TY - JOUR
T1 - Specific Arabidopsis thaliana malic enzyme isoforms can provide anaplerotic pyruvate carboxylation function in Saccharomyces cerevisiae
AU - Badia, Mariana Beatriz
AU - Mans, Robert
AU - Lis, Alicia V.
AU - Tronconi, Marcos Ariel
AU - Arias, Cintia Lucía
AU - Maurino, Verónica Graciela
AU - Andreo, Carlos Santiago
AU - Drincovich, María Fabiana
AU - van Maris, Antonius J.A.
AU - Gerrard Wheeler, Mariel Claudia
N1 - Accepted Author Manuscript
Title Manuscript differs from the publishers version
PY - 2017
Y1 - 2017
N2 - 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.
AB - 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.
KW - anaplerotic role
KW - C4 organic acids
KW - malate synthesis
KW - plant metabolism
KW - Saccharomyces cerevisiae
UR - http://resolver.tudelft.nl/UUID: 739f55d3-6ace-4d2b-97d5-457a94043aab
UR - http://www.scopus.com/inward/record.url?scp=85011286357&partnerID=8YFLogxK
U2 - 10.1111/febs.14013
DO - 10.1111/febs.14013
M3 - Article
AN - SCOPUS:85011286357
SN - 1742-464X
VL - 284
SP - 654
EP - 665
JO - The FEBS Journal
JF - The FEBS Journal
IS - 4
ER -