FAD-Binding Site and NADP Reactivity in Human Renalase: A New Enzyme Involved in Blood Pressure Regulation
FAD-Binding Site and NADP Reactivity in Human Renalase: A New Enzyme Involved in Blood Pressure Regulation
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DOI：
10.1016/j.jmb.2011.06.010
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发表时间：
2011-08-12
影响因子：
5.6
通讯作者：
Aliverti, Alessandro
中科院分区：
文献类型：
--
作者：
Milani, Mario;Ciriello, Francesco;Aliverti, Alessandro
肾酶是最近发现的一种黄素蛋白，其调节血压，调节钠和磷酸盐排泄，并通过迄今为止几乎不了解的机制显示心脏保护作用。它被认为是一种儿茶酚胺降解酶，通过O-2依赖性或NADH依赖性机制。在这里，我们报告肾酶的晶体结构在2.5埃的分辨率连同新的数据，其相互作用与烟酰胺二核苷酸。肾酶采用对羟基苯甲酸羟化酶折叠拓扑结构，包括基于罗斯曼折叠的黄素腺嘌呤二核苷酸（FAD）结合结构域和推定的底物结合结构域，后者含有五链反平行β折叠。一个大的空腔（228埃（3）），面对黄素环，推测代表活性位点。与单胺氧化酶或多胺氧化酶相比，肾酶活性位点是完全暴露于溶剂的，并且缺乏用于结合底物氨基的“芳香笼”。肾酶具有极低的心肌黄酶活性，与NADPH相比，对于NADH显示出较低的k（cat）但较高的k（cat）/K-m。此外，当在厌氧条件下与NADPH孵育时，其FAD辅基缓慢还原，并以约2 mM的Kd值结合NAD（+）或NADP（+）。蛋白质结构中缺乏可识别的NADP结合位点，并且其对NADH和NADPH的亲和力和反应性差，表明它们不是肾酶的生理配体。虽然我们的研究没有回答肾酶的催化活性的问题，它提供了一个坚实的框架，以测试其行动的分子机制的假设。(C)2011爱思唯尔有限公司保留所有权利。
Renalase is a recently discovered flavoprotein that regulates blood pressure, regulates sodium and phosphate excretion, and displays cardioprotectant action through a mechanism that is barely understood to date. It has been proposed to act as a catecholamine-degrading enzyme, via either O-2-dependent or NADH-dependent mechanisms. Here we report the renalase crystal structure at 2.5 angstrom resolution together with new data on its interaction with nicotinamide dinucleotides. Renalase adopts the p-hydroxybenzoate hydroxylase fold topology, comprising a Rossmann-fold-based flavin adenine dinucleotide (FAD)-binding domain and a putative substrate-binding domain, the latter of which contains a five-stranded anti-parallel beta-sheet. A large cavity (228 angstrom(3)), facing the flavin ring, presumably represents the active site. Compared to monoamine oxidase or polyamine oxidase, the renalase active site is fully solvent exposed and lacks an 'aromatic cage' for binding the substrate amino group. Renalase has an extremely low diaphorase activity, displaying lower k(cat) but higher k(cat)/K-m for NADH compared to NADPH. Moreover, its FAD prosthetic group becomes slowly reduced when it is incubated with NADPH under anaerobiosis, and binds NAD(+) or NADP(+) with K-d values of ca 2 mM. The absence of a recognizable NADP-binding site in the protein structure and its poor affinity for, and poor reactivity towards, NADH and NADPH suggest that these are not physiological ligands of renalase. Although our study does not answer the question on the catalytic activity of renalase, it provides a firm framework for testing hypotheses on the molecular mechanism of its action. (C) 2011 Elsevier Ltd. All rights reserved.