tothat of another yeast, Candida albicans82 (see Section 5).

S. pombe is highly diverged evolutionarily from S. cerevisiaeand C. albicans,223,224 and this is further demonstrated in lysinemetabolism.82 Lys7 groups with the Sfp-type enzyme fromClostridium acetobutylicum instead of Lys5-like enzymes fromother yeast species. This might further explain the differentcharacteristics of Lys PPTases, even though they are thought tobe mainly involved in lysine biosynthesis.

Two PPTases were initially described in S. pombe.1 BesidesLys7, therein referred to as 1314154, an integrated PPTase(1842 aa) was grouped with the FAS-integrated enzymes, similarto S. cerevisiae. However, experimental characterization islacking. One further putative PPTase might be new8 (132 aa).81

Sequence similarity of new8 is higher to the integrated PPTaseand Ppt2, than to Lys7 of S. cerevisiae. Further, it closerresembles AcpS than Sfp andmight therefore be described as anAcpS-type PPTase. The knockout of new8 caused slow growth inS. pombe.81 This supports the hypothesis of its activity onmitochondrial type II FAS, since these cells would otherwise notbe viable.

Further indications for the conservation of the lys geneswithin fungi were given by Guo et al.225 The Lys2 enzyme of thepathogenic yeast C. albicans was only activated in vitro uponaddition of cell extract. Comparing their results to the previousstudies on S. cerevisiae and S. pombe, the authors concluded thisas strong evidence for the existence and requirement of a Lys5PPTase in C. albicans. This hypothesis was conrmed by adetailed characterization study of Lys5.74 In vitro, it activates itscognate Lys2 protein, but also Lys2 of S. cerevisiae, and to alesser extent Lys1 from S. pombe. Site-directed mutagenesiscould even reveal the essential PPTase residues for Lys2 acti-vation (see Section 5). Besides Lys2, a second PPTase wasidentied in C. albicans1 which genetically also represents anintegrated enzyme.

Recently, a PPTase gene was discovered in the lipid-producing yeast Rhodosporium toruloides.226 Zhu and co-workersused a transcriptomic and proteomic approach to track downthose enzymes absent in non-oleaginous yeasts. An AcpS-typePPTase is encoded within a novel FAS system (GenBank acc. no.EMS21268), and they were transcribed simultaneously underthe tested conditions.

80 | Nat. Prod. Rep., 2014, 31, 61–108

Aspergillus. Similar to yeast, A. nidulans contains the gene ofan integrated PPTase,1 which has not been further character-ized. A second putative PPTase gene npgA (null-pigmentmutant,npg, also named cfwA) (Table 1 and Fig. 14) was identiedduring mapping of a melanin-negative mutant.227 The samephenotype was observed in a wA-knockout strain, a gene refer-ring to a PKS that is involved in pigment production,228,229

leading to the hypothesis that the ACP domains of PKS wA aresubstrates of NpgA.

The npgA gene was rst isolated and further characterized byKim et al.230 While the deletion mutant again was defective ingrowth and pigmentation, overexpression did not result in avariation of the growth or pigmentation phenotype, though theconidiophores were found to be formed at an earlier stage.Conidiophores are specialized stalks presenting the conidia, theasexual spores. Later, NpgA was evaluated for its ability tocomplement Lys5 from S. cerevisiae.220 NpgA complemented Lys5activity, demonstrating its potential function in lysine metabo-lism. By in vivo studies, NpgA has been shown to be essential forpenicillin biosynthesis (which is produced by an NRPS).67 In thisstudy, the two alleles (version of the same gene) of npgA, namelycfwA+ and cfwA2, were characterized. Allele cfwA2 differs fromcfwA+ at two positions, which results in functional changes of theenzyme, and appears to be the more important allele for peni-cillin production. cfwA2 is further essential for the production ofthe siderophores ferricrocin and triacetylfusarinene C.68 Under-lining this versatility, addition of triacetylfusarinene C did68 (butsupplementation of lysine did not) restore the growth deciencyof the cfwA2 knockout strain.67 Besides non-ribosomal peptideand lysine biosynthesis, NpgA is essential for polyketide biosyn-thesis (shamixanthone, emericellin, dehydroaustinol), butdispensable for the sterol (ergosterol, peroxiergosterol, cer-evisterol) and fatty acid production.69

PptA, a homologue of NpgA, was shown to be essential forpolyketide and non-ribosomal peptide biosynthesis in A.niger.231 The homolog in A. fumigatus activates the native NRPSAfpes1 and the non-cognate Lys2 from C. albicans.71,72 A. fumi-gatus further uses the PPTase PptB which is specic for themitochondrial AcpP.73



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Penicillium. Like other fungi, Penicillium patulum containsthe gene of an integrated PPTase,1 which has not been studiedto date. Penicillium chrysogenum further contains the PPTasePc13g04050, which is a homolog of NpgA from A. nidulans.77 ThePPTase-defective mutant was auxotrophic for lysine and lackedboth pigmentation and penicillin production. Interestingly,biosynthesis of roquefortine C was not inuenced by thismutation, even though both penicillin and roquefortine C areproduced viaNRPS pathways,77 suggesting further PPTase geneswithin the genome. Considering fatty acid biosynthesis, the FASgene (GenBank acc. no. CAP74216) contains an integratedPPTase. This explains why the Pc13g04050 mutant did notrequire fatty acids. Garcıa-Estrada et al. could further show thehigh conservation of NpgA homologs within representatives ofthe fungal classes Eurotiomycetes, Ascomycetes, and Sordar-iomycetes using sequence alignment.77

Other fungi. Homologs of NpgA were discovered in otherfungal species, such as the plant pathogens Cochliobolus sativus,Colletotrichum graminicola, Fusarium fujikuroi and the root-colonizing fungus Trichoderma virens.75,76,83,232

Ppt1-decient mutants of C. sativus, C. graminicola, andT. virens were auxotrophic for lysine, unable to producemelanin, hypersensitive to oxidative stress, and had signi-cantly reduced virulence resulting from the defective polyketidebiosynthesis.75,83,232 In comparison to other studies, nomorphological defect or germination delay of conidia in C.sativus was observed besides the loss of pigmentation and theproduction of fewer conidia.69,75,232 Conidia of Ppt1-decientmutants in C. graminicola had a reduced size in comparison tothe wild type and exhibited strong morphological defects.232 InT. virens, spore formation was severely compromised. However,the mycelia grew faster in comparison to the wild type.83 Ppt1-decient mutants were still able to colonize plant roots, butcould not prevent growth of phytopathogenic fungi in vitro.

FfPpt1 of F. fujikuroi is essential for viability and is involvedin lysine biosynthesis and production of some, but not all,natural products, made by this species.76 Interestingly, theffPpt1-deletionmutant showed enhancement in terpene-derivedmetabolites and volatile substances. During infection of rice,lysine biosynthesis and iron acquisition are required, but thebiosynthetic pathways of other PKS and NRPS seem lessimportant. Further, FfPpt1 was shown to be involved in con-idation and sexual mating recognition.

In conclusion, NpgA and its homologues might cover slightlydifferent functionalities within the disparate fungal species, butthe conidiation pathways themselves are still not well under-stood and demand further investigation.

3.6 Type I integrated PPTases

In most cases, PPTases are stand-alone proteins, separatelyencoded or genetically part of the biosynthetic cluster. However,in a few cases the PPTase is part of the megasynthase, as alreadyidentied by Lambalot et al. in 1996.1 This became furtherapparent when yeast apo-FAS was isolated and, upon addition ofCoA, spontaneously formed holo-FAS.20 Interestingly, thePPTase domain of the FAS megasynthase is located on the


outside of the barrel and cannot activate ACP aer organization,necessitating post-translational modication of ACP before thesynthase is fully formed (see Section 2.3).

Apart from the primary FAS synthase, fungi also expressdedicated FAS or PKS for secondary metabolites.233 For example,norsolorinic acid and enediyne biosynthesis require a FAS orPKS (or combination), using integrated PPTases in the mega-synthases. The PPTase from the FAS domain of the norsolorinicacid synthase was cloned out of the synthase and shown to havevery narrow substrate specicity (only its cognate ACP wasrecognized).234 Recently, it was shown that this PPTase even hasa very narrow CoA substrate specicity, accepting only CoA andno acyl-CoAs.235

Enediynes are made by iterative PKSs and decoratingenzymes. The iterative PKS from the bacteriumMicromonosporaechinospora ssp. calichensis contains a PPTase domain at its C-terminus.236 Detailed characterization revealed that this 330 aadomain forms a pseudo-trimer, showing similarity to EcAcpS.Furthermore, this PPTase has very low activity for its cognatecarrier protein, suggesting that this enzyme is not optimized forsecondary metabolism.237

Another integrated PPTase has been found in bacteriumS. erythraea (SePptI) which is most likely part of a PKS mega-synthase.44 Copp and Neilan identied several novel integratedPPTases in large megasynthases: cyanobacteria G. violaceus(GenBank acc. no. BAC92166) and A. vinelandii (GenBank acc.no. ZP_00089517) have an Sfp-like domain at the C-terminus ofa PKS megasynthase, and the plant Arabidopsis thaliana (Gen-Bank acc. no. AAC05345) has an Sfp-like PPTase at the C-terminus of a COP1 interactive partner 4 domain.14

3.7 Plants and algae

AcpS activity in plants was evaluated directly aer the initialdiscovery of AcpS in E. coli.3,9 In plants and algae, the fattybiosynthesis is located in the chloroplasts (type II FAS) andmitochondria (type II FAS).238,239 For the spinach chloroplastfatty acid metabolism, Elhussein et al. proposed that phos-phopantetheinylation mainly takes place in the cytosol and thenuclear-encoded chloroplast ACP is phosphopantetheinylatedbefore entering the chloroplast.9 This was questioned by studiesthat suggested a dedicated PPTase located within the chloro-plast.240,241 However, later on, it was shown that both apo- andholo-ACP could be efficiently taken up by the chloroplast.242,243

In contrast to the large renewed interest in algae- and plant-derived biofuels, no PPTase from these groups has been func-tionally described. Metabolic engineering of plants has insteadinvestigated co-expression of a foreign PPTase gene (seeSection 6).244–247 One study in tobacco, however, demonstratedthat a heterologously expressed PKS did not need the co-expression of a PPTase for successful biosynthesis of the naturalproduct 6-methylsalicylic acid.248 The authors propose thepresence of an endogenous enzyme that activates the integratedsynthase.

Even though current knowledge of PKSs and NRPSs and theirfunction in plants and algae is limited, a growing number ofalgal and plant genomes have been analyzed with bioinformatic

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tools (see ref. 127 and others) that predict the presence of“green” natural-product gene clusters.249–251 Further research isneeded to describe these enzymes functionally as well as theiractivation mechanism by PPTases, particularly with regard torenewable fuel and chemical production.

