Extending the capabilities of nature’s most versatile catalysts: Directed evolution of mammalian xenobiotic-metabolizing P450s

ABB Archives of Biochemistry and Biophysics 464 (2007) 176–186 www.elsevier.com/locate/yabbi

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Extending the capabilities of nature’s most versatile catalysts: Directed evolution of mammalian xenobiotic-metabolizing P450s Elizabeth M.J. Gillam

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School of Biomedical Sciences, The University of Queensland, St. Lucia, Brisbane 4072, Australia Received 19 April 2007 Available online 15 May 2007

Abstract Cytochrome P450 enzymes are amongst the most versatile enzymatic catalysts known. The ability to introduce a single atom of oxygen into an organic substrate has led to the diversification and exploitation of these enzymes throughout nature. Nowhere is this versatility more apparent than in the mammalian liver, where P450 monooxygenases catalyze the metabolic clearance of innumerate drugs and other environmental chemicals. In addition to the aromatic and aliphatic hydroxylations, N- and O-dealkylations, and heteroatom oxidations that are common in drug metabolism, many more unusual reactions catalyzed by P450s have been discovered, including reductions, group transfers and other biotransformations not typically associated with monooxygenases. A research area that shows great potential for development over the next few decades is the directed evolution of P450s as biocatalysts. Mammalian xenobiotic-metabolizing P450s are especially well suited to such protein engineering due to their ability to interact with relatively wide ranges of substrates with marked differences in structure and physicochemical properties. Typical characteristics, such as the low turnover rates and poor coupling seen during the metabolism of xenobiotics, as well as the enzyme specificity towards particular substrates and reactions, can be improved by directed evolution. This mini-review will cover the fundamental enabling technologies required to successfully engineer P450s, examine the work done to date on the directed evolution of mammalian forms, and provide a perspective on what will be required for the successful implementation of engineered enzymes. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Cytochrome P450; Directed evolution; Biocatalysis; DNA shuffling; Indole

Cytochrome P450 enzymes perform essential functions in an unequalled range of organisms by virtue of the powerful chemistry that can be catalyzed by the iron-oxo (compound I) and other activated oxygen species formed at the P450 active site [1]. These enzymes have diversified in all branches of the evolutionary tree, from bacteria and fungi to plants and higher animals [2]. They are essential for the biosynthesis of sterols and structurally related signalling molecules such as eicosanoids [3], but also for the generation of structurally dissimilar defensive allelochemicals, pigments and growth regulators such as plant auxins [4,5]. On the other hand, P450s have been exploited for the utilization of carbon sources by microorganisms [4], as well as the catabolism of environmental and dietary *

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chemicals in higher organisms, a role that has been most intensively studied in humans and mammalian models of human drug metabolism. While the typical reaction catalyzed by P450s is a monooxygenation, ever more unusual reactions catalyzed by P450s are being discovered, including reductions, dehalogenations, group transfers, and ring formation, coupling and contraction [6]. Nowhere is the catalytic versatility of P450s more apparent than in the mammalian liver, where P450s catalyze the metabolic clearance of innumerate drugs and environmental chemicals [7]. This volume celebrates the achievements of Professor F.P. Guengerich and several of the most important human P450s were first isolated and characterized in the Guengerich laboratory, including P450s 1A2, 2A6, 2C8, 2C9, 2D6 and 3A4 [8–12]. Since then, a formidable number of studies published by, or in collaboration with, the Guengerich laboratory have provided essential

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information for the understanding of human drug metabolism and bioactivation. One research area that shows considerable potential for development over the next few decades is the directed in vitro evolution of P450s as biocatalysts. Mammalian xenobiotic-metabolizing P450s are especially well suited to protein engineering due to their ability to interact with relatively wide ranges of substrates possessing marked differences in structure and physicochemical properties. They represent a starting point from which substrate specificity, regioselectivity, catalytic efficiency and other desirable properties can be optimized to derive industrially useful and economically viable biocatalysts. While certain characteristics of mammalian P450s are decidedly non-ideal for biotechnological application (such as the relatively low turnover rates, high degree of uncoupling and lack of stability over lengthy incubations), these limitations can be addressed by directed evolution. Directed evolution is an iterative, semi-random method of protein engineering. Importantly, the success of the technique does not rely upon a good understanding of the features to be engineered, in contrast to traditional approaches involving rational engineering such as by sitedirected mutagenesis [13,91]. Genetic diversity is introduced into a population of genes, and the resultant library is screened or subjected to a selection in order to identify mutants with the desired activity or property. These selected mutants are then subjected to further rounds of mutagenesis and screening/selection in order to further enhance the desired characteristic until a useful biocatalyst is obtained. Enzymes have evolved naturally to fill a particular physiological niche rather than to fit an industrial process. Taking them out of their natural environment frees them from the requirement to conform to certain conditions, e.g., the need to respond to specific concentration ranges of substrates, or in the example of xenobiotic-metabolizing enzymes, to deal with many chemicals. By directed evolution, enzymes can be optimized to particular and often unnatural substrate/reaction/cofactor combinations, and their physicochemical properties, for example thermostability and solvent tolerance, can be engineered to fit the requirements of the biotechnological process. Also, side activities or other steps in a pathway that decrease product yield can be eliminated, for example by expression in a different host [14]. This mini-review will review the fundamental enabling technologies required to successfully engineer P450s, examine the work done to date on the directed evolution of mammalian forms, and provide a perspective on what will be required for the successful biotechnological implementation of engineered enzymes. Fundamental requirements There are three fundamental requirements of a successful directed evolution strategy: first an efficient, economical

