Miller S S, Driscoll B T, Gregerson R G, Gantt J S, Vance C P
Department of Agronomy, University of Minnesota, St. Paul 55108, USA.
Plant J. 1998 Jul;15(2):173-84. doi: 10.1046/j.1365-313x.1998.00192.x.
Malate dehydrogenase (MDH) catalyzes the readily reversible reaction of oxaloacetate reversible malate using either NADH or NADPH as a reductant. In plants, the enzyme is important in providing malate for C4 metabolism, pH balance, stomatal and pulvinal movement, respiration, beta-oxidation of fatty acids, and legume root nodule functioning. Due to its diverse roles the enzyme occurs as numerous isozymes in various organelles. While antibodies have been produced and cDNAs characterized for plant mitochondrial, glyoxysomal, and chloroplast forms of MDH, little is known of other forms. Here we report the cloning and characterization of cDNAs encoding five different forms of alfalfa MDH, including a plant cytosolic MDH (cMDH) and a unique novel nodule-enhanced MDH (neMDH). Phylogenetic analyses show that neMDH is related to mitochondrial and glyoxysomal MDHs, but diverge from these forms early in land plant evolution. Four of the five forms could effectively complement an E. coli Mdh- mutant. RNA and protein blots show that neMDH is most highly expressed in effective root nodules. Immunoprecipitation experiments show that antibodies produced to cMDH and neMDH are immunologically distinct and that the neMDH form comprises the major form of total MDH activity and protein in root nodules. Kinetic analysis showed that neMDH has a turnover rate and specificity constant that can account for the extraordinarily high synthesis of malate in nodules.
苹果酸脱氢酶（MDH）利用NADH或NADPH作为还原剂，催化草酰乙酸与苹果酸之间易于逆转的反应。在植物中，该酶对于为C4代谢、pH平衡、气孔和叶枕运动、呼吸作用、脂肪酸的β-氧化以及豆科植物根瘤功能提供苹果酸至关重要。由于其作用多样，该酶以多种同工酶的形式存在于各种细胞器中。虽然已经制备了针对植物线粒体、乙醛酸循环体和叶绿体形式的MDH的抗体并对其cDNA进行了表征，但对其他形式了解甚少。在此，我们报告了编码苜蓿MDH五种不同形式的cDNA的克隆和表征，包括一种植物胞质MDH（cMDH）和一种独特的新型根瘤增强型MDH（neMDH）。系统发育分析表明，neMDH与线粒体和乙醛酸循环体MDH相关，但在陆地植物进化早期就与这些形式分化。五种形式中的四种能够有效地互补大肠杆菌Mdh-突变体。RNA和蛋白质印迹显示，neMDH在有效的根瘤中表达最高。免疫沉淀实验表明，针对cMDH和neMDH产生的抗体在免疫上是不同的，并且neMDH形式构成了根瘤中总MDH活性和蛋白质的主要形式。动力学分析表明，neMDH的周转速率和特异性常数可以解释根瘤中苹果酸的极高合成。
