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The crystal structure of phenylpyruvate decarboxylase from Azospirillum brasilense at 1.5 Åresolution. Implications for its catalytic and regulatory mechanism.Versees, Wim; Spaepen, S; Vanderleyden, J; Steyaert, Jan

Published in:The FEBS Journal

Publication date:2007

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Citation for published version (APA):Versees, W., Spaepen, S., Vanderleyden, J., & Steyaert, J. (2007). The crystal structure of phenylpyruvatedecarboxylase from Azospirillum brasilense at 1.5 Å resolution. Implications for its catalytic and regulatorymechanism. The FEBS Journal, 274, 2363-2375.

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The crystal structure of phenylpyruvate decarboxylasefrom Azospirillum brasilense at 1.5 A resolution

Implications for its catalytic and regulatory mechanism

Wim Versees1,2, Stijn Spaepen3, Jos Vanderleyden3 and Jan Steyaert1,2

1 Department of Ultrastructure, Vrije Universiteit Brussel, Brussels, Belgium

2 Department of Molecular and Cellular Interactions, VIB, Brussels, Belgium

3 Centre of Microbial and Plant Genetics, INPAC, Katholieke Universiteit Leuven, Heverlee, Belgium

Azospirillum is a genus of plant growth promoting bac-

teria that colonize the rhizosphere of certain tropical

and subtropical crop plants [1,2]. The production of

phytohormones such as the auxin indole-3-acetic acid

(IAA) by the bacterium has been recognized as an

important factor in its plant growth promoting abilities

[3]. The ipdC gene of Azospirillum brasilense was iden-

tified as a key gene in the production of IAA in the

presence of tryptophan [4–6]. The same gene was later

also found to be implicated in the production of phe-

nylacetic acid (PAA), an auxin-like molecule with anti-

microbial activity [7]. In A. brasilense, IAA and PAA

originate primarily from the amino acids l-tryptophan

and l-phenylalanine, respectively, and are synthesized

in a three-step pathway (Fig. 1) consisting of (a)

conversion of l-phenylalanine or l-tryptophan to

Keywords

allosteric enzyme activation; enzyme

mechanism; nonoxidative decarboxylation;

protein crystallography; thiamine

diphosphate

Correspondence

W. Versees, Department of Ultrastructure,

Vrije Universiteit Brussel, Oefenplein,

Gebouw E, Pleinlaan 2, 1050 Brussel,

Belgium

Fax: +32 2629 19 63

Tel: +32 2629 19 49

E-mail: [email protected]

(Received 27 November 2006, revised 22

February 2007, accepted 6 March 2007)

doi:10.1111/j.1742-4658.2007.05771.x

Phenylpyruvate decarboxylase (PPDC) of Azospirillum brasilense, involved

in the biosynthesis of the plant hormone indole-3-acetic acid and the anti-

microbial compound phenylacetic acid, is a thiamine diphosphate-depend-

ent enzyme that catalyses the nonoxidative decarboxylation of indole- and

phenylpyruvate. Analogous to yeast pyruvate decarboxylases, PPDC is sub-

ject to allosteric substrate activation, showing sigmoidal v versus [S] plots.

The present paper reports the crystal structure of this enzyme determined

at 1.5 A resolution. The subunit architecture of PPDC is characteristic for

other members of the pyruvate oxidase family, with each subunit consisting

of three domains with an open a ⁄ b topology. An active site loop, bearing

the catalytic residues His112 and His113, could not be modelled due to

flexibility. The biological tetramer is best described as an asymmetric dimer

of dimers. A cysteine residue that has been suggested as the site for regula-

tory substrate binding in yeast pyruvate decarboxylase is not conserved,

requiring a different mechanism for allosteric substrate activation in PPDC.

Only minor changes occur in the interactions with the cofactors, thiamine

diphosphate and Mg2+, compared to pyruvate decarboxylase. A greater

diversity is observed in the substrate binding pocket accounting for the dif-

ference in substrate specificity. Moreover, a catalytically important glutam-

ate residue conserved in nearly all decarboxylases is replaced by a leucine

in PPDC. The consequences of these differences in terms of the catalytic

and regulatory mechanism of PPDC are discussed.

Abbreviations

AbPPDC, Azospirillum brasilense PPDC; BFD, benzoylformate decarboxylase; EcIPDC, Enterobacter cloacae IPDC; IPDC, indolepyruvate

decarboxylase; KlPDC, Kluyveromyces lactis PDC; PDC, pyruvate decarboxylase; POX, pyruvate oxidase; PP domain, pyrophosphate domain;

PPDC, phenylpyruvate decarboxylase; PYR domain, pyrimidine domain; R domain, regulatory domain; ScPDC, Saccharomyces cerevisiae

PDC; ThDP, thiamine diphosphate; ZmPDC, Zymomonas mobilis PDC.

FEBS Journal 274 (2007) 2363–2375 ª 2007 The Authors Journal compilation ª 2007 FEBS 2363

phenyl- or indolepyruvate, catalysed by an amino-

transferase, (b) decarboxylation to the corresponding

aldehydes, and (c) oxidation of the aldehyde to phenyl-

acetic acid or indole-3-acetic acid [4,8,9]. The second

step in this pathway is catalysed by the ipdC gene

product, phenylpyruvate decarboxylase (PPDC;

EC 4.1.1.43), a thiamine diphosphate (ThDP)-depend-

ent enzyme that catalyses the nonoxidative decarboxy-

lation of indole- and phenylpyruvate [10,11].

