Mutagenic Definition of a Papain-Like Catalytic Triad, Sufficiency of the N-Terminal Domain for Single-Site Core Catalytic Enzyme Acylation, and C-Terminal Domain for Augmentative Metal Activation of a Eukaryotic Phytochelatin Synthase1
Mutagenic Definition of a Papain-Like Catalytic Triad, Sufficiency of the N-Terminal Domain for Single-Site Core Catalytic Enzyme Acylation, and C-Terminal Domain for Augmentative Metal Activation of a Eukaryotic Phytochelatin Synthase1
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类木瓜蛋白酶催化三联体的诱变定义、单位点核心催化酶酰化的 N 端结构域的充分性以及真核植物螯合素合酶增强金属活化的 C 端结构域1
作者：
N. Romanyuk;D. Rigden;O. Vatamaniuk;A. Lang;Rebecca E. Cahoon;J. Jez;P. A. Rea
植物螯合素 (PC) 合酶是 γ-谷氨酰半胱氨酸 (γ-Glu-Cys) 二肽基转肽酶，可催化从谷胱甘肽 (GSH) 和/或更短链 PC 合成重金属结合 PC、(γ-Glu-Cys)nGly 聚合物。这里通过对来自拟南芥 (Arabidopsis thaliana; AtPCS1) 的酶的研究表明，尽管仅蛋白质的 N 末端一半足以通过形成单位点酶酰基中间体进行核心催化，但不足以在第二个位点进行酰化和游离 Cd2+ 的增强刺激。仅含有酶的前 221 个 N 端氨基酸残基 (HIS-AtPCS1_221tr) 的纯化 N 端六组氨酸基标记的 AtPCS1 截短物能够在含有 Cd2+ 的介质中从 GSH 合成 PC，或在不含重金属离子的介质中从 S-甲基谷胱甘肽合成 S-甲基-PC。然而，虽然其全长六组氨酸基标记的等效物 HIS-AtPCS1 在从 GSH 依赖 Cd2+ 合成 PC 的过程中在两个位点经历 γ-Glu-Cys 酰化，并且在从 S-甲基谷胱甘肽合成 S-甲基-PC 时受到游离 Cd2+ 的刺激，但 HIS-AtPCS1_221tr 仅在一个位点经历 γ-Glu-Cys 酰化当 GSH 为底物时，当 S-甲基谷胱甘肽为底物时，游离 Cd2+ 不会直接刺激，而是被抑制。通过应用能够检测远距离同源性的序列搜索算法（我们之前简要报道过但未完整报道过的工作），已经确定 AtPCS1 的 N 端一半及其来自其他来源的等同物具有类木瓜蛋白酶 Clan CA Cys 蛋白酶的特征。然而从这些分析中推导出的折叠分配，证实并通过最近确定的来自蓝细菌发菜的远距离原核PC合酶同源物的晶体结构得到证实，能够解释对保守的Cys残基（在AtPCS1的情况下的Cys-56）的严格要求，以形成具有生物合成能力的γ-Glu-Cys酶酰基中间体，来自直接确定的实验的主要数据AtPCS1 假定的木瓜蛋白酶样催化三联体的另外两个残基 His-162 和 Asp-180 是否对于催化至关重要尚未得到证实。我们对 AtPCS1 基本理解中的这一缺陷通过系统定点诱变研究的结果得到了解决，该研究表明，网络 PC 合成确实需要 Cys-56，而且 His-162 和 Asp-180。因此，实验证实 AtPCS1 以及其他真核 PC 合酶都是木瓜蛋白酶 Cys 蛋白酶超家族成员，但与原核对应物不同，AtPCS1 除了具有经历初级 γ-Glu-Cys 酰化的木瓜蛋白酶样 N 端催化结构域外，还包含经历次级 γ-Glu-Cys 酰化的辅助金属感应 C 端结构域。
Phytochelatin (PC) synthases are γ-glutamylcysteine (γ-Glu-Cys) dipeptidyl transpeptidases that catalyze the synthesis of heavy metal-binding PCs, (γ-Glu-Cys)nGly polymers, from glutathione (GSH) and/or shorter chain PCs. Here it is shown through investigations of the enzyme from Arabidopsis (Arabidopsis thaliana; AtPCS1) that, although the N-terminal half of the protein, alone, is sufficient for core catalysis through the formation of a single-site enzyme acyl intermediate, it is not sufficient for acylation at a second site and augmentative stimulation by free Cd2+. A purified N-terminally hexahistidinyl-tagged AtPCS1 truncate containing only the first 221 N-terminal amino acid residues of the enzyme (HIS-AtPCS1_221tr) is competent in the synthesis of PCs from GSH in media containing Cd2+ or the synthesis of S-methyl-PCs from S-methylglutathione in media devoid of heavy metal ions. However, whereas its full-length hexahistidinyl-tagged equivalent, HIS-AtPCS1, undergoes γ-Glu-Cys acylation at two sites during the Cd2+-dependent synthesis of PCs from GSH and is stimulated by free Cd2+ when synthesizing S-methyl-PCs from S-methylglutathione, HIS-AtPCS1_221tr undergoes γ-Glu-Cys acylation at only one site when GSH is the substrate and is not directly stimulated, but instead inhibited, by free Cd2+ when S-methylglutathione is the substrate. Through the application of sequence search algorithms capable of detecting distant homologies, work we reported briefly before but not in its entirety, it has been determined that the N-terminal half of AtPCS1 and its equivalents from other sources have the hallmarks of a papain-like, Clan CA Cys protease. Whereas the fold assignment deduced from these analyses, which substantiates and is substantiated by the recent determination of the crystal structure of a distant prokaryotic PC synthase homolog from the cyanobacterium Nostoc, is capable of explaining the strict requirement for a conserved Cys residue, Cys-56 in the case of AtPCS1, for formation of the biosynthetically competent γ-Glu-Cys enzyme acyl intermediate, the primary data from experiments directed at determining whether the other two residues, His-162 and Asp-180 of the putative papain-like catalytic triad of AtPCS1, are essential for catalysis have yet to be presented. This shortfall in our basic understanding of AtPCS1 is addressed here by the results of systematic site-directed mutagenesis studies that demonstrate that not only Cys-56 but also His-162 and Asp-180 are indeed required for net PC synthesis. It is therefore established experimentally that AtPCS1 and, by implication, other eukaryotic PC synthases are papain Cys protease superfamily members but ones, unlike their prokaryotic counterparts, which, in addition to having a papain-like N-terminal catalytic domain that undergoes primary γ-Glu-Cys acylation, contain an auxiliary metal-sensing C-terminal domain that undergoes secondary γ-Glu-Cys acylation.
DOI：
10.1073/pnas.94.1.42
发表时间：
1997-01-07
影响因子：
11.1
作者：
Li, ZS;Lu, YP;Rea, PA
通讯作者：
Rea, PA
DOI：
10.1046/j.1432-1327.2001.02293.x
发表时间：
2001-07-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Clemens, S;Schroeder, JI;Degenkolb, T
通讯作者：
Degenkolb, T
叙利亚仓鼠多功能蛋白CAD酰胺转移酶结构域的催化机制。
DOI：
--
发表时间：
1991
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Chaparian,MG;Evans,DR
通讯作者：
Evans,DR