3.8 Animals

Although higher eukaryotes, in general, do not appear toproduce NRPS- or PKS-derived secondary metabolites, homolo-gous biosynthetic pathways seem to be conserved. The fruit yDrosophila melanogaster encodes a 98.5 kDa protein called Ebonythat exhibits homology to NRPSs and has b-alanyl-dopaminesynthase functionality (Fig. 15).252 It was shown that Ebonyactivates b-alanine by an adenylation domain and loads it onto aPCP (thiolation) domain. In a second reaction, amines such asdopamine are selected by a small amine selection domain,which then cleaves the activated amino acid from the synthase.Ebony requires 40-phosphopantetheinylation for activity and, invitro, Sfp can install the PPant arm. Sequence homology betweenconserved PPTase regions and the genome ofDrosophila revealeda PPTase. This PPTase (now called Ebony activating protein,NP_729788.1) was cloned and expressed in E. coli and shown tohave similar in vitro activity as Sfp (“unpublished results”).24

The peptidoamines produced by Ebony are involved inseveral processes, including b-alanyl-histamine biosynthesis inthe eye as part of neurotransmitter metabolism. AlthoughEbony closely resembles an NRPS, it is not characterized as aclassical NRPS, since it does not catalyze the formation of apeptide bond, via a condensation domain, and lacks a thio-esterase domain. Ebony-like proteins are found by homology inmany higher eukaryotic species, suggesting that the NRPS-likechemistry is evolutionarily preserved.249,253 However, it remainsunclear why (or if) classical non-ribosomal peptides are foundonly in bacteria and fungi. Horizontal gene transfer betweenspecies and “inventive evolution” could be an explanation forthis phenomenon, although it remains a mystery why someplants and other eukaryotes seem to contain large NRPS- andPKS-like genes in their genomes.249,253

Fig. 15 Ebony from Drosophila resembles an NRPS and catalyzes thebiological amines are used to produce b-alanine–amines. AS representsprotein.

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3.9 Homo sapiens

In mammals, 40-phosphopantetheinylation is required forseveral enzymes, including type I FAS, type II mitochondrialFAS, lysine metabolism and 10-formyltetrahydrofolate dehy-drogenase (Fig. 16). Surprisingly, mammals only encode oneunique PPTase in their genome which is called AASDHPPT. Thisgene was discovered by homology to Lys5 from S. cerevisiae.26 S.cerevisiae synthesizes the amino acid lysine by the enzyme Lys2,an a-aminoadipate reductase, which is activated by Lys5, aPPTase.1,79 In contrast to yeast, mammals catabolize lysine viatwo different pathways, involving a-aminoadipate semi-aldehyde or pipecolic acid. The enzyme responsible for thelatter of these processes is the bifunctional enzyme a-amino-adipate semialdehyde synthase (AASS), which transforms lysineinto saccharopine and a-aminoadipate semialdehyde, which isthen oxidized by AASDH to a-aminoadipate. In a yeast Lys5mutant, the mammalian AASDHPPT could efficiently comple-ment, suggesting that this enzyme is also involved in a similarreaction in mammals. Indeed, AASDH contains a PCP domainthat undergoes 40-phosphopantetheinylation by AASDHPPT.Later, it was shown that AASDHPPT has a broad substratescope.25

The mammalian fTHF-DH also requires a PPant arm forcatalysis (Fig. 16).254 This enzyme is phosphopantetheinylatedby AASDHPPT as well, and siRNA silencing this enzymecompletely prevents the modication of fTHF-DH. A mito-chondrial homolog of fTHF-DH was found to be activated by thesame PPTase.255 This suggests that there are no other PPTasespresent in humans.

3.10 Evolution and phylogeny

PPTases have been identied and classied based on theirprimary sequence. For example, Lambalot et al.1 proposed thatPPTases can be identied by two conserved motifs, namely (V/I)G(V/I)D(x)40–45(F/W)(S/C/T)xKE(A/S)hhK, where ‘h’ is an aminoacid with a hydrophobic chain. Recently, a rened analysis ofthese signature sequences wasmade and found to be (I/V/L)G(I/V/L/T)D(I/V/L/A)(x)n(F/W)(A/S/T/C)xKE(S/A)h(h/S)K(A/G), in which n

formation of b-alanine–amines, like b-alanine–dopamine. Also, otheran amine-selecting domain; A, adenylation domain; and CP a carrier



Fig. 16 Activities of human PPTase AASDHPPT. (A) 10-THF involved in 10-fTHF metabolism, (B) AASDH involved in catabolism of lysine, (C)mitochondrial FAS, and (D) cytosolic FAS. DH represents a dehydrogenase; A, an adenyltransferase; R, a reductase; ACP, acyl carrier protein; MAT,malonyl-CoA acyltransferase; KS3, ketoacyl synthase III; KR, ketoreductase; DH, dehydratase; ER, enoyl reductase; KS, ketoacyl synthase; and TE,thioesterase.

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is 42–48 aa for AcpS and 38–41 aa for Sfp-type PPTases.18 Betweenthese two conserved regions resides a third region which is lessconserved, but contains a highly conserved glutamate (E127 inSfp). Additional sequence alignments and analyses14,18,56 give riseto four more signature sequences for different subclasses of Sfp-type PPTases and AcpS-type PPTases. However, PPTases span alarge variety of primary sequences, thus making sequence align-ments and BLAST analyses difficult to interpret. We propose thata more powerful tool to compare, identify and classify PPTases isan evolutionary analysis by phylogeny.

The rst phylogenetic tree constructed for PPTases showed aclose relationship between PPTases involved in secondarymetabolism, but also distinct differences, including the evolu-tionary separation between Sfp, EntD and JadM.55 In 2003, Joshiet al. identied the human PPTase AASDHPPT and constructedan updated phylogenetic tree, including the primary metabo-lism (fatty acid) PPTases, showcasing that the three PPTasesfound in S. cerevisiae (Lys5, type I FAS and mitochondrial FASPPTases) are very distant from each other.25,82 Apicomplexanparasites have AcpS, Sfp-type or both PPTases. P. falciparumuses an AcpS-type, C. parvum uses an Sfp-type, and T. gondiiboth types of PPTases.84 Phylogenetic analysis of these proteinsputs them in separate clades: the Sfp-types are close to humanand fungal PPTases, whereas the AcpS-types are closely relatedto bacterial FAS PPTases. Copp and Neilan construct a detailedphylogenetic tree on Sfp-like PPTases, focusing on cyanobac-teria.14 Interestingly, two major Sfp-like PPTases seem to exist,utilizing conserved sequence motifs F/KES and W/KEA.


Here, we construct an unbiased phylogenetic tree from�1700 unique (putative) PPTase sequences (see Fig. S1†), aswell as a neighbor-joining tree of the currently characterized (�60) PPTases (Fig. 17). To our surprise, several clades showrelatively good affinity. For example, Sfp, Gsp, AASHDPPT,MtaA, JadM, HetI, PptT, PcpS, EntD and AcpT form separatebranches of the tree, whereas AcpS-type PPTases, includingthose from mitochondrial fatty acid synthesis, are far removedfrom the main trunk. Although we cannot discuss in detail, afew observations can be made. Whereas on one branch of thetree EntD, PcpS, PptT and others group together, on theopposite branch AASDHPPT, Lys5, Sfp and MtaA grouptogether. This division crosses Gram-positive/Gram-negativeand bacterial families, and so far the origin of the clearphylogenetic division of these two groups of PPTases has beenelusive, since both “groups” are involved in secondary metab-olism of various natural products (which do not seem tocluster). A more detailed look into the smaller tree (Fig. 17)shows a similar division. Fungal, animal, protista and humanPPTases group together, in close proximity to a Sfp clade and aclade that contains dedicated cyanobacterial, E. coli, Pseudo-monas and Stigmatella PPTases. Adding more sequences pullsthis clade apart in three major branches, characterized bycyanobacterial HetI, Stigmatella MtaA and E. coli AcpT.Completely opposite, EntD-like PPTases group together fromvarious bacteria. In between, two branches are far removedfrom the two described major clades, showing type I FASintegrated PPTases and AcpS-type PPTases.

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Fig. 17 Neighbour Joining Method phylogenetic tree of annotated �60 PPTases (see Table 1), constructed using MEGA.256

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4 Carrier protein(s)

Carrier proteins represent a large family of small (70–100 aa)proteins essential in primary and secondary metabolism.These proteins are either part of a single-chain multi-domainsynthase or exist as monomers and shuttle cargo betweenenzymes that act on the substrate. All carrier proteins have aconserved serine which requires 40-phosphopantetheinylationby a PPTase in order to tether cargo via a exible,labile, thioester linkage. In this section we will discuss thevariety of carrier proteins and their cognate PPTases, the non-carrier proteins that are phosphopantetheinylated, theamino acids essential in carrier proteins and peptides forPPTase activity and ACP hydrolases (the reversal of PPTaseactivity).

84 | Nat. Prod. Rep., 2014, 31, 61–108

4.1 Specicity and activity for carrier proteins

The kinetic data on PPTases and their carrier protein or CoAsubstrates are limited to only six different PPTases. WhichPPTase acts on what carrier protein (and to what extent) iscrucial for in vitro applications and understanding the reactionmechanism. In Table 3 we summarize kinetic data obtained forseveral PPTases and partner carrier proteins, showcasing therange of kinetic parameters found in vitro.

AcpS is the ubiquitous trimeric PPTase responsible forinstalling the PPant arm on the ACP involved in prokaryoticfatty acid biosynthesis. AcpS is relatively specic to ACPsinvolved in FAS, as the polyketide granaticin- (gra), frenolicin-(fren), oxytetracycline- and tetracenomycin- (tcm) ACPswere poorly phosphopantetheinylated when overexpressed in



Table 3 Overview of kinetic parameters for different PPTases and different carrier protein substrates

PPTase Target apo-CP kcat/Km (mM�1 min�1) Ref

EcAcpS E. coli ACP 10–50 1,11EcAcpS Gra ACP 6 11EcAcpS Fren ACP 1.6 11EcAcpS Tcm ACP 0.25 11EcAcpS Otc ACP 0.26 11Sfp B. subtilis SrfB1 80 28Sfp B. subtilis SrfB2 31 28Sfp E. coli EntB 4 28Sfp S. cerevisiae Lys2-PCP >14 28Sfp E. coli ACP 1 28PaPcpS P. aeruginosa ACP (1.6–12.5 mM) 32.5 41PaPcpS P. aeruginosa ACP (23–234 mM) 2.6 41PaPcpS B. subtilis ACP (2.2–25 mM) 8.6 41PaPcpS B. subtilis ACP (25–206 mM) 2.9 41PaPcpS TycC3 PCP (0.9–12 mM) 0.5 41PaPcpS TycC3 PCP (25–150 mm) 0.04 41PaPcpS pchE ArCP (1–10 mM) 1.1 41PaPcpS pchE ArCP (21–155 mM) 0.13 41EcAcpS B. subtilis ACP (25–206mM) 1.8 41EcAcpS B. subtilis ACP (2.2–25 mM) 110 41Sfp B. subtilis ACP (25–206 mM) 1.2 41Sfp B. subtilis ACP (2.2–25 mM) 0.3 41Sfp TycC3 PCP (25–150 mM) 21.6 41AASDHPPT H. sapiens ACP 3.6 25AASDHPPT H. sapiens mitochondrial ACP 1.0 25AASDHPPT B. brevis PCP 0.6 25AASDHPPT B. subtilis ACP 0.05 25Lys5 Lys2-PCP 3 79Svp BlmI 2.8 56Svp Tcmm 28 56Sfp BlmI 1.9 56Sfp Tcmm 0.08 56FdmW FdmH 8.1 53FdmW Tcmm 0.6 53Svp FdmH 0.4 53Svp Tcmm 7.6 53Sfp YbbR13 0.091 257Sfp A1 0.00049 257EcAcpS YbbR13 0.0033 257EcAcpS A1 0.015 257EcAcpS E. coli ACP 50 258EcAcpS NodF 0.05 258EcAcpS Hybrid E. coli ACP-NodF 0.01 258

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E. coli.11 Overexpression of tcm-ACP and induction in theexponential phase lead to no holo-ACP, but induction in thestationary phase leads to a small percentage of the modiedprotein. The apo/holo ratio of gra-, fren- and act-ACP uponoverexpression in E. coli gave 30% holo-gra-ACP, 2% holo-act-ACP and no modied holo-fren-ACP. Apo-act-ACP was convertedto 80–90% holo-act-ACP when the post-induction period wasincreased to 12 h. However, in vitro AcpS was active on theaforementioned ACPs and the transformations go to comple-tion.11 AcpS also catalyzes the in vitro 40-phosphopantetheiny-lation of NodF and D-alanyl carrier protein from L. casei, butdoes not act on the PCP from tyrocidine A synthase or apo-PCPsof E. coli enterobactin synthase.