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and technically straightforward expression system; second a means to generate diversity; and third, appropriate, sensitive screens for the desired activity or property. For each of these aspects, a means by which to manage large numbers of samples is required. Importantly, however, it is generally not necessary (or indeed feasible) to screen entire libraries in order to find forms with enhanced activity. A review of the literature suggested only a small proportion of each library is typically screened in any given directed evolution experiment [15]. In the case of libraries made from mammalian P450s, this has been of the order of a few hundred [16] to 12,000 [17]. Thus, medium to high throughput methods are satisfactory for most current studies to find mutants with improved activities. Fundamental, enabling, expression technologies Initial work on the heterologous expression of mammalian P450s resulted principally from the need to address problems with sourcing and working with native enzymes to study human drug metabolism. Preclinical in vitro drug metabolism studies are essential to understanding the metabolism of new drugs, specifically to anticipate adverse drug interactions and to detect possible problems with candidates before use in clinical trials. Four classes of host have been used for heterologous expression of mammalian P450s: mammalian cells, yeast, baculovirus and bacteria. While mammalian cells and baculovirus systems offer some advantages for in vitro drug development, they are technically more difficult and more expensive than yeast and bacteria, and hence not amenable to generating and screening large libraries of mutants. Heterologous expression in yeast Heterologous expression in yeast provides moderate amounts of P450, sufficient to measure characteristic COreduced versus reduced difference spectra in microsomes [18]. Successful expression of mammalian P450s in yeast can be achieved without significant sequence modifications, and yeast express an endogenous reductase that can couple to recombinant P450s. However, this interaction may not always be optimal, and coexpression of alternative redox partners can enhance catalytic activities [19,20]. Yeast can be transformed with up to five different mutant cDNAs at once, and such libraries can be screened directly, allowing proportionally more of the library to be investigated than would be suggested by the number of clones tested. However to analyse single clones independently, or positively identify mutants with enhanced activity, it may be necessary to segregate different mutants in some other host such as Escherichia coli [21]. Heterologous expression in bacteria Bacterial expression is a robust and cost-effective means of generating sufficient amounts of enzyme for traditional studies of drug metabolism, and has enabled the crystallization and structure determination of several mammalian

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P450s [22–32]. Bacteria are also useful for creating and characterizing libraries due to the ease with which they can be cultured and manipulated. While modification of N-terminal sequences or codon optimization throughout the open-reading frame may be necessary to achieve high levels of hemoprotein expression (a disadvantage when using recombinant enzymes to model human drug metabolism), this requirement presents few if any problems when engineering biocatalysts. Most P450 expression studies have used E. coli and lac-based induction/repression systems [33]; however, some groups have explored the use of other enterobacteria such as Acinetobacter [34]. Typically the recombinant protein is directed to the bacterial inner membrane but high levels of P450 1A1 expression have been reported using a system for directing protein to the periplasmic space [35]. The means to generate diversity All directed evolution experiments require a source of genetic diversity from which libraries of variant forms are generated, then screened for a desired property or activity. The genetic diversity may be introduced by random or semi-random mutagenesis, such as by error-prone PCR or saturation mutagenesis at specific codons. A process of ‘‘rational evolution’’ [36] is often used, whereby specific residues or regions previously shown to be important in determining substrate specificity are targeted for further mutagenesis. Alternatively a pool of existing site-directed mutants shown to have enhanced activity or a set of naturally diverse ‘‘parental’’ sequences may provide the genetic variation. Recombinatorial strategies such as DNA shuffling [37] are commonly used to accelerate the search for the best biocatalyst since they allow rapid identification of the optimal combination of beneficial mutations. Random and saturation mutagenesis Various strategies have been used for random mutagenesis, principal amongst which are error-prone PCR and saturation mutagenesis of specific sites. In the former, the natural error rate of lower fidelity DNA polymerases such as Taq and ‘‘Mutazyme’’ is enhanced by addition of Mn2+ or altering the relative concentrations of pyrimidine deoxynucleotide triphosphates [38]. In practice there is usually some degree of mutational bias, meaning certain transversions are underrepresented [39] and a smaller range of amino acid changes are seen than are theoretically possible [40]. In saturation mutagenesis of specific regions, oligonucleotides with programmed degeneracy at chosen positions are used to randomize particular codons [41]. Alternatively sequence saturation mutagenesis (SeSaM)1, a PCR method 1 Abbreviations used: SeSaM, sequence saturation mutagenesis; SiSDC, site-directed chimeragenesis; StEP, staggered extension process; MROD, methoxyresorufin O-demethylation; SRSs, substrate recognition sequences.

