Mechanistic and Structural Insights into the Specificity and Biological Functions of Bacterial Sulfoglycosidases

Glycan sulfation is an important modification supporting the functionalities of many proteins in biology. Exo-acting 6S-GlcNAcases from human microbiota are glycosidases that participate in the removal of 6-sulfo-GlcNAc from host glycans and thereby play an important role in human health and disease. Nonetheless, mechanisms underlying their ability to recognize the sulfate group remain poorly understood. Using structural and kinetic analyses, we here reveal the catalytically important amino acids directly involved in the recognition and cleavage of 6S-GlcNAc, but not of 6-phospho-GlcNAc, in BbhII from Bifidobacterium bifidum, Bt4394 from Bacteroides thetaiotaomicron, and SGL from Prevotella spp. The defining features of their sulfate recognition motifs underpin a genomic enzymological exploration of 6S-GlcNAcases to identify a wider range of human health-associated bacterial species having 6S-GlcNAcase activity. Our data provide significant insights into distinct molecular mechanisms of sulfated sugar recognition employed by 6S-GlcNAcases from both Gram-positive and Gram-negative bacteria along with valuable information for the exploration of extensive interactions between microbiota and their host glycans.