title: A single mutation in cytochrome P450 BM3 changes substrate orientation in a catalytic intermediate and the regiospecificity of hydroxylation.
abstract: Phenylalanine 87 of Bacillus megaterium cytochrome P450 BM3, a residue close to the heme in the substrate binding pocket, has been replaced by alanine by site-directed mutagenesis. The substitution had no effect on the rate of hydroxylation of laurate and increased the affinity for laurate of both the intact enzyme and its heme domain by 2.6-6-fold in the ferric state. NMR paramagnetic relaxation measurements showed that in the initial ferric enzyme-substrate complex, where the substrate binds relatively far from the heme, the substitution had no effect on the position or orientation of the bound substrate. However, in the next intermediate in the catalytic cycle, the reduced enzyme, the position of the bound substrate was altered so that the terminal methyl group was 3.1 A from the iron in the mutant, compared to 5.1 A in the wild-type enzyme. Analysis of the products of the action of the enzyme on laurate and myristate showed that the mutant catalyzed hydroxylation almost exclusively at the omega position, in marked contrast to the wild-type enzyme, with which no hydroxylation at this position was observed.
authors.affiliation: Department of Biochemistry, University of Leicester, U.K.
authors.forename: C F
authors.initials: CF
authors.lastname: Oliver
authors.forename: S
authors.initials: S
authors.lastname: Modi
authors.forename: M J
authors.initials: MJ
authors.lastname: Sutcliffe
authors.forename: W U
authors.initials: WU
authors.lastname: Primrose
authors.forename: L Y
authors.initials: LY
authors.lastname: Lian
authors.forename: G C
authors.initials: GC
authors.lastname: Roberts
mesh_terms: D001410:Bacillus megaterium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics*; D006900:Hydroxylation; D009682:Magnetic Resonance Spectroscopy; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics*; D016296:Mutagenesis; D009251:NADPH-Ferrihemoprotein Reductase; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases