X-ray crystal structure of leukocyte type core 2 beta1,6-N-acetylglucosaminyltransferase. Evidence for a convergence of metal ion-independent glycosyltransferase mechanism.
Leukocyte type core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT-L) is a key enzyme in the biosynthesis of branched O-glycans. It is an inverting, metal ion-independent family 14 glycosyltransferase that catalyzes the formation of the core 2 O-glycan (Galbeta1-3[GlcNAcbeta1-6]GalNAc-O-Ser/Thr) from its donor and acceptor substrates, UDP-GlcNAc and the core 1 O-glycan (Galbeta1-3GalNAc-O-Ser/Thr), respectively. Reported here are the x-ray crystal structures of murine C2GnT-L in the absence and presence of the acceptor substrate Galbeta1-3GalNAc at 2.0 and 2.7A resolution, respectively. C2GnT-L was found to possess the GT-A fold; however, it lacks the characteristic metal ion binding DXD motif. The Galbeta1-3GalNAc complex defines the determinants of acceptor substrate binding and shows that Glu-320 corresponds to the structurally conserved catalytic base found in other inverting GT-A fold glycosyltransferases. Comparison of the C2GnT-L structure with that of other GT-A fold glycosyltransferases further suggests that Arg-378 and Lys-401 serve to electrostatically stabilize the nucleoside diphosphate leaving group, a role normally played by metal ion in GT-A structures. The use of basic amino acid side chains in this way is strikingly similar to that seen in a number of metal ion-independent GT-B fold glycosyltransferases and suggests a convergence of catalytic mechanism shared by both GT-A and GT-B fold glycosyltransferases.
Pak
John E
JE
Department of Molecular, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Arnoux
Pascal
P
Zhou
Sihong
S
Sivarajah
Prashanth
P
Satkunarajah
Malathy
M
Xing
Xuekun
X
Rini
James M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D007477:Ions / Q000737:chemistry*; D008670:Metals / Q000737:chemistry*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism
D007477:Ions; D008670:Metals; D009844:Oligosaccharides; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D014535:Uridine Diphosphate Glucuronic Acid; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase