title: Structural and mechanistic characterization of leukocyte-type core 2 β1,6-N-acetylglucosaminyltransferase: a metal-ion-independent GT-A glycosyltransferase.
abstract: Leukocyte-type core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT-L) is an inverting, metal-ion-independent glycosyltransferase that catalyzes the formation of mucin-type core 2 O-glycans. C2GnT-L belongs to the GT-A fold, yet it lacks the metal ion binding DXD motif characteristic of other nucleoside disphosphate GT-A fold glycosyltransferases. To shed light on the basis for its metal ion independence, we have solved the X-ray crystal structure (2.3 Å resolution) of a mutant form of C2GnT-L (C217S) in complex with the nucleotide sugar product UDP and, using site-directed mutagenesis, examined the roles of R378 and K401 in both substrate binding and catalysis. The structure shows that C2GnT-L exists in an "open" conformation and a "closed" conformation and that, in the latter, R378 and K401 interact with the β-phosphate moiety of the bound UDP. The two conformations are likely to be important in catalysis, but the conformational changes that lead to their interconversion do not resemble the nucleotide-sugar-mediated loop ordering observed in other GT-A glycosyltransferases. R378 and K401 were found to be important in substrate binding and/or catalysis, an observation consistent with the suggestion that they serve the same role played by metal ion in all of the other GT-A glycosyltransferases studied to date. Notably, R378 and K401 appear to function in a manner similar to that of the arginine and lysine residues contained in the RX(4-5)K motif found in the retaining GT-B glycosyltransferases.
authors: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors: John E
authors: JE
authors: Pak
authors: Malathy
authors: M
authors: Satkunarajah
authors: Jayaraman
authors: J
authors: Seetharaman
authors: James M
authors: JM
authors: Rini
mesh_terms: D020816:Amino Acid Motifs; D057809:HEK293 Cells; D006801:Humans; D007962:Leukocytes / Q000201:enzymology*; D008958:Models, Molecular; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation
chemical_list: D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase