P450 BM3 Mutants Research Articles

Selective whole-cell biosynthesis of the designer drug metabolites 15- or 16-betahydroxynorethisterone by engineered Cytochrome P450 BM3 mutants

In the present study, the whole-cell biotransformation using strains expressing CYP BM3 mutants has been evaluated for the stereoselective hydroxylation of the steroid norethisterone (NET), a widely used contraceptive. First, an in vitro CYP BM3 mutant library screen was performed to identify mutants with high activity, as well asstereoselectivity. Subsequently, two different whole-cell setups (resting and growing cells) were tested in two different host organisms (Escherichia coli and Saccharomyces cerevisiae) expressing CYP BM3. It was found that resting E. coli whole cells produced the highest amounts of products and therefore this biocatalytic setup was further optimized for application in a laboratory-scale fermentor. In the optimized fermentor setup, high product yields (0.3g/L 15β-OH-NET and 0.16g/L 16β-OH-NET) were achieved while it was also shown that the regio- and stereoselectivities of the steroid hydroxylations, as determined during the in vitro library screen, were preserved in the whole-cell system. The combination of a mutant CYP BM3 library and the optimized whole-cell oxidation system represents a promising and cost-effective alternative to a wide range of in vitro biosynthetic routes.

Biosynthesis of a steroid metabolite by an engineered Rhodococcus erythropolis strain expressing a mutant cytochrome P450 BM3 enzyme.

In the present study, the use of Rhodococcus erythropolis mutant strain RG9 expressing the cytochrome P450 BM3 mutant M02 enzyme has been evaluated for whole-cell biotransformation of a 17-ketosteroid, norandrostenedione, as a model substrate. Purified P450 BM3 mutant M02 enzyme hydroxylated the steroid with >95% regioselectivity to form 16-β-OH norandrostenedione, as confirmed by NMR analysis. Whole cells of R. erythropolis RG9 expressing P450 BM3 M02 enzyme also converted norandrostenedione into the 16-β-hydroxylated product, resulting in the formation of about 0.35g/L. Whole cells of strain RG9 itself did not convert norandrostenedione, indicating that metabolite formation is P450 BM3 M02 enzyme mediated. This study shows that R. erythropolis is a novel and interesting host for the heterologous expression of highly selective and active P450 BM3 M02 enzyme variants able to perform steroid bioconversions.

Activation of the anticancer drugs cyclophosphamide and ifosfamide by cytochrome P450 BM3 mutants

Cyclophosphamide (CPA) and ifosfamide (IFA) are widely used anticancer agents that require metabolic activation by cytochrome P450 (CYP) enzymes. While 4-hydroxylation yields DNA-alkylating and cytotoxic metabolites, N-dechloroethylation results in the generation of neuro- and nephrotoxic byproducts. Gene-directed enzyme prodrug therapies (GDEPT) have been suggested to facilitate local CPA and IFA bioactivation by expressing CYP enzymes within the tumor cells, thereby increasing efficacy.We screened bacterial CYP BM3 mutants, previously engineered to metabolize drug-like compounds, for their ability to catalyze 4-hydroxylation of CPA and IFA. Two CYP BM3 mutants showed very rapid initial bioactivation of CPA and IFA, followed by a slower phase of product formation. N-dechloroethylation by these mutants was very low (IFA) to undetectable (CPA). Using purified CYP BM3 as an extracellular bioactivation tool, cytotoxicity of CPA and IFA metabolism was confirmed in U2OS cells. This novel application of CYP BM3 possibly provides a clean and catalytically efficient alternative to liver microsomes or S9 for the study of CYP-mediated drug toxicity.To our knowledge, the observed rate of CPA and IFA 4-hydroxylation by these CYP BM3 mutants is the fastest reported to date, and might be of potential interest for CPA and IFA GDEPT.