in which the ‘‘universal base’’, deoxyinosine, is added to the ends of random or specific fragments of genes, can be used to introduce mutations through a wider range of the sequence [42]. In each of these methods, completely new areas of sequence space can be explored: mutations can be introduced that are not found in any natural form of the enzyme, and, in theory, any of the 20 natural amino acids can be introduced at any particular position. However, random amino acid changes may compromise the activity or structural integrity of the enzyme. Strategic design of degenerate oligonucleotides used for saturation mutagenesis can reduce the probability of introducing premature stop codons, usually at the cost of mutational diversity, but other destabilizing mutations are still possible. Low error rates, while preserving function, yield few unique clones [43]. High error rates generate disproportionately more unique clones and greater diversity but fewer clones retain function. Thus it is necessary to trade-off functional integrity against mutant diversity by adjusting the mutation rate. Typically protocols are optimized to limit the error rate so that only one or two base changes are introduced per enzyme sequence. However with such small changes to the target protein, improvements in activity are usually only modest. This means that the process of evolving specialized biocatalysts is a lengthy and linear one, involving many rounds of mutation and selection to get a substantial improvement in properties [13]. Also, screening methods must be sensitive to detect incremental improvements in activity. This, coupled with the linearity of the process can mean promising lines of development are abandoned prematurely if deleterious mutations occur. Recombinatorial techniques Recombinatorial techniques involve splicing together segments of different genes. The most common technique used here is DNA shuffling (Fig. 1), in which mosaic structures are formed by subjecting a number of ‘‘parental’’ DNA sequences to fragmentation, allowing them to reassemble at random in a primerless PCR, and then amplifying the full-length sequences. The resultant library is cloned into an appropriate expression vector for screening and selection. In the original form of DNA shuffling [37], parental forms were either available site-directed mutants of the same form or were generated by random mutagenesis on a single parent. Thus, the progeny contained only a limited number of changes from each parent, but still typically more than would be achieved by a further round of random mutagenesis. In an alternative strategy, DNA family shuffling [44], evolutionarily related forms drawn from nature are used as the parental sequences. These may be related members of protein families or orthologous forms of a given protein cloned from different species. Since such parental forms usually contain many differences from each other, and the number of recombination sites is theoretically very high, mutants with a significant number of mutations compared to each parent are common in each round

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Fig. 1. Directed evolution by iterative DNA shuffling. A set of homologous genes is subjected to random or semi-random fragmentation by nucleases then allowed to hybridize and reassemble in a primerless PCR. Full-length mosaic sequences are amplified in a conventional PCR step using primers flanking the open-reading frame then cloned into an appropriate vector for heterologous expression in an appropriate host. The resultant library of clones is screened for the desired activity or property and improved mutants are used for subsequent iterative rounds of shuffling and screening until a mutant with the desired characteristics is obtained.

of shuffling. Thus, shuffled libraries can be very diverse. In practice, around 5–10 recombinations are usually seen within a P450 open-reading frame [21,45,46]. In vivo recombination in yeast can be used as an extra source of genetic diversity as well as a facile cloning step, a characteristic exploited to great advantage by the CLERY (Combinatorial Libraries Enhanced by Recombination in Yeast) technique [21], where ‘‘value is added’’ to a typical DNA family shuffling experiment by subsequent recombination of the mutant open-reading frames with the expression vector in the yeast cell. DNA family shuffling involves recombination between different parts of homologous structures from the same structural superfamily. With this approach, important structural elements are more often conserved and the mutations that are introduced are generally those found in at least one orthologous, parental form, and have been retained by the evolutionary process. Typically then, they will not have a frank destabilizing effect on the general fold of the protein. Other chimeragenic or recombinatorial approaches carry most of the same advantages as DNA shuffling, but differ in their technical feasibility and suitability to sequences of greater or lesser diversity. In the staggered extension process (StEP) approach [47], PCR cycling conditions are adjusted to promote limited extension of primers in each cycle, facilitating template switching between cycles. This achieves the same overall effect as a reassembly

of randomized fragments, in that mosaic structures are formed between a numbers of parents. SHIPREC (Sequence Homology Independent Protein Recombination) [48], RACHITT (Random Chimeragenesis on Transient Templates) [49] and ITCHY (Incremental Truncation for the Creation of Hybrid Enzymes) [50] facilitate recombinations between less closely related parents, and usually with limited number of recombination sites. Chimeragenesis between distantly related sequences can be problematic in that interactions between residues that are essential for maintaining structural or functional integrity of the protein may be disrupted. Evolutionarily divergent forms may not share many of the same intra-protein interactions. Arnold and colleagues have addressed this issue and developed SCHEMA, an algorithm for improving recombinatorial strategies by minimizing the number of residue contacts that are broken when chimeric or mosaic proteins are constructed [51]. In combination with sequence-independent site-directed chimeragenesis (SiSDC) [52], this should facilitate rational design of chimeras to maximize sequence diversity without compromizing structural integrity in even distantly related orthologues. High throughput screens for the desired activity or property A fundamental principle underpinning directed evolution is that ‘‘you get what you screen for’’ [91]. Thus, careful attention must be paid to the choice of screening