The A. brasilense PPDC (AbPPDC) is a homo-

tetramer of about 58 kDa subunits. Steady-state kinetic

analysis showed that the enzyme converts phenyl-

pyruvate with a 10 times higher specificity than indole-

pyruvate despite a slightly higher affinity for the latter,

and is completely inactive toward benzoylformate

(S. Spaepen, W. Versees, D. Gocke, M. Pohl, J. Steyaert

& J. Vanderleyden, unpublished results). This clearly dis-

tinguishes AbPPDC from indolepyruvate decarboxylase

(IPDC; EC 4.1.1.74) of Enterobacter cloacae (EcIPDC),

which showed a high activity toward indolepyruvate

and benzoylformate, while phenylpyruvate was found to

function as a competitive inhibitor [12,13]. Moreover, in

contrast to the EcIPDC, the PPDC enzyme is subject to

substrate activation, showing sigmoidal v versus [S]

plots as often observed in pyruvate decarboxylases

(PDC, EC 4.1.1.1), with the exception of most bacterial

PDCs (e.g., Zymomonas mobilis PDC) [14–18].

The amino acid sequence of A. brasilense PPDC

shows relatively low identity with the sequence of

other well studied ThDP-dependent decarboxylases

with, for instance, 26% identity with EcIPDC, 23%

and 25% identity with PDCs from Saccharomyces

2-oxoketoglutarate

glutamate

aminotransferase

RCH2

O

O

O-

phenyl/indole- pyruvatedecarboxylase

H+

CO2

RCH2

O

H

RCH2

O

O-

RCH2

NH3+

O

O-

oxidase

O2

H2O

N

H

R1=R1: L-phenylalanine

R2: L-tryptophan

R1: phenylpyruvate

R2: indole-3-pyruvate

R1: phenylacetaldehyde

R2: indole-3-acetaldehyde

R1: phenylacetate

R2: indole-3-acetate

R2=

Fig. 1. Postulated indole-3-acetate (IAA) and

phenylacetate (PAA) biosynthesis route in

A. brasilense, starting from L-tryptophan and

L-phenylalanine, respectively. The involved

enzymes are indicated in blue. This pathway

is proposed on the basis of Koga et al. [70]

and Somers et al. [7].

Structure of Azospirillum brasilense PPDC W. Versees et al.

2364 FEBS Journal 274 (2007) 2363–2375 ª 2007 The Authors Journal compilation ª 2007 FEBS

cerevisiae (ScPDC) and Zymomonas mobilis (ZmPDC)

and 20% identity with benzoylformate decarboxylase

(BFD, EC 4.1.1.7) from Pseudomonas putida.

This paper reports the first crystal structure of a

PPDC, with the structure of A. brasilense PPDC being

solved at 1.5 A resolution. The general fold of the sub-

units is similar to this of prototypical yeast PDCs

[19,20], while the biological tetramer is present in an

asymmetric conformation. The cofactor-binding site is

fairly conserved compared to IPDC and PDCs. How-

ever, some important differences occur in active site

and proposed regulatory site residues. The potential

consequences of these changes will be discussed in the

light of the currently proposed catalytic and regulatory

mechanisms.

Results and Discussion

Structure determination and subunit description

The Azospirillum brasilense PPDC crystal structure

was solved to 1.5 A resolution using molecular

replacement with the E. cloacae IPDC monomer (PDB

1OVM) as a search model [21]. The asymmetric unit

contains two polypeptide chains, two ThDP molecules,

two Mg2+ ions, two Cl– ions, three glycerol molecules

from the cryo solution, and 1337 water molecules (see

Table 1 for refinement statistics). Clear electron den-

sity is present for the cofactors and for most of the

polypeptide backbone. However, neither the residues

of the N-terminal histidine-tag, nor the seven to eight

C-terminal residues of both subunits were included in

the model due to a lack of electron density. Also a

large peptide region spanning residues 104–119 has

very poorly defined electron density in both subunits.

Hence, this region could only be modelled partially

(residues 106–107 and 112–118 in subunit A, and

105–108 and 111–118 in subunit B were omitted from

the model).

Each subunit consists of three domains with an open

a ⁄ b class topology (Fig. 2). The N-terminal PYR

domain (pyrimidine or a domain, residues 1–160) con-

sists of a central six-stranded parallel b-sheet surroun-ded by six a-helices. The topology of the R domain

(regulatory or b domain, residues 185–330) differs

from the PYR domain and consists of a mixed six-

stranded b-sheet (five parallel and one antiparallel

strand) surrounded by six a-helices, while the C-ter-

minal PP domain (pyrophosphate or c domain, resi-

dues 350–539) is clearly homologous to the PYR

domain and contains a six-stranded parallel b-sheetsurrounded by eight a-helices. Only relatively small

differences are observed between the two subunits in

the asymmetric unit (rmsd of 0.63 A for superposition

of 516 Ca atoms of subunit A on B).