Mini-dialysis tubes as tools to prepare drug-protein adducts of P450-dependent reactive drug metabolites

The modification of critical cellular proteins by reactive metabolites (RMs) resulting from P450-dependent drug bioactivation is considered essential to the onset of many idiosyncratic drug reactions. In this study, we report a novel method that can be used to prepare and study drug-protein adducts. Drug bioactivation by P450s was performed in a small container containing a mini-dialysis tube with the model target protein human glutathione-S-transferase P1-1 (hGST P1-1), allowing RMs to translocate from P450 to hGST P1-1 via a semi-permeable membrane (6–8kDa). GST P1-1 modification was evaluated by LC–MS analysis of intact protein adducts and following digestion of protein with trypsin. As proof of principle, the described methodology was first applied to the direct electrophile monochlorobimane. A highly active P450 BM3 mutant (CYP102A1M11H) was subsequently used for bioactivation of acetaminophen, clozapine, diclofenac (DF) and mefenamic acid (MFA), but hGST P1-1 adducts were only observed for the latter two drugs. CYP2C9 and CYP3A4, which metabolize DF to p-benzoquinone imines, were tested to investigate the applicability of human P450s. Finally, it was evaluated whether bioactivation of MFA by human and rat liver microsomes resulted in modification of hGST P1-1. The results show that our adduct preparation method can also be used in combination with membrane-bound P450 bioactivation systems, as long as formed RMs have sufficient life-time to reach hGST P1-1 inside the dialysis tube.

Application of engineered cytochrome P450 mutants as biocatalysts for the synthesis of benzylic and aromatic metabolites of fenamic acid NSAIDs

Cytochrome P450 BM3 mutants are promising biocatalysts for the production of drug metabolites. In the present study, the ability of cytochrome P450 BM3 mutants to produce oxidative metabolites of structurally related NSAIDs meclofenamic acid, mefenamic acid and tolfenamic acid was investigated. A library of engineered P450 BM3 mutants was screened with meclofenamic acid (1) to identify catalytically active and selective mutants. Three mono-hydroxylated metabolites were identified for 1. The hydroxylated products were confirmed by NMR analysis to be 3′-OH-methyl-meclofenamic acid (1a), 5-OH-meclofenamic acid (1b) and 4′-OH-meclofenamic acid (1c) which are human relevant metabolites. P450 BM3 variants containing V87I and V87F mutation showed high selectivity for benzylic and aromatic hydroxylation of 1 respectively. The applicability of these mutants to selectively hydroxylate structurally similar drugs such as mefenamic acid (2) and tolfenamic acid (3) was also investigated. The tested variants showed high total turnover numbers in the order of 4000–6000 and can be used as biocatalysts for preparative scale synthesis. Both 1 and 2 could undergo benzylic and aromatic hydroxylation by the P450 BM3 mutants, whereas 3 was hydroxylated only on aromatic rings. The P450 BM3 variant M11 V87F hydroxylated the aromatic ring at 4′ position of all three drugs tested with high regioselectivity. Reference metabolites produced by P450 BM3 mutants allowed the characterisation of activity and regioselectivity of metabolism of all three NSAIDs by thirteen recombinant human P450s. In conclusion, engineered P450 BM3 mutants that are capable of benzylic or aromatic hydroxylation of fenamic acid containing NSAIDs, with high selectivity and turnover numbers have been identified. This shows their potential use as a greener alternative for the generation of drug metabolites.

Human P450-like oxidation of diverse proton pump inhibitor drugs by ‘gatekeeper’ mutants of flavocytochrome P450 BM3

Production of drug metabolites is one area where enzymatic conversion has significant advantages over synthetic chemistry. These high value products are complex to synthesize, but are increasingly important in drug safety testing. The vast majority of drugs are metabolized by cytochromes P450 (P450s), with oxidative transformations usually being highly regio- and stereo-selective. The PPIs (proton pump inhibitors) are drugs that are extensively metabolized by human P450s, producing diverse metabolites dependent on the specific substrate. In the present paper we show that single mutations (A82F and F87V) in the biotechnologically important Bacillus megaterium P450 BM3 enzyme cause major alterations in its substrate selectivity such that a set of PPI molecules become good substrates in these point mutants and in the F87V/A82F double mutant. The substrate specificity switch is analysed by drug binding, enzyme kinetics and organic product analysis to confirm new activities, and X-ray crystallography provides a structural basis for the binding of esomeprazole to the F87V/A82F enzyme. These studies confirm that such 'gatekeeper' mutations in P450 BM3 produce major perturbations to its conformation and substrate selectivity, enabling novel P450 BM3 reactions typical of those performed by human P450s. Efficient transformation of several PPI drugs to human-like products by BM3 variants provides new routes to production of these metabolites.

