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J. Biol. Chem., Vol. 281, Issue 36, 26693-26701, September 8, 2006

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X-ray Crystal Structure of Leukocyte Type Core 2 1,6-N-Acetylglucosaminyltransferase

EVIDENCE FOR A CONVERGENCE OF METAL ION-INDEPENDENT GLYCOSYLTRANSFERASE MECHANISM^*

From the Departments of Molecular and Medical Genetics and Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Leukocyte type core 2 1,6-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 (Gal1-3[GlcNAc1-6]GalNAc-O-Ser/Thr) from its donor and acceptor substrates, UDP-GlcNAc and the core 1 O-glycan (Gal1-3GalNAc-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 Gal1-3GalNAc at 2.0 and 2.7Å resolution, respectively. C2GnT-L was found to possess the GT-A fold; however, it lacks the characteristic metal ion binding DXD motif. The Gal1-3GalNAc 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 disphosphate 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.

Received for publication, April 12, 2006 , and in revised form, July 6, 2006.

The atomic coordinates and structure factors (codes 2GAK and 2GAM) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by the Canadian Institutes of Health Research, the Protein Engineering Network of Centres of Excellence, and GLYCODesign Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Current address: Laboratoire de Bioénergétique Cellulaire, CEA/Cadarache, 13108 Saint Paul Lez Durance, France.

² To whom correspondence should be addressed: Dept. of Molecular and Medical Genetics, University of Toronto, Medical Sciences Bldg., Rm. 5360, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-0557; Fax: 416-978-6885; E-mail: james.rini{at}utoronto.ca.

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