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

• Published in: The Journal of biological chemistry Vol. 279; no. 31; pp. 32882 - 32896
• Main Authors: Michel, Gurvan, Projasek, Kevin, Li, Yunge, Sulea, Traian, Linhardt, Robert J, Raman, Rahul, Prabhakar, Vikas, Sasisekharan, Ram, Cygler, Miroslaw
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 30.07.2004
• Subjects: Amino Acid Sequence; Animals; Arginine - chemistry; BASIC BIOLOGICAL SCIENCES; Binding Sites; calcium; Calcium - chemistry; Calcium - metabolism; Catalysis; CHONDROITIN; Chondroitin Lyases - chemistry; chondroitinase B; Chondroitinases and Chondroitin Lyases - chemistry; crystal structure; Crystallography, X-Ray; dermatan sulfate; Dermatan Sulfate - chemistry; Disaccharides - chemistry; Dose-Response Relationship, Drug; Electrons; Electrophoresis, Capillary; Glycosaminoglycans - chemistry; Ions; Kinetics; LYASES; Lysine - chemistry; MACHINERY; Models, Chemical; Models, Molecular; Molecular Sequence Data; Mutagenesis; Mutagenesis, Site-Directed; NATIONAL SYNCHROTRON LIGHT SOURCE; OLIGOSACCHARIDES; Oligosaccharides - chemistry; Pedobacter heparinus; pharmaceutical; Polymerase Chain Reaction; Polysaccharide-Lyases - chemistry; Protein Conformation; Sequence Homology, Amino Acid; Swine; X-ray
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M403421200

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 β-helical polysaccharide lyase belongs to family PL-6 and cleaves the β(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-Ca 2+ -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 Ca 2+ in enzymatic activity. Given these results, we propose that the Ca 2+ 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.