Human NAD(P)H:quinone Oxidoreductase 1 (NQO1)-Mediated Inactivation of Reactive Quinoneimine Metabolites of Diclofenac and Mefenamic Acid

quinone oxidoreductase 1 (NQO1) is an enzyme capable of reducing a broad range of chemically reactive quinones and quinoneimines (QIs) and can be strongly upregulated by Nrf2/Keap1-mediated stress responses. Several commonly used drugs implicated in adverse drug reactions (ADRs) are known to form reactive QI metabolites upon bioactivation by P450, such as acetaminophen (APAP), diclofenac (DF), and mefenamic acid (MFA). In the present study, the reductive activity of human NQO1 toward the QI metabolites derived from APAP and hydroxy-metabolites of DF and MFA was studied, using purified bacterial P450 BM3 (CYP102A1) mutant M11 as a bioactivation system. The NQO1-catalyzed reduction of the QI metabolites was quantified relative to spontaneous glutathione (GSH) conjugation. Addition of NQO1 to the incubations strongly reduced the formation of all corresponding GSH conjugates, and this activity could be prevented by dicoumarol, a selective NQO1 inhibitor. The GSH conjugation was strongly increased by adding human GSTP1-1 in a wide range of GSH concentrations. Still, NQO1 could effectively compete with the GST catalyzed GSH conjugation by reducing the QIs. In conclusion, we identified the QI metabolites of the 4'- and 5-hydroxy-metabolites of DF and MFA as novel substrates for human NQO1. NQO1-mediated reduction proves to be an effective pathway to detoxify these QI metabolites in addition to GSH conjugation. Genetically determined deficiency of NQO1 therefore might be a risk factor for ADRs induced by reactive QI drug metabolites.

CH-activating oxidative hydroxylation of 1-tetralones and related compounds with high regio- and stereoselectivity.

Mutants of P450-BM3 evolved by directed evolution are excellent catalysts in the CH-activating oxidative hydroxylation of 1-tetralone derivatives and of indanone, with unusually high regio- and enantioselectivity being observed. Similar results were achieved in the oxidative hydroxylation of tetralin and indane. The products are useful building blocks in the synthesis of a number of biologically active compounds.

Effect of human glutathione S-transferase hGSTP1-1 polymorphism on the detoxification of reactive metabolites of clozapine, diclofenac and acetaminophen

Recent association studies suggest that genetically determined deficiencies in GSTs might be a risk factor for idiosyncratic adverse drug reactions resulting from the formation of reactive drug metabolites. hGSTP1-1 is polymorphic in the human population with a number of single nucleotide polymorphisms that yield an amino acid change in the encoded protein. Three allelic variants of hGSTP1-1 containing an Ile105Val or Ala114Val substitution, or a combination of both, have been the most widely studied and showed different activity when compared to wild-type hGSTP1-1*A (Ile105/Ala114). In the present study, we studied the ability of these allelic variants to catalyze the GSH conjugation of reactive metabolites of acetaminophen, clozapine, and diclofenac formed by bioactivation in in vitro incubations by human liver microsomes and drug metabolizing P450 BM3 mutants. The results show that effects of the change of amino acid at residue 105 and 114 on conjugation reactions were substrate dependent. A single substitution at residue 105 affects the ability to catalyze GSH conjugation, while when both residue 105 and 114 were substituted the effect was additionally enhanced. Single mutation at position 114 did not show a significant effect. The different hGSTP1-1 mutants showed slightly altered regioselectivities in formation of individual GSH conjugates of clozapine which suggests that the binding orientation of the reactive nitrenium ion of clozapine is affected by the mutations. For diclofenac, a significant decrease in activity in GSH-conjugation of diclofenac 1′,4′-quinone imine was observed for variants hGSTP1-1*B (Val105/Ala114) and hGSTP1-1*C (Val105/Val114). However, since the differences in total GSH conjugation activity catalyzed by these allelic variants were not higher than 30%, differences in inactivation of reactive intermediates by hGSTP1-1 are not likely to be a major factor in determining interindividual difference in susceptibility to adverse drug reactions induced by the drugs studied.

