Accepted Manuscript

Determinants of thermostability in the cytochrome P450 fold

Kurt L. Harris, Raine E.S. Thomson, Silja J. Strohmaier,Yosephine Gumulya, Elizabeth M.J. Gillam

PII: S1570-9639(17)30180-2DOI: doi: 10.1016/j.bbapap.2017.08.003Reference: BBAPAP 39984

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Received date: 17 May 2017Revised date: 19 July 2017Accepted date: 7 August 2017

Please cite this article as: Kurt L. Harris, Raine E.S. Thomson, Silja J. Strohmaier,Yosephine Gumulya, Elizabeth M.J. Gillam , Determinants of thermostability in thecytochrome P450 fold, (2017), doi: 10.1016/j.bbapap.2017.08.003

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Determinants of thermostability in the cytochrome P450 fold

Kurt L. Harris, Raine E.S. Thomson, Silja J. Strohmaier, Yosephine Gumulya and Elizabeth

M.J. Gillam*

School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia,

4072, Australia

* Author to whom correspondence should be addressed at:

Elizabeth M.J. Gillam

School of Chemistry and Molecular Biosciences,

The University of Queensland, St. Lucia, 4072, Australia

Tel: +61-7-3365-1410

Email: e.gillam@uq.edu.au

Keywords: Cytochrome P450, thermostability, directed evolution, biocatalysis, extremophile

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Abstract

Cytochromes P450 are found throughout the biosphere in a wide range of environments,

serving a multitude of physiological functions. The ubiquity of the P450 fold suggests that it

has been co-opted by evolution many times, and likely presents a useful compromise between

structural stability and conformational flexibility. The diversity of substrates metabolized and

reactions catalyzed by P450s makes them attractive starting materials for use as biocatalysts

of commercially useful reactions. However, process conditions impose different requirements

on enzymes to those in which they have evolved naturally. Most natural environments are

relatively mild, and therefore most P450s have not been selected in Nature for the ability to

withstand temperatures above ~ 40 °C, yet industrial processes frequently require extended

incubations at much higher temperatures. Thus, there has been considerable interest and

effort invested in finding or engineering thermostable P450 systems. Numerous P450s have

now been identified in thermophilic organisms and analysis of their structures provides

information as to mechanisms by which the P450 fold can be stabilized. In addition, protein

engineering, particularly by directed or artificial evolution, has revealed mutations that serve

to stabilize particular mesophilic enzymes of interest. Here we review the current

understanding of thermostability as it applies to the P450 fold, gleaned from the analysis of

P450s characterized from thermophilic organisms and the parallel engineering of mesophilic

forms for greater thermostability. We then present a perspective on how this information

might be used to design stable P450 enzymes for industrial application.

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Introduction

A degree of stability is essential to the ability of enzymes to function as biological catalysts.

The evolution of enzymes represents a trade-off between stability of the protein fold, which

enables enzymes to exist (for the most part at least) in a finite number of stable, predictable

structures, and flexibility, which makes possible the conformational changes that facilitate

stabilization of the transition state of a chemical reaction. Stability to temperature typically

determines both the ability of enzymes to remain folded at elevated temperatures and the

half-life at more moderate temperatures. While the majority of contemporary enzymes are

from mesophilic organisms, inhabiting relatively mild environmental conditions, the ability

of certain enzymes to operate within organisms inhabiting extreme environments, such as hot

springs, can tell us much about the determinants of stability and catalysis.

As well as presenting intriguing case studies for protein structure-function investigations,

enzymes from extremophiles are also often better suited to use in biotechnology than their

mesophilic counterparts. To work efficiently under industrial conditions, enzymes must often

be stable to elevated temperature and organic solvent composition, altered pH, oxidizing

conditions, and the presence of high concentrations of substrates, products or other

chemicals. Thermostability, in particular, enables reactions to be undertaken at higher

temperatures, increasing reaction rates, enhancing substrate solubility (i.e. substrate loading)

and reducing the potential for microbial contamination of biochemical processes. Moreover,

all things equal, a greater total turnover of substrate can be achieved per unit enzyme since

thermostable enzymes are more durable at moderate temperatures as well as tolerating high

temperatures. This has led to considerable interest in the determinants of thermostability in

proteins and to efforts to engineer stability into enzymes of industrial relevance. Throughout

this review we will focus on thermostability and use the terms stability and stabilization to

refer to elevated temperature rather than e.g. solvent or oxidizing conditions.

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Cytochrome P450 enzymes (P450s) are highly versatile monooxygenases found throughout

all domains of life, and are believed to have been present in the last universal common

ancestor (LUCA). Thus, it is not unexpected that the genomes of some extremophiles encode

P450s. Indeed, there are numerous examples, mostly of thermophilic archaea, that have been

found to contain functional P450 proteins capable of enduring significantly higher

temperatures than those found in mesophilic organisms. The mere existence of these proteins

is proof of the ability of the P450 fold to be stable, and retain activity, at high temperatures.

Through studying the sequences and structures of P450s from thermophiles, it is possible to

glean some understanding of the structural characteristics required for a P450 to be

thermostable. Moreover, having access to thermostable forms can make possible more

fundamental studies into the chemical biology of these proteins, such as the characterization

of the ultimate oxidant in the P450 catalytic cycle, compound I, which was done using

CYP119A1, an enzyme isolated from a thermophilic organism [1].

This review will examine what is known about the structures of P450s characterized from

thermophilic organisms in order to draw conclusions as to the features that stabilize the P450

fold. We also review the attempts that have been made to date to stabilize mesophilic P450

enzymes by directed evolution, since comparing the results of natural and artificial evolution

of thermostability can reveal complementary solutions to the problem of stabilizing the P450

fold. Finally, we present a perspective on how this information can be used to design P450

proteins for enhanced stability.

The place of thermostability in the evolution and ecology of P450 systems

Recent progress in metagenomics has revealed much about organisms capable of living under

extreme conditions such as high and low temperature, and P450s have been isolated from

organisms living at each extreme [2, 3]. Thermophiles can be defined as those organisms

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capable of enduring or even thriving under conditions of extreme temperatures (50-121 °C,

usually over 60 °C) [4]. Hyperthermophiles thrive at temperatures of ~ 80 °C or more. At the

other end of the temperature scale, psychrophiles (otherwise known as cryophiles) grow at

temperatures between -20 ºC and 10 ºC. Few P450s have been reported to date from

psychrophiles, purported to be alkane hydroxylases, but none has been characterized in detail

[3]. By contrast a number of P450s have been investigated from thermophilic organisms.

Both types have the potential to inform about the structural adaptations to P450s necessitated

by changes in ambient temperature.

Comparing metagenome analyses of samples from various high thermal habitats indicates

that, in general, there is a decrease in biodiversity with increasing temperature [5, 6].

Microbiota commonly represented in such extreme environments (e.g. hot springs) are the

bacterial taxa Thermotogae (Fervidobacterium), Deinococcus-Thermus, Proteobacteria

(Acidithiobacillus), Aquificae, Dictyoglomi, Nitrospirae (Thermodesulfovibrio), Firmicutes

(Clostridium, Geobacillus) and archaeal taxa such as Crenarchaeota (Pyrobaculum,

Sulfolobus, Stygiolobus), Euryarchaeota (Thermococcus, Methanococcus, Archaeoglobus,

Ferroplasma) and Nanoarchaeota [7, 8]. The abundance of certain microbial taxa in these

extreme environments differs depending on the ambient temperature, pH and availability of

organic materials.

Various genome projects have revealed a remarkable and unexpectedly large number of

P450s in a variety of organisms. Genome and metagenome sequencing has generated large

amounts of sequence information; however, the functional characterization of proteins from

such organisms is mostly lacking. Only five thermophilic P450s have been characterized to

date, namely CYP119A1 from Sulfolobus acidocaldarius [9], CYP119A2 from Sulfolobus

tokodaii strain 7 (Sulfolobus sp. strain 7) [10], CYP175A1 from Thermus thermophilus [11],

CYP231A2 from Picrophilus torridus [12], and CYP154H1 from Thermobifida fusca [13].

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Little is known about the catalytic activity, physiological role or natural substrates of even the

best-characterized forms. CYP119A1 has been shown to carry out H2O2-supported styrene

epoxidation [14-16], ω-1 hydroxylation of lauric acid [17, 18], dehalogenations of

halogenated solvents [19] and the reduction of nitrite or nitrous oxide supported by either

H2O2 or the CYP101A1 (P450cam) redox system (putidaredoxin (Pdx) and putidaredoxin

reductase (PdR)). However, the physiological significance of these is unknown [20]. Even

less is known about the natural function of CYP119A2, which has primarily been studied by

direct electrochemistry, but H2O2-driven styrene epoxidation and ethylbenzene hydroxylation

have been reported [21].

CYP175A1 from T. thermophilus shows some similarity to CYP102A1 (P450BM3), but does

not bind or turn over saturated fatty acids (C10-C20) due to steric clashes with key residues in

the active site [11]. However, H2O2- and Pdx/PdR-supported hydroxylase activity towards

unsaturated monoenoic acids has been reported [22], and MD analysis has revealed that these

substrates adopt a “U-shaped” conformation within the active site, centered around the C=C

double bond. This may suggest that the native substrate for CYP175A1 also assumes such a

conformation [22]. Other substrates turned over by CYP175A1 include β-carotene [23-25],

napthalenes [26], and colorimetric substrates guaiacol and ABTS (2,2′-azino-bis(3-

ethylbenzothiazoline-6-sulfonic acid)) [27].

To date, no information is available about the endogenous substrate or activity of

CYP231A2, whereas CYP154H1 was shown to catalyze the Pdx/PdR-supported conversion

of ethylbenzene, propylbenzene, styrene and organic sulphides [13].

Other potential thermostable P450s are being revealed as the genomes of more thermophiles

become available. Another nine open reading frames (ORFs) encoding potential P450 genes

have been identified in Thermobifida fusca, the source of CYP154H1 [13]. A P450 with

progesterone hydroxylase activity has been identified in Geobacillus thermoglucosidasius

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(growth range 42-69 °C) [28, 29]. Geobacillus stearothermophilus (growth range 35-60 °C)

has also demonstrated activity towards progesterone and testosterone, and its genome

encodes a potential P450 gene [2, 30, 31].

