Combining substrate dynamics, binding statistics, and energy barriers to rationalize regioselective hydroxylation of octane and lauric acid by CYP102A1 and mutants

Authors

  • K. Anton Feenstra,

    1. Leiden/Amsterdam Center for Drug Research, Division of Molecular Toxicology, Vrije Universiteit, 1081HV Amsterdam, The Netherlands
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  • Eugene B. Starikov,

    1. Leiden/Amsterdam Center for Drug Research, Division of Molecular Toxicology, Vrije Universiteit, 1081HV Amsterdam, The Netherlands
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  • Vlada B. Urlacher,

    1. Institute for Technical Biochemistry, University of Stuttgart, Stuttgart, Germany
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  • Jan N.M. Commandeur,

    1. Leiden/Amsterdam Center for Drug Research, Division of Molecular Toxicology, Vrije Universiteit, 1081HV Amsterdam, The Netherlands
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  • Nico P.E. Vermeulen

    Corresponding author
    1. Leiden/Amsterdam Center for Drug Research, Division of Molecular Toxicology, Vrije Universiteit, 1081HV Amsterdam, The Netherlands
    • Leiden/Amsterdam Center for Drug Research (LACDR), Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083, 1081HV Amsterdam, The Netherlands; fax: 31 20 5987610.
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Abstract

Hydroxylations of octane and lauric acid by Cytochrome P450-BM3 (CYP102A1) wild-type and three active site mutants—F87A, L188Q/A74G, and F87V/L188Q/A74G—were rationalized using a combination of substrate orientation from docking, substrate binding statistics from molecular dynamics simulations, and barrier energies for hydrogen atom abstraction from quantum mechanical calculations. Wild-type BM3 typically hydroxylates medium- to long-chain fatty acids on subterminal (ω−1, ω−2, ω−3) but not the terminal (ω) positions. The known carboxylic anchoring site Y51/R47 for lauric acid, and hydrophobic interactions and steric exclusion, mainly by F87, for octane as well as lauric acid, play a role in the binding modes of the substrates. Electrostatic interactions between the protein and the substrate strongly modulate the substrate's regiodependent activation barriers. A combination of the binding statistics and the activation barriers of hydrogen-atom abstraction in the substrates is proposed to determine the product formation. Trends observed in experimental product formation for octane and lauric acid by wild-type BM3 and the three active site mutants were qualitatively explained. It is concluded that the combination of substrate binding statistics and hydrogen–atom abstraction barrier energies is a valuable tool to rationalize substrate binding and product formation and constitutes an important step toward prediction of product ratios.

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