P450 BM3 Mutants Research Articles

Application of CYP102A1M11H as a Tool for the Generation of Protein Adducts of Reactive Drug Metabolites

Covalent binding of reactive metabolites (RMs) to proteins is considered to be one of the important mechanisms by which drugs can cause tissue damage. To facilitate the study of drug-protein adducts, we developed a potentially generic method for producing high levels of covalently modified proteins. A highly active drug metabolizing P450 BM3 mutant (CYP102A1M11H) is used for drug bioactivation. Because of its His-tag, CYP102A1M11H is easily removed by nickel affinity chromatography, facilitating subsequent characterization of the modified target protein. The applicability of our procedure is demonstrated by the trapping of RMs of acetaminophen (APAP), clozapine (CLOZ), and troglitazone (TGZ) with human glutathione-S-transferase P1-1 (hGST P1-1) as the model target protein. Tryptic digests of hGST P1-1 were subjected to analysis by LC-MS/MS and modified peptides identified by the comparative analysis of tryptic peptides of adducted and nonadducted hGST P1-1. Characteristic MS/MS ions of drug-modified peptides were identified by first searching for expected adduct-masses. Unanticipated drug-peptide adducts were subsequently identified in an unbiased manner by screening for diagnostic MS/MS ions of modified peptides. Reactive intermediates of APAP and CLOZ adducted to cysteine-47 and mass shifts corresponded to the alkylation of N-acetyl-p-benzoquinone imine (NAPQI) and the CLOZ nitrenium ion, respectively. Adduction of TGZ appeared more complex, yielding three different types of adducts to cysteine-47, two adducts to cysteine-14, and a single adduct to cysteine-101. Together, these findings show that P450 BM3 mutants with high capacity to activate drugs into relevant RMs can be employed to produce protein adducts to study the nucleophilic selectivity of highly reactive electrophiles.

Efficient Screening of Cytochrome P450 BM3 Mutants for Their Metabolic Activity and Diversity toward a Wide Set of Drug-Like Molecules in Chemical Space

In the present study, the diversity of a library of drug-metabolizing bacterial cytochrome P450 (P450) BM3 mutants was evaluated by a liquid chromatography-mass spectrometry (LC-MS)-based screening method. A strategy was designed to identify a minimal set of BM3 mutants that displays differences in regio- and stereoselectivities and is suitable to metabolize a large fraction of drug chemistry space. We first screened the activities of six structurally diverse BM3 mutants toward a library of 43 marketed drugs (encompassing a wide range of human P450 phenotypes, cLogP values, charges, and molecular weights) using a rapid LC-MS method with an automated method development and data-processing system. Significant differences in metabolic activity were found for the mutants tested and based on this drug library screen; nine structurally diverse probe drugs were selected that were subsequently used to study the metabolism of a library of 14 BM3 mutants in more detail. Using this alternative screening strategy, we were able to select a minimal set of BM3 mutants with high metabolic activities and diversity with respect to substrate specificity and regiospecificity that could produce both human relevant and BM3 unique drug metabolites. This panel of four mutants (M02, MT35, MT38, and MT43) was capable of producing P450-mediated metabolites for 41 of the 43 drugs tested while metabolizing 77% of the drugs by more than 20%. We observed this as the first step in our approach to use of bacterial P450 enzymes as general reagents for lead diversification in the drug development process and the biosynthesis of drug(-like) metabolites.

First steps towards a Zn/Co(III)sep-driven P450 BM3 reactor

Cytochrome P450s are synthetically attractive hydroxylation catalysts. For cell-free applications, a constant supply of NAD(P)H can be very costly. Mediators such as Zn/Co(III)sep can be an alternative cofactor system to NAD(P)H. Several mutants of cytochrome P450 BM3 with improved electron transfer rate to Zn/Co(III)sep have been obtained in our group. P450 BM3 M7 (F87A V281G M354S R471C A1011T S1016G Q1022R) was immobilized on DEAE-650S, further entrapped with k-carrageenan together with zinc dust which function as electron source and catalase which removes produced hydrogen peroxide instantly. Immobilized P450 BM3 M7 were treated with 0.05% (v/v) glutaraldehyde to enhance operational stability. P450 BM3 M7 retained around 76% of its activity and conversions stayed above 80% in 10 batch cycles, indicating a high stability of immobilized P450 BM3 M7. To explore the synthetic potential, a small-scale bioreactor was developed to investigate the stability and efficiencies of P450 BM3 M9 (R47F F87A M238K V281G M354S D363H W575C A595T). P450 BM3 M9 was used for the continuous conversion of 3-phenoxytoluene in a plug flow reactor (PFR) since P450 BM3 M9 has a 3-fold higher activity for 3-phenoxytoluene compared to P450 BM3 M7 which was used for optimizing immobilization conditions with the highest activity for 12-pNCA assay. The reactor could be operated for 5 days with total turnover numbers (TTNs) over 2,000.

