A structural basis for the strain-dependent UDP-sugar specificity of glycosyltransferase C from the <i>Limosilactobacillus reuteri</i> accessory secretion system

The accessory secretion (aSec) system is a protein export pathway that is uniquely present in Gram-positive bacteria and is dedicated to the secretion of large, glycosylated cell wall-anchored adhesins called serine-rich repeat proteins (SRRPs). Strain-specific glycosylation of SRRPs has previously been reported in Limosilactobacillus reuteri and attributed to GtfC, a glycosyltransferase belonging to family 113, with LrGtfC_100-23 from L. reuteri rat strain 100-23C showing specificity for UDP-Glc, while LrGtfC₅₃₆₀₈ from L. reuteri pig strain ATCC 53608, which differs at only ten amino-acid positions, shows a preference for UDP-GlcNAc. However, the structural basis underpinning GtfC sugar-donor specificity remains unclear. Here, we report X-ray crystal structures of the tetrameric LrGtfC_100-23 in the apo form and its complexes with UDP and with the noncognate sugar donor UDP-N-acetylglucosamine (UDP-GlcNAc). Analysis of the LrGtfC_100-23 structures identified candidate residues implicated in donor-sugar substrate specificity, which were supported by site-directed mutagenesis. Reciprocal swaps of candidate residues combined with thermal shift assays revealed that the W240C variant of LrGtfC_100-23 could bind both UDP-sugar donors, while the P243S variant of LrGtfC₅₃₆₀₈ became specific for UDP-Glc, opening the door for glycoengineering approaches in bacteria.