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都有毒的化合物，通过细胞代谢大量产生。如果草酸在人体中积累，可能会出现许多病理情况，包括高草酸尿症、肾脏中草酸钙结石的形成（尿石症）、肾功能衰竭、心肌病和心传导障碍。此外，草酸盐的高水平似乎与外阴痛有关，外阴痛是一种目前尚无治疗方法的女性疼痛疾病。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），这种酶在依赖锰的反应中将草酸转化为甲酸和二氧化碳，其催化机制尚不清楚。作为我们促进OxDC在草酸盐相关疾病治疗中使用的长期目标的一部分，该项目寻求使用生物无机化学、分子光谱、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对自由基介导的酶催化的一般理解，并对(i)蛋白质环境在调节金属反应性中的作用和（ii）金属酶进化提供新的见解。本项目的具体目的是：1)利用稳态动力学研究细菌草酸脱羧酶的催化机制

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.