Structure-Function Relationship Studies of the Four Arabidopsis thaliana NADP-Malic Enzyme Isoforms
The Arabidopsis thaliana genome contains four genes encoding NADP-malic enzymes (NADP-ME1−4). NADP-ME4 is localized to plastids whereas the other three isoforms are cytosolic. NADP-ME2 and −4 are constitutively expressed in mature organs, while NADP-ME1 and −3 are restricted to secondary roots and to trichomes and pollen, respectively. Although the four isoforms share a high degree of identity, the recombinant NADP-ME1 to 4 show well-distinct kinetic and structural properties. NADP-ME2 exhibits the highest specific activity, while NADP-ME3 and −4 present the highest catalytic efficiency for NADP and malate, respectively. When analyzing the activity of each isoform in the presence of possible metabolic effectors, the results obtained indicate that NADP-ME2 is the most highly regulated isoform, especially by activation. The four isoforms behave differently in terms of reversibility, presenting NADP-ME4 the highest ratio between the reverse — carboxylation and reduction of pyruvate — to the forward reaction. In order to identify residues or segments of the primary structure of each NADPME isoform that could be involved in the differences in kinetic and regulatory properties among the isoforms, NADP-ME2 mutants and deletions were constructed and analysed. The results obtained show that Arg115 is involved in fumarate activation, while the regions involved in aspartate and CoA modulation are located at the amino-terminal part of the protein. On overall, these studies show that minimal structural changes are responsible for the different kinetic behaviour of each AtNADP-ME isoform.
KeywordsNADP-malic enzyme Arabidopsis thaliana isoforms function-structure relationship recombinant expression metabolic regulation
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