Identification of Critical Residues in Novel Drug Metabolizing Mutants of Cytochrome P450 BM3 Using Random Mutagenesis

Abstract

Previously, we've described a site-directed triple mutant of cytochrome P450 BM3 (BM3) that is able to convert various drugs (van Vugt-Lussenburg, B. M. A., et al. Biochem. Biophys. Res. Commun. 2006, 346, 810-818). In the present study, random mutagenesis was used to improve the activity of this mutant. With three generations of error-prone PCR, mutants were obtained with 200-fold increased turnover toward drug substrates dextromethorphan and 3,4-methylenedioxymethylamphetamine. The initial activities of these mutants were up to 90-fold higher than that of human P450 2D6. These highly active drug metabolizing enzymes have great potential for biotechnology. Using sequencing analysis, the mutations responsible for the increase in activity were determined. The mutations that had the greatest effects on the activity were F81I, E267V, and particularly L86I, which is not located in the active site. Computer modeling studies were used to rationalize the effects of the mutations. This study shows that random mutagenesis can be used to identify novel critical residues, and to increase our insight into P450s.

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

“…One of the CYP102A1 mutants that show high activity in drug metabolism is CYP102A1 M11H, which contains 10 different amino acid substitutions compared with wild-type CYP102A1. This CYP102A1 mutant was shown to be highly active in metabolizing various drugs to human-relevant metabolites, including reactive intermediates (van Vugt-Lussenburg et al, 2007;Damsten et al, 2008;Dragovic et al, 2010). We have recently performed a saturation mutagenesis study in which the active-site residue at position 87 was mutated to all 20 possible amino acids (Vottero et al, 2011).…”

“…In particular, mutants of the bacterial cytochrome P450 CYP102A1 (P450 BM3) are considered to have good perspective for the largescale production of human-relevant drug metabolites because this very stable enzyme possesses the highest activity ever recorded for a P450 (Ost et al, 2000). By combinations of site-directed and random mutagenesis, many CYP102A1 mutants have been obtained that are able to convert drugs and drug-like molecules to human-relevant metabolites (van Vugt-Lussenburg et al, 2007;Yun et al, 2007;Sawayama et al, 2009;Kim et al, 2011). In our previous work, four mutants of CYP102A1 were evaluated as biocatalysts for the bioactivation of several drugs to reactive intermediates (Damsten et al, 2008).…”

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].…”

Differences in Functional Clustering of Endogenous and Exogenous Substrates Between Members of the CYP1A Subfamily

“…Our results give credit to the new approach of focused directed evolution which combines site-directed mutagenesis and DNA shuffling on site-directed mutants having increased promiscuous activities [47]. It is more and more grounded that surface residues mostly control the spectrum of promiscuous activities that any enzyme exhibits.…”