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

• Published in: Journal of the American Chemical Society Vol. 136; no. 49; pp. 17350 - 17354
• Main Authors: Al-Mestarihi, Ahmad H., Villamizar, Germán, Fernández, Javier, Zolova, Olga E., Lombó, Felipe, Garneau-Tsodikova, Sylvie
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 10.12.2014
• Subjects: Cysteine - chemistry; Cysteine - metabolism; Depsipeptides - biosynthesis; Depsipeptides - chemistry; Methylation; Methyltransferases - chemistry; Methyltransferases - metabolism; Molecular Conformation
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja510489j

The antitumor agent thiocoraline is a nonribosomally 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(T4). The valuable insights gained into the pathway(s) followed for the production of S-Me-l-Cys-S-TioS(T4) will serve as a guide for the development of novel engineered interrupted adenylation enzymes for combinatorial biosynthesis.