Journal
BIOCHEMICAL JOURNAL
Volume 386, Issue -, Pages 57-62Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BJ20040490
Keywords
cytochrome P450 mono-oxygenase; epoxidase; pimaricin; polyene; polyketide synthase
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The biosynthesis of the antifungal agent pimaricin by Streptomyces natalensis has been proposed to involve a cytochrome P450 encoded by the gene pimD. Pimaricin is derived from its immediate precursor de-epoxypimaricin by epoxidation of the C-4-C-5 double bond on the macrolactone ring. We have overproduced PimD with a N-terminal HiS(6) affinity tag in Escherichia coli and purified the enzyme for kinetic analysis. The protein showed a reduced CO-difference spectrum with a Soret maximum at 450 rim, indicating that it is a cytochrome P450. Purified PimD was shown to catalyse the in vitro C-4-C-5 epoxidation of 4,5-de-epoxy-pimaricin to pimaricin. The enzyme was dependent on NADPH for activity with optimal pH at 7.5, and the temperature optimum was 30 degreesC. The k(cat) value for the epoxidation of de-epoxypimaricin was similar to the values reported for other macrolide oxidases. Enzyme activity was inhibited at high substrate concentration. This is the first time that a polyene macrolide P450 mono-oxygenase has been expressed heterologously and studied. The unique specificity of this epoxidase should be useful for the oxidative modification of novel polyene macrolide antibiotics.
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