The activity of GT-B glycosyltransferases depends on their conformational flexibility and high substrate specificity, but the molecular basis of these features is still not well defined. The GT70 family contains a single well-characterized enzyme, GumK, a glucuronosyltransferase from Xanthomonas campestris required for xanthan gum biosynthesis. Here, we applied multiscale molecular simulations and sequence analysis to probe GumK dynamics and substrate specificity. We show that GumK undergoes twisting and bending motions constrained by interdomain contacts and modulated by membrane anchoring. Acceptor-substrate binding within an amphiphilic clamp promotes opening, while donor-substrate binding stabilizes closure, defining a substrate-dependent catalytic cycle. Specificity for glucuronate-UDP is mediated by a conserved electrostatic environment centered on Lys307 and a hydrophobic triad that orients the sugar moiety. On the acceptor side, the binding site selectively accommodates polyisoprenyl carriers up to three isoprene units in length and wraps around the substrate, constraining the trisaccharide moiety in a catalytically competent conformation. Comparative analysis highlights GumK-specific motifs distinguishing it from homologous GTs. This work provides mechanistic insight into the GT70 family and the dynamic behavior of GT-B enzymes, establishing principles for rational engineering of GumK to modify the monosaccharide composition of xanthan gum.
Luciano et al. (Tue,) studied this question.