A screen of the genomes of the thermophilic fungi, Thielavia terrestris (growth range 22-55

°C) [32] and Myceliophthora thermophila (growth range 38-54 °C) [33], identified 79 and 70

putative P450 genes respectively, 14 and 11 of which were identified as likely to be

thermostable [34]. These organisms do not tolerate temperatures as high as bacterial and

archaean thermophiles, so the proteins they produce may be less stable. However other

enzymes from thermophilic fungi have shown temperature optima in the range of 45-70 °C

[34], with one xylanase from T. terrestris demonstrating optimal activity at 85 °C [35].

In addition to these forms, members of other cytochrome families such as CYP107, CYP109

and CYP132 are found in thermophilic organisms. The genome database annotation of these

putative thermophilic P450s revealed their functions as variously cholest-4-en-3-one 26-

monooxygenase activity, steroid hydroxylase activity (at the 15-, 6, 6- 9, and 11-

positions on various steroids), pentalenene oxygenase, erythromycin C-12 hydroxylase, and

2-hydroxy-5-methyl-1-napthoate 7-hydroxylase. However, it is unclear whether these

annotations result from any functional characterization or simply from sequence similarity

with other P450s showing the listed activities. Extrapolation of functional properties between

even closely related P450s is fraught with error as demonstrated by the functional changes

seen in even very closely related isoforms from laboratory animals and humans.

Bioinformatic analyses of “CYPomes” suggest that a large number of thermostable P450s

remain to be explored and that genome mining may lead to the discovery of novel, highly

thermostable P450 biocatalysts. A BLAST search of existing bacterial and archaeal genome

databases for homologs of CYP119 forms, CYP175A1, CYP231A2 and CYP154H1 retrieved

45 P450s from thermophilic organisms within the bacterial phyla Deinococcus-Thermus,

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Firmicutes, Actinobacteria, Acidobacteria, Chloroflexi and the archaeal phyla Crenarchaeota

and Euryarchaeota (Supplementary Table 1). Some of these thermophilic P450s occupy

branches near the base of both bacterial and archaeal domains of the evolutionary tree. In

light of the hypothesis that LUCA was a thermophile or a hyperthermophile [36, 37], we can

speculate that ancestral forms of cytochrome P450 families were thermophilic (or

hyperthermophilic) proteins. However, the existence of thermophilic P450s in many diverse

phyla of the bacterial and archaeal kingdoms may also be a result of convergent evolution,

especially as extremophiles are well-known for participating in rampant lateral gene transfer

[38]. Importantly, this search would not reveal possible thermostable forms that have poor

homology to the existing characterized thermostable P450s. With advances in metagenomics

and single-cell genomics, the number of thermostable P450s found in the so-called

“microbial dark matter” (i.e. unclassified bacteria and archaea including putative

thermophiles) is likely to grow.

P450s from thermophilic organisms

To date, the crystal structures of four “thermophilic P450s” have been solved. Three are from

archaea: CYP119A1 from S. acidocaldarius [39]; CYP119A2 (P450St), a related enzyme

from S. tokedaii strain 7 [40]; and CYP231A2, from Picrophilus torridus [12]. A fourth

enzyme, CYP175A2 comes from a eubacterium, T. thermophilus [11]. An additional

thermostable P450, CYP154H1, was isolated and characterised from the moderately

thermophilic bacterium T. fusca, which has optimum growth conditions of 50-55 °C [13].

The melting temperature (Tm) of this protein was 67 °C but it has not yet been characterized

structurally. (While strictly only organisms, not enzymes, can be described as thermophilic or

mesophilic, these terms will be used for simplicity and since their use in this manner is

widespread in the literature.)

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CYP119A1

CYP119A1 was first reported to have been isolated from the genome of S. solfataricus by

Kennelly et al. (1996), while attempting to clone a thymidylate synthase gene from the

extremophile [9]. However, more recent reports have revealed that the enzyme was actually

derived from the closely related acidothermophile S. acidocaldarius, and that the source was

misattributed due to contamination of the cell stock (DSM 1616) [12, 14]. This protein was

initially designated CYP119, and then CYP119A1, when related enzymes were characterized

[9, 41].

The optimal growth temperature of S. acidocaldarius is commonly 70-75 °C with some

strains growing at temperatures up to 85 °C [42]. Given the extreme growth conditions of this

organism, the high Tm of CYP119A1 (~88-92°C) is not unexpected [11, 39, 43]. By

comparison, P450s derived from mesophilic microorganisms such as bacterial CYP101A1

show significantly lower Tm values (~54-61°C) [11, 44]. In addition to its high

thermostability, CYP119A1 is able to withstand greater hyperbaric pressure than most

mesophilic P450s, with a P1/2 (pressure required to inactivate half the protein) of 320 MPa at

5 °C compared to ~110-140 MPa for CYP101A1 [45, 46]. A greater stabilizing effect was

observed at increased temperatures, with a P1/2 of 380, 430 and 480 MPa at temperatures of

20, 35 and 50 °C respectively. Furthermore, once returned to normal pressure, CYP119A1

was able to completely revert from the P420 species to the P450 state in the absence of any

stabilizing agents [45].

Overall structural changes in CYP119A1 compared to mesophilic P450s

Crystal structures have been solved for the aqua-ligated enzyme [47] plus the complexes with

imidazole, 4-phenylimidazole (4-PI) [39] and various other phenyl-imidazole derivatives

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[48]. Structurally, CYP119A1 conforms to the typical P450 fold (Figure 1). However, at just

368 residues, it is considerably shorter and more compact than P450s from mesophilic

microorganisms, such as CYP101A1 and CYP107A1 (P450eryF) at 414 and 403 residues

respectively. Much of this difference in length can be attributed to the N-termini of the

proteins (Figure 2). While CYP101A1 and CYP107A1 contain N-terminal sequences prior to

any well-defined secondary structure elements, CYP119A1 begins promptly with -helix A.

Truncations occur at other positions: the β1-1/β1-2 hairpin loop of CYP119A1 (residues 13-

24) is four residues shorter than CYP101A1 (52-66); and five residues are missing from the

β5-turn, situated between helices H and I (191-195 in CYP119A1, 225-234 in CYP101A1).

The latter region is typically involved in redox partner interaction, and it has been shown to

make direct contact with the FMN-binding reductase domain in crystal structures of the

CYP102A1 heme and FMN domains [49]. In CYP119A1 this region closely resembles that

of the self-sufficient nitric oxide reductase, CYP55 (P450nor), which uses NADH without the

assistance of a redox partner [50]. The related CYP119A2 is capable of self-sufficient

catalysis, suggesting that CYP119A1 may do the same [51]. CYP119A1 has also been shown

to have nitrite/nitrous oxide reductase activity, underscoring the similarity to CYP55 [20].

F/G-loop/B’-helix and substrate-dependent conformational changes

A significant structural difference from mesophilic P450s occurs in the repositioning of the

B’-helix and F/G-loop [39, 47]. The B’-helix occurs at residues 63-66 in CYP107A1 and 67-

77 in CYP101A1. However, the cognate region in CYP119A1 does not comprise a full helix.

Instead a helix is found downstream at residues 49-53, corresponding to an insertion in

CYP101A1 between residues 88-89. This alternative B’-helix is preceded by a long loop,

resulting in a shift of the B’-helix away from the active site and towards the surface of the

protein. To accommodate the void typically filled by the B’-helix, the F/G-loop, which is

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usually oriented away from the heme and towards the surface, instead dips down to fill this

space, resulting in a similar overall solvent accessibility to the heme between the substrate-

free forms of the two proteins (~24 Å2 and ~18 Å2 for CYP119A1 and CYP101A1

respectively) [39].

Molecular dynamics simulations have shown that the F/G-loop of CYP119A1 can move

independently of the remainder of the protein, allowing it to transition in and out of the active

site to form the open (substrate-free) and closed (ligand-bound) forms without affecting the

structure of the rest of the protein[52]. Conformational changes in this area to accommodate

different ligands are not uncommon amongst P450s[53-55], however other P450s that exhibit

such changes, for example CYP102A1 [53, 54], CYP3A4 [56] and CYP2B4 [55] do so via

displacement of the F-and G-helices, requiring interactions with the I-helix to be broken.

Despite large conformational changes in the F/G-loop through unwinding of these helices,

CYP119A1 retains inter-helical contacts between the F/G- and I-helices. The F- and G-

helices of CYP119A1 remain in relatively constant positions, the F/G-loop extending and

retracting via unwinding of the F- and G-helices [12] (Figure 3). This difference from other

P450s may be due to CYP119A1 having more branched chain amino acids in the region,

which form a tight network of inter-helical contacts between the G- and I-helices. This could

allow CYP119A1 to endure harsher temperatures, with the F/G-loop taking on the role of

controlling substrate entry and release [12].

The I-helix and conserved Thr residue

The most highly conserved regions of CYP119A1 with respect to mesophilic P450s are

located near the heme group, namely the conserved Cys thiolate ligand (Cys317), and to a

degree, the I- and L-helices. The CYP119A1 I-helix is slightly unusual in that it contains an

additional two Thr residues following the highly conserved Thr213 (Thr252 in CYP101A1).

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Thr213 residue has been implicated in the transfer of protons during P450 catalysis, and the

prevention of auto-inactivation by H2O2 generated by uncoupling during catalysis [57, 58]. In

CYP101A1, Thr252 donates an H-bond to the peptide oxygen atom of nearby Gly248.

However, the H-bonding network of CYP119A1 is dissimilar, with Thr213 apparently

unbonded and Thr214 instead forming an H-bond with Gly210. This bonding pattern instead

resembles that of CYP102A1. Mutagenesis has revealed that Thr213 is catalytically

important for CYP119A1, while Thr214 may help to control the spin state of the heme and

also play a role in substrate binding [18, 59]. The role of Thr215 has not been investigated.

