Biochemical Studies of Oxalate Decarboxylase

描述（由申请人提供）：能够催化草酸分解的酶在治疗与该化合物在血液和/或尿液中积累相关的人类病理状况方面具有潜在的治疗应用。该提案概述了继续进行综合实验和计算研究，旨在了解草酸脱羧酶（OxDC）的基本生物化学和调节，草酸脱羧酶是一种催化草酸转化为二氧化碳和甲酸的酶。这两种产品都是无毒的，因此 OxDC 具有临床用于治疗尿石症和/或预防草酸钙结石形成的潜力。此外，该酶采用的锰依赖性化学机制在已知的化学中几乎没有先例，因此它的阐明将增加关于过渡金属如何参与质子耦合电子转移以产生反应性自由基中间体的知识，从而允许裂解草酸的化学惰性C-C键。在我们的第一个具体目标中，将使用先进的计算方法、X射线晶体学以及一系列定点OxDC突变体的动力学和光谱表征来测试OxDC催化脱羧催化机制的建议。更具体地说，我们将进行 X 射线晶体学研究，旨在获得有关草酸盐如何在活性位点内结合、酶中催化活性位点的数量以及 OxDC/草酸盐复合物在有氧条件下翻转时分子氧结合模式的详细结构信息。此外，还将进行 DFT 和 DFT/MM 计算，以评估假设的中间体及其相关过渡态是否与 OxDC 的动力学性质一致。最后，将测量酶的位点特异性突变体的动力学特性，以描述它们在催化和/或活性位点动力学中的功能作用。这些发现将与基于 X 射线晶体学和计算研究的预测相关联，一个关键目标将是检查 Mn(III) 和 Mn(IV) 介导催化作用的程度。第二个具体目标将侧重于了解蛋白质环境如何调节细菌 OxDC 中 Mn(II) 中心的内在化学反应性。因此，在植物草酸氧化酶 (OxOx) 和 OxDC 中观察到的 Mn 结合基序的相似性提出了一个问题：Mn(II) 如何与相同的配体协调，但在两种酶中催化相同底物的不同化学转化。将进行一系列 OxOx/OxDC 嵌合体的系统生物物理、同位素效应和计算研究，以验证现有的假设，这些假设涉及观察到关键活性位点环的改变消除脱羧活性并伴随氧化功能的获得的分子基础。最后，在第三个目标中，我们将研究OxDC在各种含盐溶液中溶解人草酸钙肾结石的能力，为后续该酶在临床应用中的使用提供基础。 公共健康相关性：能够催化草酸分解的酶在治疗与草酸在血液和/或尿液中积累相关的人类疾病（包括肾结石的形成）方面具有潜在的治疗应用。我们的研究小组在表征草酸脱羧酶（OxDC）的结构和催化机制方面发挥了主导作用，草酸脱羧酶是一种存在于真菌和一些细菌中的草酸代谢酶。除了提供有关 OxDC 催化草酸盐中化学惰性 C-C 键裂解的分子机制的新见解外，该项目还将提供有关 (i) 蛋白质环境如何调节过渡金属化学，以及 (ii) 氨基酸突变如何用于进化新酶活性的详细信息。还将进行实验来评估在未来长期转化研究中使用 OxDC 的可行性，旨在开发用于临床治疗和/或预防草酸盐相关疾病的新疗法。

DESCRIPTION (provided by applicant): Enzymes that can catalyze the breakdown of oxalic acid have potential therapeutic application in the treatment of human pathological conditions associated with the accumulation of this compound in the blood and/or urine. This proposal outlines the continuation of integrated experimental and computational studies aimed at understanding the fundamental biochemistry and regulation of oxalate decarboxylase (OxDC), an enzyme that catalyzes the conversion of oxalate to carbon dioxide and formate. Both of these products are non-toxic and so OxDC has the potential for clinical use in treating urolithiasis and/or preventing the formation of calcium oxalate-based stones. Moreover, the manganese-dependent chemical mechanism employed by the enzyme has little precedent in known chemistry, and so its elucidation will add to knowledge concerning how the transition metal might participate in proton-coupled electron transfer to yield reactive radical intermediates that permit cleavage of the chemically inert C-C bond of oxalate. In our first specific aim, proposals for the catalytic mechanism of OxDC-catalyzed decarboxylation will be tested using advanced computational methods, X-ray crystallography, and the kinetic and spectroscopic characterization of a series of site-directed OxDC mutants. More specifically, we will pursue X-ray crystallographic studies aimed at obtaining detailed structural information on how oxalate is bound within the active site, the number of catalytically active sites in the enzyme, and the mode of dioxygen binding when the OxDC/oxalate complex is turning over under aerobic conditions. In addition, DFT and DFT/MM calculations will be carried out to assess whether hypothetical intermediates, and their associated transition states, are consistent with the kinetic properties of OxDC. Finally, the kinetic properties of site-specific mutants of the enzyme will be measured to delineate their functional roles in catalysis and/or active site dynamics. These findings will be correlated with predictions made on the basis of X-ray crystallography and computational studies, and a key goal will be to examine the extent to which Mn(III) and Mn(IV) mediate catalysis. The second specific aim will focus on understanding how the protein environment can modulate the intrinsic chemical reactivity of the Mn(II) center(s) in bacterial OxDC. Thus, the similarity of the Mn-binding motifs observed in plant oxalate oxidases (OxOx) and OxDC raises the question of how Mn(II) can be coordinated by identical ligands but catalyze different chemical transformations of the same substrate in each of the two enzymes. Systematic biophysical, isotope effect and computational studies of a series of OxOx/OxDC chimeras will be undertaken to validate existing hypotheses concerning the molecular basis for the observation that changes to a critical active site loop abolish decarboxylative activity with concomitant gain of oxidative function. Finally, in the third aim, we will investigate the ability of OxDC to dissolve human, calcium oxalate-based kidney stones in various types of salt-containing solutions so as to provide a basis for subsequent use of the enzyme in clinical applications. PUBLIC HEALTH RELEVANCE: Enzymes that can catalyze the breakdown of oxalic acid have potential therapeutic application in the treatment of human diseases associated with the accumulation of this compound in the blood and/or urine, including the formation of kidney stones. Our research group has played a leading role in characterizing the structure and catalytic mechanism of oxalate decarboxylase (OxDC), an oxalate-metabolizing enzyme that is present in fungi and some bacteria. In addition to providing new insights into the molecular mechanism by which OxDC can catalyze cleavage of the chemically unreactive C-C bond in oxalate, this project will also provide detailed information on how (i) protein environment can modulate transition metal chemistry, and (ii) amino acid mutations can be used to evolve new enzyme activities. Experiments will also be undertaken to assess the feasibility of employing OxDC in future, long-term translational research studies aimed at developing novel therapies for the clinical treatment and/or prevention of oxalate-related disease.