Sfp is a highly promiscuous PPTase from B. subtilis surfactinsynthase, capable of activating PCPs and FAS apo-ACPs alike


(Table 3). Interestingly, both Sfp and EntD also show promis-cuity towards their CoA substrates,259 discussed in Section 5.Although Sfp is promiscuous to both its ACP and its CoAsubstrate, it prefers carrier proteins from secondary metabo-lism. The genome of B. subtilis also contains AcpS. Deletion ofthe acpS gene has no apparent effect on the bacteria, despite thelow in vitro activity of Sfp on FAS apo-ACP, suggesting that AcpSis not essential in B. subtilis.

P. aeruginosa has only one PPTase, named PaPcpS.41 PaPcpShas 13% sequence similarity to Sfp and shows higher similarityto E. coli EntD, but is responsible for both primary andsecondary metabolism synthase modication.100 PaPcpS alsoshows catalytic behavior more typical of AcpSs (Table 3). BothAcpS from S. pneumoniae and B. subtilis show different catalyticparameters at high and low apo-ACP substrate concentrations,

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presumably due to allosteric regulation (or cooperativity) of itsthree active sites or some conformational change(s). Sfp doesnot show this behavior, but the monomeric PaPcpS surprisinglydoes,41 possibly making PaPcpS a unique subclass of PPTases.

The human PPTase AASDHPPT shows promiscuous behaviorto a variety of apo-carrier proteins (Table 3), in line with thevarious synthases it needs to activate. Recently, the humanPPTase has been crystallized and structure solved, shiningdetailed light on substrate binding and reaction mechanism,discussed in Section 5.260

Cyanobacteria are producers of many PKS/NRPS secondarymetabolites that require PPTases. Interestingly, cyanobacteriaonly express one PPTase and seem to be devoid of an AcpS-type.For example, N. spumigena NSOR10 expresses a PPTase whichcan act on the carrier protein responsible for glycolipidbiosynthesis, ArCPNp, the PCP involved in mycrocystinbiosynthesis, MPCP and the PKS carrier protein for nosto-peptolide, ACPNp.60 In contrast, the PPTase from Synechocystissp. PCC6803 has very narrow carrier protein substratespecicity.66

The stunning range of carrier proteins modied by PPTasesis matched by a lack of understanding for what determinesspecicity or selectivity on either PPTase or carrier protein side.We further discuss this in the context of the published X-raycrystal structures of PPTases in Section 5.

4.2 The other phosphopantetheinylated proteins and theirPPTases

Not only traditional synthases (FAS, PKS and NRPS) requirePPTases, but there are many more carrier proteins and syn-thases that require a PPant cofactor. The biosynthesis ofD-alanyl-lipoteichoic acid by Gram-positive bacteria requires thecarrier protein Dcp,261,262 which is phosphopantetheinylatedin vitro by EcAcpS. L. casei contains only an AcpS-type PPTase inits genome, which presumably can install the PPant on AcpPand Dcp.

NodF is another carrier-protein-like protein that is involvedin the biosynthesis of lipo-chitin nodulation factor in Rhizobia.

Fig. 18 Sequence alignment of several carrier proteins from Rhizobia. Ecfactor biosynthesis, rkpF a CP involved in capsular polysaccharide biosynttwo other CPs present in R. leguminosarum.

86 | Nat. Prod. Rep., 2014, 31, 61–108

Although NodF has 25% sequence identity to E. coli AcpP, thisAcpP cannot replace NodF in vivo.258 Interestingly, EcAcpS andmalonyl-CoA-acyltransferase can interact with NodF, but notketoacyl synthase III. A chimera of E. coli AcpP and NodF cancomplement a NodF deciency in vivo. In the genome ofRhizobium leguminosarum, only an AcpS-type PPTase is identi-ed which presumably can phosphopantetheinylate both itsAcpP and NodF.

In Rhizobia, four other carrier proteins are found, calledACPXL (involved in lipid A biosynthesis), Rkpf (involved incapsular polysaccharide biosynthesis), SMb20651 (unknownfunction)263 and SMc01553 (unknown function).264 Thesepresumably require a PPant arm for activity, show very littlesequence identity (Fig. 18), and are modied by its endogenousAcpS. Only upon co-overexpression of E. coli or Sinorhizobiummeliloti AcpS, holo-SMb20651 was formed in E. coli, whereas thebasal expression of EcAcpS was not sufficient to modify thecarrier protein. This suggests that poor catalysis maybe due tothe presence of a DST motif instead of a DSL motif.

M. tuberculosis is an example of an organism that contains awide range of carrier proteins that require 40-phosphopante-theinylation.36 These bacteria encode >18 type I PKS, NRPSs andtwo fatty acid synthases. De novo fatty acid synthesis in M.tuberculosis is encoded by a bacterial type I FAS,265 but thesecond FAS (type II) is responsible for elongating C18 to C52–C60

fatty acids. These very long chain fatty acids, and additional PKSand acyl transferases, are required for mycolic acid biosyn-thesis. The second FAS requires a dedicated ACP, calledAcpM,266,267 which has a 35 aa C-terminal extension which ispresumably involved in substrate binding and dimerization.268

E. coli AcpS quantitatively transforms apo-AcpM into holo-AcpM.The genome of M. tuberculosis contains two PPTases, namelyAcpS and PptT. Chalut and co-workers constructed AcpS andPptT knockouts in the model bacteria Corynebacterium gluta-micum, which is easier to genetically manipulate than M.tuberculosis but lacks the type II fatty acid synthase. The AcpSknockout shows fatty acid auxotrophy but can still makemycolic acid, whereas the PptT knockout shows no mycolicacid, but wild-type levels of C16/C18 fatty acids.36 This suggests

_acpp is E. coli ACP (AcpP), NodF a CP involved in lipochitin nodulationhesis, R1_acpp the AcpP of R. leguminosarum, SM_b20651 and R02304



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that AcpS is responsible for phosphopantetheinylating both ofthe AcpPs and PptT for all the other (>20) carrier proteins inmycobacteria.

The mammalian enzyme fTHF-DH has two independentcatalytic domains located at its C- and N-termini, with a carrierprotein domain in between. The function of this protein is theconversion of 10-formyltetrahydrofolate (fTHF) to the importantco-factor THF. One domain is responsible for hydrolysis of theformyl group off fTHF and the other domain for NADP+-dependent oxidation of formyl to CO2.269 Only when thedomains are fused is activity observed, but how the formylgroup is transferred from one buried active site to the next wasunknown until 2007, when the presence of a phosphopante-theinylated carrier protein was shown to link the two domains.AASDHPPT installs the PPant arm on fTHF-DH and when thePPTase is silenced, fTHF-DH is inactivated, and cells showreduced proliferation and cell cycle arrest.254 Recently, a mito-chondrial targeted fTHF-DH was identied in mammals. Whenpuried from pig liver and shown to be active, another proteinwas added to the growing number of substrates of themammalian PPTase.270

Besides the above-discussed PPTases and their unusualcarrier protein substrates, other substrates include the previ-ously discussed (see Section 3) human AASDH, fungal lysinebiosynthesis, cyanobacterial acyl-ACP reductases, Nocardiacarboxylic acid reductase and Drosophila Ebony.

4.3 Carrier protein recognition by PPTases

Both protein–protein interactions and protein–substrate inter-actions are important in type II synthases. In this section we willdiscuss in detail how a PPTase recognizes a carrier protein andhow it is possible that one enzyme recognizes >20 differentcarrier proteins, as is the case in M. tuberculosis. The rst co-crystal structure of B. subtilis AcpS with its cognate ACP shedslight on these protein–protein interactions (Fig. 19).271 AcpS rstbinds CoA and Mg2+, followed by the ACP, which initiates thetransfer of PPant to ACP.

AcpS is a homotrimeric protein with active sites on theprotein–protein interfaces. All contacts between ACP and AcpS

Fig. 19 (Left) AcpS-ACP co-crystal structure, showing the B. subtilisAcpS trimer and three B. subtilis ACPs (in orange) binding to the PPTase(PDB: 1F80). (Right) Zoom in on the interaction between helix I of AcpSand helix III of ACP. Crucial amino acids are labeled.


seem to occur between helix I of AcpS and helix III of ACP(Fig. 19 and Section 5). Two hydrophobic residues of ACP (Leu37and Met44) protrude into AcpS, where Leu37 extends into apocket formed by Met18, Phe25, Phe54 and Ile15, and Met44binds into a pocket consisting of Phe25, Arg28 and Gln22. Arg14forms a salt bridge with Asp35 of ACP (in close proximity to the“active site” Ser36), and Arg21 forms a salt bridge with Glu41.The other end of helix III is locked in place by interaction ofArg24 and Gln22 of AcpS with Asp48 of ACP.271 When 14 aa inhelix II of a PCP were mutated into those present in AcpP, AcpSwas able to act on this hybrid carrier protein.41 More elaboratemutagenesis studies show that the closer the sequence of PCPapproached that of the ACP, the higher the activity of AcpS is onthese hybrid ACP/PCPs.272

There are some clear differences between PCPs and ACPswhich determine whether AcpS or Sfp-type PPTases can act onthese carrier proteins (Fig. 20). For example, position X in themotif (D/H)SLX is in PCPs a positive residue like Lys or Arg, butin ACPs almost always an Asp. Based on the co-crystal structureof AcpS-ACP and overlays with a Sfp-PCP model, mutations wereintroduced into AcpS. Residues R14 and K44 of AcpS areimportant for carrier protein recognition, but introduction ofpoint-mutations at those sides did not result in PPTase activityon PCPs.272 Interestingly, except for R14K, all mutants do notshow allosteric activation anymore, which is observed in wild-type AcpS (see Section 2.1).

