Novel Antibiotic Targets: Mechanistic Studies on Dinuclear Metallohydrolases

无机化学、生物无机化学和金属有机化学项目的这一奖项支持犹他州州立大学的理查德·C·霍尔茨教授的研究，目的是加深对含有双核活性中心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。这项研究将探索为什么有些水解酶利用单核中心，而另一些则以单核或双核活性中心发挥作用，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。这些体系在离散的双核中心使用不同的金属离子Lewis酸，以i)结合和定位底物，ii)结合和激活水分子以产生活性中心的氢氧化物亲核试剂，和/或iii)稳定水解反应的过渡态。详细的结构催化机制将被开发的亮氨酸氨基肽酶(AAP)，编码DAPE的N-琥珀酰基-L，L-二氨基吩酸脱琥珀酸脱琥珀酸酶(DAPE)和编码的N-乙酰-L-鸟氨酸脱乙酰酶(ARGE)的大肠杆菌。该实验方法结合了生化、光谱、X射线结晶学方法、催化重要氨基酸残基的定点突变和小分子抑制剂。具体目标是：1)AAP中的哪些金属离子起催化金属离子的作用；2)位于第二配位球上的活性中心残基起什么催化作用；3)DAPE和ARGE活性中心上的哪些残基起催化作用？4)底物和过渡态类似物如何与DAPE和ARGE相互作用；5)合成可作为抗菌剂的新型DAPE和ARGE抑制剂。这项研究探讨了双核金属蛋白酶的作用机制，这些酶在中风、糖尿病、癌症、艾滋病毒、细菌感染和与谷氨酸能神经传递失调相关的神经精神障碍，如精神分裂症、癫痫和肌萎缩侧索硬化症(ALS)等疾病中发挥核心作用。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开发。此外，这些机理数据将有助于设计具有新抗菌剂功能的小分子。

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.