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technique, and the end application will ultimately determine the screening method to be used. Most studies performed to date on mammalian xenobiotic-metabolizing P450s have focussed on assessing changes in activity. However other parameters can be (and frequently need to be) assessed to obtain full information on the basis to improvements in activity. These include relative P450 hemoprotein expression levels and coupling to reductase. For simplicity and speed, an ideal library screening method should work with intact cells. Kumar et al. make the point that whole cell screens are also more likely to reveal mutants with satisfactory expression and stability [53]. Total P450 levels have been detected in whole bacterial cells in high throughput or medium throughput mode using a straightforward dithionite/CO treatment of bacteria in microtitre plates [54]. This was originally developed for the assessment of libraries created from bacterial P450s, for which very high levels of expression can be achieved. However with some modifications [55], the method can be reliably applied to mutant libraries created from mammalian P450s expressed at much lower levels (Fig. 2). Direct measurement of P450 levels in whole yeast cells has not been reported to date to our knowledge. The method should be directly applicable to yeast cultures, but the generally lower levels of P450 expression in yeast may make sensitivity an issue. CO and dithionite are small, readily diffusible molecules which can pass easily through the cell. However the bacterial or yeast cell wall may present a more significant barrier to the passage of larger molecules such as typical P450 substrates [56]. Nonetheless, there are a number of existing whole cell screens used for libraries generated from bacterial or mammalian P450s. Several fluorescent assays typically used with microsomes have been adapted to whole bacterial cells: 7-methoxyresorufin O-demethylation (MROD) and coumarin 7-hydroxylation were assessed in P450 1A2 and

Fig. 2. High throughput measurement of whole cell P450 spectra. Escherichia coli coexpressing P450 2A mutants or P450 2A6 with recombinant human reductase or expressing reductase alone were cultured in microplates for 55 h under conditions facilitating P450 expression. Fe(II)ÆCO vs. Fe(II) P450 difference spectra were recorded on resuspended, washed cells in a 96 well microplate [54,55]. Triplicate measurements from each mutant and control are shown.

2A6 mutant libraries by a high throughput endpoint method employing a NaOH/NaCl quench step [57,58]. We have omitted the quench step for MROD and other alkoxyresorufin dealkylase measurements in a continuous assay [46]. When screening is undertaken with peroxide as the oxygen donor, naphthalene and potentially other aromatic compounds can be converted to hydroxylated derivatives that can be oxidatively coupled by coexpressed horseradish peroxidase to generate fluorescent derivatives [59]. This method can be used on bacterial colonies on solid media allowing screening of relatively large numbers of mutants. 7-Ethoxy-4-trifluoromethylcoumarin and 7-benzyloxyquinoline have been used to measure peroxide-supported activity in permeabilized whole cells [53,60]. Colorimetric assays are often the most straightforward assays and can in some cases be adapted to detection of colonies on plates. Naphthalene hydroxylation has been detected by a diazo-coupling method using solid phase extraction of culture supernatants [21]. The formation of the blue pigment, indigo and other related indigoid pigments [61,62], can be used to measure the oxidation of indoles in liquid cultures, on solid media (Fig. 3) and in subcellular fractions of cells [63,64]. p-Nitrophenol is another standard P450 substrate useful for screening P450 2A and P450 2E forms in whole cells [65]. Arnold and colleagues have developed colorimetric screens for examining libraries derived from bacterial P450s that are amenable to use with mammalian P450s. Purpald-based methods have been used for the measurement of formaldehyde formation in demethylation reactions [66] and 4-aminoantipyrine has been used for detecting phenol production [67]. These methods have been used on clarified cell lysates rather than whole cells to date. Recent studies in our own laboratory have employed luminogenic substrates to advantage in assessing library integrity and diversity. In this system, the P450-dependent metabolism of luciferin derivatives (‘‘P450glo’’ substrates) liberates luciferin which can be quantified by the light emitted during a subsequent luciferase-mediated reaction [68]. The principal benefits of bioluminescent techniques such as this are the effective absence of background signal and the correspondingly high sensitivity that can be achieved with appropriate form-selective substrates. Selection methods (as distinct from screening techniques) where feasible, can enable the detection of clones of interest amongst very large populations of mutants. Selection for activation of promutagens, e.g., 3,5-dimethylimidazo[4,5-f]quinoline (MeIQ) by reversion to auxotrophy on suitably selective culture media [69] allowed the investigation of 12,000 clones in successive rounds of a random mutagenesis evolution experiment [17]. Despite a clear rationale for their use, selections based on improved survival in the presence of toxic concentrations of xenobiotics have proven unhelpful (F.P. Guengerich, personal communication). This may be due to a lag in expression of the P450 relative to development of toxicity, or since such a selection pressure may provoke the development

