Tryptophan 2C methyltransferase (TsrM) methylates C2 of the indole ring of l-tryptophan during biosynthesis of the quinaldic acid moiety of thiostrepton. TsrM is annotated as a cobalamin-dependent radical S-adenosylmethionine (SAM) methylase; however, TsrM does not reductively cleave SAM to the universal 5ʹ-deoxyadenosyl 5ʹ-radical intermediate, a hallmark of radical SAM (RS) enzymes. Herein, we report structures of TsrM from Kitasatospora setae, which are the first structures of a cobalamin-dependent radical SAM methylase. Unexpectedly, the structures show an essential arginine residue that resides in the proximal coordination sphere of the cobalamin cofactor, and a [4Fe–4S] cluster that is ligated by a glutamyl residue and three cysteines in a canonical CXXXCXXC RS motif. Structures in the presence of substrates suggest a substrate-assisted mechanism of catalysis, wherein the carboxylate group of SAM serves as a general base to deprotonate N1 of the tryptophan substrate, facilitating the formation of a C2 carbanion.

色氨酸 2C 甲基转移酶 (TsrM)在硫链丝菌肽的喹乙醇酸部分的生物合成过程中甲基化l-色氨酸吲哚环的 C2。TsrM 被注释为钴胺素依赖性自由基S-腺苷甲硫氨酸 (SAM) 甲基化酶；然而，TsrM 不会将 SAM 还原切割成通用的 5′-脱氧腺苷 5′-自由基中间体，这是自由基 SAM (RS) 酶的标志。在这里，我们报告了来自Kitasatospora setae 的 TsrM 的结构，这是钴胺素依赖性自由基SAM甲基化酶的第一个结构。出乎意料的是，这些结构显示了一个必需的精氨酸残基，它位于钴胺素辅因子的近端配位球中，以及一个 [4Fe-4S] 簇，它由一个典型的 CXXXCXXC RS 基序中的谷氨酰残基和三个半胱氨酸连接。底物存在下的结构表明了底物辅助的催化机制，其中 SAM 的羧酸根基团作为一般碱基使色氨酸底物的 N1 去质子化，从而促进 C2 碳负离子的形成。