Active Site of Chondroitin AC Lyase Revealed by the Structure Of
Biochemistry 2001, 40, 2359-2372 2359 Active Site of Chondroitin AC Lyase Revealed by the Structure of Enzyme-Oligosaccharide Complexes and Mutagenesis†,‡ Weijun Huang,§ Lorena Boju,§ Lydia Tkalec,|,⊥ Hongsheng Su,|,# Hyun-Ok Yang,3 Nur Sibel Gunay,3 Robert J. Linhardt,3 Yeong Shik Kim,O Allan Matte,§ and Miroslaw Cygler*,§ Biotechnology Research Institute, 6100 Royalmount AVenue, Montre´al, Que´bec H4P 2R2 Canada, Montreal Joint Centre for Structural Biology, Montre´al, Que´bec, Canada, IBEX Technologies Inc., 5485 Pare Street, Montre´al, Que´bec H4P 1P7 Canada, Department of Chemistry, DiVision of Medicinal Chemistry and Department of Chemical and Biochemical Engineering, The UniVersity of Iowa, 115 South Grand AVenue, PHAR S328, Iowa City, Iowa 52242-1112, and Natural Products Research Institute, Seoul National UniVersity, Seoul 110-460, Korea ReceiVed October 17, 2000; ReVised Manuscript ReceiVed December 18, 2000 ABSTRACT: The crystal structures of FlaVobacterium heparinium chondroitin AC lyase (chondroitinase AC; EC 4.2.2.5) bound to dermatan sulfate hexasaccharide (DShexa), tetrasaccharide (DStetra), and hyaluronic acid tetrasaccharide (HAtetra) have been refined at 2.0, 2.0, and 2.1 Å resolution, respectively. The structure of the Tyr234Phe mutant of AC lyase bound to a chondroitin sulfate tetrasaccharide (CStetra) has also been determined to 2.3 Å resolution. For each of these complexes, four (DShexa and CStetra) or two (DStetra and HAtetra) 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 that subsites -2 and -1 together represent the substrate recognition area, +1 is the catalytic subsite and +1 and +2 together represent the product release area.
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