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Fig. 3. Screening for indole hydroxylation on solid media. Representative culture plates are shown for screening of randomly selected colonies from third generation P450 2A libraries. Escherichia coli coexpressing P450 2A mutants or parental P450 2A forms with recombinant human reductase or reductase alone were cultured on LB media containing additives facilitating P450 expression and left at room temperature for 3 days. Indigo formation is visible as dark blue coloration of streaks corresponding to individual mutants.

of other, unrelated mechanisms by which the host cells can survive. For substrates excluded from the cell or where the development of the color reaction, for example, is not feasible with whole cells, partial permeabilization or complete disruption of the cell is needed. Permeabilization of E. coli has been achieved by suspension in hypotonic media [70], partial lysis of cells in 100 mM potassium phosphate buffer (pH 8) [71] and with polymyxin B [53,60]. Escherichia coli can be disrupted by sonication, lysozyme treatment and freeze–thaw cycling, or other mechanical methods [70] in high throughput fashion. However, subcellular fractionation of yeast is more difficult. Permeabilization of yeast with agents such as digitonin or cetyltrimethylammonium bromide is yet to be used widely and may overcome the diffusion barrier but the effect on enzyme activity is difficult to predict [70,72]. One important aspect is the measurement of enzyme kinetics. While whole cells provide a simple system for a first tier screen, the additional barriers to substrate access to the P450 and the opportunities for non-specific binding to cellular components mean that the kinetics measured in whole cells may be markedly different to those seen in subcellular fractions such as microsomes [45]. Thus, for a more detailed characterization of selected mutants, a second tier screen with membranes or other partially purified enzyme preparations is necessary. While many of the methods described above are very useful for assessing overall library diversity and obtaining information on structure–function relationships in mutants, few represent screens for industrially useful compounds. Many desired products do not have easily measured chromophore or fluorophores, nor can they confer a selective growth advantage that can be detected by exposure to a selection pressure. In these cases, high-throughput LC–MS analysis offers the most practical solution. High throughput NADPH depletion assays have also been used, which allow concurrent assessment of uncoupling (to

hydrogen peroxide) [66,71]; however the high cost of NADPH may limit the applications in which such assays prove to be economical screens. In addition to assessment of activity, directed evolution can be used to develop improved candidates for crystallization (e.g., by mutagenesis of regions thought to be responsible for protein aggregation) or to elucidate the basis to properties such as thermostability and piezostability. Screens for protein solubility (e.g., by assessing expression of hemoprotein in supernatants of cells fractionated by centrifugation at 100,000g) or activity at different temperatures or in the presence of solvents can be developed by modifying the conditions or sample work-up for routine high throughput catalytic or spectroscopic methods (e.g., [60]). Directed evolution of mammalian forms: progress to date To date, directed evolution techniques have mostly been applied to the elucidation of determinants of substrate specificity in mammalian xenobiotic-metabolizing P450s, rather than for the explicit purpose of engineering biocatalysts. This probably reflects the early stage of such studies with mammalian P450s, and the fact that the methodology is still being established. Several groups have targeted particular forms or subfamilies and used alternative techniques, so these studies will be discussed separately in the sections below. Random and semi-random mutagenesis of P450 1A2 and P450 2A6 The Guengerich group has adopted the approach of semi-random mutagenesis of P450 1A2 targetting six substrate recognition sequences (SRSs) for saturation mutagenesis [73]. SRSs, which border the P450 active site, are believed to be major determinants of substrate binding specificity in xenobiotic-metabolizing P450s [74], although

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mutations elsewhere can also have profound effects by subtle rearrangements of major structural elements [57]. Twenty-seven mutants were obtained based on activation of 3,5-dimethylimidazo[4,5-f]quinoline (MeIQ). After a single round of mutagenesis, a 3- to 4-fold improvement was obtained in P450 1A2 mutant activity towards typical substrates, and different relative activity profiles were seen towards a range of substrates [73]. In another study using the same mutagenicity screen [17], error prone PCR, optimized to introduce only 1–2 changes per clone, was used to mutate the whole open-reading frame and 12,000 mutants were screened per generation. After three such rounds of mutagenesis, activity towards MeIQ N-hydroxylation was increased more than 12-fold (based on enzyme efficiency values i.e., kcat/Km). While turnover was enhanced, simple binding of MeIQ was not significantly changed [17]. In a P450 1A2 triple mutant obtained using the same mutagenesis strategy but after screening three rounds of 10,000 mutants by a fluorimetric method, activity (kcat) towards 7-methoxyresorufin was improved 5-fold [57]. Again the binding affinity for the substrate was not improved and the residues affected lay outside the SRSs. P450 2A6 mutants were obtained with novel catalytic properties towards the oxidation of various indoles using a combination of saturation mutagenesis of individual SRSs and StEP to combine mutants at SRS3 and SRS4 [63]. The development of blue colonies was used as the screen and different proportions of colonies showing indigo formation were seen by mutating SRS3 (21% colonies) compared to other SRSs (4% SRS4, 2.5% SRS5 and 1% for the other three SRSs). A mutant was isolated that showed improved indigo formation as well as enhanced kcat values for coumarin 7-hydroxylation and naphthol 1hydroxylation but unchanged 7-ethoxycoumarin deethylation activity. A double mutant showing enhanced indole metabolism was used subsequently in experiments to identify novel indigoid inhibitors of cyclin dependent kinases and in plant engineering [75,76]. Random mutagenesis was again applied to the full openreading frame of P450 2A6 and the double mutant in a study designed to attempt to expand the naturally small and compact active site of this form to accommodate bulky indole analogs [64]. After screening 3000 colonies, one mutant carrying a total of five mutations compared to the wild type, was isolated from the library derived from the double mutant that turned blue in the presence of 5hydroxybenzylindole. Notably one of the two mutations that proved essential to the extension of activity in the mutant was derived from the initial saturation mutagenesis study, and would not have been readily introduced by error prone PCR since it necessitated two positions being changed in a single codon [64]. This observation argues for the use of recombinatorial methods or saturation mutagenesis where practicable. In a third study, random mutagenesis of the entire P450 2A6 open-reading frame was used to identify mutants with