The subunit architecture of PPDC is conserved

among other ThDP-requiring enzymes of the pyruvate

oxidase group (POX group, named after the first estab-

lished structure) [19,22] of which the structure has been

solved, including pyruvate decarboxylase [23,24],

indolepyruvate decarboxylase [21], benzoylformate

decarboxylase [25], oxalyl-CoA decarboxylase [26],

pyruvate oxidase [22] and benzaldehyde lyase [27].

Superposition of the PPDC subunit onto its closest

neighbours yields rmsd values of 2.7 A for 511 Caatoms of ScPDC, 2.8 A for 514 Ca atoms of ZmPDC

and 2.9 A for 501 Ca atoms of EcIPDC.

An asymmetric ‘open’ dimer of dimers

In the asymmetric unit, two subunits of PPDC (desig-

nated A and B) are tightly packed to form a dimer

related by noncrystallographic two-fold symmetry

(Fig. 3A). This dimer interface buries a surface area of

about 2821 A2 and is exclusively formed by interactions

between residues of the PYR and PP domains. The

Table 1. Data collection and refinement statistics. Rsym ¼ S | Ii (hkl)

– ÆIi (hkl)æ | ⁄ S Ii (hkl), where Ii (hkl) are the intensities of multiple

measurements and ÆIi (hkl)æ is the average of the measured intensi-

ties for the ith reflection. Rcryst ¼ 100 · (Sh |Fobs, h – Fcalc, h | ⁄ Sh

Fobs, h), where Fobs and Fcalc are observed and calculated structure

amplitudes, respectively. Rfree ¼ Rcryst calculated for the test set of

reflections not used in refinement. a.u., asymmetric unit.

Diffraction Data

Space group C2221

a (A) 99.4

b (A) 179.0

c (A) 120.9

Resolution range (A) 50.0–1.5 (1.55–1.50)a

Rsym (%) 8.4 (38.2)a

I ⁄ rI 21.4 (3.5)a

Completeness (%) 99.5 (99.1)a

Redundancy 5.6 (4.7)a

Structure Refinement

Resolution range (A) 50.0–1.5

Rcryst (%) 17.5

Rfree (%) 19.6

rmsd for bond lengths (A) 0.0081

rmsd for bond angles (deg) 1.406

Ramachandran plot (% in favoured,

allowed, outlier regions)

98.58, 99.7, 0.3

Number of atoms per a.u. 9305

Average B-factors (A2)

(protein, water, ligands)

19.0, 34.2, 17.6

a Values in parentheses are for the highest resolution shell.

W. Versees et al. Structure of Azospirillum brasilense PPDC


cofactors ThDP and Mg2+ are also bound at this inter-

face. ThDP interacts with its aminopyrimidine moiety

to the PYR domain of one subunit and with its diphos-

phate moiety to the PP domain of another subunit.

A tetrameric assembly is expected for AbPPDC from

elution profiles on size exclusion chromatography per-

formed at pH 6.5 in the presence of the cofactors

ThDP and Mg2+ (S. Spaepen, W. Versees, D. Gocke,

M. Pohl, J. Steyaert & J. Vanderleyden, unpublished

results). This tetramer can be generated from two di-

mers, related by a two-fold crystallographic axis (with

the two-fold rotation axis transforming subunit A onto

C, and B onto D; see Fig. 3B for subunit nomencla-

ture). However, the tetramer has an architecture far

from 222 symmetry, caused by the nonperpendicular

arrangement between the noncrystallographic axis rela-

ting the two subunits in the dimer and the crystallo-

graphic axis relating the two dimers. As illustrated in

Fig. 3B, the noncrystallographic axis does not intersect

the crystallographic axis but is offset by about 6.5 A.

The noncrystallographic axis is moreover inclined to

the crystallographic two-fold axis by 80.4�, instead of

the 90� expected for 222 symmetry. The asymmetric

character of the PPDC tetramer is illustrated by the

difference in distance between corresponding residues

of the A and C subunits on the one hand and the

Fig. 2. Subunit of the A. brasilense PPDC crystal structure. The

PYR, R and PP domains are coloured blue, green and red, respect-

ively, with intervening loops in yellow. The two cofactors, ThDP

and Mg2+, are shared by two noncrystallographic symmetry-related

subunits and are shown as space-fill models. The positions of the

N- and C-termini, as well as the edges of a flexible active site loop

(residues 105–119) are also indicated.

A

BA C

DB

A C

DB

Fig. 3. Quaternary structure of A. brasilense

PPDC. (A) Stereo picture of a tight dimer of

AbPPDC as found in the asymmetric unit of

the crystal. The subunits are colour coded

red (subunit A) and green (subunit B). The

cofactors are shown as space-fill models,

with the carbon atoms of ThDP coloured

gold, and the Mg2+ ion depicted as a grey

sphere. The position of the flexible loop

(amino acids 105–119) of subunit B and the

C-terminus of subunit A are indicated. (B)

Stereo picture of an asymmetric dimer of

dimers of AbPPDC. The colour codes for

the A and B subunits of the tight dimer are

the same as above. The subunit nomencla-

ture is indicated. The crystallographic two-

fold axis relating the two dimers (A to C, B

to D) is indicated as an orange bar. The two

non-crystallographic symmetry axes relating

the subunits in the asymmetric unit are

indicated as blue and red bars. Note the

nonperpendicular arrangement of crystallo-

graphic and non-crystallographic symmetry

axes resulting in a nonsymmetric arrange-

ment of the tetramer.



