Characterization of a Sugar-O-methyltransferase TiaS5 Affords New Tiacumicin Analogues with Improved Antibacterial Properties and Reveals Substrate Promiscuity
Characterization of a Sugar-O-methyltransferase TiaS5 Affords New Tiacumicin Analogues with Improved Antibacterial Properties and Reveals Substrate Promiscuity
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糖-O-甲基转移酶 TiaS5 的表征提供了具有改进抗菌特性的新型台勾霉素类似物，并揭示了底物混杂性
DOI：
10.1002/cbic.201100129
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发表时间：
2011-07-25
期刊：
影响因子：
3.2
通讯作者：
Zhang, Changsheng
中科院分区：
文献类型：
--
作者：
Niu, Siwen;Hu, Tao;Zhang, Changsheng
18元大环糖苷硫库米星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。Dactylosporangium aurantiacum subsp.tiacumicin B生物合成基因簇的最新特征Hamdenens NRRL 18085揭示了两种糖基转移酶、一种C-甲基转移酶、一种酰基转移酶、两种细胞色素P450酶和一种定制二卤化酶在硫菌素生物合成中的功能。在这里，我们报道了TiaS5作为糖-O-甲基转移酶的遗传确认和生化特性，该酶是合成硫库米星B所必需的。TiaS5失活突变体能够产生14个硫库米星类似物(其中11个是新的)，它们都缺乏内部鼠李糖部分上的2‘-O-甲基。值得注意的是，两个硫库米星类似物表现出更好的抗菌性能。我们还通过生化方法验证了TiaS5是一种依赖S-腺苷-L-蛋氨酸的O-甲基转移酶，需要二价金属离子才能发挥活性。底物检测发现TiaS5是一种杂交酶，可识别12种硫菌素类似物。这些发现明确地证实了TiaS5作为2‘-O-甲基转移酶的功能，并提供了直接的生化证据，证明TiaS5催化的甲基化是硫库米星B生物合成中糖基偶联之后的一个剪裁步骤。
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.