1OFM

CRYSTAL STRUCTURE OF CHONDROITINASE B COMPLEXED TO CHONDROITIN 4-SULFATE TETRASACCHARIDE

Summary for 1OFM

• Entry DOI: 10.2210/pdb1ofm/pdb
• Related: 1DBG 1DBO 1OFL
• Descriptor: CHONDROITINASE B, alpha-D-galactopyranose-(1-3)-[beta-D-glucopyranose-(1-4)]2-O-methyl-alpha-L-fucopyranose-(1-4)-beta-D-xylopyranose-(1-4)-alpha-D-glucopyranuronic acid-(1-2)-[alpha-L-rhamnopyranose-(1-4)]alpha-D-mannopyranose, 4-deoxy-alpha-L-threo-hex-4-enopyranuronic acid-(1-3)-2-acetamido-2-deoxy-4-O-sulfo-beta-D-galactopyranose-(1-4)-beta-D-glucopyranuronic acid-(1-3)-2-acetamido-2-deoxy-4-O-sulfo-beta-D-galactopyranose, ... (4 entities in total)
• Functional Keywords: active site, beta-elimination, chondroitin 4-sulfate, lyase
• Biological source: PEDOBACTER HEPARINUS
• Total number of polymer chains: 1
• Total formula weight: 55726.58

Authors

Michel, G.,Cygler, M. (deposition date: 2003-04-15, release date: 2004-04-19, Last modification date: 2024-11-13)

Primary citation

Michel, G.,Pojasek, K.,Li, Y.,Sulea, T.,Linhardt, R.,Raman, R.,Prabhakar, V.,Sasisekharan, R.,Cygler, M.
The Structure of Chondroitin B Lyase Complexed with Glycosaminoglycan Oligosaccharides Unravels a Calcium-Dependent Catalytic Machinery
J.Biol.Chem., 279:32882-, 2004

PubMed Abstract: 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.

PubMed: 15155751
DOI: 10.1074/JBC.M403421200

Experimental method

X-RAY DIFFRACTION (1.8 Å)