Arabidopsis thaliana NADP-malic enzyme isoforms: high degree of identity but clearly distinct properties.

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.