Question: The structurally unique antibiotic tropodithietic acid (TDA) is produced by various gram-negative marine bacteria like Phaeobacter inhibens and contains two sulfur atoms, which appear to be incorporated non-canonically. Focus of this work was the investigation of the cryptic sulfur incorporation during TDA biosynthesis.
Methods: Candidate enzymes were chosen based on bioinformatic predictions and knockout studies. These candidate enzymes were heterologously produced, and their biochemical functions assessed using in vitro assays as well as biophysical methods. To test biosynthetic hypotheses structural analogues of postulated intermediates were synthesized and used as surrogate substrates for these investigations. Complementary structural studies were conducted using X-ray crystallography to scrutinize the underlying enzymology.
Results: We were able to successfully reconstitute a three‑enzyme cascade in vitro, leading to the integration of a thiol group into a model substrate. More precisely, it could be shown that a GSH moiety attached to an aromatic backbone via a C-S bond is sequentially degraded by a γ-glutamyl-cyclotransferase, metallopeptidase and cysteine-S-conjugate-β-lyase. Additionally, the structures of the γ-glutamyl-cyclotransferase aswell as the β‑lyase could be elucidated using X-ray crystallography, granting insight into the catalytic mechanism behind GSH degradation and C-S bond cleavage.
Conclusion: These findings corroborate the notion of GSH as the source of sulfur during TDA biosynthesis and pave the way for the biotechnological production of analogous compounds. Interestingly, the degradation of GSH to a reactive thiol has been described in the biosynthesis of the infamous fungal epipolythiodiketopiperazine type toxins during formation of a transannular disulfide bridge, making this the second report of GSH involvement in microbial secondary metabolism in general and the first one specifically in bacteria [1].
References
[1] Scherlach K, Kuttenlochner W, Scharf DH, Brakhage AA, Hertweck C, Groll M, u. a. Structural and Mechanistic Insights into C−S Bond Formation in Gliotoxin. Angew Chem Int Ed Engl. 14. June 2021;60(25):14188–94.