Serine-rich repeat glycoproteins (SRRPs) are highly conserved in streptococci and staphylococci. Glycosylation of SRRPs is important for bacterial adhesion and pathogenesis.Streptococcus agalactiaeis the leading cause of bacterial sepsis and meningitis among newborns. Srr2, an SRRP fromS. agalactiaestrain COH1, has been implicated in bacterial virulence. Four genes (gtfA,gtfB,gtfC, and gtfD) located downstream ofsrr2share significant homology with genes involved in glycosylation of other SRRPs. We have shown previously thatgtfAandgtfBencode two glycosyltransferases, GtfA and GtfB, that catalyze the transfer of GlcNAc residues to the Srr2 polypeptide. However, the function of other glycosyltransferases in glycosylation of Srr2 is unknown. In this study, we determined that GtfC catalyzed the direct transfer of glucosyl residues to Srr2-GlcNAc. The GtfC crystal structure was solved at 2.7 Å by molecular replacement. Structural analysis revealed a loop region at the N terminus as a putative acceptor substrate binding domain. Deletion of this domain rendered GtfC unable to bind to its substrate Srr2-GlcNAc, concurrently abolished the glycosyltransferase activity of GtfC, and also altered glycosylation of Srr2. Furthermore, deletion of the corresponding regions from GtfC homologs also abolished their substrate binding and enzymatic activity, indicating that this region is functionally conserved. In summary, we have determined that GtfC is important for the glycosylation of Srr2 and identified a conserved loop region that is crucial for acceptor substrate binding from GtfC homologs in streptococci. These findings shed new mechanistic insight into this family of glycosyltransferases.
The linker-loop region of Escherichia coli chaperone Hsp31 functions as a gate that modulates high-affinity substrate binding at elevated temperatures