Vibrio harveyi AcpP (VhACP) shares 86% sequence identitywith E. coli AcpP. Mutagenesis of Asp35 or Asp56 of VhACP has alarge effect on the ability of EcAcpS to activate the ACP.273Con-struction of mutants D30N/D35N/D38N, E47Q/D51N/E53Q/D56N and the combination of both, showcases the importanceof these residues. The VhACP mutants containing Asp35mutations are not phosphopantetheinylated by EcAcpS. Aproperly folded carrier protein also seems to be important foractivity, as shown with the I54A mutant of VhACP, which formsa highly dynamic ACP and cannot be activated by EcAcpS.274

S. coelicolor produces 22 known natural products thatutilizes synthases requiring 40-phosphopantetheinylation.Despite this large number of CP targets, the genome of S. coe-licolor contains only three PPTases: SCO5883, SCO667 andScAcpS. Actinorhodin is a polyketide natural product and it wasshown that SCO5883 and SCO667 are not required for itsproduction, suggesting that ScAcpS is responsible for 40-phos-phopantetheinylation of its synthase.50,126 Indeed, in vitroScAcpS acts on a range of FAS and PKS ACPs.50 Further muta-genesis and structural studies shed more light on the source ofpromiscuity of ScAcpS for different carrier proteins, albeit basedon modeling.126

To this day, Sfp has not been co-crystallized with any carrierprotein. However, by mapping the AcpS-ACP structure on Sfp,it was shown that the binding helix of AcpS (K13-Q22) is a loopin Sfp (T111-S124). Mutagenesis of these residues in Sfp led tomutants with 15–24-fold lower Km values for PCP, whereas CoAbinding was only reduced by 3–6-fold.275 It is speculative butthe increased promiscuity of Sfp might arise from the exi-bility of that binding loop versus the rigidity of the a-helix inAcpS.

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Fig. 20 Sequence alignment of carrier proteins and peptide mimics. TycA_PCP is the PCP1 from tyrocidine synthase TycA, SrfB_PCP1 is thePCP1 from surfactin synthase SrfB, GrsA_PCP is the PCP from gramicidin synthase GrsA, EntF and EntB are the PCPs from enterobactin synthase,EcACPP is E. coli AcpP, hACPP is the excised human ACP; YbbR, peptideS, peptideA and 8mer are the short peptides found to be substrates ofPPTases.257,276,278

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The human PPTase AASDHPPT, which falls in the samesubclass as Sfp, has been co-crystallized with its cognate excisedACP mutant Ser2156Ala. The excised ACP binds in the clebetween the two domains of the PPTase, and three hydrophobicpatches on the surface of the PPTase are responsible forprotein–protein interactions. Only a few polar interactions areobserved between ACP and PPTase, and binding seems to begoverned by shape complementarity instead of specicinteractions.

There appears to be a fundamental difference between type Iand type II synthase ACPs regarding their interactions with theirPPTases. In type II synthase ACPs, conserved negatively-chargedsurface residues mediate interactions with PPTases, whereastype I synthase ACPs seem to have a lower overall negativecharge, and both hydrophobic interactions and shape seem tobe the dominant factors in productive protein–protein interac-tions.260 With access to two ACP-PPTase co-crystal structureslight has been shed on how these proteins interact and howNature regulates their specicity. However, with such limitedstructural information, it remains unknown how general are theobservations made for these two structures.

4.4 Peptide mimics of carrier proteins as substrate ofPPTases

Another approach to probe the recognition of carrier proteins byPPTases is the study of small peptides as ACP-mimics. Synthesisof a 19 aa consensus peptide of the SrfB-PCP made it possible toinvestigate whether the activity of Sfp depends on its partner’sprimary sequence or requires structure. Even at very highconcentrations, the peptide was not a substrate for Sfp, sug-gesting that a folded apo-carrier protein is necessary.

Walsh and co-workers showed that PCPs displayed on thesurface of M13 phages (phage display) were substrates for thepromiscuous PPTase Sfp.276 Various other proteins were phage-displayed and besides PCPs and ACPs, truncated forms of the484 aa protein YbbR were found. The shortest phosphopante-theinylated YbbR fragment was 49 aa and has no sequencehomology to any ACP or PCP. Synthesis of a shorter fragmentidentied an 11 aa YbbR-derived fragment (DSLEFIASKLA) thatwas a substrate of Sfp (Fig. 20). Amino acids can be added to theN-terminus without inuencing labeling, but the C-terminuswas very sensitive to truncation. Interestingly, the residues

88 | Nat. Prod. Rep., 2014, 31, 61–108

ASKLA are not encoded by the YbbR ORF but are a linkerintroduced between the hexahistidine tag and the peptide. Inthe phage display-selected clones, ASKLG was present at the C-terminus, but again also part of a linker introduced betweenphage particle and peptide. The short peptide with the originalYbbR C-terminal sequence is not a substrate for Sfp, whichcould indicate that full-length YbbR may not be a substrate forSfp. Indeed, the original YbbR protein has not been shown to bea substrate for either AcpS or Sfp, and it is unclear whetherYbbR is phosphopantetheinylated in Nature.277

All identied YbbR-derived peptides that are substrates ofSfp show low catalytic efficiency and strong helical propensity.None of the helical wheel representations of these peptidesmatches PCPs or ACPs. This suggests that these YbbR-derivedpeptides have no structural relevance to carrier protein–PPTaseinteractions. Based on the prior YbbR-derived peptide results,phage display was used to select for small (12 aa) peptides asspecic substrates for Sfp or AcpS, respectively. Aer multiplerounds of selection, two short peptide tags (S- and A-peptide)were identied that have a 442-fold greater kcat/Km for Sfp overAcpS, or a 30-fold greater kcat/Km for AcpS over Sfp, respectively(Table 3).257 Interestingly, only the S-peptide shows structuredhelical propensity. Nevertheless, the authors compare the shortS- and A-peptides with helix II of ACPs and PCPs and tentativelyassign the differences in catalytic activity to certain residues inthese selected peptides. For example, glutamic acid at position8 of peptide A seems prominent in distinguishing whether AcpSor Sfp can act on the peptide. Further minimization of thepeptide gave an eight-residue peptide as a substrate for AcpS.278

4.5 Regulation by 40-phosphopantetheinylation

PPTases convert the inactive apo-form into the active holo-formof a synthase. The levels of apo- and holo-carrier proteins in aliving organism can therefore regulate synthase activity.243

In vitro, the disulde-bonded dimeric form of holo-carrierproteins is oen observed. The crystal structure of P. falciparumapicoplastic ACP, involved in FAS, showed that the disulde-bond is deeply buried and difficult to access, even for smallmolecule reductants.279 This raised the question of whether thisdimeric form is present in the parasite with some regulatoryfunction. However, in blood-stage parasites, no dimeric form isobserved, suggesting that the apicoplast is a sufficiently



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reducing environment to prevent disulde-bond formationbetween the two thiols of phosphopantetheinylated PfACP, andthus holo-ACP dimerization was ruled out as being involved inregulation.

The presence of large amounts of apo- or holo-carrierproteins can inuence many metabolic processes. The ratio (orpresence) of apo- over holo-ACP is directly linked to the activity(and presence) of PPTase. Early studies on ACPs oen foundboth holo- and apo-forms in cell lysate. For example, whenspinach ACP was overexpressed in tobacco leaves, a 50/50mixture of apo- and holo-spinach ACP was found, whereas thenative tobacco ACPs were all in the holo-form.243 In E. coli, nodetectable amounts of apo-ACP are found, suggesting that theactive/inactive ACP ratio is not the point of regulation inNature.280 Upon overexpression of EcACP in E. coli, growth rateswere severely retarded, suggesting some toxic effect.281 Indeed,apo-ACP is a potent inhibitor of cell growth, somehow regu-lating sn-glycerol-3-phosphate acyltransferase.281 Recently, itwas shown that C18:1-loaded ACP regulates a plastidic acetylCoA carboxylase.282 Taken together, it is still unclear whetherPPTases, which directly control the apo/holo ratio of carrierproteins, regulate cellular processes.

In E. coli, acyl carrier protein hydrolase (AcpH, also calledACP phosphodiesterase) removes the PPant arm from thecarrier protein (Fig. 21), and it seems likely that this enzyme isactivated by decreasing CoA levels.283 Vagelos and Larrabeedescribe in 1967 the isolation of AcpH from E. coli and study itsenzymatic activity in detail.284 Mn2+ appeared to be a requisitefor the reaction, although alsoMg2+, Co2+, Fe2+ and Zn2+ showedrestoration of activity. Increasing concentration of reductant(DTT, bME) also improved the hydrolytic activity. Interestingly,AcpH seemed to be highly specic for full-length ACP, since itwas unable to cleave the phosphopantetheine arm from largepeptides of proteolytically digested ACP (fragments of 43 or 62of the total 86 aa). However, it deactivated ACP from Clostridiumbutyricum, but not the ACP of mammalian type I FAS.284

The in vivo turn-over rate of the PPant arm is higher than theturnover of the ACP itself, both in E. coli7 and in rat liver,285

suggesting that perhaps PPTase or AcpH activity serves a

Fig. 21 AcpH, ACP hydrolase or ACP phosphodiesterase. (A) Reaction caafter SpoT).290 SpoT (PDB: 1VJ7) in grey, EcAcpH in orange and PaAcpH inthe Mn2+ ion as a purple sphere.


regulatory function. A crude enzyme preparation from rat liverwas able to hydrolyze 40-phospho[14C]pantetheine from the ratfatty acid synthase.286 Later, a puried enzyme preparation wasunambiguously shown to hydrolyze radioactive-pantetheinefrom labeled rat type I FAS (holo-FAS).287 This was also the rstproof that the large type I FAS only carries one ACP, since themolar ratio of pantetheine released from the protein was 1 : 1.The puried enzyme preparation was not able to cleave the PPantarm from CoA or from pigeon liver holo-FAS, and the expressionof the enzyme seems to vary with nutrition: the enzymatic activitywas high in 3-day fasted rats, whereas no hydrolase activity wasdetected in 2-day fasted or normally fed rats.287

E. coli AcpH was recently expressed and puried.288,289

EcAcpH appears to be a poorly behaving protein. It aggregatesand expresses mostly in the insoluble fraction. Holo-ACPs fromAquifex aeolicus, B. subtilis, Lactococcus lactis, and the mito-chondrial ACP of Bos taurus were tested for hydrolysis by AcpH.A. aeolicus and B. subtilis ACPs were hydrolyzed, but both L.lactis and B. taurus ACPs were not. This behavior matched thatof EcAcpS, suggesting that PPTase and AcpH recognize similarACP features.

Further, AcpH activity must somehow be regulated in vivo,since based on the cellular levels of AcpH and its activity, itwould transform all cellular holo-ACP into apo-ACP within oneminute.289 AcpH is non-essential in E. coli, so it remains unclearwhat physiological role it plays. The acpH gene is also found inother Gram-negative bacteria, cyanobacteria, and, surprisingly,in Ricinus communis (castor bean). Whether the latter is anartifact or contamination has not been discussed. Protein-BLAST does not reveal other plant AcpHs, however nucleotide-BLAST does show a few hits in other plants (e.g. Oryza sativa andZea mays).

AcpH is a non-canonical member of the HD phosphatase/phosphodiesterase family.290 Currently, there is no structure ofAcpH available, but sequence alignments identied a proteinwith homology: the N-terminal portion of SpoT protein. SpoTalso catalyzes the cleavage of a phosphoester and requires Mn2+

for activity (Fig. 21). Murugan et al.291 isolated and expressed theAcpH (Uniprot protein PA4353) from P. aeruginosa, which is a

talyzed by AcpH. (B) Model of E. coli and P. aeruginosa AcpH (modeledturquoise. The natural substrate of SpoT, ppGpp, is shown in sticks and

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soluble protein and hydrolyzes multiple holo-ACPs, as well as anacylated ACP in a multidomain polyketide.