reduced activity in a whole cell screen for coumarin 7hydroxylation and identify residues affecting catalytic specificity [58]. Several mutants showed enhanced kcat values but no mutants with improved enzyme efficiency (kcat/ Km) were reported. Random mutagenesis and shuffling of P450 2B1 and P450 3A4 Halpert and colleagues have focused on engineering P450 2B1 and P450 3A4 by random mutagenesis coupled with conventional site directed mutagenesis or chimeragenesis [16,53,60]. In the initial round of random mutagenesis and screening of a known mutant of P450 2B1 (L209A), two of several hundred screened mutants showed activity towards 7-ethoxy-4-trimethylcoumarin in a peroxide supported assay above the range seen for parental clones and 30–40% of mutant clones showed <10% of the average parental activity. A quadruple mutant (V183L/F202L/ L209A/S334P) isolated after a further round of mutagenesis and screening and one round of site-directed mutagenesis, showed a 6-fold improvement in kcat. The authors concluded no further improvements were likely with further rounds of mutagenesis. Analysis of individual mutations suggested the V183L mutation was not beneficial whereas the triple mutant, F202L/L209A/S334P, showed a 2.5-fold enhancement of kcat/Km in the NADPH-supported reaction and enhanced activity towards 7-benzyloxyresorufin, benzphetamine and testosterone. Mutants with certain combinations of these four mutations showed enhanced activation of the chemotherapeutic prodrugs, cyclophosphamide and ifosphamide, results which have been used to rationally reengineer the activity of an orthologous P450 2B enzyme, P450 2B11 [77]. The quadruple mutant isolated in the initial study on P450 2B1 [16] was further optimized for thermostability and DMSO tolerance [60]. An initial screen of 3000 clones generated by error-prone PCR of the whole plasmid showed a similar small proportion of improved mutants as seen in the earlier study [16], while a targeted screen of 150 clones at elevated temperature and DMSO content proved more useful in revealing mutations which enhanced thermostability and activity in the presence of DMSO [60]. In a study designed to improve P450 3A4 activity supported by peroxides [53], a double mutant was isolated showed an 11-fold improvement in kcat/KmCuOOH for 7-benzyloxyquinoline debenzylation and enhanced peroxide-supported activity was also seen for several other substrates. Recombinatorial mutagenesis of P450 1A subfamily enzymes Pompon and colleagues have used CLERY, an approach combining conventional DNA family shuffling with in vivo recombination in yeast to generate a library of mutants derived from human P450 1A1 and P450 1A2 [21]. The degree of mosaicism of the library was assessed

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by hybridization of individual clones with probes for either P450 1A1 or P450 1A2. Of the screened mutants, 13.8% showed apparent parental patterns of hybridization, a frequency which could be explained by the different probabilities of shuffling between neighbouring segments, suggesting the real rate of parental contamination was lower. Sequencing of a small sample of clones revealed that each mutant was composed on average of 5.4 +/ 2.2 recombined parental fragments. The rate of random mutations was 14 +/ 4 per 1.5 kbp sequence in non-functional and 8 +/ 3 in functionally competent clones. 12% of clones appeared to express activity towards naphthalene, a common substrate of both parents. In a further exploration of this library, the mosaic forms were shown to be catalytically diverse with respect to five different substrates [56,78]. In a correlation analysis with methoxy- and ethoxyresorufins, some, but not all mutants, aligned with either P450 1A1 or 1A2. The examination of a third structurally dissimilar substrate served to discriminate between mutants that showed similar phenotypes with respect to the alkoxyresorufins [78]. By contrast, mutants made by saturation mutagenesis of a specific region of P450 1A1 that had been predicted to influence substrate specificity from a previous study [79], showed the same relative activities as the parental form with respect to multiple substrates [56,78]. Thus the recombinatorial strategy appeared to generate more useful diversity in the library in comparison to the ‘‘rational evolution’’ strategy limited to a single region of the sequence, a result that was not unexpected. In mosaic libraries there appeared to be some bias towards particular types of mosaic structure [56]. The six oligo pairs used for the probe hybridization method of sequence analysis could detect 64 different mosaic types and several were overrepresented in recombinatorial libraries. This was most significant in the library cloned into E. coli, which was also compromised by a high level of contamination (24% of sequences) by parental P450 1A2, arguing for direct expression and screening within yeast [56].

