Directed evolution of cytochrome P450 enzymes for biocatalysis: exploiting the catalytic versatility of enzymes with relaxed substrate specificity.

Biochem. J. (2015) 467, 1–15 (Printed in Great Britain)

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doi:10.1042/BJ20141493

James B.Y.H. Behrendorff*, Weiliang Huang† and Elizabeth M.J. Gillam†1 *Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, Brisbane, QLD 4072, Australia †School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Brisbane, QLD 4072, Australia

Cytochrome P450 enzymes are renowned for their ability to insert oxygen into an enormous variety of compounds with a high degree of chemo- and regio-selectivity under mild conditions. This property has been exploited in Nature for an enormous variety of physiological functions, and representatives of this ancient enzyme family have been identified in all kingdoms of life. The catalytic versatility of P450s makes them well suited for repurposing for the synthesis of fine chemicals such as drugs. Although these enzymes have not evolved in Nature to perform the reactions required for modern chemical industries, many P450s show relaxed substrate specificity and exhibit some degree of activity towards non-natural substrates of relevance to applications such as drug development. Directed evolution and other protein engineering methods can be used to improve upon this low level of activity and convert these promiscuous

generalist enzymes into specialists capable of mediating reactions of interest with exquisite regio- and stereo-selectivity. Although there are some notable successes in exploiting P450s from natural sources in metabolic engineering, and P450s have been proven repeatedly to be excellent material for engineering, there are few examples to date of practical application of engineered P450s. The purpose of the present review is to illustrate the progress that has been made in altering properties of P450s such as substrate range, cofactor preference and stability, and outline some of the remaining challenges that must be overcome for industrial application of these powerful biocatalysts.

INTRODUCTION

aliphatic epoxidations, heteroatom dealkylations, and heteroatom oxidations. In addition, reductive reactions are possible by electron transfer from the ferrous P450 to the substrate [6]. The haem-thiolate chemistry and the P450 scaffold have also been adapted in Nature for isomerizations that do not require molecular oxygen, reducing cofactors or redox partners (reviewed in [8]), as well as for peroxygenase reactions [9]. The ubiquity of P450s in Nature also testifies to the ability of the protein scaffold to adapt to different substrate structures. P450s defy the classical idea of an enzyme as a protein that has evolved to catalyse one particular chemical reaction with a high degree of chemo-, regio- and stereo-selectivity on a single substrate, or a discrete set of structurally related substrates, with high efficiency. Even allowing for a more realistic understanding of enzymes as moderately efficient rather than highly specialized catalysts [10] and the importance of physiological context in determining the degree of specialization and catalytic efficiency achieved by an enzyme subject to natural evolutionary pressures [11], the catalytic versatility of P450s is exceptional. Where P450s fulfil defined roles in primary metabolism, such as in fatty acid oxidation by P450 102A1 (P450 BM3) in Bacillus megaterium, they show high catalytic efficiencies and refined substrate selectivities as befits enzymes with roles essential for the viability of the organisms or in pathways with high metabolic flux [11]. However, the majority of P450s show more substrate ambiguity and poorer turnover rates, and even the more specialized bacterial P450s can be engineered to accept alternative substrates as shown by the ample experiments on P450 BM3

The P450 fold is an ancient one that has been exploited in all kingdoms of life for innumerable physiological functions [1]. The principal reason for this is that cytochrome P450s catalyse very useful chemistry. P450s are members of the haem-thiolate family of proteins in which the haem prosthetic group is linked to the protein via a conserved cysteine residue. Most act as monooxygenases using molecular oxygen and a reducing cofactor to catalyse the insertion of one oxygen atom into an organic substrate with the reduction of the second to water. In the classical P450 catalytic cycle, a series of steps leads ultimately to the generation of a highly reactive Fe(IV)-oxo species coupled to a ligand-based radical (compound I) [2] which is responsible for abstraction of a hydrogen atom or electron from the substrate. Oxygen rebound then occurs, generating the mono-oxygenated product. The electronic properties of the thiolate linkage to the haem are thought to enable this chemistry [3,4]. Typically, this reaction requires the assistance of one or more ancillary redox partners such as NADPH-cytochrome P450 reductase (CPR). However, peroxides can be used as oxygen surrogates to bypass the electrontransfer steps, and other oxidizing species have been proposed for certain P450-mediated reactions (reviewed in detail by Hrycay and Bandiera [5]). Through this fundamental chemistry, P450s can catalyse a diverse array of different chemical reactions as detailed in previous reviews [6,7]. Common biotransformations are aliphatic or aromatic carbon hydroxylations, aromatic or

Key words: biocatalysis, cytochrome P450, directed evolution, DNA shuffling, drug discovery, substrate ambiguity.

