Role of residue 87 in substrate selectivity and regioselectivity of drug-metabolizing cytochrome P450 CYP102A1 M11

Abstract

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.

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

“…Site-directed mutants of CYP102A1 M11H at position 87 were constructed by a mutagenic polymerase chain reaction (PCR) using the Stratagene QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) using 20 complementary pairs of mutagenesis primers (Vottero et al, 2011). The mutagenic PCR was applied to a pBluescript vector containing the gene of the drug-metabolizing CYP102A1 M11H flanked by EcoR1 and BamH1 restriction sites.…”

“…Expression of the P450 CYP102A1 M11H mutants was performed by transforming competent Escherichia coli BL21 cells with the pET28ϩ vectors as described previously (Vottero et al, 2011). Proteins were purified using nickel-nitrilotriacetic acid agarose, after which P450 concentrations were determined using a carbon monoxide (CO) difference spectrum assay.…”

“…A saturation mutagenesis library with a different residue at position 87 of CYP102A1 M11H was recently created in our laboratory (Vottero et al, 2011). All 20 mutants were expressed in E. coli BL21 with pET28ϩ vectors; the P450 quantification was done by a CO difference spectrum.…”

“…As a consequence, GSH conjugates derived from these metabolites were also produced, which strongly complicates isolation of CG-4 and CG-5. We recently showed that by changing the active site residue at position 87 of CYP102A1 M11H, the regioselectivity of testosterone hydroxylation was strongly modified (Vottero et al, 2011). Therefore, in the present study, we first evaluated the effect of mutation at position 87 on the regioselectivity of CLZ metabolism to identify the most suitable biocatalyst for the bioactivation of CLZ and subsequent structural characterization of the formed GSH conjugates.…”

Stereoselective Enzymatic Synthesis of Heteroatom-Substituted Cyclopropanes

“…We note, however, the important role of position 87 in controlling diastereo-and enantioselectivity, as previously reported. 39,48,49 Given the malleability of the P411 BM3 active site observed in our studies, we expect that the variants described here can be optimized further for highly selective cyclopropanation of other, related substrates.…”