Orthogonal Biosynthesis of Pyrrolamides through Dehydration by a Pathway-Specific Glycoside Hydrolase-Like Enzyme in...

Pyrrolamides, defined by a pyrrole-2-carboxamide core, represent a family of natural products produced by Streptomyces species. They exhibit potent yet promiscuous bioactivities, including antimicrobial, antifungal, antiviral, and anticancer effects. The characteristic pyrrole moiety is derived from 4-acetamidopyrrole-2-carboxylate, which was previously proposed to originate most likely from N-acetylglucosamine-1-phosphate. This unusual transformation of a primary metabolite sugar into an aromatic peptide building block is of interest mechanistically; however, the pathway preceding pyrrole formation remains unclear. We herein report the discovery of manlipyrrolamides A-F from Bacillus manliponensis, a new class of pyrrolamides with distinctive structural features. Gene disruption and in vitro assays allowed us to identify the functions of key enzymes in the pyrrole formation pathway. Man17, a nucleotidyltransferase, functions as a gatekeeper that activates the precursor N-acetylglucosamine-1-phosphate, through guanosine diphosphate (GDP) derivatization for subsequent modifications. Man18, a glycosyltransferase-like enzyme, and Man15, a glycoside hydrolase-like enzyme, function as a hydrolase and dehydratase, respectively, forming a pyrrole from a mature GDP-linked sugar. After pyrrole formation, a xanthine dehydrogenase-like enzyme complex is utilized in oxidation to produce carboxylate, distinct from the previously proposed Streptomyces pathway. The pathway-specific enzyme Man15, which is widely distributed in Gram-positive bacteria, is encoded in diverse biosynthetic gene clusters, indicating that this unprecedented transformation is utilized for unknown classes of pyrrolamides. The use of a GDP derivative suggests an orthogonal strategy distinct from that of the canonical UDP-linked sugar pathway. The present study reveals novel sugar skeletal conversion in secondary metabolism, which provides novel insights into bacterial metabolic strategies.