Abbreviations: CPR, NADPH-cytochrome P450 reductase; [DMSO]50 , DMSO concentration at which the enzyme retains 50 % of the original activity; 7EC, 7-ethoxycoumarin; 7-EFC, 7-ethoxy-4-trifluoromethylcoumarin; ER, oestrogen receptor; epPCR, error-prone PCR; MeIQ, 2-amino-3,5-dimethylimidazo[4,5f ]quinolone; SHIPREC, sequence homology-independent recombination; SRS, substrate-recognition site; StEP, staggered extension process; T 50 , temperature at which the enzyme retains 50 % of the original activity. 1 To whom correspondence should be addressed (email [email protected]). c The Authors Journal compilation c 2015 Biochemical Society

Biochemical Journal

Directed evolution of cytochrome P450 enzymes for biocatalysis: exploiting the catalytic versatility of enzymes with relaxed substrate specificity

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REVIEW ARTICLE

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J.B.Y.H. Behrendorff, W. Huang and E.M.J. Gillam

reviewed elsewhere [12]. The focus of the present review will be on P450s showing relaxed substrate specificity, such as those involved in the detoxification of environmental chemicals and certain novel bacterial forms. Substrate diversity of xenobiotic-metabolizing P450s

The P450s that mediate drug metabolism in the human liver show an exceptional degree of substrate ambiguity: between them, P450s mediate the metabolic clearance of almost three-quarters of drugs in clinical use. P450 3A forms taken together metabolize 46 %, P450 2C9 metabolize 16 %, P450 2D6 metabolize 12 %, P450 2C19 metabolize 12 %, P450 1A forms metabolize 9 %, and P450 2E1 and P450 2B6 each metabolize 2 % of these drugs [13]. An extreme example is P450 3A4, the drug substrates of which include an unparalleled variety of chemical scaffolds, sizes and physicochemical characteristics [14]. Many of the specific interactions identified between P450s and their substrates identified from crystal structures are between hydrophobic groups on the substrate and enzyme, as might be expected since a high proportion of P450 substrates are lipophilic; however, specific hydrogen-bonding and electrostatic interactions are also seen. Considerable effort has been invested over the last two decades or more in the identification of pharmacophores for the principal drug-metabolizing P450s [15–27] and many different methods have been developed for the in silico prediction of P450 substrate specificity and regioselectivity with more or less success (reviewed in [28–33]). The drug-metabolizing P450s, like other enzymes with relaxed substrate specificity, appear to show a high degree of conformational diversity, which allows the enzyme to accommodate different substrate structures by subtle relocations of secondary structural elements. This was most clearly demonstrated by comparison of structures of P450 3A4, P450 2D6 and P450 2B forms with various substrates and inhibitors bound [34–40] (Figure 1). Jung et al. [41] have speculated that a preponderance of hydrophobic residues in the vicinity of the haem means that it is principally weak van der Waals interactions between residues that are broken and re-formed in transitions between conformational states. An analysis of the interactions between residues in P450 1A2 shows that ∼65 % of inter-residue interactions within a cut-off distance of 4.5 Å (1 Å = 0.1 nm) occur between residues that are less than ten amino acids apart in the linear sequence, principally interactions stabilizing secondary-structural elements such as β-hairpins and α-helices (W. Huang and E.M.J. Gillam, unpublished work) (Figure 2). By contrast, approximately 25 % of interactions occur between amino acids more than 50 residues apart in the linear sequence [42]. The fact that Nature has selected the P450 fold for a multitude of physiological functions suggests that the P450 scaffold is robust in an evolutionary sense, i.e. relatively tolerant to mutations. This may be related to the ability of the P450 scaffold to fold effectively with only a limited number of conserved long-range stabilizing interactions. However, this premise has not yet been tested by any comprehensive bioinformatic analysis of P450 sequence– structure relationships. Practical problems with industrial application of existing enzymes

The major factors determining the economic viability of a biotechnological process are the costs of the biocatalyst, substrate and any required cofactor, balanced against the process yields. Not surprisingly then, the three principal targets for biocatalyst engineering are volumetric productivity or activity (i.e. enzyme c The Authors Journal compilation c 2015 Biochemical Society

Figure 1

Structural plasticity of the P450 fold

Crystal structures determined for P450 2B4 in the absence and presence of various ligands are superimposed. Ligand-free structures are shown for the open (pale blue, PDB code 1PO5 [142]) and closed (grey, PDB code 3MVR [143]) forms, plus structures obtained for the wild-type enzyme bound to the ligands indicated (PDB codes 1SUO [144], 2BDM [40], 2Q6N [145], 3G93 [146] and 3R1B [147]), superimposed on the closed form (grey). The ligand-free closed form contains the H226Y mutation, introduced to prevent complexation of the protein in the open form with His226 co-ordinated to the haem of a different protein chain (PDB code 3MVR). The haem moiety of the ligand-free structures is displayed as red sticks and ligands are shown as magenta spheres.

efficiency), biocatalyst stability and the production cost of biocatalyst [43]. Despite their catalytic versatility, P450s have yet to be widely exploited in biotechnology or synthetic biology in industrial settings. Like other mesophilic enzymes, they have limited stability and solvent tolerance. In a research context, it is satisfactory for an enzyme to operate for only a few hours at its optimal temperature (whether 37 ◦ C for mammals or nearer ambient temperatures for ectothermic animals), but process conditions typically require enzymes to be stable for extended times in order to maximize productivity per unit of expensive biocatalyst. Thus enhanced thermostability is desirable to enable the enzyme to remain catalytically active over longer incubations. It is advantageous to add substrate at high concentrations in order to maximize yields, meaning that substrate delivery frequently becomes limiting with lipophilic substrates added to enzymes operating in an aqueous environment. P450s carry the additional disadvantage of requiring a reducing cofactor and one or more ancillary redox partner proteins to undertake the full catalytic cycle. Commonly P450s have been used as peroxygenases with activity supported by hydrogen peroxide or an organic peroxide to address this issue and conditions have been found to minimize oxidative damage to the P450 due to co-incubation with peroxides [44,45]. Given the limitations to the use of naturally occurring enzymes for industrial applications such as in drug discovery and development, protein engineering can be explored as a means of improving P450s with regard to substrate specificity, turnover rates, thermostability, solvent tolerance, cofactor preference and expression in the desired host (Figure 3). This process can be envisaged as taking place in two phases: selection and evolution of a candidate enzyme with the desired substrate and reaction specificity, followed by optimization of that enzyme to suit the requirements of the biotechnological process. To date, published studies have mostly focused on the first phase; however, some attempts have been made to enhance the thermostability, solvent tolerance and cofactor requirements of selected P450s.

