Hydroxylation of macrolactones YC-17 and narbomycin is mediated by the pikC-encoded cytochrome P450 in Streptomyces venezuelae.
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. Inactivation 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 coli 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.
Xue
Y
Y
Department of Microbiology Biological Process Technology Institute University of Minnesota Minneapolis MN 55455 USA.
Wilson
D
D
Zhao
L
L
Liu
H w
Hw
Sherman
D H
DH
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001483:Base Sequence; D003470:Culture Media; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D007700:Kinetics; D018942:Macrolides / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*
D000900:Anti-Bacterial Agents; D003470:Culture Media; D018942:Macrolides; C116391:YC-17 macrolide; C010488:narbomycin; D003577:Cytochrome P-450 Enzyme System