Direct evidence for a covalent ene adduct intermediate in NAD(P)H-dependent enzymes
Direct evidence for a covalent ene adduct intermediate in NAD(P)H-dependent enzymes
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DOI：
10.1038/nchembio.1385
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发表时间：
2014-01-01
影响因子：
14.8
通讯作者：
Erb, Tobias J.
中科院分区：
文献类型：
--
作者：
Rosenthal, Raoul G.;Ebert, Marc-Olivier;Erb, Tobias J.
吡啶核苷酸 NADH 和 NADPH (NAD(P)H) 是所有活细胞中普遍存在的氧化还原辅酶。尽管约 16% 的所有特征酶在催化过程中使用吡啶核苷酸作为氢化物供体或受体，但许多酶缺乏对氢化物如何在 NAD(P) H 和相应底物之间转移的详细了解。在这里，我们以巴豆酰辅酶 A 羧化酶/还原酶 (Ccr) 作为案例研究，提供了一种新机制的证据，该机制在酶促氢化物转移过程中起作用。我们使用 NMR、高分辨率 MS 和停流光谱法观察到 NADPH 和底物巴豆酰辅酶 A 之间的共价烯中间体。烯中间体的制备进一步允许以前所未有的方式直接进入其他 NADPH 依赖性酶（包括来自 II 型脂肪酸生物合成的酶）的催化循环，这表明 NAD(P) H 烯中间体的形成是催化中更普遍的原理。
The pyridine nucleotides NADH and NADPH (NAD(P)H) are ubiquitous redox coenzymes that are present in all living cells. Although about 16% of all characterized enzymes use pyridine nucleotides as hydride donors or acceptors during catalysis, a detailed understanding of how the hydride is transferred between NAD(P) H and the corresponding substrate is lacking for many enzymes. Here we present evidence for a new mechanism that operates during enzymatic hydride transfers using crotonyl-CoA carboxylase/reductase (Ccr) as a case study. We observed a covalent ene intermediate between NADPH and the substrate, crotonyl-CoA, using NMR, high-resolution MS and stopped-flow spectroscopy. Preparation of the ene intermediate further allowed direct access to the catalytic cycle of other NADPH-dependent enzymes-including those from type II fatty acid biosynthesis-in an unprecedented way, suggesting that formation of NAD(P) H ene intermediates is a more general principle in catalysis.