Despite significant effects on activity and heme coordination states, mutation of Thr213 and

Thr214 had little effect on thermal stability, with the Tm of single mutants remaining within

2.4 °C of the Tm of the WT [59].

Role of the Cys-thiolate ligand and Cys-ligand loop in stabilization of the CYP119A1 fold

The highly conserved thiolate Cys317 and Thr213 residues were mutated in CYP119A1

(C317H/T213A) and the structure was solved [60]. Compared with the wild type (WT) this

mutant only suffered a 1.2 °C loss in 10T50 (the temperature at which half the protein remains

folded after heating for 10 minutes). Mutants of the conserved cysteine to all 19 other amino

acids showed a maximal decrease in 10T50 of ~8 °C, with an average of 6 °C. Considering the

structural importance of this residue to the P450 fold and its conservation throughout the

clade, these decreases in stability can be considered minor [60]. The heme was retained to at

least some degree in all mutants, however the typical P450 Fe(II).CO Soret peak was shifted

suggesting a different coordination environment, which in some cases may have involved

coordination to His315. This is the first example of a P450 Cys-thiolate mutant crystal

structure to be solved, and it is a testament to the stability of CYP119A1 that it can withstand

such a mutation. High temperature molecular dynamics (MD) simulations showed that the

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Cys ligand loop, which unfolds during thermal denaturation of CYP176A1 (P450cin) and

CYP101A1, is stabilized by tight nonpolar interactions between Tyr26 and Leu308. A double

mutant of these two residues (Y26A/L308A) was constructed and exhibited a 16 °C decrease

in Tm compared to the WT [61]. Individual mutants Y26A and L308A resulted in a 12 and 10

°C decrease in Tm respectively [61].

Other features that may augment stability

Many theories have been proposed to explain the stability of proteins from thermophilic

organisms based on the relative contributions of individual amino acids. CYP119A1 contains

a higher proportion of buried isoleucines and fewer alanines than mesophilic P450s,

potentially resulting in an overall increase in hydrophobic interactions [62]. Yano et al.

identified fewer 2-residue salt bridges (8-10) in CYP119A1 than CYP101A1 (19) and

CYP107A1 (15), but more salt-bridged networks involving 3 or more residues (four in

CYP119A1 vs. three for CYP101A1 and one in CYP107A1) [39]. However, a comparison of

the thermophilic structures available currently and representative mesophilic structures

(Supplementary table 2) revealed less of a difference in the number of 2-residue salt bridges

(10 ± 3 for CYP119A1 vs. the mesophilic average of 11 ± 3). Nevertheless, CYP119A1 does

contain almost double the number of salt-bridged networks (8 ± 2) compared to the

mesophilic average (4 ± 1). A greater proportion of the total residues involved in salt bridges

are involved in networks (56 % compared to 44 %). The salt-bridged networks in CYP119

also span greater distances compared to those in the mesophilic forms, potentially

contributing to the compactness of the structure [39]. Mutagenesis of Glu114 which is

involved in a salt-bridged network with Arg363 and Glu342 resulted in a decrease in Tm of

3.8 °C [43]. Similarly, mutation of Arg259 to Lys to disrupt a salt link to the propionate

group of the heme caused a 5.9 °C decrease [43]. However, mutagenesis of Arg154 and

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Glu212 to disrupt a salt bridge formed in the imidazole and water-bound forms of

CYP119A1 (but not present in mesophilic P450s) did not result in a significant decrease in

stability [63].

CYP119A1 has unique clusters of aromatic residues that have been proposed to contribute to

its stability. Two clusters, linked together by the guanidinium group of an Arg residue, form

an aromatic/nonpolar “ladder” spanning a total distance of ~39 Å along the side of the protein

(Figure 4). The first cluster comprises Tyr2, Trp4, Phe5, Phe24, Trp281 and Tyr15 (the latter

via co-association with Phe24 to Met8) spanning ~11.3 Å. The second contains Phe225,

Phe228, Trp231, Trp250, Phe298, Phe334 and Phe338, spanning ~24 Å [39]. In comparison,

CYP107A1 has a single cluster of aromatic residues spanning just ~11 Å. Targeted and

random mutagenesis studies revealed that the mutation of individual aromatic residues

(Phe24Ser, Trp231Ala, Tyr250Ala, Trp281Ala) each resulted in an approximate 10 °C

decrease in Tm, with double mutants Tyr2Ala/Tyr250Ala and Trp4Ala/Trp281Ala resulting

in a 12-15 °C decrease [43, 63]. By contrast, mutation of an aromatic residue (Tyr168) that

was buried to a similar degree but on the other side of the protein caused no change from the

WT Tm value [63]. These results suggest that the extended aromatic clusters of CYP119A1

play an important role in its thermal stability.

The structure of the Phe24Leu mutant revealed no obvious structural deviations from the

WT, however this mutant was found to be only ~4 °C less stable than the WT [47]. Besides

the Phe24Ser involved in aromatic clusters, and Glu114Asp and Arg259Lys involved in salt

bridges, a set of other single and multiple mutants demonstrated Tm decreases in the range of

0.7-8.4 °C: Lys176Arg/Ile329Met (0.7 °C), Asp52Val/Asp72His/Glu273Gly/Lys348Arg (1.5

°C), Ile272Val/Asn367Arg/Glu368Ile (2.6 °C), Arg80Gly (4.6 °C),

Ser40Cys/Thr67Ala/Val118Leu (5.3 °C), Arg235Gly/Ile282Val/Ile299Val/Glu52Lys (7.4

°C), and Gly313Glu (8.5 °C) [43].

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CYP119A2

CYP119A2 (P450St) was isolated from S. tokodaii (Sulfolobus sp. strain 7), an

acidothermophilic archaeon that is closely related to S. acidocaldarius, with optimal growth

conditions of pH 2-3 and 75-80 °C [10]. CYP119A2 has been shown to remain redox-active

in a didodecyldimethylammonium bromide (DDAB) film at temperatures up to 80 °C [40].

The temperature has been increased to 120 °C when experiments were carried out with the

electrochemical solvent poly(ethylene oxide) (PEO) [64].

Sequence and structure

At 367 residues, CYP119A2 is one residue shorter than CYP119A1, with the two proteins

sharing 64% sequence identity (Figure 2). The crystal structure of CYP119A2 was solved to

3.0 Å [40] and was highly similar to the previously solved CYP119A1 water- and imidazole-

bound structures with respective root mean squared deviations (RMSDs) of 1.3 and 1.4 Å

[39, 47] (Figure 1). The substrate-free structure has a water molecule coordinated to the

heme. A second water molecule forms a bridge between the coordinated water molecule and

residues of the I-helix (Ala210, Thr214), mirroring the H-bonding network found in the

water-bound structure of CYP119A1 [47].

One key difference identified between the structures of CYP119A2 and CYP119A1 occurs in

the F/G-region, which undergoes significant conformational rearrangements in CYP119A1 to

accommodate ligands of different sizes (Figure 3). While the F/G-loop of CYP119A1 appears

to adopt a conformation either enclosing the coordination site (CYP119A1-imidazole), or

directed away from the heme group (CYP119A1-H2O), the loop appears to adopt an

intermediary conformation in the case of CYP119A2-H2O, partially covering the

coordination site of the heme. This discrepancy is due to CYP119A2 containing additional

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residues in the F-helix and G/H-loop in comparison to CYP119A1, resulting in a relative

displacement of the G-helix by ~4 Å. An interesting difference noted in this region is the

presence of a Cl- anion in CYP119A2 crystal structure at the N-terminal end of the G-helix.

This ion binds to both the side and main-chain atoms of Arg162, preventing the unravelling

of this end of the helix as observed in CYP119A1, and potentially affecting the affinity of the

active site for the ligand. The CYP119A2 structure has not yet been solved in the presence of

a substrate, so it is difficult to interpret its potential conformational changes.

Other features that may augment stability

CYP119A2 contains a total of 13 2-residue salt bridges and 7 salt-bridged networks

compared to the mesophilic averages of 11 ± 3 and 4 ± 1 respectively (Supplementary table

2), an increase which may play a role in the stability of CYP119A2. All 13 aromatic residues

involved in the aromatic clusters of CYP119A1 were conserved in CYP119A2, either directly

or through mutation to another aromatic residue in the case of Phe228Tyr and Tyr250Phe.

However, these mutations do not appear to influence the orientation or alignment of the side

chain, with the two structures superimposing very closely (Figure 4). The only exception to

this is Phe338, which is rotated by ~90° in the CYP119A2 structure due to a change in the

secondary structure of the β3 sheet.

Like CYP119A1, CYP119A2 has shorter β5-turn than most P450s, except for CYP55. When

tested for its ability to turn over substrate in the absence of a redox partner, CYP119A2 was

found to catalyze styrene epoxidation supported only by NADPH or NADH [51].

CYP175A1

CYP175A1 was the second P450 derived from a thermophilic organism to be crystallized,

and the first from a thermophilic eubacterium. T. thermophilus strain HB27 is a gram-

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negative bacterium with an optimum growth range of 65-72 °C, but which can grow at

temperatures up to 85 °C [65, 66]. CYP175A1 has a Tm of 88 °C, comparable to that of

CYP119A1 [39].

Sequence and structure

The structure of CYP175A1 was solved to 1.8 Å using the heme domain of CYP102A1 as a

molecular replacement model [11] (Figure 1). A crystal structure of the homolog from T.

thermophilus HB8, which differs only by 10 residues, has also been deposited in the PDB

library (1WIY) [67], and its structure superimposes closely with that of the HB27 form.

