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J. Biol. Chem., Vol. 282, Issue 19, 14300-14308, May 11, 2007

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Structural and Mechanistic Analyses of endo-Glycoceramidase II, a Membrane-associated Family 5 Glycosidase in the Apo and G_M3 Ganglioside-bound Forms^*

Matthew E. C. Caines, Mark D. Vaughan, Chris A. Tarling, Susan M. Hancock, R. Antony J. Warren^¶, Stephen G. Withers, and Natalie C. J. Strynadka¹

From the Departments of Biochemistry and Molecular Biology, Chemistry, and ^¶Microbiology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

endo-Glycoceramidase, a membrane-associated family 5 glycosidase, deviates from the typical polysaccharide substrate specificity of other soluble members of the family, preferentially hydrolyzing glycosidic linkages between the oligosaccharide and ceramide moieties of gangliosides. Here we report the first x-ray crystal structures of an endo-glycoceramidase from Rhodococcus sp., in the apo form, in complex with the ganglioside G_M3 (Svennerholm ganglioside nomenclature (Svennerholm, L. (1964) J. Lipid Res. 5, 145-155)), and trapped as a glycosyl-enzyme intermediate. These snapshots provide the first molecular insight into enzyme recognition and association with gangliosides, revealing the structural adaptations necessary for glycosidase-catalyzed hydrolysis and detailing a novel ganglioside binding topology. Consistent with the chemical duality of the substrate, the active site of endo-glycoceramidase is split into a wide, polar cavity to bind the polyhydroxylated oligosaccharide moiety and a narrow, hydrophobic tunnel to bind the ceramide lipid chains. The specific interactions with the ceramide polar head group manifest a surprising aglycone specificity, an observation substantiated by our kinetic analyses. Collectively, the reported structural and kinetic data provide insight toward rational redesign of the synthetic glycosynthase mutant of endo-glycoceramidase to enable facile synthesis of nonnatural, therapeutically useful gangliosides.

Received for publication, December 14, 2006 , and in revised form, February 8, 2007.

The atomic coordinates and structure factors (code 2OSW, 2OSX, and 2OSY) 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 Neose Technologies and by the Royal Society (United Kingdom), Howard Hughes Medical Institute, Canadian Institutes of Health Research, Natural Sciences and Engineering Research Council of Canada, and the Protein Engineering Network of Centres of Excellence. 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 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada. Tel.: 604-822-0789; Fax: 604-822-5227; E-mail: natalie{at}byron.biochem.ubc.ca.

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