Type-II NADH:quinone oxidoreductase from Staphylococcus aureus has two distinct binding sites and is rate limited by quinone reduction
Type-II NADH:quinone oxidoreductase from Staphylococcus aureus has two distinct binding sites and is rate limited by quinone reduction
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DOI：
10.1111/mmi.13120
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发表时间：
2015-10-01
影响因子：
3.6
通讯作者：
Pereira, Manuela M.
中科院分区：
文献类型：
--
作者：
Sena, Filipa V.;Batista, Ana P.;Pereira, Manuela M.
任何合理的药物设计策略的先决条件是了解蛋白质-配体相互作用的模式。这促使我们探索金黄色葡萄球菌的II型NADH：醌氧化还原酶（NDH-2）中的蛋白质-底物相互作用，由于其多种耐药形式，这是临床医学中的一个世界性问题。NDH-2参与呼吸链，被认为是新型抗微生物疗法的合适靶标，因为它们是在许多病原生物体中表达的唯一具有NADH：醌氧化还原酶活性的酶。测定了NDH-2的晶体和溶液结构。金黄色葡萄球菌，表明其在溶液中是二聚体。我们报告的蛋白质的快速动力学分析，并检测到NAD（+）和还原黄素，这是由醌解离之间形成的电荷转移复合物。我们观察到醌还原是限速步骤，也是受HQNO（抑制剂）存在影响的唯一半反应。我们通过荧光和STD-NMR光谱分析了蛋白质-底物相互作用，这表明NADH和醌结合到不同的位点。总之，我们的综合结果显示了两种底物存在不同的结合位点，确定醌还原为限速步骤，并表明NAD（+）-蛋白质复合物的建立，该复合物由醌释放。
A prerequisite for any rational drug design strategy is understanding the mode of protein-ligand interaction. This motivated us to explore protein-substrate interaction in Type-II NADH:quinone oxidoreductase (NDH-2) from Staphylococcus aureus, a worldwide problem in clinical medicine due to its multiple drug resistant forms. NDHs-2 are involved in respiratory chains and recognized as suitable targets for novel antimicrobial therapies, as these are the only enzymes with NADH:quinone oxidoreductase activity expressed in many pathogenic organisms. We obtained crystal and solution structures of NDH-2 from S. aureus, showing that it is a dimer in solution. We report fast kinetic analyses of the protein and detected a charge-transfer complex formed between NAD(+) and the reduced flavin, which is dissociated by the quinone. We observed that the quinone reduction is the rate limiting step and also the only half-reaction affected by the presence of HQNO, an inhibitor. We analyzed protein-substrate interactions by fluorescence and STD-NMR spectroscopies, which indicate that NADH and the quinone bind to different sites. In summary, our combined results show the presence of distinct binding sites for the two substrates, identified quinone reduction as the rate limiting step and indicate the establishment of a NAD(+)-protein complex, which is released by the quinone.