Structural and functional studies of enzymes involved in glycosaminoglycan synthesis and degradation

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This proposal aims at a structural and functional characterization of enzymes involved in synthesis and degradation of glycosaminoglycans (GAGs). GAGs are negatively charged linear polysaccharides that are essential for providing resilience to connective tissues, storage of growth factors and other proteins, participate in tissue repair and signaling through receptors. The presence of GAGs is usually associated with higher organisms; however, they are not unique to eukaryotes. Several specialized microorganisms also produce unsulfated forms of these polymers. Mammalian GAGs undergo rapid turnover through action of hydrolases. Bacteria evolved enzymes that use a lytic mechanism to degrade GAGs, namely polysaccharide lyases. These enzymes help bacteria to gain access to mammalian cells by degrading the surface polysaccharides or to utilize GAGs as a food source. Here we propose to expand our research into GAG processing enzymes and provide structural, functional and enzymological characterization of hyaluronan synthase, GAG sulfotransferases and polysaccharide lyases. All these proteins may also potential applications in synthesis of designer oligosaccharides for treatment of diseases and specific tools for characterization of GAGs. The short-term objectives are: 1. To determine the structures and catalytic mechanisms of polysaccharide lyases from families PL12 and PL17 and establish their evolutionary relationship to other PL families. 2. To establish through structural investigations and site-directed mutagenesis the mechanism of a concerted alternate addition of a glucuronate and a glucosamine by two independent glycosyltransferase active sites within hyaluronan synthase. 3. To determine the structures of lumenal fragments of mammalian sulfotransferases and identify features which define their positional specificity for sulfate addition.