The Arabidopsis thaliana genome contains two genes encoding NAD-MEs [NAD-dependent malic enzymes; NAD-ME1 (TAIR accession number At4G13560) and NAD-ME2 (TAIR accession number At4G00570)]. The encoded proteins are localized to mitochondria and assemble as homo- and hetero- dimers in vitro and in vivo. In the present work, the kinetic mechanisms of NAD-ME1 and -ME2 homodimers and NAD-MEH (NAD-ME heterodimer) were studied as an approach to understand the contribution of these enzymes to plant physiology. Product-inhibition and substrate-analogue analyses indicated that NAD-ME2 follows a sequential ordered Bi-Ter mechanism, NAD being the leading substrate followed by L-malate. On the other hand, NAD-ME1 and NAD-MEH can bind both substrates randomly. However, NAD-ME1 shows a preferred route that involves the addition of NAD first. As a consequence of the kinetic mechanism, NAD-ME1 showed a partial inhibition by L-malate at low NAD concentrations. The analysis of a protein chimaeric for NAD-ME1 and -ME2 indicated that the first 176 amino acids are associated with the differences observed in the kinetic mechanisms of the enzymes. Furthermore, NAD-ME1, -ME2 and -MEH catalyse the reverse reaction (pyruvate reductive carboxylation) with very low catalytic activity, supporting the notion that these isoforms act only in L-malate oxidation in plant mitochondria. The different kinetic mechanism of each NAD-ME entity suggests that, for a metabolic condition in which the mitochondrial NAD level is low and the L-malate level is high, the activity of NAD-ME2 and/or -MEH would be preferred over that of NAD-ME1.
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Research Article|
August 13 2010
NAD-malic enzymes of Arabidopsis thaliana display distinct kinetic mechanisms that support differences in physiological control
Marcos A. Tronconi;
Marcos A. Tronconi
*Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Universidad Nacional de Rosario, Suipacha 531, Rosario, Argentina
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Mariel C. Gerrard Wheeler;
Mariel C. Gerrard Wheeler
*Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Universidad Nacional de Rosario, Suipacha 531, Rosario, Argentina
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Verónica G. Maurino;
Verónica G. Maurino
†Botanisches Institut, Universität zu Köln, Zülpicher Str. 47b, 50674, Cologne, Germany
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María F. Drincovich;
María F. Drincovich
*Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Universidad Nacional de Rosario, Suipacha 531, Rosario, Argentina
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Carlos S. Andreo
Carlos S. Andreo
1
*Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Universidad Nacional de Rosario, Suipacha 531, Rosario, Argentina
1To whom correspondence should be addressed (carlosandreo@cefobi-conicet.gov.ar)
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Publisher: Portland Press Ltd
Received:
April 01 2010
Revision Received:
June 01 2010
Accepted:
June 09 2010
Accepted Manuscript online:
June 09 2010
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2010 Biochemical Society
2010
Biochem J (2010) 430 (2): 295–303.
Article history
Received:
April 01 2010
Revision Received:
June 01 2010
Accepted:
June 09 2010
Accepted Manuscript online:
June 09 2010
Citation
Marcos A. Tronconi, Mariel C. Gerrard Wheeler, Verónica G. Maurino, María F. Drincovich, Carlos S. Andreo; NAD-malic enzymes of Arabidopsis thaliana display distinct kinetic mechanisms that support differences in physiological control. Biochem J 1 September 2010; 430 (2): 295–303. doi: https://doi.org/10.1042/BJ20100497
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