B and D subunits on the other. For example, the dis-

tance between the Mg2+ ions bound to the A and C

subunits is 68 A, while the corresponding distance

between the B and D subunits amounts to 83 A. The

asymmetry moreover imposes very different dimer–

dimer interfaces on both subunits of the dimer in the

asymmetric unit. The dimer–dimer interface involving

the B and D subunits is mainly formed by an exten-

sion of the six-stranded b-sheet across the R domains,

forming a 12-stranded twisted b-sheet. The interface

involving the A and C subunits is different, with resi-

dues from different secondary structure elements

involved. The total accessible surface area buried in

the dimer–dimer interface is only 876 A2, much less

than in the interface within the dimer so that the entire

tetramer assembly is best described as an asymmetric

dimer of dimers. A similar asymmetric tetramer was

also observed in pyruvamide-activated ScPDC (PDB

1QPB) and in Kluyveromyces lactis PDC (KlPDC;

PDB 2G1I) [28,29]. Apart from a relatively small rota-

tion, of about 6�, of one dimer vis-a-vis the other, the

PPDC tetramer has the same overall organization as

these asymmetric PDC tetramers.

Substrate activation and signal transduction in

PPDC: comparison to the existing models

The AbPPDC enzyme displays pronounced substrate

activation with indolepyruvate as a substrate, typified

by sigmoidal plots of the reaction rate versus substrate

concentration (Hill coefficient of 1.85; with phenylpyru-

vate only marginal cooperativity is observed). This type

of regulation of enzyme activity has been observed in

nearly all PDCs characterized to date with the excep-

tion of most bacterial PDCs such as the ZmPDC

[30,31]. In contrast, most other ThDP-dependent de-

carboxylases, such as BFD and IPDC seem to exhibit

normal hyperbolic Michaelis–Menten kinetics without

any indication of allostery [13,32]. Although substrate

activation was first described by Davies in 1967 for

PDC from wheat germs [33], and has since been subject

of detailed kinetic investigations, the exact molecular

mechanism of this phenomenon remains still enigmatic.

Essentially two models have been proposed.

In the first model Baburina et al. proposed Cys221

in yeast PDC as the covalent binding site of the regu-

latory substrate molecule and the starting point of a

signal transduction pathway to the active site [34–37].

In this for the moment most generally accepted activa-

tion mechanism, Cys221 (in the R domain) would

undergo hemiketal formation with the pyruvate sub-

strate. The negative charge of the bound pyruvate is

transferred to His92 (PYR domain) via a salt bridge.

The information is than proposed to be transmitted

via Glu91 onto Trp412 (PP domain) and the loop

comprising residues 410–415. This loop provides two

conserved hydrogen bonds with the aminopyrimidine

ring of ThDP (Gly413 and Ile415) and its rearrange-

ment could induce the observed increase in exchange

rate of the proton on the C2 of ThDP, leading to an

increase in catalytic rate [38,39]. Evidence has been

accumulating in favour of this mechanism as muta-

tions in Cys221 or other residues on the proposed

information transfer route abolishes substrate activa-

tion or decreases the Hill coefficient significantly. The

pivotal Cys221 residue of yeast PDC is, however, not

conserved in AbPPDC and is replaced by a glutamate

residue (Glu212). The histidine (His92), which was

thought to interact with the Cys221-bound pyruvate in

PDC, is not conserved either (Fig. 4) [35,38]. In

AbPPDC this residue structurally aligns with Lys90.

Analogous to the situation in PPDC, an acidic residue

(Asp210) was found on the position corresponding to

Cys221 in the nonallosterically regulated BFD from

Pseudomonas putida [25]. In this enzyme a direct

hydrogen bond occurs between Asp210 and His89

(homologous to His92 in PDC). This direct hydrogen

bond is considered as a mechanism for permanent

activation of BFD, without the need for a bridging

regulatory pyruvate molecule. In AbPPDC a similar

‘permanent’ hydrogen bond occurs between Glu212

and Lys90, without losing the need for a regulatory

substrate molecule. These arguments seem to disfavour

this pocket as the primary site of regulatory substrate

binding in PPDC, although it would not a priori rule

out the use of common residues on the proposed infor-

mation transfer route. An alternative trigger position

in PPDC could as a first site be provided by a cysteine

residue at position 210, only two residues separated

from the position corresponding to Cys221 in PDC.

This residue could in theory fulfil a similar role as

Cys221 in PDC. A closer look, however, shows that

this cysteine is not readily accessible from solvent.

