Mechanistic enzymology of quinolinic acid biosynthesis

说明(申请人提供)：烟酰胺腺嘌呤二核苷酸(NAD)及其磷酸(NADP)是所有生命系统中必不可少的氧化还原辅因子。令人惊讶的是，这种辅因子的关键催化部分-喹啉酸衍生的吡啶环的生物合成仍然知之甚少。我们最近发现了一个细菌系统，其中包含将色氨酸转化为喹啉酸所需的酶。与真核酶形成鲜明对比的是，所有细菌酶都以可溶性稳定酶的形式高水平过度表达，为详细描述这一系统打开了大门。在这个方案中，我们描述了色氨酸双加氧酶、甲酰犬尿氨酸甲酰胺酶、犬尿氨酸单加氧酶、犬尿氨酸酶和羟基邻氨基苯甲酸双加氧酶的机理研究。这些研究将包括结构研究、每种酶稳态前动力学的表征、设计用于捕捉反应中间体的底物类似物的合成和测试以及ESR研究。色氨酸到喹啉酸途径的中间体在许多生物过程中起着重要的作用。犬尿氨酸是犬尿酸的前体，犬尿酸是谷氨酸受体的拮抗剂。在艾滋病相关痴呆、亨廷顿病和肝性脑病患者中，喹啉酸和3-羟基犬尿氨酸浓度均升高。3-羟基犬尿氨酸诱导大鼠纹状体神经细胞凋亡。这种中间体在眼睛中也起到滤光器的作用，保护视网膜免受紫外线的破坏。3-羟基犬尿氨酸介导的交联反应可能在白内障的形成中起一定作用。对喹啉酸生物合成酶的机理研究将有助于识别控制这些疾病状态的小分子抑制剂。

DESCRIPTION (provided by applicant): Nicotinamide adenine dinucleotide (NAD) and its phosphate (NADP) are essential redox cofactors in all living systems. Surprisingly, the biosynthesis of the key catalytic part of this cofactor, the quinolinic acid derived pyridine ring, is still poorly understood. We have recently discovered a bacterial system containing the enzymes required for the conversion of tryptophan to quinolinate. In dramatic contrast to the eukaryotic enzymes, all of the bacterial enzymes overexpress at a high level as soluble stable enzymes, opening up this system for detailed characterization. In this proposal, we describe mechanistic studies on tryptophan dioxygenase, formylkynurenine formamidase, kynurenine monooxygenase, kynureninase and hydroxyanthranilate dioxygenase. These studies will include structural studies, the characterization of the presteady state kinetics of each enzyme, the synthesis and testing of substrate analogs designed to trap reaction intermediates and ESR studies. The tryptophan to quinolinic acid pathway intermediates play an important role in several biological processes. Kynurenine is a precursor to kynurenic acid, an antagonist of the glutamate receptor. Both quinolinic acid and 3-hydroxykynurenine concentrations are elevated in patients with AIDS related dementia, Huntington's disease and hepatic encephalopathy. 3-Hydroxykynurenine induces apoptosis of neurons prepared from rat striatum. This intermediate also functions as a filter in the eye, protecting the retina from the damaging effects of UV-light. Crosslinking reactions mediated by 3-hydroxykynurenine may play a role in cataract formation. Mechanistic studies on the quinolinic acid biosynthetic enzymes will facilitate the identification of small molecule inhibitors to control these disease states.