Enantioselective α-Hydroxylation of 2-Arylacetic Acid Derivatives and Buspirone Catalyzed by Engineered Cytochrome P450 BM-3

Landwehr, Marco; Hochrein, Lisa M.; Otey, Christopher R.; Kasrayan, Alex; Bäckvall, Jan‐E.; Arnold, Frances H.

doi:10.1021/ja061261x

Cited by 149 publications

(102 citation statements)

References 35 publications

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“…These mutants have a small and apolar residue in position 87. This seems consistent with the previous hypothesis that replacement of the bulky Phe87 in wild-type CYP102A1 by smaller amino acids creates space for the bulky substrates that allows for better positioning with respect to the activated oxygen species, resulting in higher activities and coupling efficiencies (Carmichael and Wong, 2001;Li et al, 2001;Landwehr et al, 2006). However, in the present study relatively high activities were also found in the CYP102A1 M11H mutants containing the relatively bulky amino acids Phe87, Tyr87, and Trp87.…”

Section: Discussionsupporting

confidence: 93%

Role of Residue 87 in the Activity and Regioselectivity of Clozapine Metabolism by Drug-Metabolizing CYP102A1 M11H: Application for Structural Characterization of Clozapine GSH Conjugates

Rea¹,

Dragović²,

Boerma³

et al. 2011

Drug Metab Dispos

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ABSTRACT:In the present study, a site-saturation mutagenesis library of drugmetabolizing CYP102A1 M11H with all 20 amino acids at position 87 was applied as a biocatalyst for the production of stable and reactive metabolites of clozapine. Clozapine is an atypical antipsychotic drug in which formation of reactive metabolites is considered to be responsible for several adverse drug reactions. Reactive intermediates of clozapine can be inactivated by GSH to multiple GSH conjugates by nonenzymatic and glutathione transferase (GST)-mediated conjugation reactions. The structures of several GST-dependent metabolites have not yet been elucidated unequivocally. The present study shows that the nature of the amino acid at position 87 of CYP102A1 M11H strongly determines the activity and regioselectivity of clozapine metabolism. Some mutants showed preference for N-demethylation and N-oxidation, whereas others showed high selectivity for bioactivation to reactive intermediates. The mutant containing Phe87 showed high activity and high selectivity for the bioactivation pathway and was used for the large-scale production of GST-dependent GSH conjugates by incubation in the presence of recombinant human GST P1-1. Five human-relevant GSH adducts were produced at high levels, enabling structural characterization by 1 H NMR. This work shows that drug-metabolizing CYP102A1 mutants, in combination with GSTs, are very useful tools for the generation of GSH conjugates of reactive metabolites of drugs to enable their isolation and structural elucidation.

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Section: Discussionsupporting

confidence: 93%

Role of Residue 87 in the Activity and Regioselectivity of Clozapine Metabolism by Drug-Metabolizing CYP102A1 M11H: Application for Structural Characterization of Clozapine GSH Conjugates

Rea¹,

Dragović²,

Boerma³

et al. 2011

Drug Metab Dispos

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“…These substrates include aromatics (Carmichael and Wong, 2001;Li et al, 2001a;Whitehouse et al, 2008), alkanes (Appel et al, 2001;Peters et al, 2003;Kubo et al, 2006), hydrocarbons (Seifert et al, 2009), and carboxylic acids (Munzer et al, 2005). Moreover, van Vugt-Lussenburg et al (2007) and others (Landwehr et al, 2006;Otey et al, 2006;Kim et al, 2008Kim et al, , 2009Kim et al, , 2011Rentmeister et al, 2009;Sawayama et al, 2009) have shown that P450 BM3 mutants can also be used for the production of both human relevant and BM3 unique drug metabolites. A study recently published by Sawayama et al (2009) describes the drug-metabolizing potential of a panel of 120 BM3 mutants and demonstrated that this panel could produce both human relevant and BM3 unique metabolites for the two marketed drugs, verapamil and astemizole (Sawayama et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

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

Reinen¹,

Leeuwen²,

Li³

et al. 2011

Drug Metab Dispos

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ABSTRACT: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.

