Adenylation and S-Methylation of Cysteine by the Bifunctional Enzyme TioN in Thiocoraline Biosynthesis

摘要

The antitumor agent thiocoraline is a non-ribosomally biosynthesized bisintercalator natural product, which contains in its peptidic backbone two S-methylated L-cysteine residues. S-Methylation occurs very rarely in nature, and is observed extremely rarely in nonribosomal peptide scaffolds. We have proposed that during thiocoraline biosynthesis, TioN, a stand-alone adenylation domain interrupted by the S-adenosyl-L-methionine binding region of a methyltransferase enzyme, is capable of performing two functions: the adenylation and S-methylation of L-cysteine. Herein, by preparation of knockouts of TioN and its MbtH-like protein partner TioT, we confirmed their role in thiocoraline biosynthesis. We also co-expressed recombinant TioN and TioT and biochemically investigated three potential pathways involving activation, methylation, and loading of L-cysteine onto the TioN partner thiolation domain, TioS(T_4). The valuable insights gained into the pathway(s) followed for the production of S-Me-L-Cys-S-TioS(T_4) will serve as a guide for the development of novel engineered interrupted adenylation enzymes for combinatorial biosynthesis.