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Specific Arabidopsis thaliana malic enzyme isoforms can provide anaplerotic pyruvate carboxylation function in Saccharomyces cerevisiae
Author(s) -
Badia Mariana Beatriz,
Mans Robert,
Lis Alicia V.,
Tronconi Marcos Ariel,
Arias Cintia Lucía,
Maurino Verónica Graciela,
Andreo Carlos Santiago,
Drincovich María Fabiana,
Maris Antonius J. A.,
Gerrard Wheeler Mariel Claudia
Publication year - 2017
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.14013
Subject(s) - nad+ kinase , biochemistry , biology , arabidopsis thaliana , arabidopsis , pyruvate carboxylase , saccharomyces cerevisiae , malic enzyme , heterologous expression , mutant , enzyme , yeast , recombinant dna , gene , dehydrogenase
NAD(P)‐malic enzyme (NAD(P)‐ME) catalyzes the reversible oxidative decarboxylation of malate to pyruvate, CO 2 , 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 CO 2 . On the other hand, NADP‐ME4, NAD‐ME1, and NAD‐ME2 could not rescue the Pyc − strains from C 4 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 C 4 compounds and redox shuttling in plant cells.