Characterization of <i>S-</i>glycosylated glycocins containing three disulfides

Glycocins are a growing family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) that are O- and/or S-glycosylated. Using a sequence similarity network of putative glycosyltransferases, the thg biosynthetic gene cluster (BGC) was identified in the genome of Thermoanaerobacterium thermosaccharolyticum. Heterologous expression in Escherichia coli showed that the glycosyltransferase (ThgS) encoded in the BGC adds N-acetyl-glucosamine (GlcNAc) to Ser and Cys residues of ThgA. The peptide derived from ThgA, which we name thermoglycocin, was structurally characterized and shown to resemble glycocin F. In addition to two nested disulfide bonds also present in glycocin F, thermoglycocin contains a third disulfide bond creating a C-terminal loop. Unexpectedly, ThgA lacks the common double glycine motif for leader peptide removal by a C39-peptidase. Based on AlphaFold3 modeling, we postulated that cleavage between the leader and core peptide would occur instead at a GK motif, which was experimentally confirmed for an orthologous BGC from Ornithinibacillus bavariensis. Its structurally similar product termed orniglycocin was also produced in E. coli and carries two GlcNAc moieties on two Cys residues. The C39 peptidase domain of the peptidase-containing ATP-binding cassette transporter (PCAT) from this BGC removed the leader peptide after a Gly-Lys motif and the orniglycocin so produced demonstrated antimicrobial activity. This study adds to the small number of characterized glycocins, employs AlphaFold3 to predict the leader peptide cleavage site, and suggests a common naming convention similar to that established for lanthipeptides. One-Sentence Summary: Thermoglycocin from Thermoanaerobacterium thermosaccharolyticum and orniglycocin from Ornithinibacillus bavariensis were produced heterologously in E. coli, shown to contain three disulfide bonds and two GlcNAcylations, and were released by a unique C39 protease that cleaves at a Gly-Lys sequence.