title: Characterization of a sugar-O-methyltransferase TiaS5 affords new Tiacumicin analogues with improved antibacterial properties and reveals substrate promiscuity.
abstract: 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.
authors.affiliation: CAS Key Laboratory of Marine Bio-resources Sustainable Utilization, RNAM Center for Marine Microbiology, Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China.
authors.forename: Siwen
authors.initials: S
authors.lastname: Niu
authors.forename: Tao
authors.initials: T
authors.lastname: Hu
authors.forename: Sumei
authors.initials: S
authors.lastname: Li
authors.forename: Yi
authors.initials: Y
authors.lastname: Xiao
authors.forename: Liang
authors.initials: L
authors.lastname: Ma
authors.forename: Guangtao
authors.initials: G
authors.lastname: Zhang
authors.forename: Haibo
authors.initials: H
authors.lastname: Zhang
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Yang
authors.forename: Jianhua
authors.initials: J
authors.lastname: Ju
authors.forename: Changsheng
authors.initials: C
authors.lastname: Zhang
mesh_terms: D000617:Aminoglycosides / Q000096:biosynthesis* / Q000737:chemistry* / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry* / Q000494:pharmacology; D000077732:Fidaxomicin; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D013379:Substrate Specificity
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D008780:Methyltransferases; D000077732:Fidaxomicin