Enzymes of the Meta-Fission Pathway

描述(由申请人提供)：儿茶酚和高原儿茶酸(HPC)代谢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机械酶学、分子生物学和X射线结晶学的结合来回答这些问题。已经取得了重大进展，可以解决有关途径的起源和其中的酶的进化的根本问题，使它们成为分解代谢途径如何组装的模型系统。这些研究还将加深我们对酶机制、烯醇和二烯醇化学的理解，并可能有助于生物修复努力、生物催化剂的设计，并导致更好地理解医学上相关的酶(例如，β-内酰胺酶)是如何进化的。在上一次资助期间，我们确定了4-草酰基转移酶(4-OT)家族的成员，这是互变消除酶超家族中的一个家族，并确定了它们的特征。大自然显然使用了β-α-β-支架，这是互变抹去酶超家族成员的关键组成部分，作为制造几种新酶的模板。4-OT是儿茶酚途径中的一种酶，也是该家族的标题成员，它将这些活性保留为低水平的活性。将确定这些活动的结构基础，并在掌握这些信息的情况下，扩大这些活动。现已鉴定出一个富马酸乙酰乙酸酯水解酶(FAH)超家族，它由儿茶酚途径和HPC途径中的脱羧酶和一个命名为YcgM的大肠杆菌同源物组成。FAH类超家族的特征基序和特征将被确定。主要的具体目标将是：1)利用诱变、立体化学探针和结晶学来确定4-OT的反式-3-卤代丙烯酸酯脱卤酶活性的结构基础；2)使用合理和随机的诱变来扩增4-OT的脱卤酶活性；3)表征4-OT的顺式-3-卤代丙烯酸酯脱卤酶和脱羧酶活性；4)利用诱变、抑制剂和结晶学表征COHED的脱羧酶和互变构型擦除酶结构域以及YcgM的脱羧酶活性；5)合成乙烯基丙酮水合酶的环氧化物抑制剂，并获得晶体结构，以发现其超家族。

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.