Peroxidase activity stabilization of cytochrome P450BM3 by rational analysis of intramolecular electron transfer

Combined quantum mechanical and molecular mechanical (QM/MM) calculations were used to explore the electron pathway involved in the suicide inactivation of cytochrome P450BM3 from Bacillus megaterium. The suicide inactivation is a common phenomenon observed for heme peroxidases, in which the enzyme is inactivated as a result of self-oxidation mediated by highly oxidizing enzyme intermediates formed during the catalytic cycle. The selected model was a mutant comprising only the heme domain (CYPBM3 21B3) that had been previously evolved to efficiently catalyze hydroxylation reactions with hydrogen peroxide (H2O2) as electron acceptor. An extensive mapping of residues involved in electron transfer routes was obtained from density functional calculations on activated heme (i.e. Compound I) and selected amino acid residues. Identification of oxidizable residues (electron donors) was performed by selectively activating/deactivating different quantum regions. This method allowed a rational identification of key oxidizable targets in order to replace them for less oxidizable residues by site-directed mutagenesis. The residues W96 and F405 were consistently predicted by the QM/MM electron pathway to hold high spin density; single and double mutants of P450BM3 on these positions (W96A, F405L, W96A/F405L) resulted in a more stable variants in the presence of hydrogen peroxide, displaying a similar reaction rate than P450BM3 21B3. Furthermore, mass spectrometry confirmed these oxidation sites and corroborated the possible routes described by QM/MM electron transfer (ET) pathways.

Combination of biotransformation by P450 BM3 mutants with on-line post-column bioaffinity and mass spectrometric profiling as a novel strategy to diversify and characterize p38α kinase inhibitors

Mutants of bacterial cytochrome P450 BM3 from Bacillus megaterium have gained increasing interest to support drug metabolism studies by producing human relevant biotransformation products and/or to support drug discovery by producing libraries of new chemical entities based on initial lead compounds. In this study, a library of 33 P450 BM3 mutants was used to diversify TAK-715, a representative (highly lipophilic) inhibitor of p38α mitogen-activated protein kinase (p38α). To simultaneously determine the individual bioaffinity and identity of the different products, an analytical high-resolution screening approach was used, based on post-column on-line bioaffinity profiling with parallel mass spectrometric identification. The screening of the product mixtures produced demonstrated that introducing mutations in the active site of the P450 BM3 resulted in different product profiles. Several P450 BM3 mutants were mimicking the metabolic profile obtained by human liver microsomes. The major biotransformation products of TAK-715 could be produced with the most active P450 BM3 mutant in sufficient amounts to enable further structure elucidation by 1H-NMR and quantification of their p38α affinity.

Hydroxylation of non-substituted polycyclic aromatic hydrocarbons by cytochrome P450 BM3 engineered by directed evolution