Recently, we have utilized this well-behaved AcpH to cleavemany different PPant probes from a variety of carrierproteins.292 Various chain-length probes were cleaved fromcarrier proteins to facilitate facile attachment and detachment,in order to study the behavior of the labeled protein by proteinNMR spectroscopy. The plastidic holo-AcpP from green micro-algae Chlamydomonas reinhardtii is also a substrate for thisAcpH and is efficiently transformed into apo-Cr-cACP.293

Regulation by 40-phosphopantetheinylation, as predicted byVagelos et al.,284 remains elusive and might not be the point atwhich Nature regulates fatty acid biosynthesis and synthaseactivity. However, the presence of AcpH in some species and itsbroad activity suggests that there is some control on the acti-vation of carrier proteins.

Fig. 22 Comparison of B. subtilis AcpS (left) and Sfp (right). (A) Top

5 Structures

Structural studies of PPTases began as early as 1999.271 Sincethen, 13 PPTases have been structurally described (Table 4).Several of these PPTases were crystallized along with CoA andcarrier protein targets, providing a substantial amount ofarchitectural information concerning the residues required forCoA binding and carrier protein recognition. From a structuralpoint of view, the AcpS-type PPTases are better understood thateither Sfp-type or Fungal Type I FAS PPTases. Of the 13 knownstructures, two are Sfp-type PPTases, and only one representsthe fungal type I FAS PPTases. Further structural characteriza-tion of additional PPTases will aid in our understanding of theevolution of function in PPTases generally and the discovery ofpotent inhibitors as well as modication of substrate prolesfor biotechnological use.

view of both enzymes. CoA is colored red, and divalent cations areshown as black spheres. Each trimer of AcpS is colored differently toshow arrangement of monomers, and each “pseudodimer” of Sfp halfis colored differently for comparison to AcpS. (B) Assignment ofsecondary structure to both AcpS and Sfp. (The a-helices are shown inin blue, and b-sheets shown in orange.)

5.1 Structural features of AcpS-type PPTases

The rst structurally characterized AcpS-type PPTase is AcpSfrom B. subtilis.271 It exhibits a trimeric quaternary structure,

Table 4 List of currently solved structures

Organism Type Ligands

M. tuberculosis AcpS Apo, 30,50-ADPB. anthracis AcpS 2� 30,50-ADPV. cholerae AcpS CoAM. smegmatis AcpS ApoS. aureus AcpS Apo, ACPC. ammoniagenes AcpS Apo, CoAB. subtilis AcpS Apo, CoA, ACP, Inhibitors

(not deposited)S. pneumoniae AcpS Apo, 30,50-ADPS. coelicolor AcpS Apo, CoA, Acetyl-CoA, H110A

D111A/CoAP. yoelii AcpS 30,50-ADPB. subtilis Sfp CoAH. sapiens Sfp Apo, CoA, hACP/CoAS. cerevisiae Part of type I FAS Apo, CoA

90 | Nat. Prod. Rep., 2014, 31, 61–108

with each monomer measuring about 15 kDa. The trimercontains three active sites, formed at the interface betweenmonomers. At each interface, a b-sheet “cle” is responsible forbinding the cofactor CoA (Fig. 22).

The active site contains a Mg2+ cation that coordinates to thepyrophosphate moiety of CoA (Fig. 23). The Mg2+ ion is held inplace by two acidic residues, Asp8 located on b-sheet 1 andGlu58 located on a-helix 4. Glu58 serves a dual purpose,responsible for both coordinating the Mg2+ ion as well asdeprotonating the conserved serine residue of the ACP. Oligo-merization of the trimer is controlled by the interaction of b-sheet 1, the sheet that contains Mg2+ coordinating residues, andb-sheet 5 of an adjacent monomer. These interactions are

Publication year PDB code Ref

2009, 2011 3NE1, 3H7Q, 3NE3, 4HC6 1512012 3HYK 2942012 3QMN 294To be published 3GWM2012 4DXE 2942011 3NE9, 3NFD 1512000, 2005(not depositied)

1F7T, 1F7L, 1F80 271,295

2000 1FTE, 1FTF, 1FTH 296/CoA, 2011 2JCA, 2JBZ, 2WDO,

2WDS, 2WDY126

To be published 2QG81999 1QR0 2972007 2YBD, 2C43, 2CG5 2602009 2WAS, 2WAT 22



Fig. 23 Comparison of the active site of B. subtilis AcpS (A) and Sfp (B).CoA is shown in stick form, divalent cations are shown as blackspheres, and important residues are shown in ball-and-stick form. (A)Residues on different monomers are colored green and yellow. (B) ThePPant moiety of CoA was removed for clarity.

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dominated by hydrophobic interactions, with Ile5 on b-sheet 1contributing many hydrophobic interactions with b-sheet 5.Additionally, the Gln113 residues of each monomer form ahydrogen-bonding network with each other in the core of theassembled trimer. The adenine base of CoA is cradled by theopposite side of an adjacent AcpS monomer, with Pro86 on b-sheet 3 supporting the aromatic base. Lys64 on a-helix 4 pointsat the diphosphate of CoA, most likely donating a proton duringPPant-transfer. While all subsequently solved structures ofAcpS-type PPTases exhibit similar overall structural features,varying crystallization conditions and ligand complexes resultin structural variations that enable a greater understanding ofstructure–function relationships.

AcpS from M. tuberculosis adopts two distinct conformerswhen crystallized under varying conditions. A structure con-taining 30,50-ADP was initially solved by Dym et al.150 Whencompared to other AcpS structures, several regions were absentin the electron density including residues 22–30, 41–44 and 75–78. Additionally, a-helix 3 extends further than other AcpSs. Alinker region between a-helix 3 and loop 1, which is longer thanthe same region of other AcpS structures, adopts an “open”conformation. When later crystallized by Gokulan et al.151 at alower pH, this linker region adopted a “closed” conformation,with a 12 A shi in a-helix 2 and a 9 A shi in the previouslydescribed a-helix 3 region. No absence of electron density wasobserved, possibly indicating that cofactor-free AcpS is morecompact and ordered than when bound to 30,50-ADP. Thisconformational change was predicted to be pH dependent whenGokulan et al. obtained an apo-AcpS structure that more closelyresembled the “open” conformation, crystallized under similarpH conditions to Dym et al.150 The same helix movementsobserved by Dym et al.150 were observed in this new structure.

Comparison of the structures of apo-, CoA-bound, and acetylCoA-bound S. coelicolor AcpS revealed large conformationalchanges upon cofactor binding.126 Conserved residue Arg44shied to bind to the 30-phosphate of CoA. Additionally, thebackbone shied to accommodate both the PPant moiety andthe adenine base of CoA. Mutational studies led to the discoveryof an H110A mutant that showed a negligible decrease in CoA


binding, but a severe loss of activity. Based on structural data,H110A greatly alters the orientation of D111, which is essentialfor Mg2+ coordination. This observation suggests that loss ofactivity does not always corroborate with the ability of a PPTaseto bind CoA.

5.2 Structural features of Sfp-type PPTases

Only two structures of Sfp-type PPTases have been characterizedto date: Sfp from B. subtilis,15 the founding member of theenzyme class, and AASDHPPT from H. sapiens.260 These PPTasesrange from �200 aa to over 340 aa in size. Unlike AcpS-typePPTases, Sfp-type PPTases exhibit a pseudodimeric fold, con-sisting of two structurally similar subdomains connected by ashort polypeptide loop (Fig. 22). This pseudodimer resemblesthe interface of two AcpS-type monomers (Fig. 24). It is possiblethat this pseudodimer is a result of a gene duplication of AcpSto form two tandem copies. Alignment of AcpS with each half ofSfp reveals an interesting conservation pattern. Residues thatcorrespond to the pseudodimer interface, as well as severalactive-site residues, are more highly conserved than the outerresidues. Additionally, the C-terminal half of Sfp more closelyresembles a monomer of AcpS, which contains the acidic resi-dues that coordinate the Mg2+ ion.

An extra loop that contains a short helix is found at the C-terminus of Sfp-type PPTases. Based on the currently solvedstructures, this confers stability to the pseudodimer, with the a-helix bound between the two pseudodimer halves (Fig. 22). Sfp-type PPTases contain only one active site in which CoA is bound.The structure of Sfp was solved with CoA and a Mg2+ ion boundin the active site. Similar to AcpS-type PPTases, an absolutelyconserved glutamate residue, which corresponds to Glu151 inSfp, serves to deprotonate the serine of the incoming ACP tofacilitate PPant transfer (Fig. 23). Sfp contains two acidic resi-dues, Asp107 and Glu109 on b-sheet 6, that coordinate the Mg2+

ion, as opposed to the single Asp residue found in AcpS typePPTases (Fig. 23). It was observed by Mod et al. that uponMALDI analysis of Sfp, both the mass for Sfp and the mass forSfp plus CoA were observed.297 It was estimated that about 20–30% of the recombinantly expressed Sfp co-puries withcellular CoA.

AASDHPPT is the only PPTase gene identied in the H.sapiens genome, and thus is likely responsible for activating allenzymes that require a PPant modication, which include theACP from type I FAS,260 aminoadipate semialdehyde dehydro-genase,26 and tetrahydrofolate reductase.269 It is almost 100residues larger than Sfp, with a long “tail” that wraps aroundthe back of the PPTase (Fig. 25). This tail may be important forrecognition of the various target proteins. In contrast to Sfp,AASDHPPT only contains two acidic residues at the Mg2+

binding site, more closely resembling AcpS-type PPTases.Sequence analysis of Sfp-type PPTases reveals conservation ofthree acidic residues in the active site in prokaryotes (resem-bling Sfp), while only two acidic residues are observed ineukaryotes (resembling AASDHPPT). In the structure of Sfp, twob-sheets of the C-terminal half extend out to form an “arm” thatwraps around an adjacent Sfp monomer. While gel ltration of

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Fig. 24 Overlay of a monomer of AcpS onto the structure of Sfp, exhibiting the similarity of the pseudodimer to the interface between two AcpSmonomers. The “arm” in the C-terminal portion of Sfp has been omitted for clarity. (A) Overlay onto the C-terminal portion of Sfp. Strongsecondary structure conservation is observed. (B) While overall shape is conserved, less similarity is observed between AcpS and the N-terminalportion of Sfp.

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Sfp indicates a monomeric quaternary structure, it is unclearwhether this is an artifact of crystallization or indicative of anative interaction between monomers. AASDHPPT exhibits amore globular overall structure.

AASDHPPT was co-crystallized with human AcpP, sub-clonedfrom the large type I FAS biosynthetic complex. In contrast toyeast type I FAS, which contains a PPTase at the C-terminus ofFASI complex,21 human type I FAS must be activated before nalassembly by AASDHPPT. To obtain a structure with both ACPand CoA bound simultaneously, a Ser to Ala mutation wasintroduced into the ACP, eliminating the serine residueinvolved in PPant transfer. This structure differs slightly fromthe structure of AASDHPPT. Mg2+ was not observed in the activesite, which may be due to the pH of the crystallization condi-tions, as the acidic residues responsible for coordinating theMg2+ ion are likely protonated at the acidic pH of crystallization.The active site Glu, responsible for both Mg2+ coordination andserine deprotonation, is rotated away from the Mg2+ bindingsite and points into a space that would normally be occupied bythe conserved serine of the ACP. The “tail” observed in the CoA-bound AASDHPPT structure is largely absent from theAASDHPPT-ACP co-structure.