Exploiting the relaxed specificity of P450 enzymes for biocatalysis

Figure 2

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Intramolecular interactions in the P450 1A2 structure

The map of interactions between amino acid residues in the crystal structure of P450 1A2 (PDB code 2HI4) within a cut-off distance of 4.5 A˚. (A) The two-dimensional interaction map showing the interactions between two residues in the primary structure. Each point represents one interaction formed between two residues. Points close to the diagonal indicate that the two residues forming interactions are close in the linear sequence. (B) The distribution of distant interactions in the primary structure.

PROTEIN ENGINEERING TECHNIQUES: DIRECTED EVOLUTION VERSUS RATIONAL DESIGN

In recent years, there have been several notable successes in the computational design of entirely novel biocatalysts including retro-aldolase [46], Kemp eliminase [47] and Diels–Alderase [48] enzymes. However, de novo biocatalyst design is yet to supersede the strategy of mining and modifying pre-existing enzymes found in Nature. Insufficient information exists on how structure determines function in P450s to design active sites, and protein engineering still largely depends on first identifying enzymes that can perform some reaction similar to the desired activity (candidate enzymes), and then optimizing that enzyme by modifying it using rational, semi-rational or evolutionary approaches. Identifying the right enzyme or enzymes to be used as starting material is important, as successes in protein engineering have mostly come from making relatively minor changes to active sites and substrate-access channels in order to reposition the compound of interest. The inherent catalytic versatility of xenobiotic-metabolizing P450s increases the likelihood that an enzyme can be found that already possesses some activity similar to the desired reaction of interest. However, the availability of structural data is critical: even when random or other evolutionary strategies are used, structural data or models built from highly similar proteins are important for rationalizing effects of the selected mutations. Crystal structures have been obtained for many P450 enzymes, including most human forms with and without various ligands bound. Rational engineering of biocatalysts, where specific changes are made to targeted amino acids, is usually limited to enzyme templates that are well-characterized with regard to their structure–activity relationships. Preferably, a crystal structure should be available with an example of a ligand bound in the active site to aid in the prediction of mutations that can be usefully targeted to alter substrate specificity. However, in some instances, mutations known to be useful from studies of one enzyme can be transposed to homologous enzymes for which no structures are available. For example, P450 106A2, a steroid-

hydroxylating P450 from B. megaterium, was altered on the basis of previous rational engineering of P450 119 from Sulfolobus solfataricus [49]. Structure-based rational engineering of P450 119 revealed that a T214V mutation enhanced the binding kinetics toward its native substrate, lauric acid [50]. The analogous mutation of T248V also had an important catalytic role in P450 106A2, improving product specificity for 15β-hydroxylation of testosterone, enhancing the 15β-/16β-hydroxylation ratio from 4.2:1 to 12.9:1 [49]. For semi-rational engineering, where specific functional regions of the candidate enzyme, such as the active site cavity or cofactor-binding residues, are targeted by methods such as iterative and combinatorial saturation mutagenesis [51], at least some knowledge is required regarding the general role of different structural features. The key substrate-recognition sites (SRSs) generally occur in the same regions of the general P450 fold [52,53], which can allow findings from mutagenesis studies to be transferred between sufficiently similar enzymes. SRSs have been popular targets for semi-rational engineering via saturation mutagenesis [54–57], often with the aim of altering substrate and product specificity. Saturation mutagenesis of 25 residues within the SRSs of P450 1A2 produced mutants with dramatic changes in reaction kinetics toward the native substrates phenacetin and 7-ethoxyresorufin [57]. Mutants were produced with kcat /K m values up to 3–4-fold greater than the wild-type, and residues of particular importance to reaction kinetics could be identified. For example, all Phe226 mutants exhibited reduced activity, whereas activity was increased in Glu225 mutants [57]. In contrast with rational approaches where individual changes are planned and tested, directed evolution strategies mimic natural selection by coupling genetic diversification to a mechanism that screens or selects for desired phenotypes. Typically, no prior knowledge of enzyme structure or function is required, but some information may be acquired by analysis of the results. A typical directed evolution experiment begins with selection of the starting material (genes encoding enzymes with some property similar to the desired outcome), a genetic diversification step and identification of mutants with improved c The Authors Journal compilation c 2015 Biochemical Society

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Figure 3


Considerations in the selection and engineering of an enzyme for biocatalysis

The initial stage of biocatalyst development involves judicious selection of a candidate enzyme and followed by evolution to produce a lead biocatalyst with the desired substrate and reaction specificity that can be expressed in a suitable host. The second stage involves optimization of that enzyme to suit the requirements of the biotechnological process.

