title: Combining substrate dynamics, binding statistics, and energy barriers to rationalize regioselective hydroxylation of octane and lauric acid by CYP102A1 and mutants.
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 (omega-1, omega-2, omega-3) but not the terminal (omega) 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.
authors.affiliation: Leiden/Amsterdam Center for Drug Research, Division of Molecular Toxicology, Vrije Universiteit, 1081HV Amsterdam, The Netherlands.
authors.forename: K Anton
authors.initials: KA
authors.lastname: Feenstra
authors.forename: Eugene B
authors.initials: EB
authors.lastname: Starikov
authors.forename: Vlada B
authors.initials: VB
authors.lastname: Urlacher
authors.forename: Jan N M
authors.initials: JN
authors.lastname: Commandeur
authors.forename: Nico P E
authors.initials: NP
authors.lastname: Vermeulen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D003627:Data Interpretation, Statistical; D006900:Hydroxylation; D007850:Lauric Acids / Q000737:chemistry*; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D008954:Models, Biological; D008958:Models, Molecular; D009251:NADPH-Ferrihemoprotein Reductase; D009795:Octanes / Q000737:chemistry*; D011485:Protein Binding; D011789:Quantum Theory; D013237:Stereoisomerism; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D007850:Lauric Acids; D009795:Octanes; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases