A single mutation in cytochrome P450 BM3 changes substrate orientation in a catalytic intermediate and the regiospecificity of hydroxylation

Catherine F. Oliver, Sandeep Modi, Michael J. Sutcliffe, William U. Primrose, Lu Yun Lian, Gordon C K Roberts

    Research output: Contribution to journalArticlepeer-review

    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87 of Bacillus megaterium cytochrome P450 BM3, a residue close to the heme in the substrate binding pocket, has been replaced by alanine by site-directed mutagenesis.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 Å from the iron in the mutant, compared to 5.1 Å 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 ω position, in marked contrast to the wild-type enzyme, with which no hydroxylation at this position was observed.
    Original languageEnglish
    Pages (from-to)1567-1572
    Number of pages5
    JournalBiochemistry
    Volume36
    Issue number7
    DOIs
    Publication statusPublished - 18 Feb 1997

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