The structure of chondroitin B lyase complexed with glycosaminoglycan oligosaccharides unravels a calcium-dependent catalytic machinery.
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 beta-helical polysaccharide lyase belongs to family PL-6 and cleaves the beta(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.
Michel
Gurvan
G
Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada.
Pojasek
Kevin
K
Li
Yunge
Y
Sulea
Traian
T
Linhardt
Robert J
RJ
Raman
Rahul
R
Prabhakar
Vikas
V
Sasisekharan
Ram
R
Cygler
Miroslaw
M
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D002808:Chondroitin Lyases / Q000737:chemistry*; D002810:Chondroitinases and Chondroitin Lyases / Q000737:chemistry; D018360:Crystallography, X-Ray; D003871:Dermatan Sulfate / Q000737:chemistry; D004187:Disaccharides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004583:Electrons; D019075:Electrophoresis, Capillary; D006025:Glycosaminoglycans / Q000737:chemistry*; D007477:Ions; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D011133:Polysaccharide-Lyases / Q000737:chemistry; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013552:Swine
D004187:Disaccharides; D006025:Glycosaminoglycans; D007477:Ions; D009844:Oligosaccharides; D003871:Dermatan Sulfate; D001120:Arginine; D002808:Chondroitin Lyases; D002810:Chondroitinases and Chondroitin Lyases; D011133:Polysaccharide-Lyases; C446037:chondroitinase B; D008239:Lysine; D002118:Calcium