Background: Streptomyces venezuelae produces two groups of antibiotics that include the 12-membered ring macrolides methymycin and neomethymycin, and the 14-membered ring macrolide pikromycin. Methymycin and pikromycin are derived from the corresponding precursors, YC-17 and narbomycin, respectively, by hydroxylation of the tertiary carbon position (C-10 in YC-17 or C-12 in narbomycin) on the macrolactone ring. In contrast, neomethymycin is derived from YC-17 by hydroxylation of the secondary carbon (C-12) of the propionyl starter unit sidechain. Results: Using a genetic and biochemical approach we have characterized a single P450 hydroxylase (PikC) in the methymycin/pikromycin biosynthetic gene cluster (pik) from S. venezuelae. Inacfivation of pikC abolished production of all hydroxylated macrolides, with corresponding accumulation of YC-17 and narbomycin in the culture medium. The enzyme was produced efficiently and purified as a His-tagged protein from recombinant Escherichia coil cells. Purified PikC effectively converts YC-17 into methymycin and neomethymycin and narbomycin into pikromycin in vitro. Conclusions: These results demonstrate that PikC is responsible for the conversion of YC-17 to methymycin and neomethymycin, and narbomycin to pikromycin in S. venezuelae. This substrate flexibility is unique and represents the first example of a P450 hydroxylase that can accept 12- and 14-membered ring macrolides as substrates, as well as functionalize at two positions on the macrolactone system. The broad substrate specificity of PikC provides a potentially valuable entry into the construction of novel macrolide- and ketolide-based antibiotics.
Bibliographical noteFunding Information:
The authors thank Leonard Katz (Abbott Laboratories) for authentic pikromycin and David E. Cane (Brown University) for authentic YC-I 7. The authors also thank Mustafa Varoolu for helpful discussions. This work was supported by NIH grant GM48562 and a’grant from the Office of Naval Research (to D.H.S.), and GM35906 and GM54346 (to H.-w. Liu). Y. Xue is a recipient of the Dennis W. Watson fellowship (University of Minnesota) in support of graduate research.
- Cytochrome P450
- Macrolide antibiotics
- Polyketide synthase