): 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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA011778-29
Application #
2610055
Study Section
Experimental Therapeutics Subcommittee 1 (ET)
Program Officer
Beisler, John A
Project Start
1979-05-01
Project End
2001-03-31
Budget Start
1998-04-01
Budget End
1999-03-31
Support Year
29
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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Chang, C F; Bray, T; Whiteley, J M (1999) Mutant PTR1 proteins from Leishmania tarentolae: comparative kinetic properties and active-site labeling. Arch Biochem Biophys 368:161-71
Kiefer, P M; Grimshaw, C E; Whiteley, J M (1997) The comparative interaction of quinonoid (6R)-dihydrobiopterin and an alternative dihydropterin substrate with wild-type and mutant rat dihydropteridine reductases. Biochemistry 36:9438-45
Wang, J; Leblanc, E; Chang, C F et al. (1997) Pterin and folate reduction by the Leishmania tarentolae H locus short-chain dehydrogenase/reductase PTR1. Arch Biochem Biophys 342:197-202
Kiefer, P M; Varughese, K I; Su, Y et al. (1996) Altered structural and mechanistic properties of mutant dihydropteridine reductases. J Biol Chem 271:3437-44
Varughese, K I; Xuong, N H; Kiefer, P M et al. (1994) Structural and mechanistic characteristics of dihydropteridine reductase: a member of the Tyr-(Xaa)3-Lys-containing family of reductases and dehydrogenases. Proc Natl Acad Sci U S A 91:5582-6
Varughese, K I; Xuong, N H; Whiteley, J M (1994) Structural and mechanistic implications of incorporating naturally occurring aberrant mutations of human dihydropteridine reductase into a rat model. Int J Pept Protein Res 44:278-87
Su, Y; Varughese, K I; Xuong, N H et al. (1993) The crystallographic structure of a human dihydropteridine reductase NADH binary complex expressed in Escherichia coli by a cDNA constructed from its rat homologue. J Biol Chem 268:26836-41
Whiteley, J M; Xuong, N H; Varughese, K I (1993) Is dihydropteridine reductase an anomalous dihydrofolate reductase, a flavin-like enzyme, or a short-chain dehydrogenase? Adv Exp Med Biol 338:115-21

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