Arabidopsis thaliana dehydroascorbate reductase 2: Conformational flexibility during catalysis.
Arabidopsis thaliana dehydroascorbate reductase 2: Conformational flexibility during catalysis.
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DOI：
10.1038/srep42494
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发表时间：
2017-02-14
影响因子：
4.6
通讯作者：
Messens J
中科院分区：
文献类型：
--
作者：
Bodra N;Young D;Astolfi Rosado L;Pallo A;Wahni K;De Proft F;Huang J;Van Breusegem F;Messens J
脱氢抗坏血酸还原酶（达尔）催化脱氢抗坏血酸的谷胱甘肽（GSH）依赖性还原，并在再生抗坏血酸中起直接作用，抗坏血酸是防御氧化胁迫的重要植物抗氧化剂。达尔酶与谷胱甘肽转移酶（GST）超家族酶具有密切的结构同源性，并含有相同的活性位点基序，但大多数GST不表现出达尔活性。半胱氨酸在活性位点的存在对于达尔的催化功能是必不可少的，因为该半胱氨酸的突变消除了活性。在这里，我们提出的晶体结构的DHAR 2从拟南芥谷胱甘肽结合到催化半胱氨酸。该结构揭示了活性位点周围的局部构象差异，其将GSH结合的DHAR 2结构与DHAR 1结构区分开。我们还揭示了达尔释放氧化型谷胱甘肽（GSSG）的酶促步骤。为了巩固我们的结构和动力学的研究结果，我们调查了潜在的构象灵活性DHAR 2正常模式分析，并发现，子域的流动性可能与GSH结合或GSSG释放。
Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational differences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release.