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9.1 ± 1.8 crossovers and 1.5 ± 0.5 spontaneous mutations per 1.5 kbp open-reading frame and 50–54% of mutants showed detectable P450 hemoprotein spectra (>80 nmoles per L culture). In each case, only a small proportion of the library has been screened (20–200 clones) but significant diversity in substrate selectivity, turnover rates, kinetic behaviour and overall P450 expression has been found [45,46]. In the case of the P450 2A library, where indole hydroxylation, an activity common to all three parents (P450 2a5, P450 2A6 and P450 2A13) can be readily visualized by the appearance of blue colonies (Fig. 3), 56% percent of first generation (F1) clones showed activity towards indole when screened on solid media. This result that supports the inference made from hemoprotein spectra that a large proportion of the library is potentially functional, correctly folded protein. Further shuffling of P450 2A mutants with high activity towards indole led to an enrichment of this activity in F2 (83%) and F3 (71%) libraries (Gillam, Soucek, Huang and Johnston, unpublished data). Recombination of P450 1A2 with P450 102 Arnold et al. used Sequence Homology Independent Protein Recombination (SHIPREC), a recombinatorial method involving single crossovers between distantly related genes to generate functional chimeras between P450 1A2 and the P450 domain of bacterial P450BM3 (P450 102) [48]. In this technique, size selection favours mutants formed by recombination at similar points in the primary structure and the two out of three predicted frameshift mutants are eliminated by an elegant preselection step, which concurrently selected for soluble mutants. While high proportions (80%) of mutants were correctly folded, a bias was observed amongst folded mutants in the (single) position of recombination. Recombination at one or other terminus appeared to be correlated with structural integrity. Two mutants showing characteristic P450 1A2 activity were comprised principally of P450 1A2 with only short N-terminal segments from P450 102. The authors concluded that recombination in central regions led to problems in protein folding.

Recombinatorial mutagenesis of P450 2C, 1A and 2A subfamily enzymes by family shuffling with restriction enzymes

Perspective: requirements for the successful implementation of engineered P450 enzymes

The approach taken in our own lab has been to exploit DNA family shuffling on multiple P450 subfamilies. To date, libraries have been generated from P450 2C [45], P450 1A [46], P450 3A and P450 2A (Gillam et al. and Soucek et al., unpublished data) subfamilies. Between two (P450 1A) and four (P450 2C, P450 3A) parents have been used, typically sharing over 80% sequence identity. Mutant P450s were subjected to fragmentation using combinations of restriction enzymes that generated fragment sizes under 300 bp, fragments were isolated by size-selective filtration then reassembled in a primerless PCR. Sequencing of a random selection of P450 2C library clones revealed

Directed evolution is yet to be fully exploited for the generation of biocatalysts from xenobiotic-metabolizing P450s. The studies performed to date have demonstrated the proof of principle that these enzymes can be successfully engineered for both catalytic activity and other industrially desirable properties; however, no study has yet succeeded in generating a mutant with activity and efficiency comparable to the highly specialized bacterial P450s acting on their natural substrates. Several interrelated characteristics of xenobiotic-metabolizing P450s will need to be addressed before the full potential of these enzymes can be realized.

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Source of reducing equivalents NADPH is costly, and both high throughput screening of large libraries and industrial scale-up of engineered enzymes will require a cost-effective means by which to supply the reducing equivalents required for the catalytic cycle. The most straightforward way to deliver these is by using the cell to provide NADPH by using whole, metabolically active cells. Maintaining enzymes in the cellular environment also avoids possible losses due to mechanical and chemical stress during permeabilization, cell lysis, or subcellular fractionation. However, substrate access to and product egress from whole cells may limit the turnover rates and product yields achieved in such bioreactor systems. Halpert et al. [16,53,60] have used the peroxide shunt to deliver electrons in permeabilized cells in the same way as has been done by Arnold and colleagues with bacterial P450s [59]. However H2O2 destroys heme at concentrations over 10–20 mM, a concentration in the same range as the KmHOOH values for P450 2B1 and P450 3A4 mutants optimized for peroxidase activity [53,60]. An alternative oxygen donor, cumene hydroperoxide, shows the same effect although the separation between Km and Ki values may be slightly greater for P450 3A4 mutants [53]. Thus, on balance, peroxygenase-based systems are not ideal. Electrocatalytic and redox mediator systems have been developed for P450s but not yet widely exploited [80–82]. In theory the electrons required for oxygen activation may be delivered from an appropriately positioned electrode; however achieving the appropriate orientation of enzyme on the electrode can be difficult and enzyme stability may be limited in such systems. Overall enzyme efficiency and other influences on product yield Batch or fed batch cultures make up roughly half the total examples of industrial biocatalysis [83]. Mostly yields are >80–90% and of the order of >50 g product per L culture [83]. In general, for optimal process efficiency, workup costs should be minimized, so there is a need to be able to add high concentrations of substrate into small volumes with modest amounts of enzyme and retrieve large amounts of product in a straightforward manner. However xenobiotic-metabolizing P450s have typically very poor turnover rates, a characteristic that has been attributed to the evolutionary need to clear a wide variety of xenobiotics. No one substrate has driven evolution of the active site, so substrate fit has not been optimized to exclude water. The presence of water molecules at the active site can potentiate uncoupling side reactions meaning that coupling of NADPH to substrate monooxygenation in such P450s will be poor. It is hoped that through directed evolution, fit of the desired substrate will be optimized, leading to a concurrent reduction in competing uncoupling reactions. However this hypothesis is yet to be properly tested.

