PYRIMIDINE BIOSYNTHESIS IN TOXOPLASMA GONDII

弓形虫中的嘧啶生物合成

• 批准号: 6271702
• 负责人: BARBARA H ZIMMERMANN
• 金额: $ 12.07万
• 依托单位: UNIVERSITY OF PUERTO RICO MED SCIENCES
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6271702
• 关键词: NADPH cytochrome c2 reductase; Toxoplasma gondii; atomic absorption spectrometry; enzyme activity; enzyme biosynthesis; gene complementation; laboratory rabbit; nucleic acid sequence; polymerase chain reaction; pyrimidines; radiotracer

The goal of the proposed research is to gain a better understanding of pyrimidine biosynthesis in Toxoplasma gondii. The presence of the pathway in this organism has been established (Schwartzman & Pefferferkorn, 1981; Asai et al., 1983), however none of the enzymes involved have been fully characterized or cloned. We will study dihydroorotase (DHOase) and dihydroorotate dehydrogenase (DHODase) which catalyze the third and fourth reactions, respectively. Large quantities of recombinant enzymes will be produced in bacterial or yeast expression systems. We will determine whether T.gondii DHOase is a monofunctional enzyme or a domain of a multifunctional enzyme. Our studies will show whether the membrane- associated T. gondii DHODase is a simple flavoprotein, as are some protozoan DHODases (Pascal et al., 1983), or whether it is a two-cofactor protein like the bovine DHODase (Hines & Johnston, 1989). Analysis of sequences, kinetic characteristics, and metal/cofactor composition of T. gondii DHOase and DHODase will lead to an understanding of the catalytic mechanisms at a molecular level. A comparison of the enzymes with their mammalian counterparts will reveal whether T. gondii DHOase and DHODase might be appropriate targets for compounds which could serve as drugs. Crystallization and structural analysis of T. gondii DHOase will allow identification of active site residues and will be useful for identifying such residues in other DHOases. Structural information will be invaluable in understanding the efficacy of inhibitors, and will aid in identifying other inhibitory compounds. We will determine whether DHODase binds to coenzyme Q homologs and hydroxynaphthoquinones which are active against T. gondii (Ellis, 1994; Araujo et al, 1991). Future directions of the proposed research are to investigate the role of individual residues in the recombinant enzymes by site-directed mutagenesis, to conduct studies with DHOase and DHODase inhibitors which have anti-cancer or antimalarial activity, to investigate potential co-regulation of purine salvage and pyrimidine synthesis, and to investigate linkage of pyrimidine biosynthesis and the election transport chain.

拟议的研究的目的是更好地理解 在弓形虫弓形虫中的嘧啶生物合成。 路径的存在 在这个生物体中已经建立了（Schwartzman＆Pefferferkorn，1981； Asai等，1983），但是涉及的酶都没有完全 特征或克隆。 我们将研究二氢酶（Dhoase）和 二氢二氢酸酯脱氢酶（DHODASE），催化第三和第四 反应分别。 大量的重组酶将是 在细菌或酵母表达系统中产生。 我们将确定 T.Gondii Dhoase是单功能酶还是一个域 多功能酶。 我们的研究将表明膜是否是否 相关的T. gondii dhodase是一种简单的黄蛋蛋白，有些也是 原生动物脱糖酶（Pascal等，1983），或者它是否是两种表现力 蛋白质如牛dhodase（Hines＆Johnston，1989）。 分析 T的序列，动力学特性和金属/辅因子组成。 Gondii dhoase和dhodase将导致对催化的理解 分子水平的机制。 将酶与它们的比较 哺乳动物对应物将揭示gondii dhoase和dhodase是否会揭示 可能是可以用作药物的化合物的适当靶标。 T. gondii dhoase的结晶和结构分析将允许 识别活动现场残留物，将有助于识别 此类残留物中的残留物。 结构信息将是无价的 了解抑制剂的功效，并将有助于识别 其他抑制性化合物。 我们将确定DHODase是否结合到 辅酶Q同源物和羟基喹酮，它们具有活性。 Gondii（Ellis，1994； Araujo等，1991）。 未来的方向 拟议的研究是研究个体残基在 通过位置定向诱变重组酶进行研究，以进行研究 具有抗癌或抗疟疾的Dhoase和dhodase抑制剂 活动，研究嘌呤救助的潜在共同调节和 嘧啶合成，并研究嘧啶生物合成的连接 和选举运输链。