CYP175A1 exhibits typical P450 structural characteristics, with a conserved heme-binding

motif and thiolate Cys residue at position 336. At 389 residues in length, this protein is again

significantly shorter than mesophilic P450s like the CYP102A1 heme domain (472 residues),

but slightly longer than CYP119A1 (368 residues) [11, 39, 68] (Figure 2). As with

CYP119A1, many of the differences to mesophilic P450s are in loops connecting secondary

structural elements. A difference of seven residues is found in the loop connecting helices E

and F, which spans residues 159-172 in CYP102A1 compared to 146-152 in CYP175A1

(Figure 2). Eight residues are also missing from the β5 turn connecting helices H and I (239-

250 in CYP102A1, 202-205 in CYP175A1), as for the thermophilic CYP119s and the self-

sufficient CYP55 [11, 39, 50, 51]. The G-helix is notably two turns shorter in CYP175A1,

however residues 176-195 align well with 198-217 of CYP102A1 with a RMSD of 1.14 Å. In

comparison to CYP102A1, the F-helix is shifted by an approximate half-turn however the

core region (157-167 in CYP175A1) aligns closely (RMSD = 1.66 Å).

CYP175A1 is highly similar to the CYP119A1 imidazole- and 4-PI bound structures, with an

RMSD of 1.7-1.8 Å. Its N-terminus is slightly longer (by ~8 residues), and contains an

additional A’-helix from residues 5-18, resembling that of the 310-helix in CYP102A1. The

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start of the A-helix of CYP119A1 corresponds to approximately residue 22 of CYP175A1.

The B’-helix of CYP175A1 is more conventional than in CYP119A1, adopting a similar

conformation to CYP102A1 as the lid of the substrate access channel. Compared to

CYP119A1, the CYP175A1 protein retains a more traditional conserved Thr(225) H-bonding

network, with no additional Thr residues in the subsequent positions.

Other features that may augment stability

CYP175A1 has a higher overall Arg content (12.1 %) and lower Lys content (2.8 %), than

many mesophilic P450s (averaging 6.1 % and 5.3 % respectively), especially compared with

CYP102A1 (4.5 % and 8.1 % respectively, Supplementary table 3). Improved stability is also

sometimes correlated with a decrease in uncharged polar residues (Asn, Glu, Ser, Thr) and an

increase in charged residues (Lys, His, Arg, Glu, Asp) [69, 70]. CYP175A1 shows a decrease

in polar uncharged residues, but only a minor increase in charged residues. Notably,

CYP175A1 does not contain the large network of aromatic residues present in CYP119A1.

The longest network resembling that of CYP119A1 spans only ~13 Å (compared to ~39 Å in

CYP119A1), and so is unlikely to play a significant role in the stability of CYP175A1.

However, CYP175A1 contains more salt-bridged networks than mesophilic P450s; 8 ± 2

compared to the mesophilic average of 4 ± 1 (Supplementary table 2). A greater proportion of

the total residues involved in salt bridges are involved in networks containing more than one

residue: 62 % of the salt-bridged residues in CYP175A1 are in networks, compared to an

average of 41 % in mesophilic P450s.

A thermodynamic analysis of the heat- and urea-driven denaturation of CYP175A1 and

CYP101A1 showed that the increased stability of CYP175A1 is the result of a higher free

folding energy (ΔGAq), due to higher enthalpy (ΔHm) [71]. The increase in enthalpy may be

due to more internal electrostatic and H-bonding interactions, since more hydrophobic

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interactions would result in entropy-driven stability. This is supported by the fact that both

proteins contain a similar content of hydrophobic residues, but CYP175A1 has more salt-

bridged networks and a greater ratio of Arg to Lys residues than CYP101A1 [71].

CYP231A2

CYP231A2 was one of two P450 genes identified in the acidothermophilic archaeon P.

torridus by a BLAST search of the available genomes of thermophilic organisms [2, 72].

While not quite as thermophilic as Solfolobus or T. thermophilus, this species thrives at

temperatures of ~60°C. It has optimal growth at pH 0.7 and can even grow at pH 0 making it

the one of the most acidophilic organisms identified to date [72]. The intracellular pH of P.

torridus is 4.6, lower than that of S. acidocaldarius at ~5.6 [72, 73].

CYP231A2 has a Tm of 65 °C when ligand free, but the more compact 4-PI-bound form has

higher stability (Tm of 73 °C) [12] . The optimal growth temperature of P. torridus is 60 °C,

so it is presumed that binding of the endogenous substrate of CYP231A2 would cause a

similar increase in stability. While CYP231A2 is more thermostable than mesophilic P450s,

its Tm is not as high as that of the “hyper-thermostable” P450s (CYP119A1/2 and

CYP175A1) from more hyperthermophilic organisms. Whereas CYP101A1 is irreversibly

denatured at pH 4.5, CYP231A2 can undergo a reversible transition to P420, returning to

P450 upon neutralization of pH [12].

Sequence and structure

The structure of CYP231A2 was solved to a resolution of 2.5-3.1 Å using both molecular

replacement, based on CYP119A1, and MAD (Figure 1) to resolution of 2.5-3.1 Å [12].

CYP231A2 shares 38-39% sequence identity with CYP119A1 and CYP119A2. At 352

residues, CYP231A2 is even shorter than previously studied thermophilic proteins, and much

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shorter than mesophilic P450s, due mostly to an N-terminal truncation. The N-terminal A-

helix present in most P450s, including the thermophilic forms studied to date, is absent and

the protein instead begins with the β1 strand (Figure 2). There is a degree of structural

ambiguity in the B’-helix, specifically from residues 44 to 52, which was attributed to either

lack of a bound substrate to stabilize the structure, or the effects of missing an A-helix on the

B’-helix [12].

Other features that may augment stability

CYP231A2 differs from the other thermophilic P450s, in that it contains fewer salt-bridged

networks (3 ± 1) than mesophilic P450s (4 ± 1) (Supplementary table 2). Additionally, as Ho

et al. point out, salt bridges are unlikely to play a role in the stabilization of CYP231A2, as

many carboxylate groups would be protonated at internal the pH of P. torridus (~ 4.6) [12].

Moreover, there was no significant difference observed in Tm values measured at both pH 7

and 4 [12]. CYP231A2 does not contain aromatic networks on the scale of those seen in

CYP119A1, with the largest network consisting of just five residues (Tyr18, His23, Tyr25,

Phe243, Tyr269) and spanning a total distance of 13.9 Å (Figure 4). Rather, the

thermostability of CYP231A2 was attributed primarily to its small size and hence lower

surface to volume ratio. Its lack of hyper-thermostability compared to the other thermophilic

P450s may be the result of the absence of salt-bridged networks and large aromatic clusters.

Structural insights from thermophilic P450 structures

Determining the structural characteristics that give rise to thermostability is complex, as these

factors can vary between and even within protein families. There have been many attempts to

establish general rules of what yields a thermostable protein, but for every rule there is at

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least one exception. The thermophilic P450s are no different, appearing to employ a variety

of stabilization mechanisms.

Size and surface turns

The first and most obvious generalization that can be drawn from the sequences and

structures of thermophilic P450s is that these proteins tend to be shorter than those derived

from mesophilic organisms (Figure 2). The thermophilic proteins are ~352-389 residues

whereas their mesophilic relatives average around 403-470 residues, for the commonly

studied mesophilic bacterial P450s: CYP107A1, CYP101A1, CYP176A1, CYP107H1

(P450BioI), CYP108 (P450terp) and the CYP102A1 heme domain). This difference is mostly

due to an N-terminal truncation, but throughout the proteins, several more residues are absent

in connecting loops and surface turns, resulting in a more compact overall structure (Figure

2). The β1-1/β1-2 hairpin loop and the β5 turn, between helices H and I, are each shortened

by 4-8 residues in multiple forms. The β5 turn truncation may be characteristic of the ability

to act independently of a reductase partner. Shortened helices are also a common feature,

with all thermophilic forms having G-helices that are shorter by ~1-3 turns compared to

CYP102A1 and CYP101A1. In the absence of sequence elements that are (evidently) not

essential to establish and maintain the P450 fold, these proteins gain a more compact, robust

structure with a higher surface area to volume ratio.

Amino acid content

Several studies have linked changes in amino acid content with improvements in overall

thermal stability [62, 69, 70, 74]. Examining the thermophilic P450s, while some trends can

be observed, multiple mechanisms appear to be able to achieve the same result

(Supplementary table 3). The one major difference that can be observed across all

thermophilic forms appears to be an increase in charged residues (Lys, His, Arg, Glu, Asp)

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compared to polar uncharged residues (Asn, Gln, Ser, Thr), with thermophiles showing a

higher charged:polar uncharged ratio (31.2:15.5 %) than the mesophiles (27.1:18.1 %). The

hyperthermophiles (CYP119A1, CYP119A2, CYP175A1) showed a significant increase of

4.7% (P=0.02) in the proportion of charged residues compared to mesophilic organisms and a

decrease of 3.5% (P=0.08, Table 2) in polar uncharged residues. CYP231A2 has an

intermediate ratio (29.7:18.4 %), concordant with its intermediary stability.

Statistical analysis of the amino acid composition of three hyperthermophilic (CYP119A1,

CYP119A2, CYP175A1) vs. representative mesophilic forms (Supplementary table 3)

revealed significant changes in individual amino acid content: higher Glu (P=0.00001), lower

Cys (P=0.02), lower His (P=0.05), lower Met (P=0.05), lower Gln (P=0.02), and lower Thr

content (P=0.03). The changes in Gln, Thr and Glu correlate with alterations in the balance of

charged to polar, uncharged residues [70]. Cys, His and Met content are slightly decreased in

the hyperthermophiles (0.7 - 1.1 %), which is in agreement with generally small decreases in

these residues across a broader comparison of mesophilic and thermophilic proteins [69].

Substitutions of Met for Leu residues have also been observed to correlate with stability [74],

and although not significant, a general increase is also seen in the proportion of Leu residues

in these hyperthermophiles (Supplementary table 3).

CYP175A1 has a higher percentage of hydrophobic residues (54.8 %) compared to all the

other P450s examined (42.9-51.0 %). Increased Ala content was originally thought to

improve stability, due to its propensity for forming helices [74]. However later studies have

associated a slight decrease in Ala content with thermophilic proteins [69]. This seems to be

true for the thermophilic P450s other than CYP175A1 (which has an Ala content of 11.6%),

as they show Ala contents of 3.3-4.9 % compared with 7.2-9.8 % in bacterial mesophiles.