properties. This process may be repeated iteratively to evolve the desired outcome over many generations. The limiting factor in directed evolution is usually the throughput of the screening method used to identify improved mutants, so it is preferable to limit library size. This can be done by enhancing the proportion of functional mutants by increasing the fidelity of the mutagenesis step and by incorporating pre-existing knowledge on structure–function relationships to focus the mutations in specific regions. Homology-dependent recombination methods such as DNA shuffling [58] (Figure 4) greatly increased the speed of in vitro evolution experiments, as compared with error-prone PCR (epPCR), by allowing the combination of otherwise infrequent c The Authors Journal compilation c 2015 Biochemical Society

mutations from multiple templates [58]. P450s belonging to the same evolutionary branch share significant nucleotide sequence homology, yet often have unique functional properties, making them excellent starting material for DNA shuffling. This approach has been repeatedly demonstrated as a useful tool for the diversification of mammalian P450s [59–62]. Libraries of P450 mutants from the P450 3A and P450 1A subfamilies capable of accepting substrates not metabolized by the parental enzymes [61,62] and P450 3A-derived mutants with altered product specificity [62] have been created. Another recombinatorial method, the staggered extension process (StEP) (Figure 4), in which the cDNAs for the parental genes are used directly as PCR templates [63], was used


Figure 4

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Schematic representation of the principal methods used for directed evolution of P450s

Random mutagenesis by PCR in the presence of Mn2 + or using error-prone (ep) polymerases introduces mutations throughout the coding sequence resulting in amino acid substitutions throughout the encoded protein. By contrast, in saturation mutagenesis, specific sites are targeted with a full or reduced set of codon substitutions. In both cases, the resultant mutants typically contain relatively few changes from the parent. Recombinatorial methods such as DNA shuffling and StEP generate mutants that are mosaics of two or more related sequences. In DNA shuffling, homologous coding sequences are fragmented and then allowed to reassemble in a primer-less PCR. In StEP, recombination is achieved by template switching in successive cycles of a PCR in which the extension stage is curtailed so that sequences cannot be fully extended in a single cycle.

to recombine PCR-generated mutants of P450 BM3 with the aim of evolving hydroxylation activity toward C3 –C8 alkanes [64]. The most efficient mutant isolated after five rounds of StEP mutagenesis and screening was the fastest known alkane hydroxylase at that time. StEP has also been used to create libraries

of P450 2A6 mutants to examine sequence determinants of indole hydroxylation activities [55,65]. Methods have also been developed for shuffling nonhomologous DNA sequences {e.g. incremental truncation of hybrid enzymes (ITCHY) [66] and sequence homology-independent c The Authors Journal compilation c 2015 Biochemical Society

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recombination (SHIPREC) [67]}. Although many proteins with 5-fold enhancement in kcat towards the probe substrate 7-methoxyresorufin were obtained with three rounds of epPCR and activity screening [73]. Because the fluorogenic probe substrate 7-methoxyresorufin crosses the E. coli cell membrane, functional mutants could be screened in intact cells. A key common feature of these studies is the decision to use a first-pass screen that discards a large proportion of the library, permitting a detailed analysis of relatively few mutants that exhibit some property of interest. Although many mutants with other interesting properties may remain undiscovered, and alternative evolutionary pathways towards an optimal enzyme may be lost [102], this strategy is essential when using random mutagenesis. The substrate specificity of P450 2B enzymes has been addressed using both evolutionary and rational approaches with the aim of improving activation of the chemotherapy prodrugs cyclophosphamide and ifosfamide by P450s for use in targeted gene therapy [103–106]. An epPCR study of P450 2B1 using 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) as a model substrate revealed mutants with improved kinetic parameters for bioactivation of cyclophosphamide and ifosfamide [75]. The relevant mutations were transferred to P450 2B11, the P450 with the lowest known K m value for 4-hydroxylation of cyclophosphamide [107]. One such transferred mutation outside the active site, V183L, almost doubled the catalytic efficiency of P450 2B11 by further decreasing the K m value for both cyclophosphamide and ifosfamide. In a contrasting approach, several P450 2B models were compared in molecular dynamics simulations with cyclophosphamide in order to target mutations that could be introduced to P450 2B6 [106]. A double mutant containing two of the best single mutations, I114V and V477W, showed 4-fold improved catalytic efficiency over wild-type P450 2B6. Two important proof-of-concept studies demonstrated the utility of directed evolution for improving catalytic efficiency and stability in P450 2B1 [75,76]. To enhance the catalytic c The Authors Journal compilation c 2015 Biochemical Society

