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J. Biol. Chem., Vol. 279, Issue 31, 32882-32896, July 30, 2004

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The Structure of Chondroitin B Lyase Complexed with Glycosaminoglycan Oligosaccharides Unravels a Calcium-dependent Catalytic Machinery^*

Gurvan Michel¶, Kevin Pojasek||, Yunge Li, Traian Sulea, Robert J. Linhardt**, Rahul Raman||, Vikas Prabhakar||, Ram Sasisekharan||, and Miroslaw Cygler

From the Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada, the Montreal Joint Centre for Structural Biology, Montreal, Quebec H4P 2R2, Canada, the ^||Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and the ^**Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York 12180

Chondroitinase B from Pedobacter heparinus is the only known enzyme strictly specific for dermatan sulfate and is a widely used enzymatic tool for the structural characterization of glycosaminoglycans. This -helical polysaccharide lyase belongs to family PL-6 and cleaves the (1,4) linkage of dermatan sulfate in a random manner, yielding 4,5-unsaturated dermatan sulfate disaccharides as the product. The previously reported structure of its complex with a dermatan sulfate disaccharide product identified the -1 and -2 subsites of the catalytic groove. We present here the structure of chondroitinase B complexed with several dermatan sulfate and chondroitin sulfate oligosaccharides. In particular, the soaking of chondroitinase B crystals with a dermatan sulfate hexasaccharide results in a complex with two dermatan sulfate disaccharide reaction products, enabling the identification of the +2 and +1 subsites. Unexpectedly, this structure revealed the presence of a calcium ion coordinated by sequence-conserved acidic residues and by the carboxyl group of the L-iduronic acid at the +1 subsite. Kinetic and site-directed mutagenesis experiments have subsequently demonstrated that chondroitinase B absolutely requires calcium for its activity, indicating that the protein-Ca²⁺-oligosaccharide complex is functionally relevant. Modeling of an intact tetrasaccharide in the active site of chondroitinase B provided a better understanding of substrate specificity and the role of Ca²⁺ in enzymatic activity. Given these results, we propose that the Ca²⁺ ion neutralizes the carboxyl moiety of the L-iduronic acid at the cleavage site, whereas the conserved residues Lys-250 and Arg-271 act as Brønsted base and acid, respectively, in the lytic degradation of dermatan sulfate by chondroitinase B.

Received for publication, March 29, 2004 , and in revised form, May 21, 2004.

The atomic coordinates and structure factors (code 1OFL and 1OFM) 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 in part by Canadian Institutes for Health Research Grant 200009MOP-84373-M-CFAA-26164 (to M. C.); National Institutes of Health Grants GM38060, HL52622, and HL62244 (to R. J. L.); and National Institutes of Health Grant GM57073 (to R. S.). 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.

^¶ Present address: Végétaux Marins et Biomolécules, UMR 7139 (CNRS/UPMC/Goömar), Station Biologique, Place Georges Teissier, BP 74, 29682 Roscoff Cedex, Bretagne, France.

To whom correspondence should be addressed: Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada. Tel.: 514-496-6321; Fax: 514-496-5143; E-mail: mirek{at}bri.nrc.ca.

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