Fig. 25 Cartoon representation of AASDHPPT. (A) The C-terminal “tail” pMg2+ in Black. (B) Human FASI ACP-AASDHPPT co-crystal structure. Aphopantetheinylation. The Ala residue is positioned closed to the phosphaup to two carbons past the diphosphate.

92 | Nat. Prod. Rep., 2014, 31, 61–108

5.3 Structural features of type I integrated PPTases

The structure of the FAS from S. cerevisiae did not exhibit strongelectron density for the PPTase located at the C-terminus of themegasynthase, which is located outside of the core of the largeFAS assembly.21 It was unclear how this PPTase interacted withthe ACP to convert it from the apo- to the holo-form, since thisPPTase resembles AcpS-type PPTases that undergo trimeriza-tion before they are active. This PPTase domain was excisedfrom the fungal FAS and structurally characterized both withand without CoA.22 Interestingly, it formed a trimer in solution,similar to AcpS-type PPTases. However, the active site moreclosely resembles the Sfp-type PPTases, since it contains threeacidic residues (E1817, D1772, E1774) that coordinate the Mg2+

ion. This excised PPTase showed phosphopantetheinylationactivity with the S. cerevisiae ACP from the same FAS complex.These data suggest that an assembly event of several mega-synthase subunits form either a dimer or trimer that can thenactivate other synthases. Since both the ACP and the PPTase arelocated on the a-subunit of the FAS, it is possible that thesesubunits associate and activate the ACP domains prior to a6b6megasynthase assembly.

resent in ASDPPT but not in Sfp is depicted in red, with CoA in grey andSer to Ala mutation was performed on the ACP to prevent 40-phos-te that is attacked by the native Ser. Density for CoA was only observed



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5.4 Identication of residues important for PPTase activity

Mutational analyses of PPTases based on structural data haveelucidated several integral residues that are required for func-tion, many of which are conserved throughout the proteinfamily. In general, acidic residues that coordinate the integralMg2+ ion are essential for activity. Additionally, the lysinepositioned behind the diphosphate of CoA is required forcatalysis (Fig. 23).

Parris et al. investigated the residues required for oligo-merization of AcpS.271 I5A and I5R mutations led to a decreasein apparent molecular weight, as measured by gel ltration.Activity of these mutants was unobservable. An N113E mutationdisplayed gel ltration behavior similar to wild type, but withreduced activity in vitro, indicating that the hydrogen-bondingnetwork important for oligomerization can be formed from theglutamate residues. An N113R mutation led to no activity andreduction of oligomerization. Residues E57, H110, and D111were mutated in S. coelicolor AcpS and assayed for CoA binding(via isothermal titration calorimetry, ITC) and activity. E57A ledto a 4–6-fold reduction in CoA binding, while H110A showedlittle change when compared to wild type. D111A, however,exhibited uncharacteristic binding behavior during ITC exper-iments, with CoA binding occurring at lower concentrations ofCoA with a stoichiometry of 0.5, and a second binding event athigher concentrations. A Kd could not be accurately calculatedfor this mutant. E57A completely abolished PPTase activity,further implicating its importance in the 40-phosphopante-theinylation reaction. H110A and D111A both showed signi-cantly reduced activity, measured at 5% and 28% of wild-typeactivity, respectively.

A comprehensive panel of active site mutants of Sfp wasassayed for carrier protein and CoA binding and activity.275

H90A and H90N, mutations of a His that facilitates CoA bindingby coordinating the diphosphate moiety, signicantly reducethe activity of Sfp. K75N, a mutation of the lysine that aids inbinding the adenine base of CoA, decreased the Km for CoA, butdid not severely affect activity. The S89L mutation caused asimilar effect. Mutation of the highly conserved E151 and D107to a Glu and Asp, respectively, resulted in a large decrease inactivity. Mutations that completely inactivated Sfp includeT44S, D109E, D107A, E109D, and E151A. The resulting effect ofthese mutations corroborate with their proposed functions.Interestingly, substitutions of Glu with Asp (and vice versa),which conserve the acidic nature of the natural amino acid, leadto catalytically inactive Sfp mutants. This indicates that bothcharge and the position of the charge are important for Sfpactivity.

A similar panel of mutants was constructed for AASDHPPTbased on the structure and proposed catalytic mechanism.The effect of these mutations was observed based on varyingthe concentration of the substrate acetyl-CoA as well as themagnesium required for catalysis. Mutation of acidic residuesD129 and E181 greatly decreased PPTase activity, but had arelatively small effect on the Km for CoA. Although Gln112 wasthought to aid in Mg2+ binding, mutation of this residue hadlittle to no effect on the enzyme. Mutation of Arg47, Arg86,


and His111 reduced the Km for both CoA and Mg. Thissuggests a cooperative binding event in which CoA facilitatesthe binding of Mg2+ to the PPTase. Alteration of these two Argresidues increased the kcat, suggesting that the release of thebyproduct 30,50-PAP is controlled by coordination of the 30-phosphate with these two arginine residues. Lys185, locatedbehind the diphosphate of CoA, did not affect the affinity ofAASDHPPT for Mg2+ or CoA, but signicantly reduced theactivity.

Mutants of Sfp-type PPTases from several species of fungihave been investigated for functional alterations in PPTaseactivity. The Lys5 gene of C. albicans was biochemically char-acterized, and residues proposed to be important for activityand function were mutated.74 The two mutations that had thegreatest negative effect on Lys5 activity were E198D, K197R,K202R, and D153E. The Glu and Asp are most likely the acidicresidues responsible for both coordination of Mg2+ anddeprotonation of the carrier protein substrate. As previouslyobserved in Sfp, mutations of acidic residues that alter thedistance between the backbone and the acid moiety severelyaltered the natural function of Lys5. This indicates that bothcharge identity and position are integral for proper PPTasefunction. The K202R mutation most likely disrupts the keyinteraction with the diphosphate of CoA. K197R, which may ormay not play a role in catalysis, might affect the positioning ofthe key acidic residue E198. Cfw/NpgA, the Sfp-type PPTasefrom the fungus A. nidulans, is required for the production ofseveral natural products, as well as conidiophore development,and was named for the phenotype of non-pigmented colo-nies.67,68 It was discovered that a single-point mutation in thisgene, L217R, resulted in a temperature-sensitive strain of A.nidulans.69 This residue is possibly required for proteinstability, and mutation from a hydrophobic residue to ahydrophilic residue severely disrupts the solubility and stabilityof the PPTase NpgA.

5.5 pH and metal effects on PPTase activity

The activity of PPTases is pH dependent. In general, PPTases aremost active at pH values ranging from 6.0 to 7.0, but stillmaintain some activity at pH values approaching 8.5.25,28,41,56

This is advantageous to in vitro studies that contain otherproteins that require higher pH conditions for stability and/oractivity.298

All PPTases require aMg2+ ion for PPant transfer, andMg2+ isa key component for PPTase reaction buffer conditions. Asdiscussed previously, acidic residues involved in coordinating aMg2+ ion are universally observed in all PPTases. Since thestructure of B. subtilis AcpS contains a Ca2+ ion instead of Mg2+,Mod et al. investigated the ability of Sfp to utilize other diva-lent cations for catalysis.275 Interestingly, only Mn2+ showedactivity when supplemented to Sfp. In fact, while having a muchlower binding affinity for Sfp, it conferred a greater catalyticefficiency to Sfp. PPTase activity with Mg2+ was only 40% of theactivity seen with manganese, with a kcat/Km almost 3 orders ofmagnitude lower. Ca2+, Zn2+, Co2+, and Ni2+ did not confer anyPPTase activity to Sfp.

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Fig. 26 CoA analogs: CoA can be broken down into several parts, including phosphopantetheine, pantetheine, pantothenate, and cysteamine.Sfp and other Sfp-type PPTases can utilize CoA analogs that bear modified pantetheine arms.

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5.6 Activity and specicity for CoA donor

As previously described, PPTases catalyze the transfer of thePPant portion of CoA onto the conserved serine residue ofcarrier proteins. Both AcpS- and Sfp-type PPTases utilize CoA invivo. However, the inherent promiscuity of some PPTasesenables the utilization of CoA analogs as alternative substratesboth in vitro and in vivo (Fig. 26).299–301

94 | Nat. Prod. Rep., 2014, 31, 61–108

The phosphoadenylate portion of CoA is indispensable forPPTase binding. Structural studies suggest strong conservationamong closely related organisms of the hydrophobic pocketsurrounding this portion of CoA. However, the residues thatform this hydrophobic pocket may vary between distantlyrelated PPTases. CoA that lacks a 30-phosphate on the ribosesugar will not bind to PPTases.28 30,50-PAP, the byproduct of



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PPTase labeling, binds strongly enough to inhibit PPTaseactivity at high concentrations, and is used as a standard for Sfpinhibition.302

While the adenylate moiety is integral for binding, currentstructural data indicate that the PPant portion does not signif-icantly contribute to CoA binding. In several crystal structures,density for the PPant arm of CoA is not completely observed,indicating that it is not locked in a specic “binding confor-mation.” Additionally, the PPant arm may shi into alternativeconformations upon carrier protein binding. The AcpS-typePPTase from S. coelicolor is capable of utilizing a variety of acylCoAs.50 The co-structure of this PPTase with CoA shows adistinct binding pocket for the PPant arm. However, closerexamination of the structure reveals a possible route for thepantetheine arm to extend over the surface of the PPTase thatwould not interfere with the phosphopantetheinylation reac-tion.126 A possible explanation involves the residues Leu70 andThr72, which are usually larger charged or polar residues inother AcpS-type PPTases. Their small size and lack of chargedoes not obstruct the exit route for the PPant arm of CoA. In thestructure of AASDHPPT (Fig. 25) with the excised human type IACP, density for the PPant arm was not observed. This suggeststhat it is projected out between the ACP-PPTase interactionsurface through an exit route similar to that observed in S.coelicolor AcpS (Fig. 25). Residues 199–204 border the PPantarm, and consist of small, non-polar amino acids that couldallow alternative substrates to protrude from the bindingpocket. These observations corroborate with the ability ofcertain PPTases to modify carrier protein domains with a widevariety of modied CoAs, in which the PPant arm bearsunnatural molecules, including substrate mimics,292,303 uo-rescent molecules,304,305 cross-linking agents,299 and affinitytags257 (Fig. 26). The ability to utilize unnatural CoA analogs hasbeen important for assessing PPTase activity, especially in thecontext of drug discovery.306 Additionally, modication ofcarrier proteins with substrate intermediates is important forelucidating carrier protein function.307

5.7 Measuring the activity of PPTases

Methods for measuring PPTase activity are integral for studyingPPTase specicity towards carrier protein targets and the abilityto use non-natural CoA substrates. Additionally, measuring theactivity of mutant PPTases reveals both important catalyticresidues and residues required for PPTase-ACP interaction.Unlike many enzyme reactions, there are no co-factor trans-formations that can be easily followed for PPTases, nor is thereaction mechanism easily coupled to a reporter system. Thus,the modication of target carrier proteins is measured usingvarious separation methods and CoA substrates.

