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 catalyze d by cytochrome P450 monooxygenases. As the specificities of members f rom this class of enzymes vary significantly among PKS gene clusters, the identification and study of new macrolide P450s are important to t he growing field of combinatorial biosynthesis. We have isolated the c ytochrome P450 gene picK from Streptomyces venezuelae which is respons ible 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 wi th a C-terminal six-His affinity tag (PicK/6-Kis) in Escherichia coli aided the purification of the enzyme for kinetic analysis. PicK/6-Kis was shown to catalyze the in vitro C-12 hydroxylation of narbomycin wi th a k(cat) of 1.4 s(-1), which is similar to the value reported for t he related C-12 hydroxylation of erythromycin D by the EryK hydroxylas e. The unique specificity of this enzyme should be useful for the modi fication of novel macrolide substrates similar to narbomycin, in parti cular, ketolides, a promising class of semisynthetic macrolides with a ctivity against erythromycin-resistant pathogens.