Structural rationalization of novel drug metabolizing mutants of cytochrome P450 BM3

Abstract

Three newly discovered drug metabolizing mutants of cytochrome P450 BM3 (van Vugt-Lussenburg et al., Identification of critical residues in novel drug metabolizing mutants of Cytochrome P450 BM3 using random mutagenesis, J Med Chem 2007;50:455-461) have been studied at an atomistic level to provide structural explanations for a number of their characteristics. In this study, computational methods are combined with experimental techniques. Molecular dynamics simulations, resonance Raman and UV-VIS spectroscopy, as well as coupling efficiency and substrate-binding experiments, have been performed. The computational findings, supported by the experimental results, enable structural rationalizations of the mutants. The substrates used in this study are known to be metabolized by human cytochrome P450 2D6. Interestingly, the major metabolites formed by the P450 BM3 mutants differ from those formed by human cytochrome P450 2D6. The computational findings, supported by resonance Raman data, suggest a conformational change of one of the heme propionate groups. The modeling results furthermore suggest that this conformational change allows for an interaction between the negatively charged carboxylate of the heme substituent and the positively charged nitrogen of the substrates. This allows for an orientation of the substrates favorable for formation of the major metabolite by P450 BM3.

Efficient Screening of Cytochrome P450 BM3 Mutants for Their Metabolic Activity and Diversity toward a Wide Set of Drug-Like Molecules in Chemical Space

“…2), which is in agreement with the observation that a high proportion of drugs are weak bases (Manallack, 2008). Three of the six BM3 mutants (M01, M02, and M11) that were chosen for the drug library screening have previously been demonstrated to display different metabolic activities (van Vugt- Lussenburg et al, 2007;Stjernschantz et al, 2008). The other three mutants (MT35, MT38, and MT43) were selected because they contained mutations at different residues in the active site, which was expected to result in changes in metabolic activity and diversity (Carmichael and Wong, 2001;Li et al, 2001b;Dietrich et al, 2009).…”

“…The four P450 BM3 mutants, M01, M02, M05, and M11 (see Supplemental Table S1), have previously been demonstrated to display good expression and activity toward drug-like compounds (van Vugt- Lussenburg et al, 2007;Damsten et al, 2008a,b;Stjernschantz et al, 2008) and were therefore included in the mutant library. Ten novel mutants (see Supplemental Table 1) using both M01 and M11 as template were additionally selected from an already existing library (J. S. van Leeuwen and E. Stjernschantz, unpublished results).…”

Generation of Human Chiral Metabolites of Simvastatin and Lovastatin by Bacterial CYP102A1 Mutants

“…In recent studies, several lines of evidence supporting our proposal have been reported. CYP102A1 mutants were found to generate human drug metabolites by oxidizing various drugs, including clozapine (Damsten et al, 2008), diclofenac (Damsten et al, 2008), acetaminophen (Damsten et al, 2008), 3,4-methylenedioxymethylamphetamine (Stjernschantz et al, 2008), dextromethorphan (Stjernschantz et al, 2008), phenacetin , verapamil (Sawayama et al, 2009), and astemizole (Sawayama et al, 2009). CYP102A1 mutants can also oxidize several human P450 substrates, including 7-ethoxycoumarin (Kim et al, 2008b), coumarin (Park et al, 2010), chlorzoxazone (Park et al, 2010), and resveratrol (Kim et al, 2009), to generate human metabolites.…”

Generation of the Human Metabolite Piceatannol from the Anticancer-Preventive Agent Resveratrol by Bacterial Cytochrome P450 BM3

“…In addition to the directed evolution, the use of rational design for predicting protein structure and calculating the precise molecular interactions between the substrate and active site of the enzyme can be successfully applied to the engineering of CYP102A1 to a specific medium chain p-nitrophenoxycarboxylic acid (Li et al, 2001). More recently, computational methods combined with experimental techniques including molecular dynamics simulations, resonance Raman, and UV-VIS spectroscopy, as well as coupling efficiency and substrate-binding experiments, could be successfully applied to the engineering of CYP102A1 to metabolize human CYP2D6 substrates, 3,4-methylenedioxymethylamphetamine and dextromethorphan (Stjernschantz et al, 2008).…”

“…Several mutants of Bacillus megaterium P450 BM3 (CYP102A1) generated through rational design or directed evolution could oxidize several human P450 substrates to produce authentic metabolites with higher activities (Otey et al, 2005;Yun et al, 2007 and references therein;Kim et al, 2008a;Stjernschantz et al, 2008). These recent ABBREVIATIONS: P450, cytochrome P450; HPLC, high-performance liquid chromatography; TTNs, total turnover numbers; GC-MS, gas chromatograph-mass spectrometry.…”