Characterization of a novel glycocin from a thermophile

Abstract

Glycocins are a growing family of ribosomally synthesized and posttranslationally modified peptides that are O- and/or S-glycosylated. Using a sequence similarity network of putative glycosyltransferases, the tht biosynthetic gene cluster was identified in the genome of Thermoanaerobacterium thermosaccharolyticum. ThtA is the precursor peptide to a member of the glycocin F family of glycocins. Like other members of this family, the glycosyltransferase (ThtS) encoded in the biosynthetic gene cluster adds N-acetyl-glucosamine to both Ser and Cys residues of ThtA. S-linked glycosylation has been shown to be chemically and enzymatically resistant to cleavage and therefore ThtS may be a valuable starting point for engineering efforts. The glycocin derived from ThtA, which we name thermoglycocin, was structurally characterized. Thermoglycocin is unique in that in addition to two nested disulfide bonds, it contains an additional disulfide bond creating a C-terminal loop. Unexpectedly, ThtA lacks the common double glycine motif that denotes a C39-peptidase leader peptide cleavage site. Based on AlphaFold3 modeling, we postulate that cleavage between the leader and core peptide occurs instead at a GK motif. This study adds to the small number of characterized glycocins, employs AlphaFold3 to aid in predicting the structure of the mature peptide product, and suggests a common naming convention similar to that established for lanthipeptides. One sentence summaryThermoglycocin is a novel glycocin derived from the thermophile Thermoanaerobacterium thermosaccharolyticum, containing three disulfide bonds, O- and S-GlcNAcylation, and is postulated to have a unique C39 protease cut site. O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/655019v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@17c74bforg.highwire.dtl.DTLVardef@1d4ddb3org.highwire.dtl.DTLVardef@27490dorg.highwire.dtl.DTLVardef@12cffd7_HPS_FORMAT_FIGEXP M_FIG C_FIG