Pauff James M, Zhang Jinjin, Bell Charles E, Hille Russ
Department of Biochemistry, University of California, Riverside, California 92521, USA.
J Biol Chem. 2008 Feb 22;283(8):4818-24. doi: 10.1074/jbc.M707918200. Epub 2007 Dec 6.
Xanthine oxidoreductase catalyzes the final two steps of purine catabolism and is involved in a variety of pathological states ranging from hyperuricemia to ischemia-reperfusion injury. The human enzyme is expressed primarily in its dehydrogenase form utilizing NAD+ as the final electron acceptor from the enzyme's flavin site but can exist as an oxidase that utilizes O2 for this purpose. Central to an understanding of the enzyme's function is knowledge of purine substrate orientation in the enzyme's molybdenum-containing active site. We report here the crystal structure of xanthine oxidase, trapped at the stage of a critical intermediate in the course of reaction with the slow substrate 2-hydroxy-6-methylpurine at 2.3A. This is the first crystal structure of a reaction intermediate with a purine substrate that is hydroxylated at its C8 position as is xanthine and confirms the structure predicted to occur in the course of the presently favored reaction mechanism. The structure also corroborates recent work suggesting that 2-hydroxy-6-methylpurine orients in the active site with its C2 carbonyl group interacting with Arg-880 and extends our hypothesis that xanthine binds opposite this orientation, with its C6 carbonyl positioned to interact with Arg-880 in stabilizing the MoV transition state.
黄嘌呤氧化还原酶催化嘌呤分解代谢的最后两步，并参与从高尿酸血症到缺血再灌注损伤等多种病理状态。人类的这种酶主要以脱氢酶形式表达，利用NAD⁺作为来自酶的黄素位点的最终电子受体，但也可以作为氧化酶存在，利用氧气来实现这一目的。理解该酶功能的核心是了解嘌呤底物在酶的含钼活性位点中的取向。我们在此报告黄嘌呤氧化酶的晶体结构，它在与缓慢底物2-羟基-6-甲基嘌呤反应过程中被困在关键中间体阶段，分辨率为2.3埃。这是与嘌呤底物形成的反应中间体的首个晶体结构，该嘌呤底物在其C8位置被羟基化，如同黄嘌呤一样，并证实了目前所支持的反应机制过程中预测会出现的结构。该结构还证实了最近的研究工作，即2-羟基-6-甲基嘌呤在活性位点中以其C2羰基与精氨酸-880相互作用的方式取向，并扩展了我们的假设，即黄嘌呤以相反的取向结合，其C6羰基定位为与精氨酸-880相互作用以稳定MoV过渡态。
