Wheeler Mariel C Gerrard, Arias Cintia L, Tronconi Marcos A, Maurino Verónica G, Andreo Carlos S, Drincovitch María F
Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina.
Plant Mol Biol. 2008 Jun;67(3):231-42. doi: 10.1007/s11103-008-9313-9.
The Arabidopsis thaliana genome contains four NADP-malic enzymes genes (NADP-ME1-4). NADP-ME4 is localized to plastids whereas the other isoforms are cytosolic. NADP-ME2 and 4 are constitutively expressed, while NADP-ME1 is restricted to secondary roots and NADP-ME3 to trichomes and pollen. Although the four isoforms share remarkably high degree of identity (75-90%), recombinant NADP-ME1 through 4 show distinct kinetic properties, both in the forward (malate oxidative decarboxylation) and reverse (pyruvate reductive carboxylation) reactions. The four isoforms behave differently in terms of reversibility, with NADP-ME2 presenting the highest reverse catalytic efficiency. When analyzing the activity of each isoform in the presence of metabolic effectors, NADP-ME2 was the most highly regulated isoform, especially in its activation by certain effectors. Several metabolites modulate both the forward and reverse reactions, exhibiting dual effects in some cases. Therefore, pyruvate reductive carboxylation may be relevant in vivo, especially in some cellular compartments and conditions. In order to identify residues or segments of the NADP-ME primary structure that could be involved in the differences among the isoforms, NADP-ME2 mutants and deletions were analysed. The results obtained show that Arg115 is involved in fumarate activation, while the amino-terminal part is critical for aspartate and CoA activation, as well as for the reverse reaction. As a whole, these studies show that minimal changes in the primary structure are responsible for the different kinetic behaviour of each AtNADP-ME isoform. In this way, the co-expression of some isoforms in the same cellular compartment would not imply redundancy but represents specificity of function.
拟南芥基因组包含四个NADP-苹果酸酶基因（NADP-ME1-4）。NADP-ME4定位于质体，而其他同工型位于胞质溶胶中。NADP-ME2和4组成型表达，而NADP-ME1局限于次生根，NADP-ME3局限于毛状体和花粉。尽管这四种同工型具有非常高的同一性（75-90%），但重组的NADP-ME1至4在正向（苹果酸氧化脱羧）和反向（丙酮酸还原羧化）反应中表现出不同的动力学特性。这四种同工型在可逆性方面表现不同，NADP-ME2具有最高的反向催化效率。在分析每种同工型在代谢效应物存在下的活性时，NADP-ME2是受调控程度最高的同工型，尤其是在某些效应物对其的激活方面。几种代谢物调节正向和反向反应，在某些情况下表现出双重作用。因此，丙酮酸还原羧化在体内可能是相关的，特别是在一些细胞区室和条件下。为了鉴定NADP-ME一级结构中可能与同工型差异有关的残基或片段，对NADP-ME2突变体和缺失体进行了分析。获得的结果表明，Arg115参与富马酸激活，而氨基末端部分对天冬氨酸和辅酶A激活以及反向反应至关重要。总体而言，这些研究表明一级结构的微小变化是导致每个拟南芥NADP-ME同工型不同动力学行为的原因。这样，一些同工型在同一细胞区室中的共表达并不意味着冗余，而是代表功能的特异性。