A second model for substrate activation proposed

by Konig and coworkers involves large scale conform-

ational changes and tetramer reassembly [28,40]. This

model was proposed on the basis of the crystal struc-

ture of ScPDC in complex with the substrate analogue

and allosteric activator pyruvamide, showing pyruva-

mide bound in a pocket 10 A away from Cys221. A

transition was observed from a symmetrical tetramer

(obeying 222 symmetry) in the unactivated state (called

form A) to an asymmetrical tetramer (called form B)

in complex with pyruvamide. In going from the nonac-

tivated to the activated form of this enzyme, one dimer

has to rotate by about 30� relative to the other. In



form B ScPDC this structural asymmetry leads to a

disorder-order transition in two active site loops (resi-

dues 106–115 and 292–303) in half of the active sites,

leading to a half-site closed, half-site open asymmetric

tetramer. Both loops also contribute to the new dimer–

dimer interface created in the asymmetric tetramer,

leading to an increase in the dimer–dimer interface

(from about 900 A2)1550 A2). From this observation

a relationship was inferred between activator binding,

tetramer asymmetry and active site loop reorganization

in half of the sites, leading ultimately to catalysis. A

recent site-directed mutagenesis study involving several

residues of loop 292–303 of ScPDC has shown that

this loop is involved in both catalysis and substrate

activation. However, although the different mutants

lower the Hill coefficient, none of the mutants is able

to completely abolish substrate activation [41]. Also,

the other active site loop of ScPDC (residues 106–115)

harbours two catalytically important residues, His114

and His115. Replacing His114 (but not His115) with a

phenylalanine has been described to reduce the Hill

coefficient, also implying a role for this flexible loop in

the substrate activation mechanism of yeast PDC [42].

As already described in the previous paragraph, the

AbPPDC also adopts a highly asymmetrical tetramer

with relative loose dimer–dimer interactions, which

might seem in agreement with this last model of sub-

strate activation. However, as previously also observed

in the KlPDC [29], the AbPPDC is present as the

asymmetric tetramer without any activator present.

This might indicate an equilibrium in solution between

the asymmetric and symmetric tetramer, shifted toward

the asymmetric architecture in AbPPDC and KlPDC.

Moreover, the active site loop comprising residues

104–119, remains flexible in all subunits, indicating no

strict relationship between tetramer asymmetry and

loop closure in AbPPDC. In AbPPDC a glycerol mole-

cule originating from the cryo solution is bound at the

site corresponding to the observed pyruvamide binding

site in yeast PDC. This glycerol, however, doesn’t seem

to be tightly bound, because apart from an interaction

with Ser156 it only interacts with the protein via brid-

ging water molecules. Whether this or yet another site

corresponds to the regulatory substrate binding site in

AbPPDC and how this information can be transferred

to the active site is currently being investigated

through cocrystallizations with substrate (analogues)

and mutational studies combined with enzyme kinetic

measurements.

Apart from the communication between a regulatory

site and the active site as described above, recent kin-

etic and structural data on certain ThDP-dependent

enzymes also suggested communication between the

active sites of these multimeric enzymes, resulting in a

half-of-the-sites reactivity. In the case of ScPDC and

BFD this type of regulation has mainly been inferred

from complex kinetic behaviour not being consistent

with independently working active sites [43]. For the

E1 component of the pyruvate dehydrogenase complex

(PDH) of both bacterial and human origin, proof for

such a regulatory mode has been accumulating from

kinetics, dissymmetry of active sites in proteolytic

Fig. 4. Putative regulatory binding site of

PDC versus PPDC. The central frame shows

a superposition of the proposed regulatory

substrate binding site of yeast PDC (PDB

1PVD), shown in grey, with the correspond-

ing site in AbPPDC, shown in green (resi-

dues from the R domain) and in blue

(residues from the PYR domain). Cys221

has been proposed as the central residue in

the substrate activation mechanism of

ScPDC [34,35]. In AbPPDC this residue is

replaced by a glutamate (Glu212) forming a

direct hydrogen bond with Lys90 (see text

for further details). In the left corner the

location of this site within the PPDC mono-

mer is indicated by a black square.



patterns and X-ray structures, and from direct obser-

vation of significantly differing ThDP C2 ionization

rates in different active sites of the multimer [44–46].

On the basis of the X-ray structure of the E1 compo-

nent of the PDH of Bacillus stearothermophilus, Per-

ham and coworkers proposed the existence of a proton

wire mediated by a tunnel of water molecules lined by

acidic residues that connect one cofactor to another.

This proton shuttle serves to reversibly transfer a pro-

ton between the active sites leading to an alternating

site mechanism with both active sites working out of

phase [46]. While waiting for further detailed kinetic

measurements on the AbPPDC, no definite conclusion

about the existence of a half-of-the-sites mechanism in

this enzyme can be drawn for the moment. From a

structural point of view, however, evidence for such a

mechanism in AbPPDC is lacking. No significant

asymmetry between the two active sites in the tight

dimer (AB) is observed. Such a structural asymmetry

would of course not be an absolute requirement for

the ‘half of the sites’ reactivity in the case of a proton

wire mechanism, where the only difference between the

active sites would be their protonation state. More sig-

nificantly no tunnel-like cavity filled with water mole-

cules that links both cofactors seems to be present.

Rather, the region spanning the two cofactors (20 A

between the N1 atoms of both ThDPs) is occupied by

the residues Glu48 (which interacts with N1 of ThDP),

His47, Pro49 and Asn78 of both subunits (not shown).

