Molecular Determinants of Substrate Affinity and Enzyme Activity of a Cytochrome P450BM3 Variant

Abstract

Cytochrome P450 catalyzes the hydroxylation and/or epoxidation of fatty acids, fatty amides, and alcohols. Protein engineering has produced P450 variants capable of accepting drug molecules normally metabolized by human P450 enzymes. The enhanced substrate promiscuity has been attributed to the greater flexibility of the lid of the substrate channel. However, it is not well understood how structurally different and highly polar drug molecules can stably bind in the active site nor how the activity and coupling efficiency of the enzyme may be affected by the lack of enzyme-substrate complementarity. To address these important aspects of non-native small molecule binding, this study investigated the binding of drug molecules with different size, charge, polar surface area, and human P450 affinity on the promiscuous R47L/F87V/L188Q/E267V/F81I pentuple mutant of P450. Binding free energy data and energy decomposition analysis showed that pentuple mutant P450 stably binds (i.e., negative ΔG°) a broad range of substrate and inhibitor types because dispersion interactions with active site residues overcome unfavorable repulsive and electrostatic effects. Molecular dynamics simulations revealed that 1) acidic substrates tend to disrupt the heme propionate A-K69 salt bridge, which may reduce heme oxidizing ability, and 2) the lack of complementarity leads to high substrate mobility and water density in the active site, which may lead to uncoupling. These factors must be considered in future developments of P450 as a biocatalyst in the large-scale production of drug metabolites.

Production of metabolites of the anti-cancer drug noscapine using a P450BM3 mutant library

“…Mutation of Leu75 to a smaller residue, such as alanine, has been shown to improve activity on several drug-like compounds [52,53]; these mutations were not tested here and may improve noscapine activity. Recent molecular dynamics simulations suggest, however, that hydrophobic interactions between Leu75 and some drug substrates are important contributors to binding stability [54], making the interactions between this residue and noscapine difficult to predict.…”

Choose Your Own Adventure: A Comprehensive Database of Reactions Catalyzed by Cytochrome P450 BM3 Variants

“…P450 BM3 naturally hydroxylates long-chain fatty acids, such as lauric acid. However, enzyme engineering has expanded the range of relevant substrates and reactions. − Selective hydroxylation is a desirable feature for synthesizing fine chemicals, including chiral compounds used in flavorings, fragrances, and pharmaceuticals, as well as for developing new antibiotics and drug precursors. , In recent years, enzyme engineering has broadened its reactivity to include various oxidation reactions, as well as carbene, , and nitrene transfer reactions, which are new-to-nature reactions. ,, …”

“…substitutions are included in the database. Positions 75,78,82,87,188,263,268, and 328 satisfy these criteria. Positions 75 and 78 (B′-Helix).…”

Electrochemical transformations catalyzed by cytochrome P450s and peroxidases

“…The dealkylation can be initiated either by single electron transfer or hydrogen atom transfer. [328][329][330][331][332][333][334][335] For oxidative N-dealkylation, [336][337][338][339] two elementary steps are involved, i.e., hydrogen atom transfer and single electron transfer. N-Oxidation proceeds through charge transfer to the oxo-ferryl group, followed by the formation of radical cation and homolysis of the iron-oxygen bond to generate the new N-O bond.…”

“…The dealkylation can be initiated either by single electron transfer or hydrogen atom transfer. 328–335 For oxidative N -dealkylation, 336–339 two elementary steps are involved, i.e. , hydrogen atom transfer and single electron transfer.…”