Abstract

Chondroitinase B from Flavobacterium heparinum is the only known lyase that cleaves the glycosaminoglycan, dermatan sulfate (DS), as its sole substrate. A recent co-crystal structure of chondroitinase B with a disaccharide product of DS depolymerization has provided some insight into the location of the active site and suggested potential roles of some active site residues in substrate binding and catalysis. However, this co-crystal structure was not representative of the actual enzyme-substrate complex, because the disaccharide product did not have the right length or the chemical structure of the minimal substrate (tetrasaccharide) involved in catalysis. Therefore, only a limited picture of the functional role of active site residues in DS depolymerization was presented in previous structural studies. In this study, by docking a DS tetrasaccharide into the proposed active site of the enzyme, we have identified novel roles of specific active site amino acids in the catalytic function of chondroitinase B. Our conformational analysis also revealed a unique, symmetrical arrangement of active site amino acids that may impinge on the catalytic mechanism of action of chondroitinase B. The catalytic residues Lys-250, Arg-271, His-272, and Glu-333 along with the substrate binding residues Arg-363 and Arg-364 were mutated using site-directed mutagenesis, and the kinetics and product profile of each mutant were compared with recombinant chondroitinase B. Mutating Lys-250 to alanine resulted in inactivation of the enzyme, potentially attributable to the role of the residue in stabilizing the carbanion intermediate formed during enzymatic catalysis. The His-272 and Glu-333 mutants showed diminished enzymatic activity that could be indicative of a possible role for one or both residues in the abstraction of the C-5 proton from the galactosamine. In addition, the Arg-364 mutant had an altered product profile after exhaustive digestion of DS, suggesting a role for this residue in defining the substrate specificity of chondroitinase B.

Highlights

  • Dermatan sulfate (DS)1 and chondroitin sulfate (CS) are related glycosaminoglycans that are composed of a disaccha

  • Interactions between Chondroitinase B and Dermatan Sulfate Substrate—The structure of a previously crystallized dermatan sulfate (DS) tetrasaccharide was docked into the chondroitinase B active site

  • When the non-reducing end of the tetrasaccharide was superimposed with the disaccharide product from the co-crystal structure, the orientation of the tetrasaccharide was such that its reducing end collided with a wall of the active site cleft (Fig. 1A)

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Summary

Introduction

Dermatan sulfate (DS) and chondroitin sulfate (CS) are related glycosaminoglycans that are composed of a disaccha-. Flavobacterium heparinum is a common source for glycosaminoglycan-degrading lyases, producing both the extensively characterized heparin-degrading heparinases (14 –16) and the DS/CS-degrading chondroitinases [17]. Extensive biochemical characterization of the catalytic mechanism and substrate specificities of the heparinases enabled their application as tools to sequence biologically important heparin oligosaccharides [13, 20]. Chondroitinase B, like the other glycosaminoglycan-degrading lyases from F. heparinum, is thought to cleave its DS substrate through a concerted ␤-elimination mechanism originally proposed by Gerlt and Gassman [21]. The first step in the proposed reaction is the abstraction of the C-5 proton on the uronic acid moiety by a basic amino acid forming an enolate intermediate The enzyme stabilizes this carbanion intermediate usually via a positively charged, hydrophilic amino acid [21, 22]. The final step of the reaction mechanism involves protonation of the anomeric oxygen by an acidic residue with consulfate; GalNAc, N-acetylgalactosamine; ⌬UA, uronic acid moiety with a 4,5 double bond; IdoUA, iduronic acid; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry; 4S, sulfation at the 4-O position of galactosamine; 6S, sulfation at the 6-O position of galactosamine; 2S, sulfation at the 2-O position of a uronic acid; ⌬Di4S, ⌬UA-GalNAC-4S

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