Biochemical Studies of Oxalate Decarboxylase

描述（由申请人提供）：草酸是一种对几乎所有生物体都有毒的化合物，通过细胞代谢大量产生。如果草酸盐在人体中积累，可能会出现许多病理状况，包括高尿酸、肾中草酸钙结石的形成（尿石症）、肾衰竭、心肌病和心脏传导障碍。此外，高水平的草酸盐似乎与外阴痛有关，外阴痛是一种目前尚无治疗方法的女性疼痛性疾病。已有证据支持草酸代谢酶在降低生物体液中草酸水平的新的、有趣的治疗策略中的临床应用。在枯草芽孢杆菌中发现的Yvrk基因编码草酸脱羧酶（OxDC），这是一种在Mn依赖性反应中将草酸转化为甲酸和CO2的酶，其催化机制尚不清楚。作为我们促进OxDC用于治疗与糖尿病相关疾病的长期目标的一部分，该项目旨在使用生物无机化学，分子光谱学，酶动力学和蛋白质工程技术来表征枯草芽孢杆菌OxDC。这些研究也可能影响自由基介导的酶催化的一般理解，并提供新的见解（i）蛋白质环境在调节金属反应性和（ii）金属酶的演变的作用。本课题的具体目标是：1）利用稳态动力学、定点突变、定点突变等方法研究细菌草酸脱羧酶的催化机理。 诱变和同位素效应，2）确定OxDC的金属依赖性，3）评估蛋白质环境在控制OxDC中金属中心的化学性质中的作用，和4）鉴定在草酸脱羧酶的稳态转换期间形成的稳态自由基。

DESCRIPTION (provided by applicant): Oxalic acid, a compound that is toxic to almost all organisms, is produced in large quantities by cellular metabolism. A number of pathological conditions can arise if oxalate accumulates in Man, including hyperoxaluria, the formation of calcium oxalate stones in the kidney (urolithiasis), renal failure, cardiomyopathy and cardiac conductance disorders. In addition, high levels of oxalate appear correlated with vulvodynia, a painful disease in women for which no treatment is currently available. Evidence has emerged to support the clinical application of oxalate-metabolizing enzymes in new, and intriguing, therapeutic strategies for lowering oxalate levels in biological fluids. The Yvrk gene found in Bacillus subtilis encodes oxalate decarboxylase (OxDC), an enzyme that converts oxalate to formate and CO2 in a Mn-dependent reaction for which the catalytic mechanism is not known. As part of our long-term aim to facilitate the use of OxDC in the treatment of oxalate-related illness, this project seeks to characterize Bacillus subtilis OxDC using the techniques of bioinorganic chemistry, molecular spectroscopy, enzyme kinetics and protein engineering. These studies are also likely to impact general understanding of radical mediated enzyme catalysis and to give new insights into (i) the role of protein environment in modulating metal reactivity and (ii) metalloenzyme evolution. Specific aims of this project are: 1) To investigate the catalytic mechanism of bacterial oxalate decarboxylase using steady-state kinetics, site-directed mutagenesis and isotope effects, 2) To determine the metal-dependence of OxDC, 3) To evaluate the effect of protein environment in controlling the chemical properties of the metal center(s) in OxDC, and 4) To identify steady-state radicals formed during steady-state turnover of oxalate decarboxylase.