PRELIMINARY CRYSTALLOGRAPHIC ANALYSIS OF CHONDROITINASE AC FROM FLAVOBACTERIUM HEPARINUM. J. Féthière, B. H. Shilton, Y. Li, M. Laliberté, B. Eggimann, M. Cygler, Biotechnology Research Institute, National Research Council of Canada, Montréal, Qué. Can., H4P 2R2, IBEX Technologies, Montréal, Qué. Can. H4P 1P7

Glycosaminoglycans (GAG's) are polymeric saccharide structures found in the extracellular matrix, and on the cell surface of many cell types where they are linked to core proteins. Their biological function ranges from serving as a protective barrier to diffusion, to the modulation of cell signalling. In vivo, there are two main mechanism for their degradation: the eliminative cleavage by lyases and the hydrolytic cleavage by hydrolases. The enzymes responsible for this degradation are specific for particular sequences in the GAG chain. Degradation of GAG's (or the lack thereof) has been linked to particular diseases, and the enzymes involved are potential targets for new pharmaceuticals. Chondroitinase AC represent one of the three categories of lyases; it is responsible for the cleavage of chondroitin-4-sulfate, and chondroitin-6-sulfate. In order to understand the structural aspect of the narrow substrate specificity of these lyases, we have undertaken the structure determination of chondroitinase AC from Flavobacterium heparinum. The enzyme expressed in E. coli is a monomer of 77 kDa with two O-glycosylation sites. Tetragonal crystals of 0.2 x 0.2 x 1.0 mm are obtained after 3 weeks from a solution containing PEG 3350. They belong to the space group P4₃2₁2 or its enantiomorph P4₁2₁2. The unit cell dimensions are a=b=87.2Å, and c=192.8Å. Assuming one 77 kDa molecule in the assymetric unit, one obtains a Matthews coefficient of 2.37, a typical value for protein crystals. The crystals are sensitive to radiation damage, thus they were flash frozen for data collection. A 2.9Å dataset was collected at 130K. We are now in the process of screening for heavy atom derivatives in order to solve the structure by the multiple isomorphous replacement method. Initial screening identified potential derivatives that will be used for the structure determination.