title: Adenylation and S-methylation of cysteine by the bifunctional enzyme TioN in thiocoraline biosynthesis.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Sciences, University of Kentucky , Lexington, Kentucky 40536-0596, United States.
authors.forename: Ahmad H
authors.initials: AH
authors.lastname: Al-Mestarihi
authors.forename: Germán
authors.initials: G
authors.lastname: Villamizar
authors.forename: Javier
authors.initials: J
authors.lastname: Fernández
authors.forename: Olga E
authors.initials: OE
authors.lastname: Zolova
authors.forename: Felipe
authors.initials: F
authors.lastname: Lombó
authors.forename: Sylvie
authors.initials: S
authors.lastname: Garneau-Tsodikova
mesh_terms: D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D047630:Depsipeptides / Q000096:biosynthesis* / Q000737:chemistry; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry / Q000378:metabolism*; D008968:Molecular Conformation
chemical_list: D047630:Depsipeptides; C109005:thiocoraline; D008780:Methyltransferases; D003545:Cysteine