): Experimental results obtained during the current period of support indicate that the superficial functional similarity of dihydropteridine reductase (DHPR) and dihydrofolate reductase (DHFR) is misleading and that the former enzyme falls into a large family of proteins known as short-chain dehydrogenases/reductases (SDR). Exploitation of this novel observation forms the basis of the present application. Each member of the SDR family contains a dinucleotide (NAD(P)(H) binding pocket close to the N-terminal and a Y(Xaa)3K motif oriented toward the active site. By selecting several biologically important members of the SDR family for investigation (human 3-beta-hydroxysteroid dehydrogenase delta-5,4-isomerase (3-beta-HSD), 15-hydroxyprostaglandin dehydrogenase (PGDH), sepiapterin reductase ((SPR) and a PTR1 protein from Leischmania) and using structural and mechanistic results obtained with DHPR as a foundation, it is intended to discover how each protein accommodates both substrate and dinucleotide allowing efficient two-hydrogen transfer, commensurate with oxidation or reduction, to occur. Each protein will be cloned, overexpressed in E. coli, purified to homogeneity, and crystallized. Kinetic and crystallographic analyis will allow comparison between each enzyme and afford a rationale for the overall integrity of the SDR family. Knowledge of the enzyme structures will also facilitate the design of inhibitors, which may allow control of their important metabolic activitiesAs a corollary to the project, a nucleotide-binding domain (NBD1) of human multiple drug resistant protein (hMDRP) will also be cloned, overexpressed in E. coli, purified to homogeneity, and crystallized in its apo- and ATP-bound form. Structural characterization of these complexes may contribute to the understanding of how drug efflux pumps function.
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