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.
Chembiochem. 2011 Jul 25;12(11):1740-8. doi: 10.1002/cbic.201100129. Epub 2011 Jun 1.
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.
18 元大环糖苷替考拉宁 B 是一种 RNA 聚合酶抑制剂，在治疗艰难梭菌感染方面具有重要的治疗意义。最近对来自 Dactylosporangium aurantiacum subsp. hamdenensis NRRL 18085 的替考拉宁 B 生物合成基因簇的特征分析揭示了两个糖基转移酶、一个 C-甲基转移酶、一个酰基转移酶、两个细胞色素 P450 和一个修饰二卤代酶在替考拉宁生物合成中的功能。在这里，我们报告了 TiaS5 作为糖-O-甲基转移酶的遗传确认和生化特征，这是替考拉宁 B 生物合成所必需的。tiaS5 失活突变体能够产生 14 种替考拉宁类似物（其中 11 种是新的），所有这些类似物都缺乏内部鼠李糖部分的 2'-O-甲基。值得注意的是，两种替考拉宁类似物表现出改善的抗菌特性。我们还通过生化方法验证了 TiaS5 是一种 S-腺苷-L-甲硫氨酸依赖性 O-甲基转移酶，需要二价金属离子才能发挥活性。底物探测表明 TiaS5 是一种混杂酶，可识别 12 种替考拉宁类似物。这些发现明确确立了 TiaS5 作为 2'-O-甲基转移酶的功能，并提供了直接的生化证据，表明 TiaS5 催化的甲基化是替考拉宁 B 生物合成中糖基偶联后的一个修饰步骤。