High temperature liquid chromatography hyphenated with ESI-MS and ICP-MS detection for the structural characterization and quantification of halogen containing drug metabolites

In this paper we describe the hyphenation of high temperature liquid chromatography with ICP-MS and ESI-MS for the characterization of halogen containing drug metabolites. The use of temperature gradients up to 200 °C enabled the separation of metabolites with low organic modifier content. This specific property allowed the use of detection methods that suffer from (significant) changes in analyte response factors as a function of the organic modifier content such as ICP-MS. Metabolites of two kinase inhibitors (SB-203580-Iodo and MAPK inhibitor VIII) produced by bacterial cytochrome P450 BM3 mutants and human liver microsomes were identified based on high resolution MS n data. Quantification was done using their normalized and elemental specific response in the ICP-MS. The importance of these kinds of quantification strategies is stressed by the observation that the difference of the position of one oxygen atom in a structure can greatly affect its response in ESI-MS and UV detection.

Process Optimization for the Electroenzymatic Hydroxylation of 3-Phenoxytoluene Catalyzed by P450 BM-3 Mutant

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Application of a Fluorescence-Based Continuous-Flow Bioassay to Screen for Diversity of Cytochrome P450 BM3 Mutant Libraries

A fluorescence-based continuous-flow enzyme affinity detection (EAD) setup was used to screen cytochrome P450 BM3 mutants on-line for diversity. The flow-injection screening assay is based on the BM3-mediated O-dealkylation of alkoxyresorufins forming the highly fluorescent product resorufin, and can be used in different configurations, namely injection of ligands, enzymes and substrates. Screening conditions were optimized and the activity of a library of 32 BM3 mutants towards the recently synthesized new probe substrate allyloxyresorufin was measured in flow-injection analysis (FIA) mode and it was shown that large activity differences between the mutants existed. Next, six BM3 mutants containing mutations at different positions in the active site were selected for which on-line enzyme kinetics were determined. Subsequently, for these six BM3 mutants affinity towards a set of 30 xenobiotics was determined in FIA EAD mode. It was demonstrated that significant differences existed for the affinity profiles of the mutants tested and that these differences correlated to alterations in the BM3 mutant-generated metabolic profiles of the drug buspirone. In conclusion, the developed FIA EAD approach is suitable to screen for diversity within BM3 mutants and this alternative screening technology offers new perspectives for rapid and sensitive screening of compound libraries towards BM3 mutants.

Control of the stereo-selectivity of styrene epoxidation by cytochrome P450 BM3 using structure-based mutagenesis

The potential of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium for biocatalysis and biotechnological application is widely acknowledged. The catalytic and structural analysis of the Ala82Phe mutant of P450 BM3 has shown that filling a hydrophobic pocket near the active site improved the binding of small molecules, such as indole (see Huang et al., J. Mol. Biol., 2007, 373, 633) and styrene. In this paper, additional mutations at Thr438 are shown to decrease the binding of and catalytic activity towards laurate, whereas they significantly increased the stereo-specificity of styrene epoxidation. Production of R-styrene oxide with 48% and 64% e.e., respectively, was achieved by the Ala82Phe-Thr438Leu and Ala82Phe-Thr438Phe mutants. These structure-based mutants of P450 BM3 illustrate the promise of rational design of synthetically useful biocatalysts for regio- and stereo- specific mono-oxygenation reactions.