The other thermophiles also generally show higher Ile content (but in CYP175A1, Ile content

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is lower). This could be an alternative mechanism of stability, resulting in better side-chain

packing in the protein core [62].

CYP175A1 was also distinguished amongst the thermophilic P450s by being the only form to

show a change in Arg/Lys ratio (12.1:2.8 % Arg:Lys) over mesophilic P450s, especially

compared to CYP102A1 (heme domain) which has high Lys content (4.4:7.9 %). However,

comparison with other representative forms (Supplementary table 3) reveals that CYP102A1

appears to be unusual amongst mesophiles, which have an average Arg:Lys ratio of 6.1:5.3%.

Structural interactions

Another common feature of thermostable P450s is the number of salt bridges; in particular,

an increase in the number of salt-bridged networks (i.e., containing more than 2 residues). It

has long been recognized that proteins from thermophilic organisms tend to have an

increased number of charged residues on the surface and more salt bridges [75-78] and

experimental work has linked individual salt bridges and salt-bridged networks with

increased stability across many different protein families [43, 76, 79, 80]. While some models

have suggested that salt bridges should have a negative or neutral effect on stability due to a

high associated desolvation penalty [81, 82], others have suggested that these parameters do

not hold true at high temperatures, such as those experienced by thermophilic organisms [78,

83]. At elevated temperatures, the dielectric content of water decreases and charged side

chains incur significantly lower desolvation penalties [11, 78].

Interestingly, most of the thermophilic P450 structures appear to have a slightly lower

average number of 2-residue salt bridges (8-10) compared to mesophilic forms (11 ± 3),

except for CYP119A2, which contains 13 (Supplementary table 2). However, compared to

the mesophilic average (4 ± 1), there are more salt-bridged networks containing 3 or more

residues in every thermophilic structure (7-8) except for CYP231A2 (3 ± 1). Of the residues

involved in salt bridges, a greater proportion are involved in networks containing more than 2

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residues (52%) compared with mesophiles (41%). CYP231A2 once again fails to follow this

trend with only 37% of salt-bridged residues belonging to networks. Salt-bridged networks

appear to be distributed more evenly across the surface of the proteins in the thermophiles,

compared to CYP101A1 and CYP102A1 [11].

Most conclusions regarding the relevance of electrostatic interactions are drawn only from

comparisons of relative abundance, but a few studies have tested this hypothesis

experimentally. Disruption of a salt-bridged network in CYP119A1 (Glu114, Arg363,

Glu342) via the mutation Glu114Asp resulted in a 3.8 °C decrease in Tm [43] and a mutation

disrupting the electrostatic interaction between Arg259 and the heme propionate resulted in a

5.9 °C decrease [43] consistent with a stabilizing effect of these salt bridges on the active site

of thermophilic proteins at high temperatures [76]. In contrast, disruption of the unique salt

bridge between surface residues Arg154 and Glu212 did not have a significant effect on

stability [63].

The impact of pH on thermostability has only been considered for CYP231A2 [12], and it

was suggested that salt bridges are unlikely to be important due to the acidic cellular

environment. Similar analyses have not been done for the other thermophilic P450s, but

would be informative concerning the role of charge-charge interactions on thermostability.

A significant factor believed to contribute to the stability of CYP119A1 and CYP119A2 are

extended networks of aromatic residues. This has been experimentally validated for CYP119,

with mutations of several individual residues in this network resulting in a decrease in Tm of

up to 10°C [43, 63]. The aromatic networks of CYP175A1 and CYP231A2 (Figure 4) are

less extensive (with the largest networks spanning ~13 Å compared to ~ 39 Å for CYP119)

and thus far no experimental data is available to test the hypothesis that they stabilize these

proteins.

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Conformational dynamics

Another feature identified in CYP119A1 that differs from other P450s is the active site

arrangement and mechanism of rearrangement during substrate binding. Large

conformational changes, depending on the size of the substrate bound, have been identified in

several P450s, including CYP102A1, CYP3A4 and CYP2B4. CYP175A1 and CYP231A2

both exhibit similar conformational movements to the mesophilic examples, with the F- and

G-helices moving together as a unit, breaking and forming new inter-peptidyl bonds with the

I-helix and other parts of the protein [39, 47, 55, 56]. By contrast, the F- and G-helices of

CYP119A1 do not move so much when different substrates are bound, but appear to remain

locked in their positions while the ends of the F- and G-helices unravel to extend and contract

the F/G-loop, enabling it to either point outward away from protein in the case of the water-

bound form, or dip down to enclose the active site varying degrees depending on the size of

the bound substrate. The F/G-loop unravelling may be an adaptation to allow the active site

to retain its conformational flexibility and ability bind different substrates, while more

branched-chain amino acids in the G- and I-helical contacts, resulting in rigidly locked core

helices, may improve the overall rigidity and stability of the core P450 structure.

Artificial evolution of thermostability in P450s

Improving the thermal tolerance of native enzymes by protein engineering has become a

popular objective in recent years with increasing appreciation of the potential to implement

enzymes as biocatalysts. With regard to P450s, attempts to increase thermostability have thus

far focused on two subfamilies: the mammalian, xenobiotic metabolizing CYP2B subfamily;

and the bacterial fatty acid hydroxylases from the CYP102A subfamily, particularly

CYP102A1 (CYP102A1).

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Engineering of CYP102A1

The interest in stabilizing CYP102A1 stems from its potential as a biocatalyst for the

production of fine chemicals and bio-remediation. In directed evolution studies targeting

CYP102A1 thermostability, random mutagenesis has been applied to identify specific

stabilizing residues. Extensive chimeric libraries have also been generated using predictive

statistical methods. All studies have used a laboratory-evolved variant of the isolated

CYP102A1 heme domain, denoted as 21B3, as the ultimate template. The 21B3 mutant is

capable of functioning as a peroxygenase, utilizing hydrogen peroxide in place of NADPH

and O2 [84], eliminating the requirement for NADPH or the reductase domain. The heme

domain alone is more stable (T50 = 57 °C) than the full-length protein (T50 = 43 °C) since the

reductase is the less stable domain of the holoprotein. The 21B3 mutant was found to have a

T50 of ~46 °C, which is less stable than the wild type (WT) heme domain, but more stable

than the full-length protein.

In the first study to report engineering of a P450 to resist thermal denaturation, five cycles of

random mutagenesis were performed starting with 21B3, followed by a round of DNA

shuffling with another heme domain peroxygenase variant containing the mutation Phe87Ala

[85, 86]. From this, a thermostable peroxygenase variant, 5H6 (T50 = 61°C), was identified

that differs from 21B3 in 8 of the total 464 amino acids. Only two non-conservative

mutations, Ser106Arg and Glu442Lys, involved a change in charge. The other six

substitutions, Leu52Ile, Met145Val (a reversion to the WT), Ala184Val, Leu324Ile,

Val340Met and Ile366Val, were conservative mutations but differed in their hydrophobicity

and size.

Mutations from 5H6 have been used for stabilizing other CYP102A1 mutants in subsequent

directed evolution studies. The Ile366Val and Glu442Lys mutations were introduced and four

other mutations reverted to the WT residues (Cys47Arg Ile94Lys Cys205Phe Ser255Arg) in

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9-10A, a mutant engineered to accommodate bulky substrates and containing 15 mutations

compared to the WT [87]. These changes increased the half-life from 3 minutes to

136 minutes at 50 °C (native CYP102A1 has a half-life of 68 minutes) [88]. Mutation

Ile366Val has also been paired with Leu52Ile and introduced into a dopamine binding mutant

that had been destabilized through the accumulation of 15 mutations (T50 = 43.4 °C) [89].

These substitutions were able to increase the T50 by 5 °C. Likewise, Ile366Val was used to

increase the T50 of a CYP102A1 mutant already containing 6 other mutations [90] from 48 °C

to 52 °C. In addition, variant 5H6 itself was subjected to error-prone PCR (epPCR) and,

although the library was screened primarily for functional improvement, three mutants were

identified with T50 values up to 3 °C higher [91]. In all three variants, a buried Phe was

converted to Leu at position 173; this substitution was not found in any other characterized

mutant.

Analysis of the individual stabilizing effects of mutations Leu52Ile, Leu324Ile, Val340Met,

Ile336Val and Glu442Lys found that each substitution alone, with the exception of

Leu324Ile, was able to increase the stability of the aforementioned dopamine binding mutant

[89] by ~1-4 °C. The Leu324Ile mutation caused a destabilization of 0.3 °C. Furthermore,

double mutants Ile366Val/Glu442Lys and Ile366Val/Val340Met, which individually were

the most stabilizing substitutions, resulted in misfolded proteins.

All these residues are relatively dispersed throughout the protein fold (Figure 5) mostly

located in peripheral regions as opposed to the heme-binding core. According to the crystal

structure of the WT CYP102A1 heme domain [92], Ser106Arg, Leu324Ile, Val340Met,

Ile366Val, and Glu442Lys are located on the protein surface and Leu52Ile, Ala184Val,

Met145Val are buried. The three buried mutations all introduce residues of increased

hydrophobicity, which may act to anchor peripheral loops and turns, preventing their

disruption. Conversely, mutations Val340Met and Ile336Val decrease hydrophobicity but are

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exposed to solvent, as is Glu442Lys, and these residues may be stabilizing through altered

interactions with the surrounding water matrix.

Chimeras of CYP102A forms have been created by structure-guided recombination, using the

SCHEMA algorithm [93]. This method identifies cross-over points at which parent proteins

can be recombined to minimize disruption (i.e., destabilization) of the three-dimensional fold.

