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Published on January 25, 2008; 10.1104/pp.107.114975

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Received December 13, 2007
Accepted January 23, 2008

Arabidopsis thaliana NAD-malic enzyme functions as a homo- and heterodimer and has a major impact on nocturnal metabolism

Marcos A. Tronconi , Holger Fahnenstich , Mariel C. Gerrard Weehler , Carlos S. Andreo , Ulf-Ingo Flugge , Maria F. Drincovich , and Veronica G. Maurino *

Centro de Estudios Fotosinteticos y Bioquimicos (CEFOBI), Universidad Nacional de Rosario, Suipacha 531, Rosario, Argentina; Botanisches Institut, Universitat zu Koln, Gyrhofstr. 15, 50931 Cologne, Germany

* Corresponding author; email: v.maurino{at}uni-koeln.de.

Although the non-photosynthetic NAD-malic enzyme (NAD-ME) was assumed to play a central role in the metabolite flux through the TCA cycle, the knowledge on this enzyme is still limited. Here, we report on the identification and characterization of two genes encoding mitochondrial NAD-MEs from Arabidopsis thaliana, AtNAD-ME1 and AtNAD-ME2. The encoded proteins can be grouped into the two clades found in the plant NAD-ME phylogenetic tree. AtNAD-ME1 belongs to the clade that includes known {alpha}-subunits with molecular masses of approximately 65 kDa whilst AtNAD-ME2 clusters with the known {beta}-subunits with molecular masses of about 58 kDa. The separated recombinant proteins showed NAD-ME activity, presented comparable kinetic properties and are dimers in their active conformation. Native-electrophoresis coupled to denaturing-electrophoresis revealed that in vivo AtNAD-ME forms a dimer of non-identical subunits in A. thaliana. Further support for this conclusion was obtained by reconstitution of the active heterodimer in vitro. The characterization of loss-of-function mutants for both AtNAD-MEs indicated that both proteins also exhibit enzymatic activity in vivo. Neither the single nor the double mutants showed a growth or a developmental phenotype suggesting that NAD-ME activity is not essential for normal autotrophic development. Nevertheless, metabolic profiling of plants completely lacking NAD-ME activity revealed differential patterns of modifications in light and dark periods and indicates a major role for NAD-MEs during nocturnal metabolism.




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