In vivo pyruvate carboxylation activity of specific Arabidopsis thaliana malic enzymes

BADIA, M. B.; MANS, R.; LIS, A.; TRONCONI, M. A.; ARIAS, C. L.; MAURINO, V. G.; ANDREO, C. S.; DRINCOVICH, M. F.; VAN MARIS, A. J. A.; GERRARD WHEELER, M. C.

Buenos Aires

Workshop; Fronteras en Biociencias; 2016

MinCyT-Sociedad Max Planck

NAD(P)-malic enzyme (NAD(P)-ME) catalyzes the oxidative decarboxylation of malate to pyruvate, CO2 and NAD(P)H and is present as a multigene family in Arabidopsis. Studies with purified recombinant A. thaliana NADP-ME proteins showed a high catalytic efficiency for the reverse reaction, i.e., the reductive carboxylation of pyruvate. In contrast, for the NAD-dependent counterparts, no reverse activity could be detected in vitro. In this work, Arabidopsis NAD(P)-ME isoforms as well as NADP-ME2del2 (with a decreased ability to carboxylate pyruvate) and NADP-ME2R115A (without fumarate activation) were tested as reductive pyruvate carboxylases in a cellular context. For this purpose, each one was functionally expressed in the cytosol of pyruvate carboxylase negative (Pyc-) Saccharomyces cerevisiae strains -incapable of growing using glucose as the sole carbon source. Experiments showed that heterologous expression of NADP-ME1, NADP-ME2 (and its mutant proteins) and NADP-ME3 restored the growth of Pyc- S. cerevisiae on glucose, and this capacity was dependent on the availability of CO2. On the other hand, NADP-ME4, NAD-ME1 and NAD-ME2 could not rescue Pyc- strains from C4 auxotrophy. In addition to the yeast complementation assays, the carboxylation reaction was evaluated with the recombinant ME isoforms under conditions that mimic the cellular environment, and in wild type and transgenic Arabidopsis plants with modified levels of NADP-ME. Together, the results indicate that specific A. thaliana NADP-ME isoforms are able to play an anaplerotic role in vivo supporting the hypothesis that the pyruvate carboxylation activity of ME would take place in plant cells, and may contribute to C4 organic acid biosynthesis, CO2 fixation and redox shuttling.