Analysis of the oxidation of short chain alkynes by flavocytochrome P450 BM3

Bacillus megaterium flavocytochrome P450 BM3 (BM3) is a high activity fatty acid hydroxylase, formed by the fusion of soluble cytochrome P450 and cytochrome P450 reductase modules. Short chain (C6, C8) alkynes were shown to be substrates for BM3, with productive outcomes (i.e. alkyne hydroxylation) dependent on position of the carbon-carbon triple bond in the molecule. Wild-type P450 BM3 catalyses ω-3 hydroxylation of both 1-hexyne and 1-octyne, but is suicidally inactivated in NADPH-dependent turnover with non-terminal alkynes. A F87G mutant of P450 BM3 also undergoes turnover-dependent heme destruction with the terminal alkynes, pointing to a key role for Phe87 in controlling regioselectivity of alkyne oxidation. The terminal alkynes access the BM3 heme active site led by the acetylene functional group, since hydroxylated products are not observed near the opposite end of the molecules. For both 1-hexyne and 1-octyne, the predominant enantiomeric product formed (up to ∼90%) is the (S)-(-)-1-alkyn-3-ol form. Wild-type P450 BM3 is shown to be an effective oxidase catalyst of terminal alkynes, with strict regioselectivity of oxidation and potential biotechnological applications. The absence of measurable octanoic or hexanoic acid products from oxidation of the relevant 1-alkynes is also consistent with previous studies suggesting that removal of the phenyl group in the F87G mutant does not lead to significant levels of ω-oxidation of alkyl chain substrates.

Screening of a minimal enriched P450 BM3 mutant library for hydroxylation of cyclic and acyclic alkanes

A minimal enriched P450 BM3 library was screened for the ability to oxidize inert cyclic and acyclic alkanes. The F87A/A328V mutant was found to effectively hydroxylate cyclooctane, cyclodecane and cyclododecane. F87V/A328F with high activity towards cyclooctane hydroxylated acyclic n-octane to 2-(R)-octanol (46% ee) with high regioselectivity (92%).

Metabolism related toxicity of diclofenac in yeast as model system

Diclofenac is a widely used drug that can cause serious hepatotoxicity, which has been linked to metabolism by cytochrome P450s (P450). To investigate the role of oxidative metabolites in diclofenac toxicity, a model for P450-related toxicity was set up in Saccharomyces cerevisiae. We expressed a drug-metabolizing mutant of cytochrome P450 BM3 (BM3 M11) in yeast. Importantly, BM3 M11 yielded similar oxidative metabolite profiles of diclofenac as human P450s. It was found that yeast strains expressing BM3 M11 grew significantly slower when exposed to diclofenac than strains without BM3 M11. Furthermore, the amount of reactive oxygen species (ROS) after incubation with diclofenac was higher in strains expressing BM3 M11 than in strains without this enzyme, confirming that P450 activity increases diclofenac toxicity. Interestingly, 4′- and 5-hydroxydiclofenac had no effect on cell growth or ROS formation in cells expressing BM3 M11, although hydroxydiclofenac-derived quinone imines were identified in these strains by detection of their glutathione conjugates. This suggests that 4′- and 5-hydroxydiclofenac, as well as their quinone imines, are not involved in toxicity in yeast. Rather, the P450-related toxicity of diclofenac is caused by primary metabolites such as arene oxides resulting in hydroxydiclofenac or radical species formed during decarboxylation.

Application of cytochrome P450 BM3 mutants as biocatalysts for the profiling of estrogen receptor binding metabolites of the mycotoxin zearalenone

The estrogenic mycotoxin zearalenone (ZEN) can undergo hepatic reductive metabolism to form the estrogenic α and β isomers of zearalenol. ZEN also undergoes cytochrome P450 monooxygenase (P450)-mediated oxidative metabolism to form monohydroxylated products, but until now nothing is known about the estrogenic potency of these metabolites.This study aimed at investigating the metabolism of ZEN by different P450 isoforms and to determine the estrogen receptor α (ERα) affinities of the in vitro P450-generated ZEN metabolites in an online high-resolution screening (HRS) setup. Human liver microsomes (HLM), recombinant P450s, and mutants of the bacterial P450 BM3 were used to investigate the oxidative metabolism of ZEN.It was shown that mutants of the bacterial P450 BM3 could be used to produce the human relevant 13- and 15-OH-ZEN catechol metabolites at such levels that their ERα affinity could be determined in an HRS setup, which was not possible with HLM. It was demonstrated that P450-mediated hydroxylation at the 13 and 15 positions of ZEN resulted in a loss of ERα affinity.The approach presented here can be used for the elucidation of the metabolism of other endocrine disrupting compounds and xenobiotics to get clear pictures of the total effects of these compounds and their metabolites.

