Abstract
Recently, the wild-type and mutant forms of cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium were found to oxidize various xenobiotic substrates, including pharmaceuticals, of human P450 enzymes. Simvastatin and lovastatin, which are used to treat hyperlipidemia and hypercholesterolemia, are oxidized by human P450 3A4/5 to produce several metabolites, including 6′β-OH, 3″β-OH and exomethylene products. In this report, we show that the oxidation of simvastatin and lovastatin was catalyzed by wild-type CYP102A1 and a set of its mutants, which were generated by site-directed and random mutagenesis. One major hydroxylated product (6″β-OH) and one minor product (6′-exomethylene), but not other products, were produced by CYP102A1 mutants. Formation of the metabolites was confirmed by HPLC, LC-MS, and NMR. Chemical methods to synthesize the metabolites of simvastatin and lovastatin have not been reported. These results demonstrate that CYP102A1 mutants can be used to produce human metabolites, especially chiral metabolites, of simvastatin and lovastatin. Our computational findings suggest that a conformational change in the cavity of the mutant active sites is related to the activity change. The modeling results also suggest that the activity change results from the movement of several specific residues in the mutants' active sites. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward simvastatin and lovastatin.
- cytochrome P450
- cytochrome P450 catalyzed oxidations
- cytochrome P450 function
- human CYP enzymes
- metabolite kinetics
- Received September 20, 2010.
- Accepted October 20, 2010.
- The American Society for Pharmacology and Experimental Therapeutics