MECHANISM AND STRUCTURE OF PROTOZOAN PTERIDINE REDUCTASE

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DESCRIPTION: This application describes proposed studies of Leishmania pteridine reductase 1 (PTR1), a newly described enzyme of pterin metabolism. PTR1 belongs to a diverse family of short chain dehydrogenases. This family includes the mammalian enzyme dihydropteridine reductase (DHPR), which catalyzes reduction of the quinoid forms of dihydropterins. Remarkably, the reactions catalyzed by PTR1 appear to more closely resemble reactions catalyzed by a structurally unrelated enzyme, dihydrofolate reductase (DHFR). Unlike DHFR or any previously described enzyme, however, PTR1 prefers to reduce fully oxidized pteridines, including folate and biopterin. PTR1 is a potential drug target, since either inhibitors of the enzyme or deletion of its gene prevent the growth of Leishmania in culture. Worldwide, 10-12 million people suffer from diseases caused by the parasitic protozoan Leishmania, and no good therapy currently exists. The long term goal of the proposed research is to decipher how the structure of PTR1 relates to substrate and inhibitor binding and catalysis of hydride transfer from NADPH to several key pteridine substrates. This will allow informative comparisons of the PTR1 mechanism to those of DHPR and DHFR. Aim 1 will involve the generation of PTR1 variants by site-directed mutagenesis, to test the sequence requirements of a region of the protein proposed to be involved, in hydride transfer catalysis. Aim 2 will test the similarity of the stereochemical and kinetic aspects of PTR1 catalysis to those of other short chain dehydrogenases and to those of DHFR. These experiments will also provide valuable tools for studying the effects of interesting mutations deriving from Aim 1, and for understanding the inhibition of PTR1 by drug candidates being identified by screening in the laboratory of our collaborator, Dr. Stephen Beverley. Dr. Beverley and his colleagues will provide quantities of the wild type enzyme needed for Aims 2 and 3. Aim 3 is to identify crystals of the PTR1 holoenzyme and complexes which are suitable for high resolution x-ray diffraction studies. Such crystals will be essential for determining the atomic structure of PTR1, leading to stereochemical models for PTR1 catalysis and inhibition.