Department of Chemistry, University of Illinois at Urbana-Champaign , 600 South Mathews Avenue, Urbana, Illinois 61801, United States.
Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign , 1206 West Gregory Drive, Urbana, Illinois 61801, United States.
J Am Chem Soc. 2017 Mar 29;139(12):4310-4313. doi: 10.1021/jacs.7b00693. Epub 2017 Mar 21.
Thiomuracin is a thiopeptide antibiotic with potent activity toward Gram-positive drug-resistant bacteria. Thiomuracin is biosynthesized from a precursor peptide, TbtA, by a complex array of posttranslational modifications. One of several intriguing transformations is the C-methylation of thiazole, occurring at an unactivated sp carbon. Herein, we report the in vitro reconstitution of TbtI, the responsible radical S-adenosyl-methionine (rSAM) C-methyltransferase, which catalyzes the formation of 5-methylthiazole at a single site. Our studies demonstrate that a linear hexazole-bearing intermediate of TbtA is a substrate for TbtI whereas macrocyclized thiomuracin GZ is not. In determining the minimal substrate for TbtI, we found that the enzyme is functional when most of the leader peptide has been removed. The in vitro reconstitution of TbtI, a class C rSAM methyltransferase, further adds to the chemical versatility of rSAM enzymes, and informs on the complexity of thiomuracin biosynthesis.
硫霉素是一种具有强效活性的噻唑肽抗生素，对革兰氏阳性耐药菌有作用。硫霉素是由前体肽 TbtA 通过一系列复杂的翻译后修饰生物合成的。其中一个引人注目的转化是噻唑的 C-甲基化，发生在未激活的 sp 碳上。在此，我们报告了负责自由基 S-腺苷甲硫氨酸 (rSAM) C-甲基转移酶 TbtI 的体外重建，该酶催化在单一位置形成 5-甲基噻唑。我们的研究表明，TbtA 的线性六唑载体中间体是 TbtI 的底物，而大环硫霉素 GZ 则不是。在确定 TbtI 的最小底物时，我们发现当去除大部分前导肽时，该酶仍然具有功能。TbtI（一种 C 类 rSAM 甲基转移酶）的体外重建进一步增加了 rSAM 酶的化学多功能性，并阐明了硫霉素生物合成的复杂性。
