Premium
Characterization of a Sugar‐O‐methyltransferase TiaS5 Affords New Tiacumicin Analogues with Improved Antibacterial Properties and Reveals Substrate Promiscuity
Author(s) -
Niu Siwen,
Hu Tao,
Li Sumei,
Xiao Yi,
Ma Liang,
Zhang Guangtao,
Zhang Haibo,
Yang Xiaohong,
Ju Jianhua,
Zhang Changsheng
Publication year - 2011
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201100129
Subject(s) - glycosyltransferase , biosynthesis , o methyltransferase , methyltransferase , biochemistry , glycosyl , transferase , stereochemistry , enzyme , gene cluster , chemistry , methylation , mutant , acyltransferase , biology , gene
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.