Molecular Determinants of Substrate Affinity and Enzyme Activity of a Cytochrome P450<sub>BM3</sub> Variant.
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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. |
Year of Publication | :
2018
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Journal | :
Biophysical journal
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Volume | :
115
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Issue | :
7
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Number of Pages | :
1251-1263
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Date Published | :
2018
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ISSN Number | :
0006-3495
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URL | :
https://linkinghub.elsevier.com/retrieve/pii/S0006-3495(18)30980-9
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DOI | :
10.1016/j.bpj.2018.08.026
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Short Title | :
Biophys J
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