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87 of Bacillus megaterium cytochrome P450 BM3, a residue close to the heme in the substrate binding pocket, has been replaced by alanine by site-directed mutagenesis.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.

Flavocytochrome P450 BM3 Mutant A264E Undergoes Substrate-dependent Formation of a Novel Heme Iron Ligand Set

“…Selected difference spectra (with progressive deviation from the base line) are shown for data recorded at 0. 66 absence of fatty acids, both the wild-type and A264E flavocytochrome P450 BM3 enzymes oxidized NADPH at a slow rate (ϳ5 min Ϫ1 ). In the presence of either lauric acid or myristic acid, there was considerable stimulation of NADPH oxidase activity, despite the fact that the addition of these fatty acids did not induce any considerable changes in the optical spectrum of the A264E mutant.…”

“…Phe 87 is known to be a conformationally flexible amino acid, which is important in controlling substrate selectivity and for interaction with the -terminal carbon of fatty acid substrates of P450 BM3, preventing hydroxylation at this position (65,66). Potentially, interactions between Phe 87 and Glu 264 in its ligated form provide further explanations for the heterogeneity in the substrate-free EPR spectrum.…”

A Single Mutation in Cytochrome P450 BM3 Induces the Conformational Rearrangement Seen upon Substrate Binding in the Wild-type Enzyme

“…The interaction with Phe 87 is particularly interesting, given the fact that this residue is absent from the CYP4 enzymes in which covalent ligation of the heme methyl group has been demonstrated. Phe 87 interacts with the -methyl group of fatty acid substrate(s) in wild-type P450 BM3 and is considered to be a critical regulatory residue that controls regioselectivity of substrate oxygenation (8,22,23). A particular difference in the behavior of P450 BM3 with respect to eukaryotic CYP4 enzymes is the inability of the former to hydroxylate at the -position (24).…”

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

“…F87 changes its orientation to shield one side of the substrates terminus and the pro-S C-H bonds promoting regioselectivity and controlling the (R)-selectivity of the enzyme [111]. A variation of the bulky F87 results (contrary to older reports [112]) in a more relaxed hydroxylation pattern due to the Table 1), (ii)…”