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这种化合物在血液和/或尿液中积累相关的人类病理疾病方面具有潜在的治疗应用。这项提议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控，该酶催化草酸转化为二氧化碳和甲酸盐。这两种产品都是无毒的，因此OxDC有潜力用于治疗尿路结石和/或防止草酸钙结石的形成。此外，该酶使用的依赖于锰的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活性自由基中间体的知识，这些中间体允许裂解草酸盐的化学惰性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射线结晶学以及一系列定点定向的OxDC突变体的动力学和光谱表征来检验关于OxDC催化脱羧基的催化机理的建议。更具体地说，我们将进行X射线结晶学研究，旨在获得有关草酸如何结合在活性中心内的详细结构信息，酶中催化活性中心的数量，以及当OxDC/草酸盐复合体在有氧条件下翻转时的氧结合模式。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的过渡态是否与OxDC的动力学性质一致。最后，将测量酶的特定位点突变体的动力学性质，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射线结晶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)在多大程度上起催化作用。第二个具体目标将集中在了解蛋白质环境如何调节细菌OxDC中Mn(II)中心(S)的内在化学反应活性。因此，在植物草酸氧化酶(Oxox)和OxDC中观察到的锰结合基序的相似性提出了一个问题：如何通过相同的配体配位锰(II)，但在两种酶中催化同一底物的不同化学转化。将对一系列oxox/OxDC嵌合体进行系统的生物物理、同位素效应和计算研究，以验证现有的关于观察到关键活性部位环改变取消脱羧基活性并伴随氧化功能增加的分子基础的假设。最后，在第三个目标中，我们将考察OxDC在不同类型的含盐溶液中溶解人草酸钙肾结石的能力，为后续该酶在临床上的应用提供基础。 公共卫生相关性：可以催化草酸分解的酶在治疗人类疾病方面具有潜在的治疗应用，这些疾病与这种化合物在血液和/或尿液中的积累有关，包括肾结石的形成。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。草酸脱羧酶是一种草酸代谢酶，存在于真菌和一些细菌中。除了对OxDC催化草酸盐中化学上非活性的C-C键断裂的分子机制提供新的见解外，该项目还将提供关于(I)蛋白质环境如何调节过渡金属化学以及(Ii)氨基酸突变可用于进化新的酶活性的详细信息。还将进行实验，以评估在未来旨在开发临床治疗和/或预防草酸相关疾病的新疗法的长期转化性研究中使用OxDC的可行性。

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.