Novel Antibiotic Targets: Mechanistic Studies on Dinuclear Metallohydrolases

这个奖项在无机，生物无机和有机化学计划支持研究教授理查德C。霍尔茨在犹他州州立大学研究了含有双核活性位点的金属水解酶催化的水解反应。这些酶催化多种反应，如DNA、RNA、磷脂和多肽的降解。这项研究将探索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中使用不同的金属离子刘易斯酸性以i）结合和定位底物，ii）结合和活化水分子以产生活性位点氢氧化物亲核试剂，和/或iii）稳定水解反应的过渡态。从结构上详细研究了溶蛋白弧菌的亮氨酸氨肽酶（AAP）、H.和argE编码的N-乙酰基-L-鸟氨酸脱乙酰酶（ArgE）E.杆菌 实验方法包括生物化学、光谱学、X射线晶体学方法、催化重要氨基酸残基的定点突变体和小分子抑制剂。 具体目标是：i）AAP中的哪种金属离子起催化金属离子的作用？ii）存在于第二配位层中的活性位点残基的催化作用是什么？iii）DapE和ArgE活性位点中的哪些残基起催化重要作用？iv）底物和过渡态类似物抑制剂如何与DapE和ArgE相互作用？v）合成可用作抗菌剂的DapE和ArgE的新型抑制剂。本研究探讨了双核金属蛋白酶的作用机制，这些蛋白酶在几种疾病状态中发挥核心作用，包括中风、糖尿病、癌症、HIV、细菌感染和与神经递质神经传递失调相关的神经精神疾病，如精神分裂症、癫痫发作和肌萎缩侧索硬化症（ALS）。 这一知识可能导致能够催化工业上重要的水解反应的配位络合物的发展。此外，这些机理数据将有助于设计作为新抗菌剂的小分子。

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.