Computational characterization of the xanthan gum glycosyltransferase GumK

Abstract 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, whereas donor-substrate binding stabilizes closure, defining a substrate-dependent catalytic cycle. Specificity for UDP-glucuronate 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 that distinguish it from homologous GTs. This work provides mechanistic insight into the GT70 family and the dynamic behavior of GT-B enzymes, establishing principles for the rational engineering of GumK to modify the monosaccharide composition of xanthan gum. Author summary Glycosyltransferases are enzymes that build many of the sugars and polysaccharides essential for life. One of them, called GumK, is responsible for a key step in producing xanthan gum — a natural polymer widely used as a thickener in food and industrial materials. Despite its importance, how GumK works at the molecular level has remained unclear. In our study, we used multiscale computer simulation strategies to explore the dynamics of this enzyme and how it interacts with the cell membrane and its sugar substrates. We found that GumK acts like a flexible clamp that opens and closes as it binds two different sugar molecules through specific residues that may control the enzyme’s selectivity. We also discovered how the surrounding membrane helps the enzyme remain in the correct orientation to perform its function. By identifying the molecular details that determine GumK’s selectivity and flexibility, our work provides a foundation for modifying this enzyme to produce xanthan gum with new properties. More broadly, our findings help explain how a large family of related enzymes controls sugar transfer in living organisms, with potential applications in biotechnology and materials science.