Chrysene and pyrene are known toxic compounds recalcitrant to biodegradation. Here directed evolution allowed us to identify two new mutants of cytochrome P450 BM3 that are able to hydroxylate both compounds. Random mutagenesis has been used to generate libraries of mutants of P450 BM3 active toward polycyclic aromatic hydrocarbons (PAHs) PAHs. After two rounds of error-prone PCR and backcross with parental DNA, three mutants were identified for improved activity toward pyrene and for the first time a new activity toward chrysene in comparison to the wild type enzyme. The mutants show higher affinity and coupling efficiency for chrysene with faster rates of product formation compared to the wild type. Furthermore, the mutants are able to hydroxylate chrysene in different positions, producing four metabolites, 1-, 3-, 4-, and 6-hydroxychrysene, and to hydroxylate pyrene to 1-hydroxypyrene.The majority of the mutation sites are found to be far from the active site, demonstrating the power of directed evolution in identifying mutations difficult to predict with a rational design approach.The different product profiles obtained for the different P450 BM3 mutants indicate that substrate orientation in the catalytic pocket of the protein can be modified by protein engineering. The mutants can be used for metabolic engineering for safe and cost-effective sustainable production of hydroxylated PAHs for industrial purposes as well as for the assessment of their carcinogenic activity in mammals.

Mass Spectrometric Characterization of Protein Adducts of Multiple P450-Dependent Reactive Intermediates of Diclofenac to Human Glutathione-S-transferase P1-1

Use of the nonsteroidal anti-inflammatory drug diclofenac (DF) is associated with serious idiosyncratic hepatotoxicity. Covalent binding of reactive intermediates of DF to proteins is considered to initiate the process leading to this severe side-effect. The aim of this study was to characterize the nature of covalent protein modifications by reactive metabolites of DF which result from bioactivation by cytochrome P450. DF and its major monohydroxylated metabolites 4'-hydroxydiclofenac (4'-OH-DF) and 5-hydroxydiclofenac (5-OH-DF) were bioactivated using a highly active P450 BM3 mutant (CYP102A1M11H) in the presence of the model target protein human glutathione-S-transferase P1-1 (hGST P1-1). Protein-adducts were subsequently identified by LC-MS/MS analysis of tryptic digests of hGST P1-1. In total, 10 different peptide adducts were observed which result from modifications of Cys-47 and Cys-14 of hGST P1-1. The majority of the protein thiol modifications appeared to be derived from 5-OH-DF, which produced seven different peptide adducts with mass increments of 289.0, 309.0, and 339.0 Da. Remarkably, no peptide adducts were observed upon the bioactivation of 4'-OH-DF. Incubations of P450 BM3 with DF also showed the peptide adducts derived from 5-OH-DF and peptide adducts that are not derived from quinone imine. A peptide adduct with a mass increment of 249.0 Da most likely results from the o-imine methide formed by oxidative decarboxylation of DF. In addition, a peptide adduct was observed with a mass increment of 259.0 Da, which corresponds to the substitution of one of the chlorine atoms of DF by protein thiol. A corresponding GSH-conjugate with a similar mass increment was only observed if incubations of DF with P450 and GSH were supplemented by human GST P1-1. The results of this study not only confirm the importance of 5-OH-DF in covalent protein-binding but also suggest that the nature of protein adduction is not necessarily reflected by chemical conjugation with GSH.

Regio‐ and Stereoselective Hydroxylation of Optically Active α‐Ionone Enantiomers by Engineered Cytochrome P450 BM3 Mutants

AbstractThe selective hydroxylation of an unactivated CH bond is a crucial step in the synthesis of fine chemicals such as hydroxylated terpenoids. In the present study, the ability of 40 cytochrome P450 BM3 mutants to perform the regio‐ and stereoselective hydroxylation of α‐ionone has been investigated. Based on their activity and selectivity to produce 3‐hydroxy‐α‐ionone from racemic α‐ionone, 6 BM3 mutants were selected. Out of these, 3 mutants (M01 A82W, M11 A82W and M11 V87I) showed high selectivity for trans‐3‐hydroxy‐α‐ionone formation while 3 other mutants (M11 L437N, M11 L437S and M11 L437T) formed almost equal amounts of both cis‐3‐hydroxy‐ and trans‐3‐hydroxy‐α‐ionone. Incubation with individual enantiomers showed that M11 L437N, M11 L437S and M11 L437T exhibited opposite stereoselectivity producing (3S,6S)‐hydroxy‐α‐ionone with the (6S)‐enantiomer and (3S,6R)‐hydroxy‐α‐ionone with the (6R)‐enantiomer. Thus for the first time, BM3 mutants that can selectively produce diastereomers of 3‐hydroxy‐α‐ionone (>90% de), with high turnover numbers and minimal secondary metabolism, have been identified. Docking studies have been performed to rationalize the basis of the experimentally observed selectivity. In conclusion, engineered P450 BM3s are promising biocatalysts for regio‐ and stereoselective production of hydroxylated α‐ionones for industrial applications.

