Structure of the polyisoprenyl-phosphate glycosyltransferase GtrB and insights into the mechanism of catalysis.
The attachment of a sugar to a hydrophobic polyisoprenyl carrier is the first step for all extracellular glycosylation processes. The enzymes that perform these reactions, polyisoprenyl-glycosyltransferases (PI-GTs) include dolichol phosphate mannose synthase (DPMS), which generates the mannose donor for glycosylation in the endoplasmic reticulum. Here we report the 3.0 Å resolution crystal structure of GtrB, a glucose-specific PI-GT from Synechocystis, showing a tetramer in which each protomer contributes two helices to a membrane-spanning bundle. The active site is 15 Å from the membrane, raising the question of how water-soluble and membrane-embedded substrates are brought into apposition for catalysis. A conserved juxtamembrane domain harbours disease mutations, which compromised activity in GtrB in vitro and in human DPM1 tested in zebrafish. We hypothesize a role of this domain in shielding the polyisoprenyl-phosphate for transport to the active site. Our results reveal the basis of PI-GT function, and provide a potential molecular explanation for DPM1-related disease.
Ardiccioni, C; Clarke, OB; Tomasek, D; Issa, HA; von Alpen, DC; Pond, HL; Banerjee, S; Rajashankar, KR; Liu, Q; Guan, Z; Li, C; Kloss, B; Bruni, R; Kloppmann, E; Rost, B; Manzini, MC; Shapiro, L; Mancia, F
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