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efficiency of P450 2B1 supported by hydrogen peroxide, a previously identified mutant, which had a 2-fold greater kcat value for the model substrate 7-EFC [108], was used as a template for epPCR [75]. Two rounds of epPCR and screening using hydrogen peroxide as an alternative oxygen donor revealed a quadruple mutant (V183L/F202L/L209A/S334P) which had a 6-fold greater kcat value for 7-EFC than the initial L209A mutant. No significant improvements were observed in a third round of mutagenesis and screening, suggesting that an evolutionary plateau had been reached. This study demonstrated that kinetics could be improved quickly even when using epPCR with a very low mutation rate, but the relatively poor performance of the ‘best’ mutant when coupled to NADPH via CPR highlighted the importance of using a screening system that mimics the desired outcome as closely as possible. To demonstrate that human P450s can be evolved for greater stability, including under industrially relevant conditions such as increased temperature and organic solvent concentration, a single round of epPCR was performed on the peroxide-driven P450 2B1 quadruple mutant, and 3000 new mutants were screened [77]. Whereas most mutants showed large losses in activity, ∼5 % of mutants had + −40 % of parental enzyme activity. Rescreening of >150 individual mutants at the T 50 and [DMSO]50 (i.e. temperature and DMSO concentration at which the enzyme retains 50 % of the original activity) determined for the parental P450 revealed eight mutants with >2-fold improvement in reaction rates under those conditions [77]. Mutants showing enhanced activity at the T 50 and [DMSO]50 of the parental form would be expected to show greater thermostability and solvent tolerance than the parental P450. A similar approach was used to improve peroxide-mediated activity of P450 3A4. Mutants from an epPCR library showing greater peroxide-supported oxidation of 7-benzyloxyquinoline than native P450 3A4 [76], and which predominantly contained mutations outside of the active site, were used as templates for further semi-rational engineering. The active-site residue Thr309 was also mutated on the basis that a previously reported T309A mutation in P450 2D6 increased peroxide-mediated activity [109]. The best mutant produced in that study contained a randomly identified T433S mutation located outside the active site.

Studies using recombinatorial approaches

The above studies demonstrated that mammalian P450 enzymes can be evolved for improved properties in recombinant systems, but the frequency of improved mutants among libraries was low, necessitating a facile high-throughput screen. Recombination of homologous xenobiotic-metabolizing P450s from the same subfamily has been shown to be an effective means for creating smaller libraries with greater functional diversity. DNA family shuffling and in vivo recombination in yeast was first used to recombine P450s 1A1 and 1A2 [59]. The amino acid sequences for these enzymes are 74 % identical over their ORFs, and 86 % of sequences in the resulting library were recombinations of P450s 1A1 and 1A2, with both parental sequences represented in roughly equal proportions and frequent recombinations. Activity against naphthalene (a substrate metabolized by both P450 1A1 and 1A2) was reported in ∼20 % of mutants. Restriction enzyme-mediated DNA family shuffling was subsequently used to recombine the same sequences for expression in in E. coli [61]. An average of six recombination events were seen per mutant, and 23 % of mutants exhibited activity against at least one of the substrates tested in assays on intact cells. Several mutants showed broadened substrate c The Authors Journal compilation c 2015 Biochemical Society

ranges that exceeded those of the parents combined and up to 9fold greater activity for particular probe substrates. Furthermore, 53 % of mutants expressed detectable haemoprotein and were therefore potentially functional. A similar degree of structural and functional integrity was achieved when P450s 2C8, 2C9, 2C18 and 2C19 were shuffled [60]. A library of 110 shuffled mutants produced from P450s 3A4, 3A5, 3A7 and 3A9 was screened against a panel of 17 probe substrates. At least one activity was observed in 96 % of mutants, which is greater than the proportion of mutants that produced intact haemoprotein at levels greater than the detection limit of the whole-cell screening assay for P450 concentration [62]. This hints that a greater proportion of mutants from previous P450 libraries [59–61] may have been catalytically active, but that activities were not observed in the absence of a suitable substrate. Multifactorial traits such as expression in E. coli are amenable to improvement by directed evolution. The use of DNA shuffling to examine sequence elements affecting P450 expression [110] highlights the broader applicability of the evolutionary approach to optimizing other traits of industrial relevance, while at the same time providing useful basic information concerning the basis to those traits.

EXPLOITING THE MICROBIAL P450 METABOLOME FOR BIOCATALYSIS

Recently, there has been renewed interest in exploiting and engineering microbial P450 enzymes beyond the heavily studied P450 BM3. The advantages of evolving bacterial P450s for biocatalysis include superior solubility in microbial hosts such as E. coli and a greater range of options with regard to redox partners. Importantly, whereas the ‘classic’ bacterial enzymes P450cam and P450 BM3 are considered to be efficient specialists, catalytically promiscuous bacterial P450s have recently been discovered, particularly in microbes that produce a variety of secondary metabolites. Moreover, there exist in Nature numerous fusion proteins analogous to P450 BM3 which may equal or surpass the catalytic efficiency of this prototypical form. To date, only limited efforts have been made to exploit these catalytically self-sufficient mono-oxygenases. However, initial results suggest that P450s may be found with superior stability and useful specificity that can be further optimized by protein engineering [111], e.g. P450 505X from Aspergillus fumigatus showed superior solvent tolerance to that of P450 BM3, as well as the potential for high turnover rates as indicated by activity towards lauric acid, suggesting that this form may be especially useful for further development. Similarly, P450 OleTJE, a P450 152 form from Jeotgalicoccus sp. ATCC 8456 [112], which converts fatty acids into terminal olefins, could be expressed in E. coli in M9 minimal medium, was functional without coexpression of a reductase partner and was highly active in vitro in the presence of hydrogen peroxide. Caffeic acid is a highly versatile pharmacophore from which a range of biologically active antioxidant, anti-inflammatory, anti-cancer and anti-retroviral compounds can be synthesized (reviewed in [113]). Caffeic acid can be produced by P450 199A2 from Rhodopseudomonas palustris by hydroxylation of the relatively cheap and commercially available p-coumaric acid [114] (Figure 4). Mutation of Phe185 (positioned directly above the haem in the crystal structure) to leucine increased p-coumaric acid hydroxylase activity 5-fold. Using whole-cell biocatalysis in a 50 ml culture, 15 mM caffeic acid was produced from a starting concentration of 20 mM p-coumaric acid [114]. Production of p-coumaric acid has also been engineered into


