Increase of human CYP1B1 activities by acidic phospholipids and kinetic deuterium isotope effects on CYP1B1 substrate oxidation.

The effect of phospholipids on the kinetic parameters of three substrates, 7-ethoxy-4 -(trifluoromethyl)coumarin (7-EFC), 7-ethoxycoumarin (7-EC) and 17ß-estradiol (E(2)), of human CYP1B1 was studied. In general, anionic phospholipids, phosphatidic acid and cardiolipin increased catalytic efficiency by increasing k(cat) values or decreasing K(m) values. The advantages of using the 7-EFC as a substrate over 7-EC and E(2) include high k(cat), low K(m) and high catalytic efficiency. Spectral binding titrations indicated that the binding affinity of 7-EFC to CYP1B1 in the presence or absence of phospholipids is higher than that of 7-EC or E(2). Furthermore, phosphatidylcholine increased the binding affinity of the substrates to the CYP1B1. High non-competitive intermolecular kinetic deuterium isotope effects (values 5.4-12) were observed for O-deethylation of 7-EFC and 7-EC with deuterium substitution at the ethoxy group, indicating that the C-H bond-breaking step makes a major contribution to the rate of these CYP1B1-catalyzed reactions. However, the intermolecular kinetic deuterium isotope effect is ~2 for the E(2) 4-hydroxylation reaction, indicating that the C-H bond-breaking step contributes only partially to the rate of this CYP1B1-catalyzed reaction. These results indicate that the reaction mechanism of CYP1B1-catalyzed reactions is distinct for each substrate.

The Flavin-Containing Reductase Domain of Cytochrome P450 BM3 Acts as a Surrogate for Mammalian NADPH-P450 Reductase.

Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium is a self-sufficient monooxygenase that consists of a heme domain and FAD/FMN-containing reductase domain (BMR). In this report, the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) by BMR was evaluated as a method for monitoring BMR activity. The electron transfer proceeds from NADPH to BMR and then to BMR substrates, MTT and CTC. MTT and CTC are monotetrazolium salts that form formazans upon reduction. The reduction of MTT and CTC followed classical Michaelis-Menten kinetics (kcat =4120 min(-1), Km =77 µM for MTT and kcat =6580 min(-1), Km =51 µM for CTC). Our continuous assay using MTT and CTC allows the simple, rapid measurement of BMR activity. The BMR was able to metabolize mitomycin C and doxorubicin, which are anticancer drug substrates for CPR, producing the same metabolites as those produced by CPR. Moreover, the BMR was able to interact with CYP1A2 and transfer electrons to promote the oxidation reactions of substrates by CYP1A2 and CYP2E1 in humans. The results of this study suggest the possibility of the utilization of BMR as a surrogate for mammalian CPR.

Generation of human chiral metabolites of simvastatin and lovastatin by bacterial CYP102A1 mutants.

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 CYP3A4/5 to produce several metabolites, including 6'ß-hydroxy (OH), 3″-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'ß-OH) and one minor product (6'-exomethylene), but not other products, were produced by CYP102A1 mutants. Formation of the metabolites was confirmed by high-performance liquid chromatography, liquid chromatography-mass spectroscopy, 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 active sites of the mutants. 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.