Flavocytochrome P450 BM3 mutant W1046A is a NADH-dependent fatty acid hydroxylase: Implications for the mechanism of electron transfer in the P450 BM3 dimer

Bacillus megaterium P450 BM3 (BM3) is a P450/P450 reductase fusion enzyme, where the dimer is considered the active form in NADPH-dependent fatty acid hydroxylation. The BM3 W1046A mutant was generated, removing an aromatic “shield” from its FAD isoalloxazine ring. W1046A BM3 is a catalytically active NADH-dependent lauric acid hydroxylase, with product formation slightly superior to the NADPH-driven enzyme. The W1046A BM3 K m for NADH is 20-fold lower than wild-type BM3, and catalytic efficiency of W1046A BM3 with NADH and NADPH are similar in lauric acid oxidation. Wild-type BM3 also catalyzes NADH-dependent lauric acid hydroxylation, but less efficiently than W1046A BM3. A hypothesis that W1046A BM3 is inactive [15] helped underpin a model of electron transfer from FAD in one BM3 monomer to FMN in the other in order to drive fatty acid hydroxylation in native BM3. Our data showing W1046A BM3 is a functional fatty acid hydroxylase are consistent instead with a BM3 catalytic model involving electron transfer within a reductase monomer, and from FMN of one monomer to heme of the other [12]. W1046A BM3 is an efficient NADH-utilizing fatty acid hydroxylase with potential biotechnological applications.

Role of Human Glutathione S-Transferases in the Inactivation of Reactive Metabolites of Clozapine

The conjugation of reactive drug metabolites to GSH is considered an important detoxification mechanism that can be spontaneous and/or mediated by glutathione S-transferases (GSTs). In case GSTs play an important role in GSH conjugation, genetically determined deficiencies in GSTs may be a risk factor for adverse drug reactions (ADRs) resulting from reactive drug metabolites. So far, the role of GSTs in the detoxification of reactive intermediates of clozapine, a drug-causing idiosyncratic drug reactions (IDRs), has not been studied. In the present study, we studied the ability of four recombinant human GSTs (hGST A1-1, hGST M1-1, hGST P1-1, and hGST T1-1) to catalyze the GSH conjugation of reactive metabolites of clozapine, formed in vitro by human and rat liver microsomes and drug-metabolizing P450 BM3 mutant, P450 102A1M11H. Consistent with previous studies, in the absence of GSTs, three GSH conjugates were identified derived from the nitrenium ion of clozapine. In the presence of three of the GSTs, hGST P1-1, hGST M1-1, and hGST A1-1, total GSH conjugation was strongly increased in all bioactivation systems tested. The highest activity was observed with hGST P1-1, whereas hGST M1-1 and hGST A1-1 showed slightly lower activity. Polymorphic hGST T1-1 did not show any activity in catalyzing GSH conjugation of reactive clozapine metabolites. Interestingly, the addition of hGSTs resulted in major changes in the regioselectivity of GSH conjugation of the reactive clozapine metabolite, possibly due to the different active site geometries of hGSTs. Two GSH conjugates found were completely dependent on the presence of hGSTs. Chlorine substitution of the clozapine nitrenium ion, which so far was only observed in in vivo studies, appeared to be the major pathway of hGST P1-1-catalyzed GSH conjugation, whereas hGST A1-1 and hGST M1-1 also showed significant activity. The second GSH conjugate, previously also only found in in vivo studies, was also formed by hGST P1-1 and to a small extent by hGST A1-1. These results demonstrate that human GSTs may play a significant role in the inactivation of reactive intermediates of clozapine. Therefore, further studies are required to investigate whether genetic polymorphisms of hGST P1-1 and hGST M1-1 contribute to the interindividual differences in susceptibility to clozapine-induced adverse drug reactions.

