Structure of the polyisoprenyl-phosphate glycosyltransferase GtrB and insights into the mechanism of catalysis

Abstract

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 3.0Å 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.

Dolichol-phosphate mannose synthase depletion in zebrafish leads to dystrophic muscle with hypoglycosylated α-dystroglycan

“…Our dpm morphants showed, grossly, the main characteristics described in the related human disease, namely dystrophic skeletal muscle, hypoglycosylation on α-DG immunostaining, and brain malformations [9]. The phenotype recently described in a morphant model of dpm1 consisted in developmental delay, microcephaly, and ocular defects [11] but knocking-down was generated using a higher level of MO and did not specifically investigate (neither histochemically nor biochemically) muscle function. The M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT severity of our morphant phenotypes grossly correlated with the putative importance of each subunit.…”

“…has also been proposed [11]. Modeling DPMS loss of function in zebrafish in its three protein components might provide us with a means of studying, in vivo, the pathological and biochemical consequences of DPMS hypofunctioning.…”

Structural basis for catalysis at the membrane-water interface

“…In summary, STS describes a protein in which a lipid substrate is taken from the membrane and moved into the catalytic domain, similar to what has been proposed for the GtrB enzyme [72], though the movement required for catalysis to occur appears to be less dramatic.…”

“…The crystal structure of GtrB from Synechocystis sp. at 3.0 Å resolution [72] shows a tetramer, with each subunit consisting of an N-terminal cytosolic glycosyltransferase domain (GT) of the GT-A fold (Fig. 4A) [73], two TM helices (TM1 & TM2) which form a compact stalk anchoring the intracellular domains to the membrane and two amphipathic JM helices (JM1 & JM2) that lie at the surface of the membrane, and connect the GT and TM domains.…”

“…Though the active site lies outside of the membrane environment, the acyl chain of lipid II does not seem to be interacting with the protein and is not drawn out of the membrane as has been hypothesized for GtrB [72] or SCD1 [9, 84]. Here, only the sugar-peptide head groups of the lipids seem to be interacting with the active site, presenting yet another distinct mode of interaction between a lipid-modifying enzyme and a membrane embedded substrate.…”