Thuricin CD Biosynthetic Pathway: Isolation and Characterization of the Precursor Peptides and Sactisynthases TrnC and TrnD

Abstract

Thuricin CD is the most potent antibiotic against C. difficile known to date. Metronidazole and other drugs currently in use strongly alter the microbiota balance in the gut, with dangerous side effects. Thuricin CD has been shown to have a potency equivalent to those antibiotics, but with far less unspecific activity. It has only been isolated in low amounts from B. thuringiensis, and now a larger scale production and advances in clinical trials are needed. Understanding of its biosynthetic pathway is fundamental in order to accomplish such goals. To date, the mature bioactive peptide has been thoroughly characterized, but none of the enzymes responsible for the post-translational modifications have been studied at all. The two subunits forming the active peptide thuricin CD, Trnα and Trnβ, each feature three cysteine to α-carbon crosslinks. This unique structure is the hallmark of the sactipeptides family, a class of ribosomal synthesized and post-translationally modified natural products counting only a few known members. These thioether crosslinks are known to be formed by radical S-adenosyl-L-methionine (SAM) enzymes, and while several mechanisms for installation of these bonds have been proposed, little enzymology data exists to support them. Usually, the sactipeptide operons contain one of these enzymes, which is responsible for one or multiple crosslinks. Thuricin CD is comprised of two different peptide subunits that works in synergy, and the gene cluster shows the presence of two radical SAM enzymes, whose specific functions are unknown. Heterologous expression in Escherichia coli, purification and initial characterization of TrnC and TrnD will be presented, in addition to methodology for the expression and purification of their substrates TrnA and TrnB, by way of a fusion protein system. Attempts to the understanding of the system will also be discussed, laying the groundwork for further investigations.