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

描述（由申请人提供）：在本提案中，我们将采用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）和来自韦氏链霉菌的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性蛋白质家族，其激活氧以进行困难的反应。这些反应对于关键功能是必不可少的，例如氧传感;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，Fe（II）-α-酮戊二酸（aKG）依赖性加氧酶的原型，催化牛磺酸羟基化为氨基乙醛和亚硫酸盐，同时aKG转化为琥珀酸盐和二氧化碳，以向细菌提供硫。单核铁酶HppE催化S-（2）-羟丙基膦酸（S-HPP）形成抗生素磷霉素，磷霉素是一种不常见的含C-P键的环氧化物。该反应是一个双电子氧化反应，并且在机理上是非典型的，因为它不依赖于aKG或任何其他辅因子，并且导致底物的羟基氧而不是双氧原子掺入环氧环中。我们的研究将采用机械晶体学来解开模糊的细节，在充分研究的氧活化反应的TauD和HppE。我们将解决TauL>Fe（II）*aKG* 牛磺酸（或牛磺酸类似物（1，1-二氘牛磺酸或1，1-二氟牛磺酸））和HPPE的一氧化氮（分子氧的催化惰性模拟物）加合物的结构“Fe（II）”S-HPP以解决氧与金属中心的结合模式，适应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HppE-Fe（II）-亚硝酰基与R-（2）-羟丙基膦酸（R-HPP）和S-（2）-羟基-（2）-苯基乙基膦酸（S-HPEP）的复合物的结构，以了解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对几种疾病状态的临床研究，例如癌症，酒精性肝硬化，Ehlers-Danlos综合征和纤维化疾病，这些疾病与该蛋白质家族中许多酶的功能障碍有关。

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.