Crystal Structure of Chondroitin AC Lyase, a Representative of a family of Glycosaminoglycan Degrading Enzymes James Fe  thie Áre 1,3 , Bernhard Eggimann 2 and Miroslaw Cygler 1,3 * 1 Biotechnology Research Institute, NRC, 6100 Royalmount Avenue, Montre Âal Que Âbec, H4P 2R2, Canada 2 Ibex Technologies, 5485 Pare  Street, Montre Âal, Que Âbec H4P 2R2, Canada 3 Montre Âal Joint Centre for Structural Biology, Montreal Que Âbec, Canada Glycosaminoglycans (GAGs), highly sulfated polymers built of hexosa- mine-uronic acid disaccharide units, are major components of the extra- cellular matrix, mostly in the form of proteoglycans. They interact with a large array of proteins, in particular of the blood coagulation cascade. Degradation of GAGs in mammalian systems occurs by the action of GAG hydrolases. Bacteria express a large number of GAG-degrading lyases that break the hexosamine-uronic acid bond to create an unsatu- rated sugar ring. Flavobacterium heparinum produces at least ®ve GAG lyases of different speci®city. Chondroitin AC lyase (chondroitinase AC, 75 kDa) is highly active toward chondroitin 4-sulfate and chondroitin-6 sulfate. Its crystal structure has been determined to 1.9 A Ê resolution. The enzyme is composed of two domains. The N-terminal domain of approxi- mately 300 residues contains mostly a-helices which form a doubly- layered horseshoe (a subset of the (a/a) 6 toroidal topology). The 370 residues long C-terminal domain is made of b-strands arranged in a four layered b-sheet sandwich, with the ®rst two sheets having nine strands each. This fold is novel and has no counterpart in full among known structures. The sequence of chondroitinase AC shows low level of hom- ology to several hyaluronate lyases, which likely share its fold. The shape of the molecule, distribution of electrostatic potential, the pattern of con- servation of the amino acids and the results of mutagenesis of hyaluro- nate lyases, indicate that the enzymatic activity resides primarily within the N-terminal domain. The most likely candidate for the catalytic base is His225. Other residues involved in catalysis and/or substrate binding are Arg288, Arg292, Lys298 and Lys299. # 1999 Academic Press Keywords: crystal structure; glycosaminoglycan; chondroitin; chondroitinase; GAG lyase *Corresponding author Introduction Glycosaminoglycans (GAGs) are heterogeneous polymers built of repeating disaccharide units which are extensively N and O-sulfated. The disac- charide units of the polymers are composed of a hexosamine and a uronic acid linked by (1,3) or (1,4) linkages. They are usually covalently linked at the reducing end to core proteins (proteoglycans) through an O-glycosidic linkage to a serine residue. GAGs are divided into four main classes: chondroi- tin sulfate and dermatan sulfate, heparin and heparan sulfate, hyaluronic acid, and keratan sul- fate. Chondroitin sulfate and dermatan sulfate con- tain D-galactosamine linked to glucuronic or iduronic acid, respectively. Sulfation occurs to var- ious degrees at the 2 position of uronic acid and 4, 6 positions of galactosamine, and the amino group of galactosamine is acetylated (Iozzo, 1998). With the exception of hyaluronic acid, GAGs are synthesized as components of proteoglycans in the Golgi where extensive modi®cations to the repeat- ing disaccharides are made prior to subsequent secretion from the cell (Lidholt, 1997). Proteogly- cans remain either bound to the cell surface or become part of the extracellular matrix (ECM). Through their interactions with other ECM E-mail address of the corresponding author: mirek.cygler@bri.nrc.ca Abbreviations used: GAG, glycosaminoglycan; ECM, extracellular matrix; MAD, multiwavelength anomalous dispersion; MIRAS, multiple isomorphous replacement with anomalous scattering; FOM, ®gure of merit. Article No. jmbi.1999.2698 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 288, 635±647 0022-2836/99/190635±13 $30.00/0 # 1999 Academic Press