Identification of domains involved in the allosteric regulation of cytosolic Arabidopsis thaliana NADP-malic enzymes
Identification of domains involved in the allosteric regulation of cytosolic Arabidopsis thaliana NADP-malic enzymes
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DOI：
10.1111/j.1742-4658.2009.07258.x
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发表时间：
2009-10-01
期刊：
影响因子：
5.4
通讯作者：
Drincovich, Maria F.
中科院分区：
文献类型：
--
作者：
Gerrard Wheeler, Mariel C.;Arias, Cintia L.;Drincovich, Maria F.
拟南芥基因组包含四个编码NADP-苹果酸酶（NADP-ME 1 -4）的基因。两种同工酶NADP-ME 2和NADP-ME 3位于胞质溶胶中，具有非常高的同一性（90%）。然而，它们显示不同的表达模式，并显示出不同的动力学特性，特别是在正向（苹果酸氧化脱羧）和反向（丙酮酸还原羧化）反应中，它们受效应物的调节。为了确定在一级结构中的域，可能是负责的监管差异，这些同工酶之间的嵌合体构建和分析。所有嵌合体版本表现出与亲本蛋白相同的天然结构。对构建的嵌合体的分析表明，从NADP-ME 2的氨基酸残基303到C-末端的区域对于富马酸盐活化是关键的。然而，NADP-ME 3两侧的氨基酸残基303和500的区域参与高苹果酸浓度的pH依赖性抑制。此外，NADP-ME 2的N-末端区域对于琥珀酸激活逆反应是必需的。总体而言，结果表明NADP-ME 2和NADP-ME 3能够区分相似的C-4酸并与相似的C-4酸不同地相互作用，这是由于最小的结构差异。因此，尽管NADP-ME 2和NADP-ME 3的活性位点高度保守，但两种同工酶获得不同的变构位点，导致产生具有独特调节机制的蛋白质，可能最适合于每种同工酶的特定器官和发育表达模式。
The Arabidopsis thaliana genome contains four genes encoding NADP-malic enzymes (NADP-ME1-4). Two isoenzymes, NADP-ME2 and NADP-ME3, which are shown to be located in the cytosol, share a remarkably high degree of identity (90%). However, they display different expression patterns and show distinct kinetic properties, especially with regard to their regulation by effectors, in both the forward (malate oxidative decarboxylation) and reverse (pyruvate reductive carboxylation) reactions. In order to identify the domains in the primary structure that could be responsible for the regulatory differences, four chimeras between these isoenzymes were constructed and analysed. All chimeric versions exhibited the same native structures as the parental proteins. Analysis of the chimeras constructed indicated that the region from amino acid residue 303 to the C-terminal end of NADP-ME2 is critical for fumarate activation. However, the region flanked by amino acid residues 303 and 500 of NADP-ME3 is involved in the pH-dependent inhibition by high malate concentration. Furthermore, the N-terminal region of NADP-ME2 is necessary for the activation by succinate of the reverse reaction. Overall, the results show that NADP-ME2 and NADP-ME3 are able to distinguish and interact differently with similar C-4 acids as a result of minimal structural differences. Therefore, although the active sites of NADP-ME2 and NADP-ME3 are highly conserved, both isoenzymes acquire different allosteric sites, leading to the creation of proteins with unique regulatory mechanisms, probably best suited to the specific organ and developmental pattern of expression of each isoenzyme.