Seventeen double-crossover chimeras were created by swapping fragments between the heme

domains of CYP102A1 and CYP102A2, that had been mutated at Phe87Ala (CYP102A1)

and Phe88Ala (CYP102A2) respectively to enhance peroxygenase activity. None of the

chimeras were more stable than CYP102A1, but more than half were more thermostable than

CYP102A2 (T50 = 44°C) [94]. This study was subsequently expanded with the inclusion of

CYP102A3, and the three CYP102A peroxygenase mutants were recombined using seven

crossover points to create a library of >600 properly folded chimeras [95]. The most stable

chimera identified from this library had a T50 of 62 °C and differed from its closest parent by

84 amino acid substitutions, making it difficult to identify the residues responsible for

stabilization. However, this mutant only differed from the second most stable mutant (T50 =

56 °C) at 30 residues within the N-terminal fragment, which was derived from CYP102A2

(T50 =44 °C) in the more stable variant and CYP102A3 (T50 =49 °C) in the less stable variant.

In a later study, a further 204 chimeras [95] were assessed for their thermostability. This data

was then used as a training set to produce a linear regression model that was used to predict

T50 values for >6000 chimeras, including 30 with T50 ≥ 60 °C. These 30 mutants were

constructed and all displayed T50 values between 58.5 °C and 64.4 °C. The most thermostable

chimera predicted by the model (T50 = 64.4 °C) also happened to be the consensus sequence,

i.e. the sequence that combined the fragments that showed the highest frequency at each

position amongst the folded chimeras. The stable chimeras differed from one another at

between 7 and 99 positions with an average of 46 differences [96]. This illustrates the

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limitation of chimeragenesis approaches, in that it is difficult to identify the stabilizing

residues and interactions in these mutants since the number of changes from any given parent

can range from tens to hundreds of residues.

Another statistical method that has been employed for predicting stable chimeric P450s is by

modelling fitness landscapes using Gaussian process regression, a Bayesian, machine-

learning approach [96]. A model was trained on sequence information and thermostability

data from all available chimeric P450s [96] and used to predict a total of 34 sequences that

were likely to be more stable than the most stable parent. Of these, 28 had a T50 ≥ 60 °C, 12

had a T50 ≥ 65 °C. The most stable variant displayed a T50 of 69.7 °C, making it the most

stable CYP102 variant identified to date [97]. In this study, the Upper Confidence Bound

(UCB) algorithm was used to iteratively improve the Gaussian process model in regions of

the landscape that were predicted to be highly optimized. These models are successful

because they were trained on experimental data, which implicitly allows consideration of all

factors that contribute to stability, including those that are unknown.

Engineering of CYP2B enzymes

Despite the abundance of industrially relevant eukaryotic P450s, the only forms to have been

the focus of engineering towards thermostability are several from the CYP2B subfamily.

Thermostable CYP2B forms are of interest to the pharmaceutical industry as they play a

significant role in the metabolism of many drugs and xenobiotics (albeit a relatively minor

one in humans compared to CYP3A, CYP2C and CYP2D forms). CYP2B forms show

conformational flexibility and substrate promiscuity while also displaying higher

thermostability than many other native mammalian P450s [98] (Thomson et al., unpublished

data) making them interesting candidates for structural studies. Both rational and random

approaches have been taken to stabilize CYP2B forms.

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Residues of interest have been selected for rational mutagenesis based on the stability

differences between native CYP2B forms. CYP2B1, CYP2B4 and CYP2B11 have been

found to have Tm values ~13 C, 10 C and 3 C higher than CYP2B6 respectively, and 25

residues are conserved between the three more stable forms but differ in CYP2B6. Eleven of

these residues that are buried in the available crystal structures were mutated in CYP2B6 to

match the common CYP2B1/CYP2B4/CYP2B11 sequence [98]. The Leu264Phe mutant was

the only variant to show an increase in stability (4 C) and it was hypothesized that this

change may increase the hydrophobicity or integrity of the H-helix.

A similar method was used to improve the thermal stability of CYP2B6 and CYP2B11.

Seven residues in these forms were mutated to match the amino acid found in the relatively

more stable CYP2B1 and CYP2B4. Mutation Pro334Ser was found to improve the Tm of

CYP2B6 by ~7 C and CYP2B11 by ~2 C. The mechanism of this stabilization was

explored with pressure-perturbation spectroscopy and it was found that CYP2B enzymes

containing a serine at position 334 are more compressible than those with a proline at this

position. Therefore, the Pro334Ser may stabilize the structure by increasing conformational

flexibility in the region of the heme pocket [99].

Random mutagenesis has also been used to produce a library >3000 variants of the CYP2B1

peroxygenase mutant, QM (Val183Leu/Phe202Leu/Leu209Ala/Ser334Pro). Two QM

mutants, containing Leu295His and Lys236Ile/Asp257Asn mutations respectively, showed

enhanced tolerance to temperature over QM with an increase in T50 of ~1 C and ~2 C

respectively [100]. However, when these three mutations were combined, the resulting

mutant displayed a T50 ~ 6 C lower than QM. The reason for this apparent deleterious

epistatic effect is not known. Residues 295, 236 and 257 are located in the I-helix, G-helix

and the G/H-loop respectively, and do not appear to interact with each other. This illustrates

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the difficulty in predicting the effect of mutations in a landscape of unknown epistatic

interactions.

Structural insights from engineering studies

Thus far, attempts to increase the thermostability of P450s using random or site-directed

mutagenesis have identified relatively few beneficial mutations that generally provide

increases in stability of less than 10 °C. The effect of these mutations on divergent forms has

yet to be explored so it is difficult to predict whether any of these changes may have a

generally stabilizing effect on the P450 fold. While chimeragenesis has proven to be a

promising alternative method for producing highly stabilized variants, particularly when

coupled with predictive modelling, the underlying structural determinants of enhancements in

stability are difficult to determine since inter-residue interactions are altered simultaneously.

Chimeragenesis may be a more successful approach for engineering thermostability due to

the increased proportion of properly folded mutants that are produced by recombination of

naturally evolved forms, compared to the proportion produced by random methods, i.e., a

greater number of properly folded variants can be effectively assessed for their stability given

the lower “kill rate” compared to random mutagenesis. Moreover, recombination samples a

broader area of sequence space.

Thermostable redox partners

Ultimately, stabilization of the P450s is only half the challenge in implementing these

monooxygenases as biocatalysts. In order to exploit the full potential of a thermostable P450

it is necessary to couple it with a stabilized redox partner. Most P450 redox systems conform

to one of ten different classes, depending on the composition and topology of the electron

transfer pathway [101]. A minority of P450s undergo direct reduction by NADH (Class IX)

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[102, 103], catalyze isomerizations (Class X) [104-106], or act as peroxygenases, which

involves the direct oxygenation of the heme without the requirement of any redox partner

[107-109]. However most P450 enzymes rely on one or more auxiliary redox partners to

carry out monooxygenations [101]. Therefore, thermostable P450 systems, rather than only

thermostable P450 enzymes, are generally required for biocatalysis. So far only a very

limited number of thermostable P450 redox partners have been identified and the

understanding of what makes them more robust at higher temperatures is poorly understood.

In some cases, the P450 and a reductase domain are assembled on a single polypeptide chain

(Classes VII and VIII) [110-112] with the best characterized being the CYP102 family of

enzymes. As noted above, the reductase domain of CYP102A1 showed lower thermostability

than the heme domain, and a more thermostable holoprotein could be engineered by

replacing the reductase domain with that of the more stable CYP102A3, creating a functional

holoprotein with an optimal temperature of 51 ºC and ten-times longer half-life than

CYP102A1 at 50 ºC [113].

Most prokaryotic P450s and the mitochondrial P450s from eukaryotes belong to Class I,

requiring a flavin-containing ferredoxin reductase (FdR) plus a ferredoxin (Fd), an iron-sulfur

protein which acts as a mediator shuttling the electrons from the FdR to the P450. Fds can be

further classified according to the configuration of the iron–sulfur as [2Fe–2S], [3Fe–4S],

[4Fe–4S], or [3Fe–4S]/[4Fe–4S] [101].

The CYP175A1 redox partners

Native T. thermophilus redox partners for CYP175A1 have been identified in the form of a

[3Fe−4S][4Fe−4S] dicluster Fd (FdTt) and an NADP-selective FdR (FdRTt) [114]. FdTt and

FdRTt supported β-carotene activity with a turnover rate ~54 times higher than the PdX/PdR-

supported system [24]. FdRTt exhibits a 30T50 value of 99 ºC and FdTt shows a thermally

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induced unfolding temperature of 114 ºC at pH 7.4 [115], making the combination of these

redox partners the most thermostable class I redox system identified so far. A fusion protein

comprised of the FdTt and FdRTt (175RF) showed an additional 25-fold increase in Vmax for

β-carotene hydroxylation at 65 ºC [25]. However the 10T50 of the fusion was lower at 80 ºC.

The crystal structure of FdTt has been solved and compared to other, homologous Fds [116]

such as the mesophilic Fd I from Azotobacter vinelandii, FdAv. The overall structure of FdTt

is defined by two iron-sulfur clusters which are sandwiched between two antiparallel alpha

helices and two beta-sheets, in a typical bacterial dicluster Fd ((βαβ)2 fold. FdTt is mostly

stabilized around the [3Fe−4S] cluster (cluster I). Cluster I is generally considered to be the

functional cluster in seven-iron Fds whereas cluster II plays a more structural role [117, 118].

Polar residues were found to predominate on the protein surface of FdTt, compared with

more negatively charged residues in FdAv, which may contribute to greater thermal stability

due to a reduction in unfavorable clustering of similarly charged groups [116]. Additionally,

the -helices in FdTt are more stable than those in FdAv. One (αA) is stabilized by

electrostatic interactions between an Asn residue and the positively charged end of the helix

macrodipole. The other helix (αB) is stabilized by the presence of alanine residues leading to

a more well defined -helix, compared to the 310-helix present in FdAv [116, 119]. FdTt also

exhibits tighter packing compared to FdAv leading to a general stabilization of secondary

structure elements in FdTt. In particular, it shows less local conformational strain than

FdAdv, which has non-glycine residues in a left handed helical conformation [116, 119]. By

contrast, FdTt and its thermostable homologue, FdBs from B. schlegelii, have glycines in

topologically related positions, which release strain in the protein backbone [116, 119, 120].

Large hydrogen bond networks were found in FdTt, involving side chain:main chain

interactions that stabilize the core structure, with the largest network around cluster I [116].