Engineering Bacterial Cytochrome P450 (P450) BM3 into a Prototype with Human P450 Enzyme Activity Using Indigo Formation

Human cytochrome P450 (P450) enzymes metabolize a variety of endogenous and xenobiotic compounds, including steroids, drugs, and environmental chemicals. In this study, we examine the possibility that bacterial P450 BM3 (CYP102A1) mutants with indole oxidation activity have the catalytic activities of human P450 enzymes. Error-prone polymerase chain reaction was carried out on the heme domain-coding region of the wild-type gene to generate a CYP102A1 DNA library. The library was transformed into Escherichia coli for expression of the P450 mutants. A colorimetric colony-based method was adopted for primary screening of the mutants. When the P450 activities were measured at the whole-cell level, some of the blue colonies, but not the white colonies, possessed apparent oxidation activity toward coumarin and 7-ethoxycoumarin, which are typical human P450 substrates that produce fluorescent products. Coumarin is oxidized by the CYP102A1 mutants to produce two metabolites, 7-hydroxycoumarin and 3-hydroxycoumarin. In addition, 7-ethoxycoumarin is simultaneously oxidized to 7-hydroxycoumarin by O-deethylation reaction and to 3-hydroxy,7-ethoxycoumarin by 3-hydroxylation reactions. Highly active mutants are also able to metabolize several other human P450 substrates, including phenacetin, ethoxyresorufin, and chlorzoxazone. These results indicate that indigo formation provides a simple assay for identifying CYP102A1 mutants with a greater potential for human P450 activity. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward coumarin: the more stable the structure in the binding site, the lower the energy barrier and the higher the catalytic efficiency.

Altering the regioselectivity of the subterminal fatty acid hydroxylase P450 BM-3 towards γ- and δ-positions

Cytochrome P450 BM-3 monooxygenase from Bacillus megaterium (CYP102A1) catalyzes the subterminal hydroxylation of fatty acids with a chain length of 12–22 carbons. Wild-type P450 BM-3 oxidizes saturated fatty acids at subterminal positions producing a mixture of ω-1, ω-2 and ω-3 hydroxylated products. Using a rational site-directed mutagenesis approach, three new elements have been introduced into the substrate binding pocket of the monooxygenase, which greatly changed the product pattern of lauric acid hydroxylation. Particularly, substitutions at positions S72, V78 and I263 had an effect on the enzyme regioselectivity. The P450 BM-3 mutants V78A F87A I263G and S72Y V78A F87A were able to oxidize lauric acid not only at δ-position (14% and 16%, respectively), but also produced γ- and β-hydroxylated products. δ-Hydroxy lauric and γ-hydroxy lauric acid are important synthons for the production of the corresponding lactones.

Trimethoprim: Novel Reactive Intermediates and Bioactivation Pathways by Cytochrome P450s

Trimethoprim (TMP) is a widely used antibacterial agent that is usually considered as a safe drug. TMP has, however, been implicated in rare adverse drug reactions (ADRs) in humans. Bioactivation to a reactive iminoquinone methide intermediate has been proposed as a possible cause for the toxicity of the drug. However, little is known about the cytochrome P450s (P450s) involved in this bioactivation and in the metabolism of TMP in general. In this study, we have investigated the metabolism and bioactivation of TMP by human liver microsomes (HLM) and rat liver microsomes, by recombinant human cytochrome P450s, and by the bacterial P450 BM3 mutant M11(his). In addition to non GSH-dependent metabolites, five GSH adducts were identified in the HLM incubations. Next to two major GSH adducts probably originating from the iminoquinone methide intermediate described previously, three minor GSH adducts were also identified, indicating that other types of reactive intermediates are formed by HLM, such as ortho-quinones and para-quinone methide intermediates. The major GSH adducts were produced by P450 1A2 and P450 3A4, while the minor GSH adducts were mainly formed by P450 1A2, P450 3A4, and P450 2D6. Although preliminary, these results might implicate that genetic polymorphisms in P450 enzymes could play a role in the onset of TMP-related ADRs in humans.