The distances between these residues and their nonio-

nisable nature (Pro and Asn) preclude this route as a

proton wire. Possibly the active sites of AbPPDC work

independently of each other as also observed for cer-

tain other ThDP-dependent enzymes such as bacterial

PDCs, transketolase and POX [44]. Alternatively, more

complex signal transduction routes would have to

underlie an active centre communication mechanism in

PPDC.

The active site: variations on a common theme

The catalytic cycle of ThDP-dependent decarboxylases

can be subdivided into a number of microscopic steps

[14]. In the first step the thiazolium C2 atom is deprot-

onated, forming a highly nucleophilic ylide. Recent

studies have shown that the N4¢ group of the cofactor,

in its imino tautomeric form, functions as the general

base in this step [22,47,48]. Upon substrate entry the

ylide attacks the substrate carbonyl forming a first

covalent tetrahedral intermediate. With phenylpyruvate

as substrate this intermediate can be called 2-(3-phe-

nyl-lactyl)-ThDP. Decarboxylation of this intermediate

leads to a second carbanion which is resonance-stabil-

ized by its enamine form. Protonation of this interme-

diate gives a second tetrahedral intermediate called

2-(1-hydroxy-2-phenyl-ethyl)-ThDP. Deprotonation of

the a-hydroxyethyl group with concomitant cleavage

of the carbon–carbon bond between C2 of ThDP and

Ca of the intermediate finally releases the product phe-

nylacetaldehyde. Assignment of specific roles to partic-

ular amino acids in this catalytic cycle has proven to

be quite challenging despite some thorough site-direc-

ted mutagenesis studies combined with in-depth kinetic

measurements. The general architecture of the active

site, and hence probably the mechanism, of AbPPDC

generally corresponds to that of PDCs and IPDC,

although some remarkable differences exist.

PPDC binds two molecules of the cofactors ThDP

and Mg2+ per tight dimer (Fig. 3A). The ThDP mole-

cules are bound in the typical V-conformation in clefts

at the interface of the PYR and PP domains. This

V-conformation brings the C2 and N4¢ atoms of the

cofactor in close proximity, at 3.11 A of each other

(Fig. 5). Typically a large hydrophobic residue is

wedged underneath the two rings of the ThDP molecule

hence enforcing the V-conformation. In PPDC this resi-

due is a methionine (Met404) in contrast to an isoleu-

cine normally found in PDCs, but analogous to the

methionine found at this position in POX and benzalde-

hyde lyase (EC 4.1.2.38) [22,27]. The diphosphate moi-

ety of the cofactor is tightly anchored by bidentate

interaction with the octahedral coordinated Mg2+ and

numerous hydrogen bonds with amino acid residues

(Fig. 5). Although some small differences exist between

ScPDC and PPDC in the interaction pattern with the

diphosphate moiety of ThDP, the diphosphate-binding

sequence fingerprint G-D-G-X24-N-N (with Asp429,

Asn455 and Asn456) is conserved [49]. The Mg2+ ion is

coordinated by the side chains of Asp429 and Asn456,

by the main chain carbonyl of Ser458, by the phos-

phates of ThDP and by a water molecule; a pattern

completely conserved among different ThDP-dependent

enzymes [27]. Three conserved and catalytically import-

ant hydrogen bonds are also formed with the amino-

pyrimidine ring of ThDP: one between Glu48 and the

N1¢ atom, one between the main chain carbonyl of

Ala402 and the N4¢, and one looser hydrogen bond

between the main chain amino group of Met404

and the N3¢. Glu48 has been proposed to induce the

1¢4¢-imino tautomer of the aminopyrimidine ring, allow-

ing this N4¢ group to alternate between the amino and

imino form [39,50]. The hydrogen bond between the

main chain carbonyl of Ala402 and the N4¢-imino

group might be used subsequently to position the lat-

ter’s lone pair electrons in suitable orientation to trans-

fer a proton from C2 to the 4¢-imine.



The current picture of the AbPPDC substrate-bind-

ing pocket is incomplete due to the flexibility of loop

104–119 and the C-terminal residues. In analogy with

other ThDP-dependent decarboxylases (e.g., form B

ScPDC, ZmPDC and EcPPDC) it can be anticipated

that the active site loop will close over the active site

at some point during the catalytic cycle, thus provi-

ding two extra catalytically important histidines

(His112 and His113) [40]. Apart from these two histi-

dine residues, only two other polar residues, Asp25

and Thr71, line the active site pocket (Fig. 6). In

recent years a number of X-ray structures of ThDP-

dependent enzymes in complex with covalent reaction

intermediate analogues have been solved [51–53].

These structures allow modelling of the intermediates

in the active site of AbPPDC to gain further insight

in the possible catalytic and binding interactions,

as shown for the 2-a-lactyl-ThDP intermediate in

Fig. 6A. However, care should be taken in the inter-

pretation because protein conformational changes are

possible (and even likely) along the reaction trajectory.

