Novel Antibiotic Targets: Mechanistic Studies on Dinuclear Metallohydrolases

新的抗生素靶点：双核金属水解酶的机制研究

• 批准号: 0549221
• 负责人: Richard Holz
• 金额: $ 39.5万
• 依托单位: Utah State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2006-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0549221&HistoricalAwards=false
• 关键词: Novel; Antibiotic; Targets; Mechanistic; Studies

This award in the Inorganic, Bioinorganic and Organometallic Chemistry program supports research by Professor Richard C. Holz at the Utah State University to develop an understanding of hydrolytic reactions catalyzed by metallohydrolases that contain dinuclear active sites. These enzymes catalyze diverse reactions such as the degradation of DNA, RNA, phospholipids, and polypeptides. The research will explore why some hydrolases utilize a mononuclear center while others function with either a mononuclear or dinuclear active site, and still others require two metal ions to catalyze the same chemical reaction. These systems use different metal ion Lewis acidities in discrete dinuclear sites to i) bind and position substrate, ii) bind and activate a water molecule to yield an active site hydroxide nucleophile, and/or iii) stabilize the transition state of the hydrolytic reaction. Structurally detailed catalytic mechanisms will be developed for the leucine aminopeptidase from Vibrio (Aeromonas) proteolyticus (AAP), the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from H. influenzae, and the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) E. coli. The experimental approach incorporates biochemical, spectroscopic, X-ray crystallographic methods.and site directed mutants of catalytically important amino acid residues, and small molecule inhibitors. The specific aims are: i) Which metal ion in AAP functions as the catalytic metal ion?, ii) What are the catalytic roles of active site residues that reside in the second coordination sphere?, iii) What residues in the active site of DapE and ArgE play catalytically important roles?, iv) How do substrate- and transition-state analog inhibitors interact with DapE and ArgE?, v) Synthesize novel inhibitors of DapE and ArgE that may function as antibacterial agents. This research addresses the mechanism of action of dinuclear metalloproteases which play a central role in several disease states including stroke, diabetes, cancer, HIV, bacterial infections, and neuropsychiatric disorders associated with the dysregulation of glutamatergic neurotransmission, such as schizophrenia, seizure disorders, and amyotrophic lateral sclerosis (ALS). This knowledge may lead to the development of coordination complexes that are capable of catalyzing industrially important hydrolytic reactions. In addition, these mechanistic data will aid in the design of small molecules that function as new anti-bacterial agents.

这个无机、生物无机和有机金属化学项目的奖项支持犹他州立大学Richard C. Holz教授的研究，以发展对含有双核活性位点的金属水解酶催化的水解反应的理解。这些酶催化多种反应，如DNA、RNA、磷脂和多肽的降解。这项研究将探讨为什么一些水解酶利用单核中心，而另一些水解酶则利用单核或双核活性位点，还有一些水解酶需要两个金属离子来催化相同的化学反应。这些系统在离散的双核位点上使用不同的金属离子路易斯酸来i)结合和定位底物，ii)结合和激活水分子以产生活性位点的氢氧亲核试剂，和/或iii)稳定水解反应的过渡状态。从结构上详细的催化机制将开发来自溶蛋白弧菌（AAP）的亮氨酸氨基肽酶，来自流感嗜血杆菌的DapE编码的n -琥珀酰- l， l-二氨基戊酸去乙酰化酶（DapE），以及大肠杆菌编码的n -乙酰基- l-鸟氨酸去乙酰化酶（ArgE）。实验方法结合了生化、光谱学、x射线晶体学方法。位点定向突变的催化重要的氨基酸残基，和小分子抑制剂。具体目的是：i) AAP中哪个金属离子起催化金属离子的作用？ii)第二配位球的活性位点残基的催化作用是什么？iii) DapE和ArgE活性位点的哪些残基起催化作用？iv)底物和过渡态模拟抑制剂如何与DapE和ArgE相互作用？5)合成新型DapE和ArgE抑制剂，可作为抗菌药物。本研究探讨了双核金属蛋白酶的作用机制，双核金属蛋白酶在多种疾病状态中发挥核心作用，包括中风、糖尿病、癌症、HIV、细菌感染和与谷氨酸能神经传递失调相关的神经精神疾病，如精神分裂症、癫痫性疾病和肌萎缩侧索硬化症（ALS）。这一知识可能会导致配合物的发展，能够催化工业上重要的水解反应。此外，这些机理数据将有助于设计作为新型抗菌剂的小分子。