Free Energy Calculations Give Insight into the Stereoselective Hydroxylation of α-Ionones by Engineered Cytochrome P450 BM3 Mutants

Previously, stereoselective hydroxylation of α-ionone by Cytochrome P450 BM3 mutants M01 A82W and M11 L437N was observed. While both mutants hydroxylate α-ionone in a regioselective manner at the C3 position, M01 A82W catalyzes formation of trans-3-OH-α-ionone products whereas M11 L437N exhibits opposite stereoselectivity, producing trans-(3S,6S)-OH-α-ionone and cis-(3S,6R)-OH-α-ionone. Here, we explore the stereoselective C3 hydroxylation of α-ionone by Cytochrome P450 BM3 mutants M01 A82W and M11 L437N using molecular dynamics-based free energy calculations to study the interaction between the enzyme and both the substrates and the products. The one-step perturbation approach is applied using an optimized reference state for substrates and products. While the free energy differences between the substrates free in solution amount to ~0 kJ mol(-1), the differences in mutant M01 A82W agree with the experimentally obtained dissociation constants K(d). Moreover, a correlation with experimentally observed trends in product formation is found in both mutants. The trans isomers show the most favorable relative binding free energy in the range of all four possible hydroxylated diastereomers for mutant M01 A82W, while the trans product from (6S)-α-ionone and the cis product from (6R)-α-ionone show highest affinity for mutant M11 L437N. Marcus theory is subsequently used to relate the thermodynamic stability to transition state energies and rates of formation.

A series of hybrid P450 BM3 enzymes with different catalytic activity in the light-initiated hydroxylation of lauric acid

We have developed a series of hybrid P450 BM3 enzymes to perform the light-activated hydroxylation of lauric acid. These enzymes contain a Ru(II)-diimine photosensitizer covalently attached to single cysteine residues of mutant P450 BM3 heme domains. The library of hybrid enzymes includes four non-native single cysteine mutants (K97C, Q397C, Q109C and L407C). In addition, mutations around the heme active site, F87A and I401P, were inserted in the Q397C mutant. Two heteroleptic Ru(II) complexes, Ru(bpy)2phenA (1) and Ru(phen)2phenA (2) (bpy=bipyridine, phen=1,10-phenanthroline, and phenA=5-acetamido-1,10-phenanthroline), are used as photosensitizers. Upon visible light irradiation, the hybrid enzymes display various total turnover numbers in the hydroxylation of lauric acid, up to 140 for the L407C-1 mutant, a 16-fold increase compared to the F87A/Q397C-1 mutant. CO binding studies confirm the ability of the photogenerated Ru(I) compound to reduce the fraction of ferric high spin species present in the mutants upon substrate binding.

Defluorination of 4-fluorophenol by cytochrome P450BM3-F87G: activation by long chain fatty aldehydes

Cytochrome P450(BM3)-F87G catalyzed the oxidative defluorination of 4-fluorophenol, followed by reduction of the resulting benzoquinone to hydroquinone via the NADPH P450-reductase activity of the enzyme. The k (cat) and K (m) for this reaction were 71 ± 5 min(-1) and 9.5 ± 1.3 mM, respectively. Co-incubation of the reaction mixture with long chain aldehydes stimulated the defluorination reaction, with the 2,3-unsaturated aldehyde, 2-decenal producing a 12-fold increase in catalytic efficiency. At 150 μM aldehyde, k (cat) increased to 158 ± 4, while K (m) decreased to 1.8 ± 0.2. The effects of catalase, glutathione and ascorbate on the reaction were all consistent with a direct oxygen insertion mechanism, as opposed to a radical mechanism. The study demonstrates the potential use of P450(BM3) mutants in oxidative defluorination reactions, and characterizes the novel stimulatory action of straight chain aldehydes on this activity.

