title: Specific Arabidopsis thaliana malic enzyme isoforms can provide anaplerotic pyruvate carboxylation function in Saccharomyces cerevisiae.
abstract: 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 C4auxotrophy. 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 C4compounds and redox shuttling in plant cells.
authors.affiliation: Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Universidad Nacional de Rosario, Argentina.
authors.forename: Mariana Beatriz
authors.initials: MB
authors.lastname: Badia
authors.affiliation: Department of Biotechnology, Delft University of Technology, The Netherlands.
authors.forename: Robert
authors.initials: R
authors.lastname: Mans
authors.affiliation: Department of Biotechnology, Delft University of Technology, The Netherlands.|Biochemical Engineering Institute, Saarland University, Saarbrücken, Germany.
authors.forename: Alicia V
authors.initials: AV
authors.lastname: Lis
authors.affiliation: Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Universidad Nacional de Rosario, Argentina.
authors.forename: Marcos Ariel
authors.initials: MA
authors.lastname: Tronconi
authors.affiliation: Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Universidad Nacional de Rosario, Argentina.
authors.forename: Cintia Lucía
authors.initials: CL
authors.lastname: Arias
authors.affiliation: Plant Molecular Physiology and Biotechnology Group, Institute of Developmental and Molecular Biology of Plants, Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich-Heine-Universität, Düsseldorf, Germany.
authors.forename: Verónica Graciela
authors.initials: VG
authors.lastname: Maurino
authors.affiliation: Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Universidad Nacional de Rosario, Argentina.
authors.forename: Carlos Santiago
authors.initials: CS
authors.lastname: Andreo
authors.affiliation: Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Universidad Nacional de Rosario, Argentina.
authors.forename: María Fabiana
authors.initials: MF
authors.lastname: Drincovich
authors.affiliation: Department of Biotechnology, Delft University of Technology, The Netherlands.|Division of Industrial Biotechnology, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Center, Stockholm, Sweden.
authors.forename: Antonius J A
authors.initials: AJ
authors.lastname: van Maris
authors.affiliation: Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Universidad Nacional de Rosario, Argentina.
authors.forename: Mariel Claudia
authors.initials: MC
authors.lastname: Gerrard Wheeler
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D003001:Cloning, Molecular; D015870:Gene Expression; D005816:Genetic Complementation Test; D005818:Genetic Engineering; D005947:Glucose / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D050538:Malate Dehydrogenase (NADP+) / Q000235:genetics* / Q000378:metabolism; D008293:Malates / Q000378:metabolism*; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D019289:Pyruvic Acid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014170:Transformation, Genetic; D019076:Transgenes
chemical_list: D029681:Arabidopsis Proteins; D007527:Isoenzymes; D008293:Malates; D011994:Recombinant Proteins; D009243:NAD; D002245:Carbon Dioxide; D009249:NADP; C030298:malic acid; D019289:Pyruvic Acid; C000609452:NADP-ME2 protein, Arabidopsis; D050538:Malate Dehydrogenase (NADP+); D005947:Glucose
keywords: 
  Saccharomyces cerevisiae
  ; C4 organic acids; anaplerotic role; malate synthesis; plant metabolism