Characterization of the Macrolide P-450 Hydroxylase from Streptomyces venezuelae Which Converts Narbomycin to Picromycin

The post-polyketide synthase (PKS) biosynthetic tailoring of macrolide antibiotics usually involves one or more oxidation reactions catalyzed by cytochrome P450 monooxygenases. As the specificities of members from this class of enzymes vary significantly among PKS gene clusters, the identification a...

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Bibliographic Details
Published in:Biochemistry (Easton) 1998-10, Vol.37 (42), p.14937-14942
Main Authors: Betlach, Melanie C, Kealey, James T, Betlach, Mary C, Ashley, Gary W, McDaniel, Robert
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Aminoglycosides
Anti-Bacterial Agents - chemistry
Anti-Bacterial Agents - metabolism
Bacterial Proteins
Catalysis
Cloning, Molecular
Cytochrome P-450 Enzyme System - chemistry
Cytochrome P-450 Enzyme System - genetics
Genes, Bacterial
Histidine - genetics
Hydroxylation
Kinetics
Macrolides
Mixed Function Oxygenases - chemistry
Mixed Function Oxygenases - genetics
Molecular Sequence Data
Streptomyces - enzymology
Streptomyces venezuelae
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Summary:The post-polyketide synthase (PKS) biosynthetic tailoring of macrolide antibiotics usually involves one or more oxidation reactions catalyzed by cytochrome P450 monooxygenases. As the specificities of members from this class of enzymes vary significantly among PKS gene clusters, the identification and study of new macrolide P450s are important to the growing field of combinatorial biosynthesis. We have isolated the cytochrome P450 gene picK from Streptomyces venezuelae which is responsible for the C-12 hydroxylation of narbomycin to picromycin. The gene was located by searching regions proximal to modular PKS genes with a probe for macrolide P450 monooxygenases. The overproduction of PicK with a C-terminal six-His affinity tag (PicK/6-His) in Escherichia coli aided the purification of the enzyme for kinetic analysis. PicK/6-His was shown to catalyze the in vitro C-12 hydroxylation of narbomycin with a k cat of 1.4 s-1, which is similar to the value reported for the related C-12 hydroxylation of erythromycin D by the EryK hydroxylase. The unique specificity of this enzyme should be useful for the modification of novel macrolide substrates similar to narbomycin, in particular, ketolides, a promising class of semisynthetic macrolides with activity against erythromycin-resistant pathogens.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi981699c