Efficient methylation of C2 in L-tryptophan by the cobalamin-dependent radical S-adenosylmethionine methylase TsrM requires an unmodified N1 amine

TsrM catalyzes the methylation of C2 in L-tryptophan (Trp). This reaction is the first step in the biosynthesis of the quinaldic acid moiety of the thiopeptide antibiotic thiostrepton, which exhibits potent activity against Gram-positive pathogens. TsrM is a member of the radical S-adenosylmethionine (SAM) superfamily of enzymes, but it does not catalyze the formation of 5' -deoxyadenosin-5' -yl or any other SAM-derived radical. In addition to a [4Fe-4S] cluster, TsrM contains a cobalamin cofactor that serves as an intermediate methyl carrier in its reaction. However, how this cofactor donates a methyl moiety to the Trp substrate is unknown. Here, we showed that the unmodified N1 position of Trp is important for turnover and that 1-thia-Trp and 1-oxa-Trp serve as competitive inhibitors. We also showed that beta-cyclopropyl-Trp undergoes C2 methylation in the absence of cyclopropyl ring opening, disfavoring mechanisms that involve unpaired electron density at C3 of the indole ring. Moreover, we showed that all other indole-substituted analogs of Trp undergo methylation at varying but measurable rates and that the analog 7-aza-Trp, which is expected to temper the nucleophilicity of C2 in Trp, is a very poor substrate. Last, no formation of cob(II) alamin or substrate radicals was observed during the reaction with Trp or any molecule within a tested panel of Trp analogs. In summary, our results are most consistent with a mechanism that involves two polar nucleophilic displacements, the second of which requires deprotonation of the indole nitrogen in Trp during its attack on methylcobalamin.