Other factors, such as the need for productive interaction between redox partners, contribute to uncoupling. It is pertinent that the most efficient P450 known, P450 102, is a fusion between tethered P450 and reductase domains. Artificial P450-reductase fusions have been trialled with mammalian P450s [84–87], however in most cases coupling was not optimal as indicated by the improvements in activity obtained upon addition of exogenous reductase. There is considerable scope for optimization of the tethering region separating the domains. Engineering of the reductase component, an enzyme that must normally support the activity of several different types of protein (different P450s as well as heme oxygenase and fatty acid desaturase), to better interact with particular families of P450s, may also enhance rates. Efficiency may also be limited by the requirement to coexpress two recombinant proteins (P450 and the cognate reductase), or one large P450-reductase fusion in an appropriate host. Bacteria have no endogenous P450 or P450 reductase, but the flavodoxin–flavodoxin reductase system can support the activity of certain mammalian P450s [33,88] opening up the possibility of engineering P450s that interact more effectively with these endogenous proteins. For certain applications, such as phytoremediation or other plant improvement strategies, it may instead be desirable to optimize the P450 to interact with other (plant) reductases in the host of interest. Substrate or product inhibition may also be a problem with P450 biocatalysts given the desirability of adding high concentrations of substrate and generating large amounts of product. Many xenobiotic-metabolizing P450s have been shown to display atypical kinetics that are thought to result from the binding of multiple ligands in the capacious active site [89]. Again optimization of the biocatalyst to one substrate by directed evolution may address this problem, but this means that assay procedures should include some means to monitor the type of kinetic behaviour shown by mutants to detect substrate or product inhibition (e.g., screens at both low and high substrate concentrations and in the presence of product). The presence of excess substrate or product may also be avoided by two phase partitioning, where substrate diffuses from the organic phase and product diffuses back [90]. The equilibrium between phases is influenced by the catalytic activity of the enzyme. However, this requires the enzyme or engineered organism to be active in the presence of solvents, raising the issue of other considerations imposed by process engineering requirements. Process engineering issues Some advantageous characteristics of industrial enzymes have already been mentioned above. It is generally desirable that biocatalysts should be stable with time and over a range of temperatures that may be encountered during the process. Immobilization or encapsulation of biocatalysts may be necessary to fulfil regulatory requirements or to

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maximize throughput or product recovery. For many hydrophobic P450 substrates and products, it may be necessary to administer substrates in higher concentrations of organic solvents than typically used for P450 studies, so solvent tolerance would be an essential requirement. With the exception of one study [60], such issues are yet to be addressed with mammalian P450 biocatalysts. However the existence of thermostable P450s suggests that there is no theoretical barrier to engineering thermal stability into other P450s. Conclusion In summary, the directed evolution of xenobioticmetabolizing P450s is in the very early stages of development. While the existing studies have shown promise that these enzymes can be diversified in useful ways, the full power of directed evolution in bringing about improvements in catalytic activity of many orders of magnitude is yet to be realized. Moreover questions remain as to the scope for improving other properties of these notoriously unstable enzymes. Nevertheless, given the diverse ways in which the basic P450 structure and chemistry has been exploited by natural evolution to fill such a great variety of niches, with time and serious research effort, artificial, directed evolution will doubtless show that P450s can be engineered to an equally diverse set of industrially important niches. Acknowledgments This work follows ultimately from studies undertaken in the Guengerich laboratory and in subsequent collaborations, in which bacterial bicistronic expression systems were developed for many mammalian P450s. An early outcome of these studies was the discovery of indigo production by mammalian P450s and the elucidation of the metabolic pathway in collaboration with the Guengerich and Arnold labs [61,62]. Indigo formation provided a facile screening tool for the initial development of the DNA family shuffling method used in current studies in the author’s laboratory. I would like to express my sincere gratitude to Prof. F.P. Guengerich for the long and productive association I have enjoyed with his laboratory and for the superlative role model he has provided for all past and present members of his laboratory. Thanks are also extended to Drs. Pavel Soucek, Wayne Johnston and Weiliang Huang for their participation in the unpublished experiments on the P450 2A libraries mentioned in this paper and assistance with preparation of the cognate figures. References [1] M. Newcomb, P.F. Hollenberg, M.J. Coon, Arch. Biochem. Biophys. 409 (2003) 72–79. [2] D.R. Nelson (2007). Available from: .

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