E. coli [115]. Tyrosine was converted directly into p-coumaric acid by phenylalanine/tyrosine ammonia lyase, although this appeared to be less effective than synthesis via phenylalanine, which was accomplished by adding P450 73A from Helianthus tuberosus to the system along with the corresponding CPR [115]. Potentially, P450 199A2 could be added to this pathway for the total biosynthesis of caffeic acid in E. coli (Figure 5). Catalytically promiscuous P450s from actinomycetes are receiving increased attention for their broad substrate specificity and potential role in producing an impressive array of secondary metabolites. Vitamin D3 hydroxylation is not a native activity of actinomycetes, but P450 107 of Pseudonocardia autotrophica [116] converted vitamin D3 into 1α,25-dihydroxyvitamin D3 via two hydroxylation reactions [117] (Figure 5). Screening of an initial epPCR library revealed eight regions of interest at which saturation mutagenesis produced a quadruple mutant with a 6fold improvement in specific vitamin D3 hydroxylase activity compared with the wild-type enzyme [117]. The evolved mutant appeared to adopt a more closed conformation than the wild-type, and the synergistic effects of all four mutations were necessary for enhanced activity [118]. P450 105 from Nonomuraea reticatena is a catalytically promiscuous fatty acid hydroxylase [119] that also metabolizes steroids [120] and aromatic compounds [121]. Following epPCR of the native P450 105 sequence [121], mutants were screened for enhanced activity in a two-tiered fashion using 7-ethoxycoumarin (7EC), diclofenac and naringenin. Initially, 800 mutants were screened in a high-throughput format against 7EC from which 11 unique mutants were found with enhanced activity. The cumulative effects of individual mutations in these mutants were examined by combining mutations and testing activity against 7EC, diclofenac and naringenin (Figure 5). A mutant with five substitutions demonstrated the greatest rate of product turnover against all three substrates, with improvements of 3–5-fold over the activity of the wild-type P450 105. Importantly, this study demonstrated that P450 105 can be evolved for enhanced reaction kinetics without loss of catalytic promiscuity.

A CASE IN POINT: APPLICATION OF ENGINEERED P450S IN DRUG DISCOVERY AND DEVELOPMENT

Given the difficulty in finding valid high-throughput screens for specific applications, the trend has been towards using small libraries of mutants (one or two 96-well plates), that show robust expression and diverse function. Structure-guided evolution strategies can now yield libraries enriched in properly folded, potentially functional mutants that can be ‘typed’ for activity. Typically, useful mutants have also been ‘cherry-picked’ from prior studies for more intensive screening against substrates of industrial importance. In one study [122], 12 of the 13 known mammalian metabolites of two drugs and a research compound were formed by at least one member of a 120-member P450 102derived library, a subset of larger libraries developed using the SCHEMA approach as well as from earlier random and targeted mutagenesis experiments. New metabolites, not previously characterized from biological extracts, were also identified. Importantly, the majority of metabolites could be isolated from preparative scale incubations without further optimization of the enzyme or biocatalytic system [122], suggesting that it may not be necessary to invest further effort in the directed evolution of substrate specificity; a ‘smart’ multifunctional library with sufficient diversity may be satisfactory for many pharmaceutical applications. The library of P450s marketed by Codexis is an example of just such a ‘smart’ small library pre-screened

