n nnnThe redox active peptide glutathione is ubiquitous in nature,but some plants also synthesize glutathione analogs in responseto environmental stresses. To understand the evolution of chemicaldiversity in the closely related enzymes homoglutathione synthetase(hGS) and glutathione synthetase (GS), we determined the structuresof soybean (Glycine max) hGS in three states: apoenzyme, boundto -glutamylcysteine (EC), and with hGSH, ADP, and a sulfateion bound in the active site. Domain movements and rearrangementof active site loops change the structure from an open activesite form (apoenzyme and EC complex) to a closed active siteform (hGSH•ADP•SO42– complex). The structureof hGS shows that two amino acid differences in an active siteloop provide extra space to accommodate the longer β-Alamoiety of hGSH in comparison to the glycinyl group of glutathione.Mutation of either Leu-487 or Pro-488 to an Ala improves catalyticefficiency using Gly, but a double mutation (L487A/P488A) isrequired to convert the substrate preference of hGS from β-Alato Gly. These structures, combined with site-directed mutagenesis,reveal the molecular changes that define the substrate preferenceof hGS, explain the product diversity within evolutionarilyrelated GS-like enzymes, and reinforce the critical role ofactive site loops in the adaptation and diversification of enzymefunction.展开▼
机译:ABSTRACTn FONT> TH> TR> TABLE> n
n TOP n <字体颜色= 464c53>抽象 FONT> n 介绍 n 结果 n 讨论 n 方法 n 参考文献 n FONT> TH> TR> TABLE> n nnn氧化还原活性肽谷胱甘肽本质上无处不在, SUP>我的植物还可以响应环境胁迫 SUP>合成谷胱甘肽类似物。为了了解紧密相关的酶同谷胱甘肽合成酶 SUP>(hGS)和谷胱甘肽合成酶(GS)中化学 SUP>多样性的演变,我们确定了其结构 SUP>大豆( Gycine max I>)hGS处于三种状态:脱辅酶, SUP>与-谷氨酰半胱氨酸( EC),以及hGSH,ADP和硫酸盐 SUP>离子结合在活性位点上。活动站点循环的域移动和重新排列 SUP>会从开放的活动 SUP>站点形式(脱辅基酶和 EC复合体)关闭的活动站点 SUP>表单(hGSH•ADP•SO 4 SUB> 2 – SUP>复合体) 。 hGS的结构 SUP>表明,活性位点 SUP>环中的两个氨基酸差异提供了额外的空间,以容纳hGSH中较长的β-Ala SUP>部分。 SUP>将Leu-487或Pro-488突变为Ala可以提高Gly的催化 SUP>效率,但会产生双重突变(L487A / P488A) SUP>是将hGS的底物偏好从β-Ala SUP>转换为Gly所必需的。这些结构结合定点诱变,揭示了定义hGS底物偏好的分子变化的分子变化,解释了进化相关的产物多样性。 GS样酶,并增强 SUP>活性位点环在酶 SUP>功能的适应和多样化中的关键作用。 SUP>
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