发菜属植物螯合合酶样酶的结构和生物物理分析。表明其蛋白酶活性对底物的氧化还原状态敏感,ACS Chemical Biology

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发菜属植物螯合合酶样酶的结构和生物物理分析。表明其蛋白酶活性对底物的氧化还原状态敏感
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2022-04-04 , DOI: 10.1021/acschembio.1c00941
Florian J Gisdon _{1,

2} , Christian G Feiler ₃ , Oxana Kempf ₁ , Johannes M Foerster ₂ , Jonathan Haiss ₁ , Wulf Blankenfeldt _{3,

4} , G Matthias Ullmann ₂ , Elisa Bombarda ₁

Affiliation

植物螯合素 (PCs) 是由谷胱甘肽 (GSH) 在由 PC 合酶 (PCS) 催化的 γ-谷氨酰半胱氨酰转肽反应中合成的富含非核糖体硫醇的寡肽。在植物中普遍存在并存在于一些无脊椎动物中，PCS 参与金属解毒和体内平衡。来自蓝藻念珠菌的类 PCS 酶sp。(NsPCS) 被认为是真正 PCS 的进化前体酶，因为它与真核 PCS 的催化结构域具有足够的序列相似性，并具有 GSH 的去甘化肽酶活性。在这项工作中，我们通过结合结构、光谱、热力学和理论技术来研究 NsPCS 的催化机制。我们报告了 NsPCS 的几种晶体结构，它们捕获了催化化学反应的不同状态：（i）野生型酶（wt-NsPCS）的结构；(ii) γ-谷氨酰-半胱氨酸酰基-酶中间体 (acyl-NsPCS) 的高分辨率结构；(iii) NsPCS 无活性变体的结构，催化半胱氨酸突变为丝氨酸 (C70S-NsPCS)。我们将 NsPCS 描述为一种相对缓慢的酶，其活性对底物的氧化还原状态敏感。即，NsPCS 对还原型谷胱甘肽 (GSH) 有活性，但会被氧化型谷胱甘肽 (GSSG) 抑制，因为酶不会释放切割产物。我们的生物物理分析使我们认为 NsPCS 的生物学功能是氧化还原传感系统的一部分。此外，我们提出了一种类似 PCS 的酶如何进化为真正的 PCS 酶的机制。

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Structural and Biophysical Analysis of the Phytochelatin-Synthase-Like Enzyme from Nostoc sp. Shows That Its Protease Activity is Sensitive to the Redox State of the Substrate

Phytochelatins (PCs) are nonribosomal thiol-rich oligopeptides synthetized from glutathione (GSH) in a γ-glutamylcysteinyl transpeptidation reaction catalyzed by PC synthases (PCSs). Ubiquitous in plant and present in some invertebrates, PCSs are involved in metal detoxification and homeostasis. The PCS-like enzyme from the cyanobacterium Nostoc sp. (NsPCS) is considered to be an evolutionary precursor enzyme of genuine PCSs because it shows sufficient sequence similarity for homology to the catalytic domain of the eukaryotic PCSs and shares the peptidase activity consisting in the deglycination of GSH. In this work, we investigate the catalytic mechanism of NsPCS by combining structural, spectroscopic, thermodynamic, and theoretical techniques. We report several crystal structures of NsPCS capturing different states of the catalyzed chemical reaction: (i) the structure of the wild-type enzyme (wt-NsPCS); (ii) the high-resolution structure of the γ-glutamyl-cysteine acyl-enzyme intermediate (acyl-NsPCS); and (iii) the structure of an inactive variant of NsPCS, with the catalytic cysteine mutated into serine (C70S-NsPCS). We characterize NsPCS as a relatively slow enzyme whose activity is sensitive to the redox state of the substrate. Namely, NsPCS is active with reduced glutathione (GSH), but is inhibited by oxidized glutathione (GSSG) because the cleavage product is not released from the enzyme. Our biophysical analysis led us to suggest that the biological function of NsPCS is being a part of a redox sensing system. In addition, we propose a mechanism how PCS-like enzymes may have evolved toward genuine PCS enzymes.

更新日期：2022-04-04

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