Mechanistic X-Ray Crystallography of Mononuclear Iron Enzymes, TauD and HppE

单核铁酶、TauD 和 HppE 的机械 X 射线晶体学

• 批准号: 7222403
• 负责人: DANNY YUN
• 金额: $ 4.68万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7222403
• 关键词: 2-hydroxypropylphosphonic acid epoxidase; Acids; Active Sites; Address; Alcoholic Liver Cirrhosis; Antibiotics; Bacteria; Binding; Binding Sites; Carbon; Carbon Dioxide; Class; Clinical Research; Collagen; Complement component C1s; Complex; Crystallography; DNA; Decarboxylation; Dioxygen; Dioxygenases; Disease; Ehlers-Danlos Syndrome; Electrons; Environment; Enzymes; Epoxy Compounds; Escherichia coli; Evaluation; Family; Fatty Acids; Fosfomycin; Functional disorder; Heme; Heme Iron; Herbicides; Human; Hydrogen; Hydroxyl Radical; Hydroxylation; Hypoxia; Iron; Malignant Neoplasms; Metals; Mononuclear; Nature; Nitric Oxide; Numbers; Object Attachment; Organism; Oxygen; Oxygenases; Personal Satisfaction; Protein Family; Proteins; Public Health; Reaction; Resolution; Site; Specificity; Streptomyces; Structure; Substrate Specificity; Succinates; Sulfites; Sulfur; Taurine; Thinking; Urine; X-Ray Crystallography; abstracting; adduct; alpha ketoglutarate; analog; cofactor; insight; member; oxidation; prototype; repaired; response; succinate; taurine-alpha-ketoglutarate dioxygenase; tool

DESCRIPTION (provided by applicant): In this proposal, we will employ X-ray crystallography as the chief tool to study the structures and mechanisms of two medically-essential enzymes: taurine/alpha-ketoglutarate dioxygenase (TauD)from Escherichia coli and S-(2)-hydroxypropylphosphonic acid epoxidase (HppE)from Streptomyces wedmorensis. Both enzymes belong to the non-heme mononuclear-iron dependent protein family, which activates oxygen to carry difficult reactions. These reactions are essential for critical functions such as oxygen sensing; hypoxia response; herbicide degradation; fatty acid, collagen, and antibiotic synthesis; and repair of alkylated DNA. TauD, the prototype of the Fe(ll)-alpha-ketoglutarate (aKG) dependent oxygenases, catalyzes the hydroxylation of taurine to aminoacetaldehyde and sulfite, concurrent with the conversion of aKG to succinate and carbon dioxide, to provide sulfur to the bacterium. The mononuclear iron enzyme, HppE, catalyzes the formation of the antibiotic fosfomycin, an unusual C-P-bond-containing epoxide, from S-(2)-hydroxypropylphosphonic acid (S-HPP). The reaction is a two-electron oxidation and is mechanistically atypical because it is independent of aKG or any other cofactor and results in incorporation of the hydroxyl oxygen of the substrate, rather than an atom of dioxygen, into the epoxide ring. Our studies will employ mechanistic crystallography to unravel vague details in the well-studied oxygen activation reactions of TauD and HppE. We will solve the structures of the nitric oxide (a catalytically inert mimic of dioxygen) adducts of TauL>Fe(ll)*aKG*taurine (or ta urine analogues (1,1-dideuterotaurine or 1,1- difluorotaurine)) and HPPE"Fe(ll)"S-HPP to address binding modes of oxygen to the metal center, accomanying conformational dynamics at the active site, and the nature of the reaction steps that follow. We will also determine the structures of the HppE-Fe(ll)-nitrosyl complex with R-(2)- hydroxypropylphosphonic acid (R-HPP) and S-(2)-hydroxy-(2)-phenylethylphosphonic acid (S-HPEP) to understand the enzyme's regio- and stereo-specificity. Relevance to Public Health: Insight obtained from our studies into the structures and mechanisms of TauD and HppE will contribute to the advancement of clinical research on several disease states, such as cancer, alcoholic liver cirrhosis, Ehlers-Danlos syndrome, and fibrotic disease, which are associated with dysfunction in a number of enzymes in this family of proteins.

描述（由申请人提供）：在本提案中，我们将使用x射线晶体学作为主要工具来研究两种医学必需酶的结构和机制：来自大肠杆菌的牛磺酸/ α -酮戊二酸双加氧酶（TauD）和来自wedmorensis链霉菌的S-(2)-羟丙基膦酸环氧酶（HppE）。这两种酶都属于非血红素单核铁依赖蛋白家族，它激活氧气来进行困难的反应。这些反应对于氧传感等关键功能至关重要；缺氧反应;除草剂降解;脂肪酸、胶原蛋白和抗生素合成；以及烷基化DNA的修复。TauD是Fe(ll)- α -酮戊二酸（aKG）依赖加氧酶的原型，它催化牛磺酸羟基化为氨基乙醛和亚硫酸盐，同时将aKG转化为琥珀酸盐和二氧化碳，为细菌提供硫。单核铁酶（HppE）催化S-(2)-羟丙基膦酸（S- hpp）生成抗生素磷霉素（一种不寻常的含c - p键的环氧化物）。该反应是一种双电子氧化反应，在机理上是非典型的，因为它不依赖于aKG或任何其他辅助因子，并导致底物的羟基氧结合，而不是一个双氧原子，进入环氧化物环。我们的研究将采用机械晶体学来解开已经被充分研究的TauD和HppE氧活化反应中的模糊细节。我们将解决一氧化氮（催化惰性模拟双氧）的TauL b> Fe(ll)*aKG*牛磺酸加合物（或尿类似物（1,1-二氘牛磺酸或1,1-二氟牛磺酸））和HPPE“Fe（ll）”S-HPP的结构，以解决氧与金属中心的结合模式，伴随活性位点的构象动力学，以及随后反应步骤的性质。我们还将确定hpe - fe (ll)-亚硝基配合物与R-(2)-羟丙基膦酸（R- hpp）和S-(2)-羟基-(2)-苯乙基膦酸（S- hpep）的结构，以了解该酶的区域特异性和立体特异性。与公共卫生的相关性：从我们对TauD和HppE的结构和机制的研究中获得的见解将有助于推进几种疾病状态的临床研究，如癌症、酒精性肝硬化、ehers - danlos综合征和纤维化疾病，这些疾病与该蛋白家族中许多酶的功能障碍有关。