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The 6-PGDH catalyzes the oxidative decarboxylation of a beta-hydroxyacid and is a member of a class of enzymes including malic enzyme and isocitrate dehydrogenase. Mechanistically this class of enzymes is thought to catalyze the oxidative decarboxylation in two steps with oxidation of the alcohol beta to the carboxyl to be eliminated, preceding decarboxylation. Recent work from the lab of the PI, however, suggests that this class of enzymes can catalyze either a concerted or stepwise oxidative decarboxylation dependent on the enzyme studied, and the dinucleotide substrate used. Interestingly, comparison of the three dimensional structures of the 6-PGDH and the isocitrate dehydrogenase, and the amino acid sequence of the malic enzyme indicates that each of these is distinct from the other at least with respect to the dinucleotide binding site. A significant amount of mechanistic information is now available for the Candida 6-PGDH. However, whether there is a Lewis acid requirement for the oxidative decarboxylation portion of the reaction (the enzyme does not require a divalent metal ion), the identity of the acid-base catalytic and binding groups and what each contributes in terms of the free energy of binding and/or catalysis is not clear. A crystal structure is available for the sheep liver enzyme, but not for the Candida enzyme, while a full length clone and expression system is available for neither. Thus, the sheep liver enzyme will be cloned, sequenced, and expressed in preparation for crystallization and mutagenesis, and studied kinetically to determine where differences exist (if they do) compared to the Candida enzyme. These general objectives will be carried out via the following Specific Aims. 1. The sheep liver 6-PGDH clone will be isolated from a sheep liver cDNA library in lambda gt10. The library will be screened initially using oligonucleotide probes synthesized based on the known sequence of the cDNA and finally via metabolic selection using an Escherichia coli mutant strain that requires the 6-PGDH gene for growth on gluconate. The isolated cDNA will be sequenced, the recombinant protein expressed and characterized. 2. Mutant 6-PDGH enzymes will be crystallized in the absence and presence of reactants and their structures solved to determine what differences have resulted from the amino acid change. This can most likely be accomplished by the difference method in most cases. 3. Kinetic studies of the sheep liver 6-PGDH will be carried out to obtain mechanistic information. Selected initial velocity, pH, and isotope effect studies will be performed to identify the differences, qualitative and quantitative, between sheep liver 6-PGDH and the enzyme from Candida. 4. Site-directed mutagenesis will be carried out to identify catalytic and binding groups and the contribution to catalysis. Emphasis will initially be placed on acid-base catalytic groups, sugar and dinucleotide binding groups. These studies should significantly increase our understanding of the mechanism of 6- phosphogluconate dehydrogenase, and beta-hydroxyacid oxidative decarboxylases in general. They should specifically provide for the 6- phosphogluconate dehydrogenase the unique opportunity to understand the kinetics of the reaction of this metal-independent oxidative decarboxylase in terms of the enzyme's structure.
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