Identification of Mutant Asp251Gly/Gln307His of Cytochrome P450 BM3 for the Generation of Metabolites of Diclofenac, Ibuprofen and Tolbutamide

The soluble, catalytically self-sufficient cytochrome P450 BM3 from Bacillus megaterium is a good candidate as biocatalyst for the synthesis of drug metabolites. To this end, error-prone polymerase chain reaction (PCR) was used to generate a library of P450 BM3 mutants with novel activities toward drugs. The double mutant Asp251Gly/Gln307His (A2) with activities towards diclofenac, ibuprofen and tolbutamide was identified by screening with the alkali method. This is based on the detection of NADPH oxidation during enzymatic turnover on whole Escherichia coli cells heterologously expressing the P450 BM3 mutants in the presence of the target substrates. The three drugs screened are marker substrates of human liver cytochromes P450 belonging to the 2C subfamily. Interestingly the mutations Asp251Gly/Gln307His are located on the protein surface and they are not directly involved in substrate binding and turnover. Dissociation constants and K(M) values of mutant A2 for diclofenac, ibuprofen and tolbutamide are in the micromolar range. Catalysis leads to hydroxylations in specific positions, producing 4'-hydroxydiclofenac, 2-hydroxyibuprofen and 4-hydroxytolbutamide, respectively.

The Role of Protein Plasticity in Computational Rationalization Studies on Regioselectivity in Testosterone Hydroxylation by Cytochrome P450 BM3 Mutants

Recently, it was found that mutations in the binding cavity of drug-metabolizing Cytochrome P450 BM3 mutants can result in major changes in regioselectivity in testosterone (TES) hydroxylation. In the current work, we report the intrinsic reactivity of TES' C-H bonds and our attempts to rationalize experimentally observed changes in TES hydroxylation using a protein structure-based in silico approach, by setting up and employing a combined Molecular Dynamics (MD) and ligand docking approach to account for the flexibility and plasticity of BM3 mutants. Using this approach, about 100,000 TES binding poses were obtained per mutant. The predicted regioselectivity in TES hydroxylation by the mutants was found to be in disagreement with experiment. As revealed in a detailed structural analysis of the obtained docking poses, this disagreement is due to limitations in correctly scoring hydrogen-bonding and steric interactions with specific active-site residues, which could explain the experimentally observed trends in regioselectivity in TES hydroxylation.

Active Site Substitution A82W Improves the Regioselectivity of Steroid Hydroxylation by Cytochrome P450 BM3 Mutants As Rationalized by Spin Relaxation Nuclear Magnetic Resonance Studies

Cytochrome P450 BM3 from Bacillus megaterium is a monooxygenase with great potential for biotechnological applications. In this paper, we present engineered drug-metabolizing P450 BM3 mutants as a novel tool for regioselective hydroxylation of steroids at position 16β. In particular, we show that by replacing alanine at position 82 with a tryptophan in P450 BM3 mutants M01 and M11, the selectivity toward 16β-hydroxylation for both testosterone and norethisterone was strongly increased. The A82W mutation led to a ≤42-fold increase in V(max) for 16β-hydroxylation of these steroids. Moreover, this mutation improves the coupling efficiency of the enzyme, which might be explained by a more efficient exclusion of water from the active site. The substrate affinity for testosterone increased at least 9-fold in M11 with tryptophan at position 82. A change in the orientation of testosterone in the M11 A82W mutant as compared to the orientation in M11 was observed by T(1) paramagnetic relaxation nuclear magnetic resonance. Testosterone is oriented in M11 with both the A- and D-ring protons closest to the heme iron. Substituting alanine at position 82 with tryptophan results in increased A-ring proton-iron distances, consistent with the relative decrease in the level of A-ring hydroxylation at position 2β.