Initially, PPTase activity was assessed using 3H labeled CoA,1

which was prepared by exposing CoA to tritium gas. A reactioncontaining this labeled CoA, E. coli AcpS, and E. coli ACP wasallowed to progress for 30 min at 37 �C. The proteins in thereaction were precipitated with 10% trichloroacetic acid andsubsequently washed to remove free, labeled CoA. The resultingpellet was redissolved and assessed for radioactivity using


liquid scintillation counting. In this way, apparent kineticvalues for AcpS were determined. In the same study, the use ofurea-PAGE allowed the authors to assess labeling by gel-shi.Urea-PAGE is conformationally sensitive and can be used todistinguish between the apo- and holo-forms of carrierproteins.308 Thus, modication of the carrier protein with AcpScauses a “shi” in the ACP band down the urea-PAGE gel.Carrier protein modications can also be detected using highpressure liquid chromatography.41,102,309 For example, apo- andholo-forms of the PCP from a module of surfactin biosynthesis,SrfB, were separated on a reverse-phase HPLC column.310 Thisallowed not only for detection of activity, but also for thequantication of apo- and holo-ACP by integration of each HPLCpeak. While these methods are useful for determining kineticparameters, they are both resource- and time-intensive. Radio-labeling can pose safety hazards, while gel and HPLC analysisrequire long runs to complete. New PPTase assays haveaddressed both the safety and time issues with these olderexperimental methods.

We have developed several assays based upon PPTase-cata-lyzed transfer of uorescent labeled CoA, described in the nextsection.

Duckworth and Aldrich have recently used uorescencepolarization to measure PPTase activity by utilizing the strongassociation of CoA with Sfp.311 For this assay, a BODIPY–CoAconjugate was synthesized by reacting CoA with a BODIPY–maleimide construct. Since the free BODIPY–CoA will tumble insolution at a greater rate than an Sfp-bound BODIPY–CoAmolecule, a difference in anisotropy between the two states canbe measured. Addition of increasing amounts of BODIPY–CoAto a xed concentration of Sfp causes an increase in anisotropysignal, corresponding to the binding of BODIPY–CoA to Sfp.Validation as an assay for discovery of PPTase inhibitors wasperformed using 30,50-PAP, and an IC50 value comparable topreviously published results was obtained.

Recently, a single module NRPS protein that is responsiblefor producing the blue pigment indigoidine was discovered,312

and subsequently utilized in the detection of PPTaseactivity.40,212 This protein utilized two molecules of L-glutamineto produce a blue, bicyclic molecule that is amenable to visible-light detection methods. There is a single PCP domain thatrequires modication by a PPTase to produce the blue pigment.Since heterologous expression of BpsA in E. coli does not yield aprotein product bearing a PPant modication on the PCP, it canbe used for in vitro assessment of PPTase activity.

6 Biotechnological use of PPTases

Some PPTases such as Sfp are promiscuous. Besides theircognate enzyme, they are able to activate a wide range of non-cognate carrier proteins. Concomitantly, the interaction of aPPTase with the carrier protein is very specic. The same is truefor the second substrate CoA. While the PPTase explicitlyrecognizes CoA, it is able to utilize CoA conjugated to a broadrange of small molecules.313 With these characteristics, theunique reaction of a PPTase-catalyzed carrier protein modi-cation has been applied for the development of site-specic

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Fig. 27 Scheme demonstrating the diversity of biotechnologicalapplications of the carrier protein–PPTase interaction.

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protein labeling techniques in vitro and in vivo (Fig. 27).90,314,315

For detailed experimental labeling procedures, we refer toSunbul et al.316

6.1 In vitro labeling

In 2004, we were the rst to utilize the PPTase-carrier proteininteraction to visualize, identify and purify modular syn-thases.313 This work used Sfp and a modied CoA to attach anon-native PPant onto AcpP, PKS ACP and PCP. Several CoAderivatives were synthesized by attaching uorescent- andaffinity reporter-labeled maleimides on the free thiol group ofCoA. The probe palette was extended by generating CoA analogswith different uorescent dyes.317 To accelerate the process ofhigh-throughput screening of small-molecule libraries for drugdiscovery, Yin et al. utilized the interaction of GrsA-PCP and Sfpin phage display (see Section 4).318 Using Sfp, small moleculeslike biotin, uorescein or glutathione were covalently linked tothe serine residue of GrsA-PCP presented on the phage surface.A similar setup of this interaction was applied to identify aspecic PCP-tag for affinity purication that can be co-expressed with the target protein.318

6.2 In vivo tagging and labeling

The concept of PPTase-catalyzed carrier protein-tagging wasimproved and patented for the in vivo study of membraneproteins.315 The specic labeling allows not only localization ofproteins such as receptors, but to further study the proteindynamics and intracellular trafficking .319–322 This techniquecould be successfully applied to various membrane proteins inE. coli, S. cerevisiae, HEK293 and TRVb cells and neurons.320–324

Offering a high signal intensity as well as excellent photo-stability, quantum dots were evaluated as replacement of uo-rescent dyes for imaging of cell surface and transmembraneproteins.325,326

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All these methods have been applied in vitro or upon the cellsurface. Clarke et al. were the rst to presen in vivo labeling.304

Using non-hydrolyzable, uorescent pantetheine analogues,overexpressed VibB-PCP was labeled in vivo using co-expressedSfp. Loading cell lysate directly onto a SDS-PAGE gel, labeledVibB-PCP was visualized by UV. These uorescent probes weremodied for additional surface-based affinity detection andpurication.327 Using a stilbene reporter tag enabled a switch-able, antibody-elicited, uorescent response in solution or onaffinity resin. The range of probes was expanded by testingvarious combinations of linker, dye, and bio-orthogonalreporter.300 This allowed for purication of the carrier proteinindependent of antibody techniques. Further in vivo labelling ofnative carrier proteins using pantetheine analogues could bedemonstrated for Gram-positive and Gram-negative bacteriaand in a human carcinoma cell line.328

Site-specic protein labeling using the Sfp system wasrened using novel protein tags such as YbbR,276,329 S6, andA1,257,278 which were identied using a phage-displayed peptidelibrary. These tags are very short (11 and 12 aa), and thus causeminimal disturbance to the target protein structure and func-tion. S6 and A1 can even be used as a pair for the sequentiallabeling of two proteins with different small-molecule probeswith very little cross-labeling, using Sfp and AcpS, respectively.To evaluate how far the peptide tag size can be reduced, 15N-HSQC-based NMR titration experiments were conducted.278 Theresulting octapeptide could be used for in vitro and cell surfacelabeling. Thus, during this process, it was shown that AcpS isable to convert a PCP that is naturally not modied into asubstrate. If this is generalizable, it would be of great impor-tance for natural-product engineering.

Powerful tools were generated by combination of the carrierprotein–PPTase interaction with other techniques, such as yeastsurface display for vaccine development330 or structural xationfor X-ray crystallographic analysis as demonstrated for a di-domain construct from EntF.331 High-throughput assays weredeveloped for the identication of novel PPTases, PKS andNRPS, or improved enzyme activities using in vitro (solid phase/phage display)316,332,333 as well as in vivo methods (metagenomiclibraries).212,334

6.3 Other applications

For solid phase applications, Wong et al. used Sfp to immo-bilize YbbR-tagged protein onto surfaces functionalized withCoA.332 Immobilization was demonstrated for ACP, luciferase,glutathione-S-transferase and thioredoxin onto PEGA resin aswell as hydrogel microarray slides and could even be per-formed directly from cell lysate. Developing this technology forcell biology and tissue engineering, Mosiewicz et al. could evenencapsulate primary mouse broblasts into a hydrogel usingthe PPTase-mediated linkage.335 In reverse, Stack et al.333 usedan immobilized synthase to measure PPTase activitywith uorescent CoA substrate analogues in a microtiter plateassay.

These advancements in utilization of the PPTase–carrierprotein interaction paved the way for the elucidation of modular



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enzyme biosynthetic pathways, including mechanism, structureand proteomic identication of the synthases.336 To study theinteraction between the carrier protein and the other synthasedomains, pantetheine analogues were developed containingterminal moieties serving as irreversible cross-linkingreagents.299 The carrier protein was labeled with the pan-tetheine probe by one-pot chemo-enzymatic synthesis.298 Thisincludes two steps: the conversion of the pantothenate into aCoA analog using CoAA, CoAD and CoAE, and attachment ofthis probe onto the carrier protein by a PPTase. Following, thesecond domain interacts with the probe, forming an irreversiblecovalent adduct with the carrier protein. Cross-linking could besuccessfully demonstrated for the ketosynthase, dehydratase,and thioesterase domains.293,301,337–339

Fluorescent and affinity reporters were further utilized foractivity-based proteomic proling.340 In combination with thechemo-enzymatic methods of carrier protein labeling, themethod was applicable for probing inhibitor specicity,assigning domain structure, and identifying natural-productproducing modular synthases in vitro and in vivo.

A further example for how to study these enzymatic pathwaysin vivo is demonstrated with GlyPan (disulde of N-pan-toylglycyl-2-aminoethanethiol).303 GlyPan is a pantetheineanalogue containing glycine, and thus one carbon shorter thanendogenous b-alanine. GlyPan was efficiently loaded, in vivo,onto E. coli AcpP presumably by an endogenous PPTase,showcasing another kind of promiscuous behavior of PPTases.

6.4 Production of natural products in heterologous hosts

Many non-ribosomal peptides and polyketides are importantpharmaceuticals and agro-chemicals. Production of thesenatural products and possibly “unnatural” natural products (bymodication or mutagenesis of the synthase) has been the goalof many research projects over the past two decades. This effortled to signicant innovations in the eld of metabolic engi-neering for drug discovery and development.341

The rst attempts to heterologously express modularnatural-product synthases in E. coli yielded primarily the inac-tive apo-form of the synthase, prohibiting actual biosynthesis ofthe natural product.342–346 Aer the discovery of PPTases andtheir function,1 co-expression of a PPTase (Gsp) with a truncatedNRPS (gramicidin S synthase) led to in vivo activation of thesynthase by detection of an intermediate that was absent whenonly the NRPS was expressed.347 Co-expression of the PKS6MSAS (6-methylsalicylic acid synthase) and the PPTase Sfp inE. coli and in S. cerevisiae by Kealey and co-authors marked therst instance of heterologous biosynthesis of a naturalproduct.348 Production of 6MSA (6-methylsalicylic acid) in yeastwas even 2-fold greater in comparison to the native producerP. patulum.