Laboratory evolution of P450 BM3 for mediated electron transfer yielding an activity-improved and reductase-independent variant

One of the main obstacles in employing P450 monooxygenases for preparative chemical syntheses in cell-free systems is their requirement for cofactors such as NAD(P)H. In order to engineer P450 BM3 from Bacillus megaterium for cost-effective process conditions in vitro, a validated medium throughput screening system based on cheap Zn dust as an electron source and Cobalt(III)sepulchrate (Co(III)sep) as a mediator was reported. In the current study, the alternative cofactor system Zn/Co(III)sep was used in a directed evolution experiment to improve the Co(III)sep-mediated electron transfer to P450 BM3. A variant, carrying five mutations (R47F F87A V281G M354S D363H, Table I), P450 BM3 M5 was identified and characterized with respect to its kinetic parameters. P450 BM3 M5 achieved for mediated electron transfer a 2-fold higher k(cat) value and a 3-fold higher catalytic efficiency compared with the starting point mutant P450 BM3 F87A (k(cat): 62 min(-1) compared with 28 min(-1); k(cat)/K(m): 62 microM(-1)min(-1) compared to 19 microM(-1)min(-1)). For obtaining first insights on electron transfer contributions, three reductase-deficient variants were generated. The reductase-deficient mutant of P450 BMP M5 exhibited a catalytic efficiency of 69% and a k(cat) value of 89% of the values obtained for P450 BM3 M5.

Cytochrome P450 BM-3 Evolved by Random and Saturation Mutagenesis as an Effective Indole-Hydroxylating Catalyst

Cytochrome P450 BM-3 with the mutations A74G, F87V, and L188Q could catalyze indole to produce indigo and indirubin. To further enhance this capability, site-directed and random mutageneses on the monooxygenase domain of P450 BM-3 mutant (A74G/F87V/L188Q; 3X) were performed. The mutant libraries created by error-prone polymerase chain reaction were screened using a colorimetric colony-based method on agar plates followed by a spectroscopic assay involving in absorption of indigo at 670 nm and NADPH at 340 nm in microtiter plate. Three mutants (K434R/3X, E435D/3X, and D168N/ A225V/K440N/3X) exhibited higher hydroxylation activity toward indole in comparison to parent enzyme. Moreover, using saturation site-directed mutagenesis at amino acid positions 168, 225, 434, 435, and 440, two P450 BM-3 variants (D168H/3X, E435T/3X) with an up to sixfold increase in catalytic efficiency (kcat/Km) were identified, and the mutant D168H/3X acquired higher regioselectivity resulting in more indigo (dimerized 3hydroxy-indole) compared to parent mutant (93 vs 72%).

Which Oxidant Is Really Responsible for Sulfur Oxidation by Cytochrome P450?

Clarifying a conundrum: The question of whether Compound I or Compound 0 (Cpd I or Cpd 0) is the reactive oxidant in the sulfoxidation of thiafatty acids by P450 is addressed by theory, which demonstrates that Cpd I leads to an extremely fast process, while Cpd 0 is at least six orders of magnitude slower. Most likely, thiafatty acids promote Cpd I formation even in the T→A mutant of P450BM3. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2007/z702867_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Structural rationalization of novel drug metabolizing mutants of cytochrome P450 BM3

Three newly discovered drug metabolizing mutants of cytochrome P450 BM3 (van Vugt-Lussenburg et al., Identification of critical residues in novel drug metabolizing mutants of Cytochrome P450 BM3 using random mutagenesis, J Med Chem 2007;50:455-461) have been studied at an atomistic level to provide structural explanations for a number of their characteristics. In this study, computational methods are combined with experimental techniques. Molecular dynamics simulations, resonance Raman and UV-VIS spectroscopy, as well as coupling efficiency and substrate-binding experiments, have been performed. The computational findings, supported by the experimental results, enable structural rationalizations of the mutants. The substrates used in this study are known to be metabolized by human cytochrome P450 2D6. Interestingly, the major metabolites formed by the P450 BM3 mutants differ from those formed by human cytochrome P450 2D6. The computational findings, supported by resonance Raman data, suggest a conformational change of one of the heme propionate groups. The modeling results furthermore suggest that this conformational change allows for an interaction between the negatively charged carboxylate of the heme substituent and the positively charged nitrogen of the substrates. This allows for an orientation of the substrates favorable for formation of the major metabolite by P450 BM3.