Characterization of a sugar-O-methyltransferase TiaS5 affords new Tiacumicin analogues with improved antibacterial properties and reveals substrate promiscuity.

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.