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“…Nature has demonstrated the 'evolvability' of this bit of protein that binds an iron-heme (along with its various electron-transfer partners), and we spent several happy years creating new versions of the bacterial enzyme that could mimic known functions of other (e.g. human) P450 family members [3,4]. We could also extend its function beyond what was known to be catalyzed by P450s, making, for example, versions that hydroxylate gaseous alkanes (propane and ethane), transformations thought to lie in the functional realm of the methane monooxygenase family [5].…”

Section: Recent Research Contributions To Creating New Enzymesmentioning

confidence: 99%

Expanding the Enzyme Universe Through a Marriage of Chemistry and Evolution

Arnold¹

2014

New Chemistry and New Opportunities From the Expanding Protein Universe

Self Cite

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Present state of research on expanding enzyme catalysis beyond natureFor more than twenty years this laboratory has used directed evolution to modify enzymes. It is now widely accepted that directed evolution can change substrate specificity or reaction selectivity in desired ways, even if it sometimes remains difficult in practice. It is no longer surprising that enzymes adapt readily by accumulating beneficial mutations. And why should it be, since this is how nature tailors them for myriad biological roles? More difficult to grasp is how nature discovers new enzyme functions, particularly new catalytic activities. We know that the biological world's diverse catalytic repertoire is the product of evolution by natural selection, but we have little understanding of how nature's tinkering generates new functions. Sometimes we are (un)lucky enough to catch them in the act-e.g. the acquisition of antibiotic resistance or the ability to degrade man-made toxins. But for the vast majority of activities, the fossil record is nonexistent or too sparse to tell the molecular story. This leaves us without much guidance for evolving new enzymes in the laboratory. Consequently we are forced to contaminate our evolution experiments with knowledge-e.g. computational design [1,2]-in order to jumpstart the discovery process.If every bad catalyst could become a good one by directed evolution, part of the problem would be solved. We would be able to take any catalytic antibody or computationally-designed enzyme (or bovine serum albumin) and convert it into a great catalyst with multiple rounds of random mutagenesis and screening. We have learned the hard way, however, that not every bad catalyst lies at the base of a tall fitness peak, at least one that can be scaled by a random uphill walk. We therefore want to know what features make a protein with a new catalytic activity the potential mother of a whole new enzyme family. And, what are good ways to find new enzyme activities in the first place? Recent research contributions to creating new enzymesMy laboratory has been directing the evolution of a remarkable enzyme, a bacterial cytochrome P450, for at least a dozen years. This particular P450, from Bacillus megaterium, is quite specific-it catalyzes subterminal hydroxylation of fatty acids. As a family, however, the P450s are wonderfully diverse, catalyzing a wide range of reactions

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Enantioselective α-Hydroxylation of 2-Arylacetic Acid Derivatives and Buspirone Catalyzed by Engineered Cytochrome P450 BM-3

Abstract: Here we report that an engineered microbial cytochrome P450 BM-3 (CYP102A subfamily) efficiently catalyzes the alpha-hydroxylation of phenylacetic acid esters. This P450 BM-3 variant also produces the authentic human metabolite of buspirone, R-6-hydroxybuspirone, with 99.5% ee.

Cited by 149 publications

References 35 publications

Role of Residue 87 in the Activity and Regioselectivity of Clozapine Metabolism by Drug-Metabolizing CYP102A1 M11H: Application for Structural Characterization of Clozapine GSH Conjugates

Role of Residue 87 in the Activity and Regioselectivity of Clozapine Metabolism by Drug-Metabolizing CYP102A1 M11H: Application for Structural Characterization of Clozapine GSH Conjugates

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

Expanding the Enzyme Universe Through a Marriage of Chemistry and Evolution

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