Glycosaminoglycan degradation---Structural and kinetic studies of heparinase II and chondroitinase ABC

Glycosaminoglycans (GAGS) are linear, heterogeneous, negatively charged polysaccharides, common within the extracellular matrix (ECM) and at the cell-surfaces of all metazoan cells. Apart from their structural importance to the integrity of the ECM, GAGs are also fundamental modulators of various biological processes at the level of cells (i.e. adhesion, signaling and proliferation), tissue (i.e. inflammation, wound repair, tissue morphogenesis and organogenesis) and organism (i.e. cancer and developmental processes). The biological activities of GAGs are intricately linked to their turnover.;These studies resulted in the first structure of a heparin/HS-degrading enzyme, namely P. heparinus heparinase II, as well as the structure of B. thetaiotaomicron chondroitin lyase ABCII. They demonstrate that both enzymes utilize a single active site for cleavage next to the uronic acid, with different subset of residues assuming catalytic roles for L-iduronic acid vs. D-glucoronic acid. Based on the accumulated experimental data, an understanding of the observed substrate specificities of these lyases and their modes of action is proposed. Finally, the B. thetaiotaomicron chondroitin lyase was biochemically characterized in order to lay the groundwork for its clinical applications.;Niche-adapted microorganisms express GAG-degrading enzymes, allowing them to process GAGS for nutritional purposes, both for themselves and for their mammalian hosts. In particular, bacterial GAG lyases cleave the glycosidic bond next to the uronic acid present in GAGs, through a beta-elimination mechanism that yields disaccharide products with a saturated 4-deoxy-alpha- L-threo-hex-4-enopyranosyluronic acid (DeltaUA). Among the lyasses, heparinase II (HepII) and chondroitinase ABC (ChonABC) possess the striking ability to degrade GAGS regardless of their uronic acid epimer. HepII degrades both heparin and heparan sulfate, whereas ChonABC degrades both chondroitin sulfate and dermatan sulfate. This research is aimed at providing insight into the catalytic mechanism and the substrate specificity of these two enzymes at the molecular level. The applied methodologies used were: (1) X-ray crystallography, aimed at obtaining the crystal structures of P. heparinus HepII and B. thetaiotaomicron ChonABCII as well as enzyme-substrate/product complexes, (2) site-directed mutagenesis of key residues identified in the structures, and (3) the enzymologie characterization of these mutants, to provide insight into the mechanistic roles of the postulated active site residues necessary for degradation of GAGs by HepII and ChonABCII.