MECHANISM AND STRUCTURE OF PROTOZOAN PTERIDINE REDUCTASE

描述：本申请描述了利什曼原虫的拟议研究 蝶啶还原酶1（PTR 1）是一种新发现的蝶啶酶 新陈代谢. PTR 1属于一个多样化的短链家族 - 是的这个家族包括哺乳动物的酶 二氢蝶啶还原酶（DHPR），其催化二氢蝶啶的还原。 醌型二氢蝶呤。值得注意的是， PTR 1看起来更类似于由一种 结构无关酶，二氢叶酸还原酶（DHFR）。不像 DHFR或任何先前描述的酶，然而，PTR 1倾向于减少 完全氧化的蝶啶，包括叶酸和生物蝶呤。PTR 1是一种 潜在的药物靶点，因为无论是酶的抑制剂或删除 阻止利什曼原虫在培养物中的生长。在世界范围内， 1000万至1200万人患有寄生虫引起的疾病 利什曼原虫，目前还没有好的治疗方法。 拟议研究的长期目标是破译 PTR 1的结构涉及底物和抑制剂结合， 催化NADPH氢转移生成几种关键蝶啶 印刷受体.这将允许PTR 1的信息比较 DHPR和DHFR的机制。目标1将涉及一代人 PTR 1变异体，以测试序列 建议参与的蛋白质区域的要求， 氢化物转移催化目标2将测试 PTR 1催化作用立体化学和动力学方面与其它催化作用的比较 短链脱氢酶和DHFR的那些。这些实验将 也提供了宝贵的工具，研究的影响， 目的1衍生的突变，并了解抑制 通过实验室筛选确定候选药物的PTR 1 我们的合作者斯蒂芬·贝弗利博士贝弗利医生和他的 同事们将提供大量的野生型酶， 目标2和3。目的3是鉴定PTR 1全酶的晶体， 适用于高分辨率X射线衍射的络合物 问题研究这种晶体将是必不可少的确定原子 PTR 1的结构，导致PTR 1催化的立体化学模型 和抑制。

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.