Specific Arabidopsis thaliana malic enzyme isoforms can provide anaplerotic pyruvate carboxylation function in Saccharomyces cerevisiae

BADIA, MARIANA BEATRIZ; MANS, ROBERT; LIS, ALICIA V.; TRONCONI, MARCOS ARIEL; ARIAS, CINTIA LUCÍA; MAURINO, VERÓNICA GRACIELA; ANDREO, CARLOS SANTIAGO; DRINCOVICH, MARÍA FABIANA; VAN MARIS, ANTONIUS J. A.; GERRARD WHEELER, MARIEL CLAUDIA; BADIA, MARIANA BEATRIZ; MANS, ROBERT; LIS, ALICIA V.; TRONCONI, MARCOS ARIEL; ARIAS, CINTIA LUCÍA; MAURINO, VERÓNICA GRACIELA; ANDREO, CARLOS SANTIAGO; DRINCOVICH, MARÍA FABIANA; VAN MARIS, ANTONIUS J. A.; GERRARD WHEELER, MARIEL CLAUDIA

FEBS JOURNAL

WILEY-BLACKWELL PUBLISHING, INC

Lugar: Londres; Año: 2017 vol. 284 p. 654 - 665

1742-464X

NAD(P)-malic enzyme (NAD(P)-ME) catalyzes the reversible oxidative decarboxylation of malate to pyruvate, CO2, and NAD(P)H and is present as a multigene family in Arabidopsis thaliana. The carboxylation reaction catalyzed by purified recombinant Arabidopsis NADP-ME proteins is faster than those reported for other animal or plant isoforms. In contrast, no carboxylation activity could be detected in vitro for the NAD-dependent counterparts. In order to further investigate their putative carboxylating role in vivo, Arabidopsis NAD(P)-ME isoforms, as well as the NADP-ME2del2 (with a decreased ability to carboxylate pyruvate) and NADP-ME2R115A (lacking fumarate activation) versions, were functionally expressed in the cytosol of pyruvate carboxylase-negative (Pyc−) Saccharomyces cerevisiae strains. The 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 the Pyc− strains from C4 auxotrophy. NADP-ME carboxylation activity could be measured in leaf crude extracts of knockout and overexpressing Arabidopsis lines with modified levels of NADP-ME, where this activity was correlated with the amount of NADP-ME2 transcript. These results indicate that specific A. thaliana NADP-ME isoforms are able to play an anaplerotic role in vivo and provide a basis for the study on the carboxylating activity of NADP-ME, which may contribute to the synthesis of C4 compounds and redox shuttling in plant cells.