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to contain mutants of most interest and represents the first commercial application of evolved P450s in drug discovery and development [123]. The ‘lowest-hanging fruit’ for implementation of P450s in drug development pipelines is in the production of authentic metabolites from existing leads. The identification of minor metabolites in drug development is especially challenging as their low concentrations in vivo, and therefore in biological extracts, can hinder identification. Native human P450s expressed in various host organisms have been used previously [124– 126], but metabolite yields are typically lower than ideal for structural characterization [127]. Moreover, native enzymes produce metabolites in the same ratios as in vivo, confounding the isolation of minor metabolites in satisfactory yields. However, directed evolution can be used to tune the biocatalyst towards one site of metabolism, improving yields of the metabolite of interest relative to other products. Using testosterone as a model substrate, a P450 3A library was screened for production of the 1β-hydroxytestosterone, a minor metabolite of P450 3A4 [62]. A mutant was identified which catalysed predominantly 1β-hydroxylation rather than 6β-hydroxylation of testosterone. Following a single preparative-scale incubation, 0.3 mg of 1β-hydroxytestosterone was collected, which was sufficient for ab initio determination of the structure via NMR (Figure 5). Weis et al. [111] compared a panel of highly expressed microbial P450-reductase fusion proteins for activity towards a number of drug substrates. Mutations were introduced at positions that aligned with ‘hotspots’ found to influence activity in P450 BM3. Preparative-scale incubations with 2 nmol of the best mutants yielded milligram quantities of the major metabolites of chlorzoxazone and diclofenac (Figure 5) in 12 % and 8 % yield respectively (with product conversion of 44 % and 15 % respectively), without optimization of the production conditions. The exploration of structure–activity relationships requires the regio- and stereo-selective modification of lead compounds, an especially challenging task with structurally complex chemicals such as those derived from many natural products. For selective modification of metabolically labile positions on the structure of artemisinin (Figure 5), a P450 102 variant with broad substrate specificity was engineered to develop catalysts capable of selectively hydroxylating particular positions of the artemisinin scaffold [128]. Site-saturation mutagenesis of seven residues in the predicted substrate-binding site allowed prioritization of a set of 522 mutants with unique and useful metabolic profiles towards a battery of probe substrates. A subset of 20 P450s from this group was characterized for activity towards artemisinin, and used as a training set to model the correlation between metabolic profiling data and activity towards artemisinin. Of the mutants from the larger library predicted to have activity towards artemisinin, 78 % were confirmed, a success rate that was markedly higher than that expected by chance (20 %). Mutants were obtained that could functionalize different parts of the artemisinin structure providing access to preparative scale amounts of useful compounds for further derivatization, resulting ultimately in fluorinated analogues in which the metabolically labile position was stabilized [128]. Pharmacological screens represent a means by which to solve the ‘numbers problem’ when catalysts are sought for improvement of pharmacodynamic properties. Conceivably, extracts from incubations of a lead compound with a targeted library of P450s could be assayed directly for the desired pharmacological activity. In a pioneering study, the generation of oestrogenic metabolites of zearalenone by P450 102 mutants was coupled to the inline detection of affinity for the oestrogen receptor (ER) [94]. c The Authors Journal compilation c 2015 Biochemical Society

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Figure 5


Biotransformation reactions that have been the target of P450 engineering

Reactions shown are catalysed by P450 enzymes unless indicated otherwise.

Metabolites produced in incubations were analysed by HPLC and mixed in line with a fluorogenic ERα ligand, coumestrol, and the receptor for a fluorimetric competitive binding assay. Many privileged scaffolds have been identified from natural products, and P450s are commonly involved in the pathways of secondary metabolism required for their biosynthesis, so it stands to reason that P450s can be exploited to produce or functionalize useful chemical scaffolds. An example is the stereoselective functionalization of N-benzylpyrrolidine, an important intermediate for the synthesis of a number of different types of drugs. P450pyr (P450 153A7) from Sphingomonas sp. HXN-200 was evolved by an iterative site-saturation approach c The Authors Journal compilation c 2015 Biochemical Society

to generate mutants that could produce either the (S)- or (R)-enantiomers of N-benzyl-3-hydroxypyrrolidine from the prochiral precursor N-benzylpyrrolidine [93] (Figure 5). P450AurH, a P450 151A form from Streptomyces thioluteus, catalyses formation of the tetrahydrofuran moiety in the biosynthesis of the antifungal and anti-proliferative compound aureothin. The active site of P450AurH was engineered so as to shift the regioselectivity from the C7 position to the neighbouring C9a position, in so doing enabling the mutant to catalyse the sequential oxidation of the methyl to an acid moiety [129]. Subsequently, a methyltransferase with altered regiospecificity was substituted for the normal homologue in the


aureothin pathway to produce a non-natural γ -pyrone substrate for P450AurH [130], leading to a final product with altered backbone structure. Subsequent oxidation of the γ -pyrone by P450AurH initiated the formation of an aureothin analogue showing a significantly altered backbone structure. Although the approach used here was rational, rather than directed, evolution, this work illustrates the potential for engineering natural biosynthetic pathways so as to generate analogues of secondary metabolites that may be useful alternative scaffolds for drug discovery. Recently, P450 BM3 102 was engineered to catalyse carbene transfer to effect the cyclopropanation of double bonds, a reaction that is outside the normal repertoire of P450s [131]. A single mutation of Thr268 to alanine was shown to promote carbene transfer to double bonds using a diazoester co-substrate, and the product enantioselectivity could be further fine-tuned by additional mutations at the active site. Mutating the proximal cysteine ligand to serine or histidine enhanced activity [132,133]. The same general approach revealed effective catalysts of other reactions that have no known biological precedent, namely intramolecular C–H bond aminations in azide substrates [134], intermolecular nitrene transfer to thioethers to form sulfimides [135] and the insertion of carbenoids into N–H bonds [136]. These studies highlight further the potential for P450s in the production of useful pharmaceutical scaffolds, by extending the range of chemistries that can be used for selective functionalization reactions [131]. P450s have also been used for functionalization reactions in chemoenzymatic approaches [137]. A library of P450 102 mutants previously identified from other studies to have good turnover rates and to be more or less selective towards various substrates was screened for the ability to selectively hydroxylate a number of cyclopentenone-containing substrates [137]. This scaffold is present in a number of different classes of natural products and difficult to functionalize. The most active and selective mutants were used in preparative incubations from which products were isolated that were hydroxylated at various positions on the structure. Chemical fluorination of the mono-oxygenated products then afforded analogues of the starting substrates that were fluorinated at specific positions at high yields inaccessible by purely chemical methods. The approach was validated further on other poorly reactive sites and structures with a variety of substituents of different sizes. Multiple fluorination steps were possible and fluorine could be substituted for alkoxy groups by exploiting the dealkylase activity of P450s to reveal a free hydroxy group [137]. The versatility of the chemoenzymatic strategy was further proven in a study on monosaccharide elaboration in which P450s were used to selectively deprotect various sites on monosaccharides [138]. This proved the principle that P450s can be incorporated into medicinal chemistry as agents to not only selectively functionalize chemicals, but also provide selectivity in deprotection steps in organic syntheses.

