This research will elucidate the biochemistry and structural biology for a class of enzymes and proteins known as glycosyl hydrolases. We will specifically study glycosyl hydrolases that hydrolyze chitin and chitin- like polymers into smaller saccharides and ultimately to monosaccharide. This is performed by a set of enzymes that cleave either in the middle of the polysaccharide or by releasing mono-or disaccharides from either the non-reducing or reducing end of the polysaccharide and its fragments. In order to accomplish our goal, we plan to use computational chemistry methods coupled with molecular and structural biological techniques to study Chitinase A, Brp39, ( breast regression protein ), and Chitobiase, all members of the same chitinolytic Family. For example molecular mechanics/dynamics will be used to investigate the binding of polysaccharides to Chitinase A while coupled quantum mechanics/molecular mechanics will be used to study the catalytic reaction mechanism. Molecular biology and biochemistry will be used to validate the computational results as well as produce Chitinase A mutants that bind a polysaccharide without cleaving. A three-dimensional crystal structure of this inactive Chitinase A mutant with the bound substrate will be determined by x-ray crystallography. Thus the combined computational, molecular biological and structural results of this effort will yield a clear detailed molecular picture and understanding of the various interactions responsible of the binding of a polysaccharide to Chitinase A and an unambiguous picture of the catalytic reaction mechanism. Similar procedures will be followed in the study of Brp39 and Chitobiase. The new knowledge from this work will be applicable across several of the over 50 glycosyl hydrolase Families. This is because there is 1] conservation of the vital catalytic and other active site residues; 2] common structural evolution of glycosidases with the (beta/alpha)8-barrel folding motif; and 3] similar acid/base or substrate-assisted catalytic reaction mechanisms. These results are medically relevant for understanding lysosomal storage disease mechanisms. For example, most of the lysosomal hydrolases whose genetic deficiencies cause devastating tissue storage diseases are glycosidases, including hexosaminidase defects being responsible for Tay-Sachs or Sandhoff disease. Additionally, Brp39 is in a subgroup of these proteins that no longer show catalytic activity but which work in a variety of developmental and differentiation processes, most likely via an ability to bind oligomers of chitin. Finally, these results are of biotechnological relevance such as in the development of antifungal agents.
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