Generation of Human Metabolites of 7-Ethoxycoumarin by Bacterial Cytochrome P450 BM3

Abstract

ABSTRACT:Recently, wild-type and mutant forms of bacterial cytochrome P450 BM3 (CYP102A1) have been found to metabolize various drugs through reactions similar to those catalyzed by human cytochromes P450 (P450s). Therefore, it has been suggested that CYP102A1 may be used to produce large quantities of the metabolites of human P450-catalyzed reactions. In this report, we show that the oxidation of 7-ethoxycoumarin, a typical human P450 substrate, is catalyzed by both wild-type and mutant forms of CYP102A1. Two major products were produced as a result of O-deethylation and 3-hydroxylation reactions. These results demonstrate that CYP102A1 mutants catalyze the same reactions as human P450s. High noncompetitive intermolecular kinetic deuterium isotope effects were observed for 7-ethoxycoumarin O-deethylation in the CYP102A1 system. These results suggest that there is a common mechanism for the oxidation reactions catalyzed by both the bacterial CYP102A1 and human P450 enzymes.

Regioselective Hydroxylation of Omeprazole Enantiomers by Bacterial CYP102A1 Mutants

“…The mutants were selected based on their activities toward a variety of human P450 substrates and drugs. Mutants 1-17 have mutations in the substrate channel and active site (Kim et al, 2008), and mutants 18-26 have mutations outside of the active site and substrate channel (Park et al, 2010). Each mutant bears amino acid substitution(s) relative to wild-type CYP102A1, as summarized in Supplemental Table 1.…”

“…Wild-type and 26 different mutants of CYP102A1 used in this study were prepared as previously described (Kim et al, 2008;Park et al, 2010). The mutants were selected based on their activities toward a variety of human P450 substrates and drugs.…”

“…Wild-type and 26 mutants of CYP102A1 were expressed in the Escherichia coli strain DH5aF9-IQ and purified as previously described (Kim et al, 2008;Park et al, 2010). The CYP102A1 concentrations were determined by CO difference spectra using « = 91 mM 21 /cm…”

“…Spectral binding titrations were used to determine the dissociation constants (K s ) for substrates as previously described (Kim et al, 2008). The binding affinities of the ligands to the CYP102A1 enzymes were determined at 23°C by titrating 1.5 mM enzyme with the ligand in a total volume of 1.0 ml of 100 mM potassium phosphate buffer (pH 7.4).…”

Subtle structural changes in the Asp251Gly/Gln307His P450 BM3 mutant responsible for new activity toward diclofenac, tolbutamide and ibuprofen

“…The direct use of human cytochrome P450 is limited by the need of a redox partner and the poor stability and activity. However, bacterial counterparts such as cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium, can be directly used [3] or engineered to metabolise drugs and to produce the same metabolites as the human enzymes [4][5][6][7][8][9][10][11][12][13][14]. This protein is a selfsufficient fatty acids monoxygenase containing a NADPH-dependent reductase (BMR) and a P450 catalytic domain (BMP) fused in a single polypeptidic chain [15,16].…”