A Heterotrimeric Dehydrogenase Complex Functions with 2 Distinct YcaO Proteins to Install 5 Azole Heterocycles into 35-Membered Sulfomycin Thiopeptides.

Sulfomycins are sulfur-rich, ribosomally synthesized, and post-translationally modified peptides (RiPPs) that are characterized by a 35-membered macrocyclic ring system with a pyridine domain central to five azoles and additional dehydroamino acids. The pathway through which these large thiopeptide antibiotics are formed in Streptomyces viridochromogene remains elusive. Starting with the cloning of the biosynthetic gene cluster of sulfomycins, we here dissect a two-stage process in which an unusual dehydrogenase heterotrimer functions with two distinct YcaO proteins to install five azole heterocycles into the core peptide sequence of the precursor peptide. The first stage involves the activity of a typical cyclodehydratase complex composed of a didomain E1-YcaO protein and an F-protein partner to heterocyclize distant residues l-Cys2 and l-Thr9 and then the activity of the heterotrimeric dehydrogenase complex that converts the resulting azolines to azoles. In the second stage, this dehydrogenase complex associates with a discrete YcaO protein to form an atypical, four-component azole synthase complex, which is capable of sequentially converting residues l-Cys7, l-Thr5, and l-Ser12 to azoles in a distinct manner. During this process, an E1-like partner protein plays a critical role and functions through the two stages to mediate a variety of specific protein-protein interactions. This partner protein participates in the formation of the active dehydrogenase heterotrimer and the engagement of discrete YcaO activity to form the azole synthase heterotetramer. The findings in this study advance the understanding in the biosynthesis of different azole-containing RiPPs and set the stage for the discovery, engineering, and creation of new thiopeptides using genome mining and synthetic biology approaches.