Enzymatic Pyridine Aromatization during Thiopeptide Biosynthesis

Abstract

Thiazole-containing pyritides (thiopeptides) are ribosomally synthesized and post-translationally modified peptides (RiPPs) that have attracted interest owing to their potent biological activities and structural complexity. The class-defining feature of a thiopeptide is a six-membered, nitrogenous heterocycle formed by an enzymatic [4 + 2]-cycloaddition. In rare cases, piperidine or dehydropiperidine (DHP) is present; however, the aromatized pyridine is considerably more common. Despite significant effort, the mechanism by which the central pyridine is formed remains poorly understood. Building on our recent observation of the Bycroft–Gowland intermediate (i.e., the direct product of the [4 + 2]-cycloaddition), we interrogated thiopeptide pyridine synthases using a combination of targeted mutagenesis, kinetic assays, substrate analogs, enzyme–substrate cross-linking, and chemical rescue experiments. Collectively, our data delineate roles for several conserved residues in thiopeptide pyridine synthases. A critical tyrosine facilitates the final aromatization step of pyridine formation. This work provides a foundation for further exploration of the [4 + 2]-cycloaddition reaction and future customization of pyridine-containing macrocyclic peptides.

A Compact Reprogrammed Genetic Code for De Novo Discovery of Proteolytically Stable Thiopeptides

“…These results highlight the remarkable substrate-level cooperativity of Laz enzymesespecially when it comes to modifying C/S/T-rich local environmentsconsidering that the pattern of C/S/T residues in w7 and other discovered sequences bears no resemblance to that of wild-type LazA. Despite the extensive efforts to unravel the substrate recruitment and discrimination by Laz and related RiPP enzymes, ,− our understanding of such a promiscuous (deep modification of random inserts) but at the same time selective (formation of one or at most a few products) processing of precursor peptides is lacking. Altogether, we narrowed our focus to 18 sequences which furnished mostly homogeneous thiopeptides (seven from the library v.t.4h and 11 from v.t.4w) for the following experiments.…”

Dehydroamino acid residues in bioactive natural products

“…52 Recent studies demonstrated that a tyrosine residue in the active site of a pyritide synthase in the thiopeptide pathway facilitates the nal aromatization step of pyridine formation. 53 Many RiPPs contain D-amino acid residues. Recent studies demonstrated that a F420-dependent reductase in the biosynthesis of lexapeptide 6 catalyses an iterative biotransformation, changing the corresponding Dha residues to D-Ala in a stereo-specic manner.…”

“… 52 Recent studies demonstrated that a tyrosine residue in the active site of a pyritide synthase in the thiopeptide pathway facilitates the final aromatization step of pyridine formation. 53 …”