The modelled structure shows a possible interaction

between the substrate’s carboxylate leaving group and

the main chain amine and side chain carboxyl of

Asp25. Recently Tittmann and coworkers used a com-

bined approach of site-directed mutagenesis and the

direct observation of reaction intermediates by rapid

quench ⁄NMR, to delineate the role of active site resi-

dues in EcIPDC. They found that the residue corres-

ponding to Asp25 is part of a Glu-Asp-His triad

Fig. 5. Cofactor binding site of A. brasilense

PPDC. (A) Schematic diagram of ThDP bind-

ing interactions in AbPPDC. Interactions and

distances (in A) are indicated in magenta.

The short distance between the C2 and N4¢atoms of the cofacter, imposed by the

V-conformation, is indicated in red.

(B) Cofactor binding site. Residues belong-

ing to different subunits are coloured grey

and yellow, respectively, while the carbon

atoms of the ThDP cofactor are shown in

green. The Mg2+ ion and an interacting

water molecule are represented as grey and

red spheres, respectively. Interactions are

indicated by magenta dotted lines. The elec-

tron density, contoured at 5 r, of an fo-fc

simulated annealed omit map calculated

without ThDP is also shown (blue mesh).



(Glu468-Asp29-His115 in EcIPDC numbering) that

forms a substrate independent carboxylate pocket also

conserved in PDCs [54]. This triad appeared to be

involved on the one hand in binding of substrates and

decarboxylation of the first tetrahedral intermediate,

and on the other hand in the protonation of the carb-

anion ⁄ enamine intermediate. Asp25 together with

His112 of the flexible loop probably fulfils the same

dual role in PPDC. A remarkable difference of PPDC

compared to most other ThDP-dependent decarboxy-

lases is the presence of a leucine at position 462, which

corresponds to the glutamate in the Glu-Asp-His triad

of IPDC and PDC (Glu477 in ScPDC and Glu468 in

EcIPDC, Fig. 6B). This leucine is located directly

underneath the carboxylate group of the lactyl-ThDP

model, on the opposite side of Asp25, and could con-

tribute to catalysis by increasing the hydrophobic

character (low dielectric constant) of the active site.

Indeed, a large fraction of the catalytic power of

ThDP-dependent enzymes is established by providing

an environment of low dielectric constant [55,56]. Such

an environment significantly stabilizes key zwitterionic

intermediates along the reaction trajectory such as the

ylide and the enamine ⁄ carbanion. Hence, Leu462 in

PPDC could compensate for the loss of one partner in

the Glu-Asp-His catalytic triad by an enhanced stabil-

ization of zwitterionic intermediates through solvent

effects. Apart from the interaction with Asp25 (and

the interactions with His112 and His113, which will

likely occur in the closed structure), no specific inter-

actions seem to occur between the enzyme and the

substrate’s carboxyl or C2a-hydroxyl group. This

agrees with the emerging view that the cofactor by

itself carries out the bulk of the catalysis with the

cofactor’s 4¢-amino ⁄ imino group probably being the

central general acid ⁄base in protonation of the sub-

strate’s carbonyl (associated with substrate binding)

and in ionization of the a-hydroxyl group during acet-

aldehyde release [50,54,57].

The aromatic moiety of the substrate can be accom-

modated in the pocket lined by the nonpolar residues

Met380, Phe385, Met461, Phe465 and Phe532 (Fig. 6A).

Although the substrate specificity of PPDC can prob-

ably not be ascribed to a single amino acid substitu-

tion, a key residue in this respect appears to be

Met380. This residue is replaced by a threonine in

ScPDC and ZmPDC (Thr388 in ScPDC) and by a glu-

tamine in EcIPDC (Gln383). A similar role in sub-

strate discrimination was already proposed for the

Gln383 in EcIPDC, because mutation of this residue

to threonine significantly increased the affinity for pyru-

vate compared to indolepyruvate [54]. The role of the

active site residues in catalysis and substrate specificity,

and in particular the implications of the amino acid

Fig. 6. Catalytic substrate binding site of

PPDC versus PDC. (A) Stereo picture of the

active site of AbPPDC with the modelled

reaction intermediate analogue 2-lactyl-

ThDP. The model of 2-lactyl-ThDP was

taken from PDB 2EZ8 [51]. The carbon

atoms of the 2-lactyl-ThDP model are col-

oured yellow, and Mg2+ is depicted as a

grey sphere. Residues from the A and B

subunit are indicated without and with an

asterisk , respectively. The phenyl or indole

ring of the substrate could be accommoda-

ted by the large hydrophobic pocket lined by

Met380, Phe385, Met461, Phe465 and

Phe532. (B) Stereo representation of the

superposition of the active sites of AbPPDC

(residues shown in green) and ScPDC (PDB

1QPB, residues shown in grey). Residues of

the A and B subunits are indicated as

above. Both cofactors are taken from the

AbPPDC structure. His114 and His115 of

ScPDC are provided by an active site loop

that is missing in the AbPPDC model due to

flexibility.



substitutions compared to PDCs and IPDC, are cur-

rently being further investigated.

