PURINE CYCLOHYDROLASE OF HISTIDINE BIOSYNTHESIS

组氨酸生物合成的嘌呤环水解酶

• 批准号: 2021055
• 负责人: ROBERT L D'ORDINE
• 金额: $ 2.96万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-22 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2021055
• 关键词: aminoacid biosynthesis; enzyme mechanism; histidine; hydrolase; microorganism metabolism; plant physiology

Histidine biosynthesis in plants and microorganisms occurs via an exclusive metabolic pathway that employs advanced metabolic precursors such as ATP and 5-phospho-D-ribosyl-1-pyrophosphate (PRPP) as carbon and nitrogen sources. Since this pathway is not present in mammals, the proposed research has important applications in the rational development of enzyme inhibitors with antibacterial, antifungal, and herbicidal applications. Abundant sequence alignment data indicate the similarity and evolutionary relationship of this enzyme in archaebacteria, eubacteria, and yeast. Thus, the specific aim of this proposal is to characterize N1-phosphoribosyl-AMP cyclohydrolase (HisI), an enzyme that catalyzes a critical and unique step in the biosynthesis of histidine: the hydrolysis of the purine ring of ATP. We will start by analyzing the specific requirements (e.g. metal ions), and steady state kinetic properties of monofunctional HisI from Methanococcus vannielii using a specific continuous UV-vis assay. The amino acid sequence alignment data and results of chemical modification experiments will be used to select specific amino acids as candidates for PCR mediated site specific mutagenesis in an effort to identify specific residues involved in catalysis. A desamino analog of N1-phosphoribosyl-AMP is proposed for use as potential inhibitor and mechanistic probe. Studies with the more complex, bifunctional E. coli HisIE enzyme will also be pursued once the details of the single function HisI are understood. The potential implication of these comparative studies into the evolution of enzyme catalysis is intriguing. Electrospray mass spectrometry, 18-O labeling studies and UV-vis will be employed in the investigation of the mechanism of this enzyme. A long range goal of this study is to evaluate HisI as a potential target for the design of specific inhibitors of histidine biosynthesis.

植物和微生物中的组氨酸生物合成通过一种 采用高级代谢前体的唯一代谢途径 例如ATP和5-磷酸-D-核糖基-1-焦磷酸（PRPP）作为碳 和氮源。 由于这种途径在哺乳动物中不存在， 这项研究在合理的 开发具有抗菌、抗真菌和 除草应用。 丰富的序列比对数据表明 该酶在植物中相似性和进化关系 古细菌、真细菌和酵母。 因此，这样做的具体目的 建议是表征N1-磷酸核糖基-AMP环化水解酶 （HisI），一种催化生物合成中关键和独特步骤的酶， 组氨酸的生物合成：ATP嘌呤环的水解。 我们将从分析具体要求开始（例如金属 离子），和稳态动力学性质的单官能HisI从 使用特异性连续UV-vis测定法测定范氏甲烷球菌。 的 氨基酸序列比对数据和化学分析结果 修饰实验将用于选择特定的氨基酸 作为PCR介导的位点特异性诱变的候选物， 以识别参与催化的特定残基。 一种脱氨酶 N1-磷酸核糖基-AMP的类似物被提议用作潜在的 抑制剂和机理探针。 更复杂， 双功能E.大肠杆菌HisIE酶也将追求一旦 可以理解单功能HisI的细节。 的潜在 这些比较研究对酶进化的影响 催化作用很有趣。 电喷雾质谱法，18-O 标签研究和紫外可见光谱将被用于调查 这种酶的作用机制。 本研究的长期目标是 评估HisI作为设计特定 组氨酸生物合成的抑制剂。