Nonribosomal peptides (NRPs) are natural products that are biosynthesized by large multi-enzyme assembly lines called nonribosomal peptide synthetases (NRPSs). We have previously discovered that backbone or side chain methylation of NRP residues is carried out by an interrupted adenylation (A) domain that contains an internal methyltransferase (M) domain, while maintaining a monolithic AMA fold of the bifunctional enzyme. A key question that has remained unanswered is at which step of the assembly line mechanism the methylation by these embedded M domains takes place. Does the M domain methylate an amino acid residue tethered to a thiolation (T) domain on same NRPS module (in cis), or does it methylate this residue on a nascent peptide tethered to a T domain on another module (in trans)? In this study, we investigated the kinetics of methylation by wild-type AMAT tridomains from two NRPSs involved in biosynthesis of anticancer depsipeptides thiocoraline and echinomycin, and by mutants of these domains, for which methylation can occur only in trans. The analysis of the methylation kinetics unequivocally demonstrated that the wild-type AMATs methylate overwhelmingly in cis, strongly suggesting that this is also the case in the context of the entire NRPS assembly line process. The mechanistic insight gained in this study will facilitate rational genetic engineering of NRPS to generate unnaturally methylated NRPs.

非核糖体肽（NRPs）是被称为非核糖体肽合成酶（NRPSs）的大型多酶组装生产线生物合成的天然产物。我们以前已经发现，NRP残基的主链或侧链甲基化是通过包含内部甲基转移酶（M）域的间断腺苷酸化（A）域进行的，同时保持了双功能酶的整体AMA折叠。尚未解决的关键问题是在组装线机制的哪一步通过这些嵌入的M结构域进行甲基化。M结构域是否使同一NRPS模块上的硫醇化（T）结构域的氨基酸残基甲基化（顺式），还是将新生肽上的氨基酸残基与其他模块的T结构域的甲基化甲基化（反式））？在这项研究中，我们研究了野生型AMAT三域结构域从两个参与抗癌二肽硫代可可碱和棘轮霉素生物合成的NRPS以及这些域的突变体（仅在反式中发生甲基化）的动力学。甲基化动力学分析清楚地表明，野生型AMAT绝大多数以顺式甲基化，强烈表明在整个NRPS组装线过程中也是如此。在这项研究中获得的机制洞察力将促进NRPS的合理基因工程，以产生非天然甲基化的NRP。