Bulky, charged, side chains are present around cluster II, which enhance the interaction with

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the N-terminus and fix its conformation. In other thermostable Fds the N-terminus is also

anchored by large side chains in the proximity of cluster II, so this may be a common

mechanism for stabilization, serving to constrain solvent access to cluster II and thereby

enhance oxidative stability. Additionally, FdTt contains fewer Asn residues than FdAv,

which may be important since peptide bonds involving Asn can be easily hydrolyzed when

exposed to solvent at high temperatures [116, 121].

The lack of a crystal structure for the FdRTt obviates identification of factors that may be

responsible for stabilization of this second component of the redox system. To date, this FdTt

/FdRTt electron transfer system has only been reported to support the activity of CYP175A1

and therefore its potential for use with other P450 enzymes remains untested.

Etp1fd

The most thermostable Fd that has been identified to interact with multiple class I P450s is

the C-terminal domain of an electron transfer protein (Etp1) from the fission yeast,

Schizosaccharomyces pombe, Etp1fd [122-126]. Etp1fd is homologous to vertebrate-type Fds

[122] and has been characterized with respect to its thermal stability as the full length

(Etp1fd(505–631)) and a truncated form (Etp1fd(516–618)) created for crystallization [127].

Both are highly robust, showing thermal transition temperatures, Tm, of 70.5 °C for Etp1fd

(505–631) and 65.6 °C for Etp1fd (516–618). The lower thermal stability of the truncated

version was surprising, since C-terminal truncation of Adx, the mitochondrial sequence

homologue, yields a more thermostable Fd [128]. Both truncated Fd forms, Adx (4-108) and

Etp1fd(516–618) have been crystallized, and revealed a large number of potential stabilizing

factors [127, 129]: five additional stabilizing salt bridges at the protein surface in Etp1fd

structure, compared to Adx; fewer glycines and more prolines in loops, which would

decrease the conformational entropy upon folding [130]; potential stabilization of the helix

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dipole by the substitution of Val34 in Adx by His546 in Etp1fd [131]; more charged and

hydrophobic residues in Etp1fd in place of uncharged polar side chains in Adx; and a two-

residue shorter loop between residues 550–560 in Etp1fd. Replacement of uncharged polar

side chains and loop shortening are both typical of thermophilic proteins compared to their

mesophilic homologues and may confer enhanced intramolecular interactions and reduced

conformational entropy, respectively [132, 133].

Thermostable diflavin reductases

Class II P450s are supported by the diflavin-containing, cytochrome P450 reductases (CPRs)

that contain an FAD-containing FdR-like domain and a linked FMN-containing flavodoxin

domain. Notably, no diflavin reductase has yet shown comparable thermostability to the class

I redox partners discussed above. The most thermostable putative diflavin reductases reported

to date are the bacterial sulphite reductase, BmCPR from B. megaterium, and CaCPR1,

isolated from Capsicum annuum, with 10T50 values of 54.1 °C and 56.1 ºC respectively.

BmCPR is known to transfer electrons to Etp1fd and various Fds but also supports the

reaction of the microsomal CYP21A2 very efficiently without any Fd mediator [134, 135]. In

contrast, CaCPR1 supports activity with microsomal CYP1A2 only at low rates relative to

the cognate mammalian cytochrome P450 reductase [136]. Given the ability of BmCPR to

support CYP21A2 reactions with high efficiency, BmCPR appears to be a more promising

thermostable redox partner to support other microsomal P450-catalyzed reactions. Neither

BmCPR nor CaCPR1 have been crystallized and therefore the factors responsible for the

stability of these forms have not yet been elucidated. However, in the case of the plant

CaCPR1, it was speculated that conditions of stress during plant development may have

imposed selection pressure to improve the supply electrons to P450s, leading to improved

stability [136].

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2-Oxoacid:ferredoxin oxidoreductase systems

The thermostable redox systems that support the CYP119 from S. acidocaldarius are unusual

in utilizing coenzyme-A and pyruvic acid rather than NADH or NADPH as the source of

electrons, and are composed of a thermostable Fd and a 2-oxoacid:ferredoxin oxidoreductase

(OFOR) [137, 138]. CYP119 has been reconstituted with a Fd and an OFOR from the

thermophilic organism, S. tokodaii strain 7 (StOFOR) [137-139] and also with the Fd and an

OFOR from the thermophilic S. solfataricus (OFOR-Ss) [137] . The latter system was found

to display a 60T50 ~70 ºC with OFOR-Ss being the least stable protein component. The

reconstituted P450 system derived from S. tokodaii strain 7 withstands temperatures of up to

70 ºC for at least 20 minutes, again with the OFOR assumed to be the temperature-limiting

component [137].

The crystal structure of StOFOR has been reported recently, but factors contributing to its

thermostability were not examined in any detail [140]. By contrast, the crystal structure of the

Fd from S. tokodaii strain 7, which shows a Tm of 109 °C [141], has been solved and has

provided some insight into potentially stabilizing elements [142]. The overall structure is

composed of two parts, a core fold and an N-terminal extension. The core is a (βαβ)2 fold that

is common to bacterial dicluster Fds and so displays a similar architecture to FdTt. However,

the N-terminal extension is distinctive, comprising a 36-residue domain and a tetrahedrally

ligated zinc ion located at the interface between the core and the N-terminal extension. Both

have been proposed to contribute additional stability to the protein, as confirmed by a

subsequent protein engineering study [141, 142]. Mutants lacking zinc (while maintaining the

iron clusters), exhibited a decrease in Tm of 20 °C compared to the WT Fd [141]. The major

role attributed to the zinc was in combining the two β-sheets and mediating indirect

interactions between β-sheets (A′ + A) and β-sheet B, thereby potentially enhancing thermal

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stability compared to bacterial Fds lacking the zinc. Notably, however, the zinc-deficient

mutants were still highly robust with Tm values of ~89 °C.

Additional thermostabilizing effects have also been attributed to the N-terminus. With zinc

bound, residues 1–11 interacted with C-terminal residues leading to a 9 °C (Tm = 98 °C)

stabilization. Deletion of these 11 residues decreased the Tm by 11 °C, while further

truncation of up to 30 residues failed to reduce the Tm further. Moreover, a similar N-terminal

additional sequence to that of S. tokodaii strain 7, has been reported for two Fds (Fd A and

Fd B) from S. metallicus, which both show similar thermal stability with respect to S.

tokodaii strain 7, despite Fd B lacking zinc [143]. Thus, both, the zinc and the N-terminal

addition may represent common but not essential stabilizing features of Fds.

Prospects for identifying broadly useful thermostable redox partners

None of the thermostable redox systems characterized to date has been studied with more

than a few different P450s. Further work is needed to explore the potential of these systems

to support industrially relevant P450s as biocatalysts. There is also considerable scope for

identifying novel thermostable redox partners. No endogenous redox systems have been

identified for CYP231A2 or CYP154H1, let alone any of the numerous other P450s in

thermophilic organisms for which sequences are now available. For example, a BLAST

search using Sulfolobus sp. Fdx and OFOR sequences identified several proteins with 42-

59% identity in P. torridus, and a homologue to T. thermophilus FdR sharing 35% identity.

Likewise, existing sequence data suggests numerous Fds and FdRs are present in

Thermobifida fusca.

Overall, analysis of the redox systems characterized to date suggests that stabilizing elements

can be diverse and are in some cases quite specific, such as an extended N-terminal or zinc

ligation. Intramolecular, electrostatic and hydrophobic interactions that stabilize helices,

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reduced local conformational strain by specific amino acids and extended intramolecular

hydrogen-bonding networks are more common features. Thermophilic Fds appear to have

more balanced electrostatic potential at the protein surface, a reduced total cavity volume and

a reduced accessible surface area [116] compared to their mesophilic homologs. The

difference in surface charge distribution may be particularly significant: mesophilic Fds are

very acidic and the surface distribution of Asp and Glu residues is relatively well conserved,

enabling recognition by, and interaction with, electron acceptors [116]. The less negative

surface potential and more balanced surface charge distribution of thermostable Fds may be

advantageous with respect to stability [116] but could limit interaction with P450s. It has

been proposed that the reduced attraction between redox partners in thermophilic systems can

be compensated to some degree by the faster diffusion of the proteins at elevated

temperature, under which conditions the viscosity of the medium is reduced [116].

Accordingly, a potential redox partner for thermostable P450s may not need to be as specific

with respect to their interaction with the P450 compared to redox systems that operate at

lower temperatures. This gives hope for the identification of heterologous thermostable redox

systems that interact with thermostable P450s at enhanced temperatures.

Implications for the engineering of P450 systems

The crystal structures of P450s from thermophilic organisms provide insight into what makes

a P450 thermostable, and the minimum structural elements required to retain the P450 fold.

While some generalizations can be made, there appear to be many alternative mechanisms

within P450 sequence space to generate a thermostable enzyme. Overall, naturally

thermophilic P450s are shorter than those obtained from mesophilic organisms, with large N-

terminal truncations and fewer residues in surface turns and loops which may reduce the

conformational entropy. The fact that these proteins remain active suggests that P450

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function is not dependent on the presence of these regions. Thermophilic P450s also tend to

have a greater proportion of charged residues and fewer polar uncharged residues in

comparison to mesophilic forms. They also typically have more salt-bridged networks, and

may have extensive aromatic networks, and/or a more rigid active site that can still undergo

the conformational changes required for substrate binding through the unravelling of the F-

and G-helices. The Cys-ligand loop may also be stabilized by hydrophobic interactions. A

greater incidence of internal electrostatic and H-bonding interactions, or an increased ratio of

Arg:Lys residues may also augment stability. However, an important caveat to all these

observations is that they are made based on only a very few, select structures.

As more CYPomes are characterized from thermophilic organisms, we are likely to gain

further insight into how these proteins manage to survive under harsh conditions. Many

putative, thermostable enzymes are emerging from genome sequencing of known

thermophiles and metagenomic sequencing of microbiota sourced from hot environments,

both in terms of P450s and their cognate redox partners. These should provide ample starting

material for the development of biocatalysts that are stable under industrial conditions.

