In vivo and In vitro reconstitution of the dehydroalanine biosynthesis in goadsporin, a linear azole containing RiPP

Dehydroamino acids are often found in ribosomally synthesized and post-translationally modified peptides (RiPPs), such as lanthipeptides and thiopeptides. These non-proteinogenic amino acids often confer antibacterial activity to these compounds, and, therefore, it is important to understand the biosynthetic pathway.

Goadsporin, is a linear azole-containing peptide produced by Streptomyces sp. TP-A0584, and it is another example of a RiPP decorated with dehydroamino acids. Dehydroalanines in goadsporin are proposed to be synthesized by GodF and GodG, which show slight homology to the N-terminal glutamylation and C-terminal elimination domains, respectively, of LanB, a class I lanthipeptide dehydratase. Although similar, separated-type LanBs are conserved among thiopeptides and indispensable for their biosynthesis and biological activities, these enzymes had not yet been characterized. Here, the function of godF and godG was characterized by identification of goadsporin B, which kept Ser4 and Ser14 unmodified, from both a godF and a godG disruptant. The godG disruptant also produced goadsporin C, Ser4-glutamylated goadsporin B. These results suggested that dehydroalanines were formed by GodF-catalyzed glutamylation and GodG-catalyzed glutamate elimination. NMR analysis revealed for the first time that the glutamyl group was attached to a serine via an ester bond by the catalysis of LanB-type enzymes. In addiotion, in vitro reconstitution of dehydroalanine formation was done with the recombinant GodF and GodG. Our findings provide insights into the function of separated-type LanBs involved in the biosynthesis of goadsporin and thiopeptides.