Adenylation and <i>S</i>‑Methylation of Cysteine by the Bifunctional Enzyme TioN in Thiocoraline Biosynthesis

The antitumor agent thiocoraline is a nonribosomally biosynthesized bisintercalator natural product, which contains in its peptidic backbone two <i>S</i>-methylated l-cysteine residues. <i>S</i>-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 <i>S</i>-adenosyl-l-methionine binding region of a methyltransferase enzyme, is capable of performing two functions: the adenylation and <i>S</i>-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 <i>S</i>-Me-l-Cys-<i>S</i>-TioS­(T₄) will serve as a guide for the development of novel engineered interrupted adenylation enzymes for combinatorial biosynthesis

Activation and Loading of the Starter Unit during Thiocoraline Biosynthesis

The initiation of the nonribosomal peptide synthetase (NRPS) assembly of the bisintercalator natural product thiocoraline involves key enzymatic steps for AMP activation and carrier protein loading of the starter unit 3-hydroxyquinaldic acid (3HQA). Gene cluster data combined with protein sequence homology analysis originally led us to propose that TioJ could be responsible for the AMP activation step, whereas TioO could act as the thiolation (T) domain, facilitating the transfer of 3HQA to the next NRPS module, TioR. Herein, we confirmed the involvement of TioJ in thiocoraline biosynthesis by <i>tioJ</i> knockout and <i>in vitro</i> activation of 3HQA studies. However, we demonstrated that TioJ-activated 3HQA is not loaded onto the T domain TioO, as originally believed, but instead onto a fatty acid synthase (FAS) acyl carrier protein (ACP) domain FabC, which is located outside of the thiocoraline gene cluster. We showed a strong interaction between TioJ and FabC. By generating TioJ point mutants mimicking the active site of highly homologous enzymes activating different molecules, we showed that the identity of the substrate activated by adenylation domains such as TioJ is not determined by only the active site residues that directly interact with the substrate. The insights gained from these enzymatic transformations are valuable in the efforts toward deciphering the complete biosynthetic pathway of thiocoraline and bisintercalators in general