However, the functional characterization of these proteins is the rate-limiting step in their

exploitation.

Directed (or artificial) evolution studies have not been as useful as naturally thermostable

P450s in suggesting means by which to stabilize the P450 fold. Effectively, the sequence

space that has been surveyed to date by directed evolution has been limited to that which is

close to the mesophilic parents used in such experiments. The problem of deconvoluting the

effects of multiple substitutions means modelling and higher-order statistical analyses are

essential to interpreting data from directed evolution. Moreover, while such studies have the

opportunity to introduce a large number of changes into a P450 simultaneously, this has not

yet been done with the aim of testing hypotheses, e.g., assessing whether altering the balance

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of charged to polar uncharged, or the proportion of hydrophobic residues, improves the

thermostability of P450s derived from mesophiles. Random and rational mutagenesis of

thermophilic P450s, with the aim of decreasing rather than increasing stability, may be more

useful in revealing which residues, interactions and structural features are important

determinants of thermostability.

From the analysis of the few, naturally thermostable forms that have been characterized

structurally, it is possible to suggest some possible “design heuristics” to employ in

stabilizing P450s. Shortening mesophilic proteins at the N-terminus, in the β1-1/β1-2 hairpin

loop and in the β5 turn might be expected to be beneficial, along with shortening the G-helix

by 1-3 turns. Altering the balance of amino acids to include more charged and hydrophobic

residues, at the expense of noncharged, polar and Ala residues respectively, might also

augment stability. Much more challenging modifications could include increasing the number

of complex, salt-bridged networks (i.e., involving 3 or more residues) and introducing

lengthy stacked arrays of aromatic side chains. Truncation of specific regions of the fold

would be a straightforward strategy involving the construction and screening of relatively

few mutants. However, altering the proportion of particular amino acids or attempting to

develop salt-bridged networks or aromatic stacks would be best done using a directed

evolution approach involving the creation and screening of many mutants, given the limited

understanding of structure-function relationships of most P450s and their conformational

dynamics in particular. Fortunately, it is possible to screen hundreds of mutants for the

residual folded P450 after a heat treatment by measuring Fe(II).CO vs Fe(II) difference

spectra in whole cells in microplate format [144, 145].

A major hurdle for the implementation of existing and engineered thermostable P450s in

biocatalysis remains the choice of redox system. However, the ability of CYP119 and

CY175A1 both to interact with the Pdx from Pseudomonas putida [18, 24] underscores the

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possible interchangeability of redox partners. It appears unlikely that diflavin CPRs (class II

systems) will turn out to be as thermostable overall as Fd/FdR (class I) systems, given the

failure to identify a diflavin reductase in a thermophilic organism to date. One promising

opportunity for the identification of novel thermostable redox systems could be BLAST

searches using the sequences of mesophilic redox partners that are known to interact with a

thermostable P450 from a given thermophilic P450 organism. Alternative options, such as the

linking of a thermostable P450 to a Fd and photosystem I [146] might provide future

opportunities, considering that photosystem I complexes from thermophilic organisms are

available [147-150] and have already successfully been applied in other fields, such as the

light-induced generation of hydrogen in artificial systems [148].

The observation that CYP119A2 can catalyze monooxygenations in the absence of a redox

partner, possibly due to the shorter 5-turn that is a structural feature shared with another

self-sufficient monooxygenase, CYP55, raises the intriguing possibility that P450s could be

engineered to use NAD(P)H directly, obviating the need for a thermostable electron transport

chain.

In summary, while much has been gleaned from the study of thermostable P450 systems to

date, much more remains to be established before we can claim to understand the basis to

thermostability in the P450 fold. However, the combination of genome sequence, data

mining, directed evolution, modelling and high throughput functional analysis should

facilitate this objective and enable P450s to be exploited more fully in the design and

development of biocatalysts that are stable under industrial conditions.

Abbreviations

ABTS, (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid; 4-BPI, 4-(4-bromophenyl)-

1H-imidazole; 4-CPI, 4-(4-chlorophenyl))-1H-imidazole; CPO, chloroperoxidase; CPR,

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NADPH-cytochrome P450 reductase; Fd, ferredoxin; FdR, ferredoxin-NADPH reductase; 4-

FPI, 4-(4-fluorophenyl))-1H-imidazole; 4-PI, 4-phenylimidazole; LUCA, last universal

common ancestor; MAD, multiple wavelength anomalous dispersion; MD, Molecular

dynamics; MeOF, [13C]p-methoxyphenylalanine; NOR, nitric oxide reductase; ORF, open

reading frame; P450, cytochrome P450, heme-thiolate protein P450; RMSD, root mean

squared deviation; T50, temperature at which half the protein is denatured; 10T50, the

temperature at which half the protein remains intact after heating for 10 minutes; Tm, melting

temperature; WT, wild type.

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Acknowledgments

This research was supported by Australian Research Council Grant DP160100865, Australian

Postgraduate Research Awards to KLH and REST and an International Postgraduate

Research Scholarship to SJS. The authors gratefully acknowledge the assistance of Julian

Zaugg with the analysis of salt bridge networks in the P450 structures. Thanks are extended

to Prof. P. Hugenholtz for access to the bacterial genome database of the Australian Centre

for Ecogenomics.

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Figure legends

Figure 1. Structural comparison of thermophilic and selected mesophilic P450s

A: P450cam (PDBID: 3I61), B: P450BM3-heme domain (1FAG), C: CYP119A1 (1IO7), D:

CYP119A2 (1UE8), E: CYP175A1 (1N97) and F: CYP231A2 (2RFB). The view looking

down the axis of the I-helix is shown. Structures are colored by spectral progression from

blue at the N-terminus to red at the C-terminus. The F-and G-helices are visible at the top of

the structures in green, the A- and A’-helices where present are in deep blue. Dashed lines are

shown where elements of the structure are not visible in the crystal structure. Figures were

generated using Chimera [151].

Figure 2. Multiple sequence alignment of thermophilic and representative mesophilic-

P450s

A multiple sequence alignment of the four thermophilic P450s for which structures have been

solved with representative sequences of mesophilic P450s. Secondary structure elements are

colored as identified in the crystal structures (apart from CYP71A2, as no plant P450 crystal

structure has yet been solved) with -helices shown in orange and -sheets in blue. The

thermophilic sequences are significantly shorter at the N-terminus than mesophilic

counterparts. CYP119A1 and CYP119A2 both lack the A’-helix found in many mesophilic

P450s, beginning immediately with the A-helix, while in CYP231A2 the A-helix is entirely

absent and the structure starts with the β1-1 strand. Compared to the bacterial mesophiles,

deletions can also be seen in the β1-1/β1-2 hairpin loop and the β5 turn of the thermophilic

sequences. PDB structures used to assign secondary structure are as follows: CYP119A1,

1IO7; CYP119A2 (P450St), 1UE8; CYP175A1, 1N97; CYP231A2, 2RFB; CYP107A1

(P450eryF), 1Z8O; CYP101A1 (P450cam), 3L61; CYP108 (P450terp), 1CPT; CYP176A1

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(P450cin), 4FB2; CYP107H1 (P450BioI), 3EJE; CYP102A1 (P450BM3) heme domain, 1FAG;

CYP51, 4LXJ; CYP2C5, 1DT6; CYP3A4, 1TQN; CYP46A1, 2Q9G. The CYP71A2

sequence was obtained from UniProt (P37118).

Figure 3. Changes in conformation of the F/G-loop in various CYP119 ligand-bound

structures

The F/G-loop region is shown in CYP119 structures solved in the presence of various

ligands: A) 4-PI (PDB ID: 1F4T), B) 4-FPI (4WPD), C) imidazole (1F4U), D) 4-CPI

(4TUV), E) 4-BPI (4TT5), F) H2O (1IO7). The view is down the axis of the I-helix and the F-

and G-helices are shown above and to the top left of the I-helix, respectively. Figures were

generated using Chimera [151]. The side chain positions of Arg154 and Leu155 are shown

for reference. Note that the relative positions of the F- and G-helices change little with

respect to the I-helix, but the ends of the helices unravel, altering the size and position of the

F/G-loop to accommodate different ligands.

Figure 4: Aromatic clusters in CYP119A1, CYP119A2 and CYP231A2

A) Structural alignment of CYP119A1 (blue, PDBID 1IO7) and CYP119A2 (magenta,

1UE8), and B) Structure of CYP231A2 (green, PDBID 2RFB) with side chains of residues

belonging to aromatic clusters shown in yellow. Black brackets indicate the largest aromatic

clusters for each protein. Twelve of thirteen residues identified in the two unique aromatic

clusters of CYP119A1 are conserved in similar positions in CYP119A2, with a single residue

from cluster 2 (Phe338) rotated by ~90° in CYP119A2 due to a change in the β3 sheet. The

largest aromatic cluster of CYP231A2 (B) contains residues Tyr18, His23, Tyr25, Phe243,

Tyr269 and spans a total distance of 13.9 Å. This cluster was identified using the selection

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criteria of aromatic residues within 4 Å of each other. Figures were generated using Chimera

[151].

Figure 5. Positions in the CYP102A1 heme domain structure (PDB ID: 4ZF6) where

stabilizing substitutions were identified in the thermostable peroxygenase variant 5H6.

Two views are shown looking down the I-helix from opposite sides of the protein. The side

chains of the WT residues corresponding to the following mutations are shown: Leu52Ile

(forest green), Ser106Arg (pink), Met145Val (yellow; a reversion to the WT from a previous

mutant), Ala184Val (dark blue), Ler324Ile (cyan), Ile366Val (purple), Val340Met (orange),

Glu442Lys (lime green).

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Highlights

Thermostability is a desirable feature for using P450s in industrial processes.

The structures of four P450s from thermophiles have been characterized to date.

P450s from thermophiles tend to be shorter overall than those from mesophiles.

Extended salt-bridged and aromatic networks may stabilize the P450 fold.

Directed evolution has been used to increase the thermostability of mesophilic P450s.

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