Structure and Enzyme Function in Glyoxylate Metabolism and Hyperoxaluria

描述（由申请人提供）：该R21提案将研究人乙醛酸盐/羟丙酮酸还原酶（GRHPR）的结构-功能关系，GRHPR是乙醛酸盐和羟丙酮酸盐代谢的关键酶。人类GRHPR缺陷存在于罕见的遗传性疾病原发性高草酸尿2型（PH2）中。这些突变最终导致草酸盐的积累以及尿路草酸钙肾结石的形成和沉积。GRHPR活性的改变也可能导致特发性结石病，这是一种常见的使人衰弱的健康问题，影响日常生活并产生巨大的医疗费用。从其他生物中对GRHPR的历史分析得出了相互矛盾的证据，即辅助因子的偏好、反应的盐依赖性和底物抑制。此外，人体GRHPR的结构和生化实验尚未见报道。本研究的长期目标是表征人类GRHPR的动力学特性，确定决定其活性的结构特征，并了解这些特性在引起PH2的突变酶中如何改变。拟建的研究将（目的1）确定人类GRHPR单独和与NADPH复合的晶体结构，（目的2）通过野生型和PH2突变型酶的生化分析确定人类GRHPR的辅因子和底物特异性。人类GRHPR的结构可以将当前和未来的PH2变异映射到该结构上，并预测其生理后果。生化数据将揭示与酶与底物、辅因子和调节阴离子相互作用相关的动力学参数。人类GRHPR的突变化将有助于确定该酶更精确的生理作用，并有助于解释突变如何导致疾病。这些研究可能最终导致改善PH2患者和特发性结石患者的治疗策略。

DESCRIPTION (provided by applicant): This R21 proposal will investigate the structure-function relationships of human glyoxylate/hydroxypyruvate reductase (GRHPR), a key enzyme in glyoxylate and hydroxypyruvate metabolism. Defects in human GRHPR are present in the rare genetic disease primary hyperoxaluria type 2 (PH2). These mutations ultimately result in the buildup of oxalate and the formation and deposition of urinary tract calcium oxalate kidney stones. An altered GRHPR activity could also contribute to idiopathic stone disease, a common debilitating health problem that impacts daily life and incurs significant health care costs. The historical analysis of GRHPR from other organisms has yielded contradictory evidence for the preference of cofactor, the salt dependence of the reaction, and substrate inhibition. In addition, no structural or biochemical experiments have been reported for human GRHPR. The long-term goals of this research are to characterize the kinetic properties of human GRHPR, to identify the structural features that determine its activity, and to understand how these properties are altered in mutant enzymes causing PH2. The proposed study will (Aim 1) determine the crystal structures of human GRHPR alone and in complex with NADPH and (Aim 2) determine the cofactor and substrate specificity of human GRHPR through the biochemical analysis of wild-type and PH2 mutant enzymes. The structures of human GRHPR will enable the mapping of current and future PH2 variants onto the structure and the prediction of the physiological consequences. The biochemical data will reveal the kinetic parameters associated with the interaction of the enzyme with substrates, cofactors and modulating anions. The cha racterization of human GRHPR will help establish a more precise physiological role for the enzyme and help explain how mutations cause disease. Such studies may ultimately lead to improved treatment strategies for individulas with PH2 and possibly those with idiopathic stone disease.