Active site of chondroitin AC lyase revealed by the structure of enzyme-oligosaccharide complexes and mutagenesis

The crystal structures of Flavobacterium heparinium chondroitin AC lyase (c hondroitinase AC; EC 4.2.2.5) bound to dermatan sulfate hexasaccharide (D-S hexa),tetrasaccharide (DStetra), and hyaluronic acid tetrasaccharide (HA(te tra)) have been refined at 2.0, 2.0, and 2.1 A resolution, respectively. Th e structure of the Tyr234Phe mutant of AC lyase bound to a chondroitin sulf ate tetrasaccharide (CStetra) has also been determined to 2.3 Angstrom reso lution. For each of these complexes, four (DShexa and CStetra) Or two (DSte tra and HA(tetra)) ordered sugars are visible in electron density maps. The lyase AC DShexa and CStetra complexes reveal binding at four subsites, -2, -1, +1, and +2, within a narrow and shallow protein channel. We suggest th at subsites -2 and -1 together represent the substrate recognition area, +1 is the catalytic subsite and ii and +2 together represent the product rele ase area. The putative catalytic site is located between the substrate reco gnition area and the product release area, carrying out catalysis at the +1 subsite. Four residues near the catalytic site, His225, Tyr234, Arg288, an d Glu371 together form a catalytic tetrad. The mutations His225Ala, Tyr234P he, Arg288Ala, and Arg292Ala, revealed residual activity for only the Arg29 2Ala mutant. Structural data indicate that Arg292 is primarily involved in recognition of the N-acetyl and sulfate moieties of galactosamine, but does not participate directly in catalysis. Candidates for the general base, re moving the proton attached to C-5 of the glucuronic acid at the +1 subsite, are Tyr234, which could be transiently deprotonated during catalysis, or H is225. Tyrosine 234 is a candidate to protonate the leaving group. Arginine 288 likely contributes to charge neutralization and stabilization of the e nolate anion intermediate during catalysis.