CONCLUSIONS AND PERSPECTIVES

P450 enzymes have been repeatedly demonstrated to be amenable to functional diversification by directed evolution. However, except for a few studies where P450s have been evolved over several generations with a specific desired outcome in mind, they remain an under-utilized resource for the directed evolution as biocatalysts. The current trend is to use smarter protein engineering and evolution strategies to create small libraries enriched in functionally diverse and robust biocatalysts [128], meaning

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that screening for particular activities of interest with lowthroughput chromatography-based methods becomes feasible. However, industrially relevant screening methodologies that can be applied to multiple different systems will be required to take full advantage of the catalytic power of P450s. To date, limited efforts have been made to analyse or address volume–time output with engineered P450s. Substrate and product concentrations of 100–250 mM are typical in industrially viable processes [139], but usually impossible to achieve with the limited solubility of P450 substrates. Biphasic systems for facilitating substrate supply and product recovery for P450-mediated reactions [140,141] and the engineering of solvent tolerance into P450s will be critical in setting up such systems. Protein engineering may also be needed to relieve product/substrate inhibition of P450s developed for large-scale chemical production [139] and to improve catalytic efficiency, whether using redox partners or oxygen surrogates. An important criterion determining the economic viability of a biocatalytic process is the cost of the biocatalyst. To date, xenobiotic-metabolizing P450s have only been expressed in E. coli in quantities sufficient for biochemical studies using extremely rich complex medium. Low and variable haemoprotein yields due to misfolding represents a considerable waste of carbon and nitrogen in an industrial context. It is highly unlikely that the perfect convergence of protein stability, correct folding, and catalytic specificity and efficiency can be found in every desired biocatalyst. Therefore, once the engineered biocatalyst fulfils its basic functional requirements, further engineering efforts should be rationalized according to the intended application. If the aim is to produce a biocatalyst for in vitro use, it may be most useful to enhance stability and select for the ability to use peroxides, whereas, for whole-cell biocatalysis, greater emphasis should be placed on improving catalytic rate and efficiency so that acceptable levels of activity can be obtained with moderate expression levels in a minimal medium. In summary, although significant challenges remain to be addressed before P450s can be fully exploited in the chemical industry, the considerable potential of these remarkable biological catalysts is steadily being harnessed by the use of directed evolution to improve and extend their properties. The next decade of P450 engineering should see a transition from platform development to application.

ACKNOWLEDGEMENTS We thank Martin A. Hayes for helpful comments concerning the industrial application of P450s.

FUNDING This work was supported by an Australian Postgraduate Award (to J.B.Y.H.B.) and International Postgraduate Research Award (to W.H.) from the Australian Federal Government.

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Received 9 December 2014/5 January 2015; accepted 6 January 2015 Published on the Internet 20 March 2015, doi:10.1042/BJ20141493

c The Authors Journal compilation c 2015 Biochemical Society

Analysis of the catalytic specificity of cytochrome P450 enzymes through site-directed mutagenesis.

Characterization of human cytochrome P450 enzymes.

Alteration of the substrate specificity of cytochrome P450 CYP199A2 by site-directed mutagenesis.

Speculations on the substrate structure-activity relationship (SSAR) of cytochrome P450 enzymes.

CADEE: Computer-Aided Directed Evolution of Enzymes.

Drug metabolism by cytochrome p450 enzymes: what distinguishes the pathways leading to substrate hydroxylation over desaturation?

Cytochrome P450-Mediated Phytoremediation using Transgenic Plants: A Need for Engineered Cytochrome P450 Enzymes.

Versatile biocatalysis of fungal cytochrome P450 monooxygenases.

Genetics, epigenetics, and regulation of drug-metabolizing cytochrome p450 enzymes.

Effects of Alismatis rhizome on rat cytochrome P450 enzymes.

Expression of mammalian cytochrome P450 enzymes using yeast-based vectors.

Effects of aescin on cytochrome P450 enzymes in rats.

Directed evolution of the substrate specificity of dialkylglycine decarboxylase.

Directed evolution of the substrate specificity of biotin ligase.

Is there more to learn about cytochrome P450 enzymes?

Cytochrome P450 reductase, antioxidant enzymes and cellular resistance to doxorubicin.

Genetic differences in cytochrome P450 enzymes and antidepressant treatment response.

Computational method for the design of enzymes with altered substrate specificity.

Elucidation of catalytic specificities of human cytochrome P450 and glutathione S-transferase enzymes and relevance to molecular epidemiology.

Development of NanoART for HIV Treatment: Minding the Cytochrome P450 (CYP) Enzymes.

High daily dose and being a substrate of cytochrome P450 enzymes are two important predictors of drug-induced liver injury.

An inducible NADPH-cytochrome P450 reductase from Picrorhiza kurrooa - an imperative redox partner of cytochrome P450 enzymes.

The usefulness of genotyping cytochrome P450 enzymes in the treatment of depression.

Effects of avermectin on microsomal cytochrome P450 enzymes in the liver and kidneys of pigeons.