Enzymes of the Meta-Fission Pathway

描述（由申请人提供）：儿茶酚和同源儿茶酸（hpc）元裂变途径的酶具有丰富的机制，结构和进化问题。这项研究的长期目标是通过结合机械酶学、分子生物学和x射线晶体学来回答这些问题。已经取得了重大进展，因此有关这些途径的起源和其中酶的进化的基本问题可以得到解决，使它们成为分解代谢途径如何组装的模型系统。这些研究还将增强我们对酶机制、烯醇和二烯醇化学的理解，并可能有助于生物修复工作、生物催化剂的设计，并使我们更好地了解与医学相关的酶（例如β -内酰胺酶）是如何进化的。在上一个资助期间，我们确定了4-草草酸互变异构体酶（4-OT）家族的成员，这是互变异构体酶超家族中的一个家族，并确定了它们的定义特征。自然显然使用了- α - β -支架，这是互变酶超家族成员的关键组成部分，作为模板来制造几种新酶。4-OT是儿茶酚途径中的一种酶，也是该家族的主要成员，它保留了这些低水平的活性。将确定这些活动的结构基础，并在掌握这些资料后扩大这些活动。一个类似富马酰乙酸水解酶（FAH）的超家族现已被确定，它由儿茶酚和hpc途径中的脱羧酶以及大肠杆菌的同源物YcgM组成。将确定类fah超家族的特征基序和特征。主要的具体目标将是：1)利用诱变、立体化学探针和晶体学确定4-OT反式3-卤代丙烯酸酯脱卤酶活性的结构基础；2)采用合理随机诱变的方法扩增4-OT脱卤酶活性；3)表征4-OT的顺-3-卤代丙烯酸酯脱卤酶和脱羧酶活性；4)利用诱变、抑制剂和晶体学表征COHED的脱羧酶和变异体酶结构域以及YcgM的脱羧酶活性；5)合成乙烯丙酮酸水合酶环氧化物抑制剂，获得晶体结构，发现其超家族。

DESCRIPTION (provided by applicant): The enzymes of the catechol and homoprotocatechuate (hpc) meta-fission pathways are rich in mechanistic, structural, and evolutionary questions. The long-term goal of this research is to answer these questions by a combination of mechanistic enzymology, molecular biology, and x-ray crystallography. Significant progress has been made so that fundamental questions about the origin of the pathways and the evolution of the enzymes in them can be addressed, making them model systems for how catabolic pathways are assembled. These studies will also enhance our understanding of enzyme mechanisms, enol and dienol chemistry, and may assist in bioremediation efforts, design of biocatalysts, and lead to a better understanding of how medically relevant enzymes (e.g., beta-lactamases) evolved. During the last funding period, we identified members of the 4-oxalocrotonate tautomerase (4-OT) family, one of the families in the tautomerase superfamily, and determined their defining characteristics. Nature has apparently used the beta-alpha-beta- scaffold, the key building block for tautomerase superfamily members, as a template to make several new enzymes. 4-OT, an enzyme in the catechol pathway and the title member of the family, retains these activities as low-level ones. The structural basis for these activities will be determined and, with this information in hand, the activities amplified. A fumarylacetoacetate hydrolase (FAH)-like superfamily has now been identified and consists of the decarboxylases in the catechol and hpc pathways and an E. coli homologue designated YcgM. The signature motif and characteristics of the FAH-like superfamily will be determined. The major specific aims will be to: 1) determine the structural basis for the trans-3-haloacrylate dehalogenase activity of 4-OT using mutagenesis, stereochemical probes, and crystallography; 2) use rational and random mutagenesis to amplify 4-OT's dehalogenase activity; 3) characterize the cis-3-haloacrylate dehalogenase and decarboxylase activities of 4-OT; 4) characterize the decarboxylase and tautomerase domains of COHED and the presumed decarboxylase activity of YcgM using mutagenesis, inhibitors, and crystallography; 5) synthesize epoxide inhibitors of vinylpyruvate hydratase and obtain a crystal structure in order to discover its superfamily.