The structure of chondroitin B lyase complexed with glycosaminoglycan oligosaccharides unravels a calcium-dependent catalytic machinery

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 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 chondroitinsse B completed 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 smbsite. 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 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.