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

Abstract

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 kcat 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.

An A245T Mutation Conveys on Cytochrome P450eryF the Ability to Oxidize Alternative Substrates

“…By adding ␦-aminolevulinic acid before the isopropyl-1-thio-␤-D-galactopyranoside induction step, the expression level was greatly improved, so that we obtained ϳ80 mg of protein/ liter of culture. The activity of wild-type P450 eryF -(His) 6 toward 6-DEB, measured as reported previously (9,10,25), showed that the turnover rate of the tagged protein was comparable to that reported earlier for the untagged protein (103 min Ϫ1 ) (2). The expression levels of the A245T, A245S, and A245V mutants were somewhat lower than that of the wild-type protein.…”

The 1.92-Å Structure of Streptomyces coelicolor A3(2) CYP154C1

“…There are also a number of P450s from different Streptomyces species having about 36% identity, including CYP107C1 from S. thermotolerans (Q60005), CYP107L1 (PikC) from Streptomyces venezuelae (O87605), PTED from Streptomyces avermitilis (Q93H80), and CYP107D1 (OLEP) from Streptomyces antibioticus (Q59819). This last group of related P450s is known to be involved in modification of macrolide antibiotics such as carbomycin (46), methymycin, neomethymycin, pikromycin (19,21,22), novamethymycin (23), avermectin (15), and oleandomycin (24).…”

“…It is capable of accepting 12-and 14-membered ring macrolides as substrates and catalyzes conversion of the 12-membered ring macrolide intermediate YC-17 to methymycin, neomethymycin (12,19,21), and novamethymycin (23). PikC also converts the 14-membered ring macrolide narbomycin to pikromycin (12,19,21,22 (Fig. 1A) led us to investigate its catalytic activity toward these important compounds.…”

“…These reactions typically occur during the late stages of biosynthesis after formation of the core ring system by a polyketide synthase. The hydroxyl or epoxide substituents provide an important layer of structural variability into the final natural product structures and often significantly influence biological activity (12,22). P450 monooxygenases are also involved in one of the initial steps in formation of the coumarin group of antibiotics (31), and of the peptidyl nucleoside antibiotic nikkomycin (32), as well as in oxidative tailoring of the vancomycin-like glycopeptides balhimycin (16) and complestatin (33).…”

Inactivation of gilGT, Encoding a C‐Glycosyltransferase, and gilOIII, Encoding a P450 Enzyme, Allows the Details of the Late Biosynthetic Pathway to Gilvocarcin V to be Delineated

“…By far the greatest homology (42% amino-acid identity) of this protein was found to be with an enzyme from Nocardioides sp., strain KP7 (orf4 gene product), which is presumably involved in phenanthrene degradation. [19] Significant homologies were also observed with EthB (34% amino-acid identity), a cytochrome-P450 monooxygenase from Rhodococcus ruber involved in the degradation of ethyl tert-butyl ether (ETBE), [20] and to a number of bacterial cytochrome-P450 enzymes (> 30% amino-acid identity), including oxygenases of macrolide antibiotic biosyntheses pathways, for example, EryF, [21] TylI, [22] PicK (PikC), [23,24] and OleP. [25] All the latter enzymes catalyze the hydroxylation of aliphatic macro-lide structures.…”