Experimental procedures

Protein expression and purification

The ipdC open reading frame from A. brasilense was cloned

into a pET28a vector (Novagen, Darmstadt, Germany) as

will be described elsewhere in detail (S. Spaepen, W. Ver-

sees, D. Gocke, M. Pohl, J. Steyaert & J. Vanderleyden,

unpublished results). The wildtype AbPPDC was expressed

with an N-terminal hexahistidine-tag in Escherichia coli

Bl21-CodonPlus (DE3)-RP cells (Stratagene, La Jolla, CA,

USA). Cells were grown at 37 �C with shaking until the

cells reached an A600 between 0.6 and 0.8. The medium was

then cooled to 30 �C and isopropyl thio-b-d-galactosidewas added to initiate the expression of the recombinant

protein. After lysis of the cells the presence of the his-tag

allowed for a simple two-step purification protocol consist-

ing of a Ni-nitrilotriacetic acid affinity chromatography

step (Qiagen, Hilden, Germany) and gel filtration on a

Superdex-200 column (GE Healthcare, Uppsala, Sweden)

using 10 mm Mes ⁄KOH pH 6.5, 150 mm KCl, 0.1 mm

ThDP, 2.5 mm MgSO4 as buffer in the latter step. The

concentration of pure protein (expressed per monomer)

was determined spectrophotometrically using a e280 of

36840 m)1Æcm)1. SDS-PAGE was used to confirm enzyme

purity. During purification the enzyme activity of the pro-

tein was monitored with phenylpyruvate as substrate using

the established coupled optical test with horse liver alcohol

dehydrogenase and NADH as described previously [13].

Crystallization and data collection

The purified AbPPDC (in crystallization buffer: 10 mm

Mes ⁄KOH pH 6.5, 150 mm KCl, 0.1 mm ThDP, 2.5 mm

MgSO4) was adjusted to a final concentration of 8 mgÆmL)1

using a Vivaspin centrigugal concentrator (Sartorius Viva-

science, Goettingen, Germany, 30 kDa cut-off). PPDC was

crystallised by the hanging drop vapour diffusion method.

Equal volumes of protein solution and precipitant contain-

ing 15% PEG4000 (w ⁄ v), 10% glycerol (v ⁄ v) in 100 mm

Hepes buffer pH 7.5 were mixed and equilibrated at 20 �C.Crystals appeared after one to three weeks and grew to

maximal dimensions of 0.5 · 0.2 · 0.05 mm. The crystals

were transferred to a cryo solution containing 20%

PEG4000 (w ⁄ v), 25% glycerol (v ⁄ v) in 100 mm Hepes

pH 7.5 and transferred immediately to the cryo stream.

X-ray diffraction data were collected at 100 K to a resolu-

tion of 1.5 A on beamline X11 (EMBL, DESY, Hamburg,

Germany) using an X-ray wavelength of 0.8131 A.

The diffraction data were indexed and integrated using

denzo and scaled using scalepack [58]. Intensities were

converted to structure factor amplitudes using truncate

[59]. Table 1 summarizes the data collection and processing

statistics.

Structure determination and refinement

Initial phases were obtained by molecular replacement with

the program phaser [60] using a monomer of the E. cloacae

IPDC (PDB 1OVM) as a search model. A solution was

found, consisting of the expected two subunits forming a

relevant tight dimer in the asymmetric unit. However, the

relatively low sequence identity between AbPPDC and

EcIPDC (26%) did not allow a straightforward refinement

of this solution. Simulated annealing refinement of this

solution in cns (R ¼ 48.5%, Rfree ¼ 54% after refinement)

yielded very poor electron density maps [61,62]. The

molecular replacement solution was subsequently used as a

starting model for 20 rounds of automated model building

(WarpNTrace) in arp ⁄ warp [63]. After 20 rounds the

model had converged at a connectivity index of 0.98 with

1030 out of the 1130 residues docked in the electron den-

sity. The warp model was subjected to the simulated

annealing procedure as implemented in cns and manual

model building, checking and inspection of electron density

was performed in coot [64]. After several cycles of posi-

tional and temperature factor refinement combined with

manual corrections, solvent molecules, cofactors and alter-

native conformations were included in the model. Structure

refinement was considered complete after crystallographic

R-factor and free R-factor had converged, and the differ-

ence density was without interpretable features. The final

model was checked with the Molprobity web server [65].

Refinement statistics are summarized in Table 1.

The structural superpositions were performed using the

DALI server [66] and ⁄ or the program lsqman [67]. Inter-

face accessible surface areas were calculated with the

program provided by the protein–protein interaction

server (http://www.biochem.ucl.ac.uk/bsm/PP/server/). Fig-

ures were prepared with pymol [68] and molscript [69].

The coordinates and structure factors have been depos-

ited in the Protein Data Bank with accession code 2NXW.

Acknowledgements

The authors acknowledge the use of synchrotron beam

time on the EMBL X11 beamline (DESY, Hamburg,

Germany) and on the ID14-2 beamline at the ESRF

(Grenoble, France). This work was supported by a

research grant of the Fund for Scientific Research-

Flanders (FWO-Vlaanderen). Wim Versees is a recipient

of a postdoctoral grant from the FWO-Vlaanderen.

Stijn Spaepen is financed in part by the FWO-Vlaand-

eren (G.0085.03) and in part by the IAP (IUAP P5 ⁄ 03).



The authors also wish to thank Lieven Buts, Remy

Loris and Klaas Decanniere for helpful discussions.

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