CYP102A1, originating from Bacillus megaterium, is a highly active enzyme which has attracted much attention because of its potential applicability as a biocatalyst for oxidative reactions. Previously we developed drug-metabolizing mutant CYP102A1 M11 by a combination of site-directed and random mutagenesis. CYP102A1 M11 contains eight mutations, when compared with wild-type CYP102A1, and is able to produce human-relevant metabolites of several pharmaceuticals. In this study, active-site residue 87 of drug-metabolizing mutant CYP102A1 M11 was mutated to all possible natural amino acids to investigate its role in substrate selectivity and regioselectivity. With alkoxyresorufins as substrates, large differences in substrate selectivities and coupling efficiencies were found, dependent on the nature of residue 87. For all combinations of alkoxyresorufins and mutants, extremely fast rates of NADPH oxidation were observed (up to 6,000 min−1). However, the coupling efficiencies were extremely low: even for the substrates showing the highest rates of O-dealkylation, coupling efficiencies were lower than 1%. With testosterone as the substrate, all mutants were able to produce three hydroxytestosterone metabolites, although with different activities and with remarkably different product ratios. The results show that the nature of the amino acid at position 87 has a strong effect on activity and regioselectivity in the drug-metabolizing mutant CYP102A1 M11. Because of the wide substrate selectivity of CYP102A1 M11 when compared with wild-type CYP102A1, this panel of mutants will be useful both as biocatalysts for metabolite production and as model proteins for mechanistic studies on the function of P450s in general.

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.

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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.

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.