Since then, multiple polyketide and non-ribosomal peptideshave been successfully expressed in further optimized, biolog-ically friendly heterologous hosts, such as E. coli, S. coelicolor,and yeast.349–353 Due to their modular nature, these synthasescan theoretically be manipulated to yield a wide range ofpossible biomolecules.354–356 Within these model organisms, the


ux can further be improved by increasing the amount ofstarting material such as CoA-derivatives.357,358 Besides theirpharmaceutical application, modular synthases have gainedincreasing importance in the elds of biofuels and nutrition.The declining availability of fossil fuels has intensied the effortto investigate novel routes to heterologously produce hydro-carbons.359 Besides in vivo modication of the native fatty acidbiosynthesis by introduction of additional domains,293,360,361 theactual synthases were evaluated for the production of valuablemolecules in more suitable host systems. Towards heterologousproduction of specic aliphatic hydrocarbons, Akhtar and co-workers engineered the carboxylic acid reductase (CAR) genefrom M. marinum into E. coli (also see Section 2.1).189 Incombination with a chain-length-specic thioesterase, thisstrain was able to convert fatty acids to fatty alcohol andalkanes. Including a fatty acid-generating lipase, E. coli evenutilized natural oils for this progress. CAR, however, requires 40-phosphopantetheinylation, as previously shown (see Section3).39 The co-expression of Sfp was the key to get this biosyntheticmachinery progressing at maximum activity. Recently, Amiri-Jami and Griffiths produced both EPA and DHA in E. coli byheterologous expression of the omega-3 fatty acid synthase genecluster from Shewanella baltica MAC1 (Fig. 10).362 One fosmidclone from S. baltica contained pfaA–D, but not the PPTase genepfaE, resulting in a clone that did not produce DHA or EPA.However, when the full pfaA–E gene cluster was expressed in E.coli, the bacteria were able to produce both omega-3 fatty acids(see Section 3.6). By heterologous expression of PUFA genes inplants, these nutritional fatty acids are produced in crop plantsthat do natively not provide these compounds.363 In general,PUFA PKS genes have been shown to require co-expression of aPPTase gene.199,244–247,364,365 Similarly, the PKS 6MSAS requiresactivation by heterologously expressed Sfp in E. coli and yeast,348

but was potentially transformed into its holo-form by anendogenous PPTase in tobacco.248 This demonstrates the powerof plant expression systems for this kind of production. Besidesplants, algae that naturally produce very valuable fatty acids andlipids366 have recently received attention for their great potentialas heterologous hosts for the biosynthesis of complex mole-cules,367–370 and should be further investigated for application inbiofuel production.

6.5 Drug discovery

Since PPTases activate important metabolic pathways, theyrepresent a new drug target in bacteria that has not been fullyexplored. Only recently has the PPTase enzyme family beeninvestigated as a potential drug target, with a small handful ofpotent inhibitors identied to date (Fig. 28).

A variety of 4H-oxazol-5-one derivatives with potent AcpSinhibition were synthesized by modication of 4-chlorophenyl4H-oxazol-5-one, an AcpS inhibitor discovered via high-throughput screening.306 Modication of the oxazol-5-one corewith an anthranilic acid moiety led to decreases in IC50 valuesagainst AcpS, with one compound reaching sub-micromolaractivity. The anthranilic acid moiety was again utilized byJoseph-McCarthy et al. to produce anthranilic acid-based AcpS

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Fig. 28 Inhibitors of AcpS-type and Sfp-type PPTases.295,306,371,372

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inhibitors.295 Using structure-based drug design, initial HTShits were optimized based on molecular modeling into theactive site of AcpS from B. subtilis. The optimized compoundsshowed a 30-fold decrease in IC50 values. Furthermore, four ofthe lead compounds were co-crystallized with B. subtilis AcpS.These structures conrmed the importance of the anthranilicacid portion of each inhibitor, which binds in the locationnormally occupied by the adenine base and ribose sugar of CoA.This success underscores the value of structural information forrational design of inhibitors.

The natural product SCH 538415, isolated from an uniden-tied bacterium, was found to inhibit the AcpS gene.371 Using aradiolabeled CoA substrate, a HTS utilizing unidentiedbacteria extracts identied this product as an AcpS inhibitor.Discovery of inhibitors that target Sfp-type PPTases began onlyvery recently with the development of a uorescence resonanceenergy transfer (FRET) assay that relied on the ability of Sfpfrom B. subtilis to label the short peptide YbbR.302 YbbR wasconjugated to uorescein isothiocyanate (FITC) at the N-terminus. Modication of the serine residue of YbbR with adifferent uorescent dye enabled the FRET interaction. FITCwas chosen for its ability to act as either a uorescence energyacceptor or donor. This assay was optimized and validatedusing 30,50-PAP as a model inhibitor. This method was latermodied to replace the N-terminal FRET molecule on YbbRwith a uorescence quenching dye, BHQ2.372 When YbbR was

98 | Nat. Prod. Rep., 2014, 31, 61–108

labeled with rhodamine-CoA, the rhodamine uorescence wasquenched by the now adjacent BHQ2. This signicantlyimproved the reliability of the signal, increasing the sensitivityof the assay. The LOPAC1280 compound library was screened forpotential Sfp inhibitors using this assay. Several hits with lowIC50 values were discovered (Fig. 28). These hits were validatedusing both the original FITC assay as well as a gel-based labelingassay. 6-Nitrosobenzopyrone (NOBP), a hit from the LOPAC1280

screen, was further utilized to inhibit phosphopantetheinyla-tion of the single module NRPS BpsA.40 NOBP was measured tohave a Ki ranging from 0.4 to 5.2 mM.

PptT, the Sfp-type PPTase from M. tuberculosis, has recentlybeen identied as a valid drug target for combating tuberculosisinfection.36,37 Leblanc and co-workers have developed a high-throughput screening method for PptT, which utilizes scintil-lation proximity to measure the extent of protein carrierlabeling. A biotin-tagged ACP excised from the Pks13 gene ofM.tuberculosis served as the target for an MBP–PptT fusion, andthe labeling substrate for PptT was [3H]-CoA. Once the reactionwas allowed to progress, a streptavidin-tagged scintillation beadwas allowed to bind to the biotin tag of the ACP. Spectral countswere then taken of the reaction samples. This assay, which istolerable to DMSO and amenable to high-throughput, couldalso be adapted to PPTases from other organisms by altering thetarget ACP.



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Recent studies of bacteria and fungi reveal that severalspecies require the action of an Sfp-type PPTase for importantsecondary metabolic pathways, that affect growth, reproduc-tion, and pathogenicity. A. nidulans requires the Sfp-type PPTaseNpgA for proper pigmentation and spore formation.69 Func-tioning Sfp-type PPTases are required in the bacteria Agro-bacterium vitis187 and the fungus T. virens83 to effectively elicitplant immune responses. Several strains of plant pathogenicfungi contain an Sfp-type PPTase that is indispensable forinfection, including maize anthracnose fungus C. graminicola232

and the cereal fungus C. sativus.75 Inhibiting the Sfp-typePPTase in these fungi is a new strategy for combating plantpathogens, but there are currently no antifungal compoundsthat target this enzyme.

Detection of the Sfp-type PPTase Lys5 and the closely relatedlysine metabolic gene Lys1 in C. albicans via PCR can be used tospecically identify pathogenic strains of this fungus.373

Amplication of the Lys5 gene in C. albicans enables rapiddetection of this opportunistic fungus, which is oen found toinfect immuno-compromised patients. Since the primarysequence of Sfp-type PPTases between organisms can varygreatly, specic detection and targeting of a pathogen and notthe infected host is possible.

7 Outlook

Natural products have been an inspiration for chemists andbiologists for hundreds of years, andmany of our currently usedmedicines are natural products or based on a naturalproduct.374 Until 1990, natural-product biosynthesis wasstudied primarily by feeding radioactive precursors to wholeorganisms, protein preparations or lysates. In 1990 and 1991,Katz and Leadlay separately published the discovery of thepolyketide 6-deoxyerythronolide B synthase gene, its modularnature and its assembly line production of the natural product.Marahiel and co-workers discovered a few years later that non-ribosomally encoded peptides are also synthesized in a similarfashion. These discoveries (among others) opened up the eldof natural-product biosynthesis and allowed for connecting thegene to the natural product.

Although it was already known since the 1960s that the FASrequires a PPant arm on a conserved serine of a carrier protein,and that a dedicated PPTase is responsible for this post-trans-lational modication, it took until 1995 to establish thatPPTases are a separate superfamily of enzymes that are essentialfor the three major metabolic pathways. In other words, withoutthis post-translational modication, these synthases are unableto let the carrier protein ferry cargo from one active site to thenext. The family of PPTases now contains many annotated orputative proteins (>1700), and the characterization of �60 (andkinetic characterization of �7) enzymes illustrates the diversityof this class of proteins, in oligomerization state, specicity forCoA analogs, specicity for carrier proteins, kinetic parameters,structure and function.

Besides AcpS, which was identied from E. coli by Vagelos inthe 1960s,3 Sfp from B. subtilis is the archetypical PPTase, usedby the bacteria to post-translationally modify surfactin


synthase. Since Sfp has a broad substrate scope, both for its CoAas well as for its carrier protein substrate, this PPTase has beenthe most used for co-expression with engineered biosyntheticpathways. However, Sfp also has its limitations. For example the19 aa consensus peptide of the SrfB PCP cannot be modied bySfp, and all FAS carrier proteins are labeled relatively slow bySfp. To the best of our knowledge, however, there is no apo-carrier protein that is not post-translationally modied by Sfp.Recently, it was shown that a carrier protein from sphingolipidbiosynthesis cannot be modied with either C16 or C18 CoAanalogs using Sfp, suggesting that there might be some limi-tation to the catalytic activity and substrate promiscuity of thePPTase.375

In recent years, we have been observing a trend of expres-sion of newly identied (by genome mining) biosyntheticclusters in heterologous hosts, and it might well be that Sfpwill not always be the ideal PPTase for the job. We hypothesizethat examining the detailed phylogeny of the PPTase familycould reveal which PPTase to overexpress with the synthase ofinterest, or even selecting a heterologous host with an endog-enous PPTase that modies the desired synthase. Thus far,biosynthetic clusters have been expressed mainly in modelbacteria and fungi. However, many new synthase genes havebeen recently found in cyanobacteria, plants, and even highereukaryotes. Expression of these synthases might well requirespecialized protein expression systems, or even designerheterologous hosts.

Many natural products that are biosynthesized by synthasesare essential to bacteria and fungi for virulence and thereforesurvival. Since these synthases require 40-phosphopantetheiny-lation, targeting the PPTase (instead of any other domain) couldbe ideal to weaken or kill these pathogens. It should be notedthat in the late 1990s and early 2000s, targeting FAS by inhib-iting AcpS was a hot topic. Two crystal structures of AcpS-typePPTases were published, and inhibitors were discovered andoptimized by Wyeth and Schering-Plough.295,306,371 Since then,industrial interest in PPTase inhibition has dwindled and mostlikely these projects were terminated.

In the past years, major strides towards the discovery anddevelopment of inhibitors that target Sfp-type PPTases havebeen made. In order to discover these, high-throughput assaysrst had to be developed. Now with these in place, we expectthat antibiotics targeting secondary metabolism will be on therise in the coming years. Finally, since the discovery of PPTasesas a broad family of post-translational carrier protein modi-fying enzymes, they have been used in vitro and in vivo foractivating carrier proteins and derivatizing carrier proteins withnatural and unnatural probes. In the near future, we may see afurther expansion of the repertoire of probes used in vitro andin vivo, including – but not limited by – uorescence, sol-vatochromic, FRET, electronic, positron electron tomography(PET), nuclear magnetic resonance (NMR), electron para-magnetic resonance (EPR) and purication tags. UtilizingNature’s promiscuity to install these and other probes ontocarrier proteins opens up avenues towards drug delivery,studying post-translational modications and protein–proteininteractions.

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8 Acknowledgements

Thanks to Nikolaus Sonnenschein for visualization of geneticloci. This work was supported by California Energy CommissionCILMSF 500-10-039 (to M.D.B.); DOE DE-EE0003373 (to M.D.B.);NIH R01GM094924 and R01GM095970 (to M.D.B.); the NationalScience Foundation under Awards EEC-0813570 and MCB-0645794 (to J.P.N.), and the Howard Hughes Medical Institute(J.P.N.).

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