期刊
CHEMBIOCHEM
卷 12, 期 11, 页码 1740-1748出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201100129
关键词
biocatalysis; biosynthesis; methylation; sugar; tiacumicin
资金
- National Basic Research Program of China [2010CB833805]
- Chinese Academy of Sciences for Key Topics in Innovation Engineering [KSCX2-YW-G-065, KZCX2-YW-JC202, LYQY200805, KSCX2-EW-G-12, KSCX2-YW-G-073]
- National Science Foundation for Young Scientists of China [30900035]
- Scientific Research Foundation for Returned Overseas Chinese Scholars, State Education Ministry
The 18-membered macrocyclic glycoside tiacumicin B, an RNA polymerase inhibitor, is of great therapeutic significance in treating Clostridium difficile infections. The recent characterization of the tiacumicin B biosynthetic gene cluster from Dactylosporangium aurantiacum subsp. hamdenensis NRRL 18085 revealed the functions of two glycosyltransferases, a C-methyltransferase, an acyltransferase, two cytochrome P450s, and a tailoring dihalogenase in tiacumicin biosynthesis. Here we report the genetic confirmation and biochemical characterization of TiaS5 as a sugar-O-methyltransferase, requisite for tiacumicin B biosynthesis. The tiaS5-inactivation mutant is capable of producing 14 tiacumicin analogues (11 of which are new), all lacking the 2'-O-methyl group on the internal rhamnose moiety. Notably, two tiacumicin analogues exhibit improved antibacterial properties. We have also biochemically verified TiaS5 as an S-adenosyl-L-methionine-dependent O-methyltransferase, requiring divalent metal ions for activity. Substrate probing revealed TiaS5 to be a promiscuous enzyme, recognizing 12 tiacumicin analogues. These findings unequivocally establish that TiaS5 functions as a 2'-O-methyltransferase and provide direct biochemical evidence that TiaS5-catalyzed methylation is a tailoring step after glycosyl coupling in tiacumicin B biosynthesis.
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