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M. A. Tronconi and others
the forward decarboxylase activity and the enzyme showed very
low affinity towards the substrates pyruvate and HCO3 [35].
In agreement with this, A. thaliana NAD-ME reverse activities
were 0.05–0.16% of the forward activity. In this way, plant
mitochondrial NAD-MEs would play an exclusive catabolic role
in vivo.
REFERENCES
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1
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Differential kinetic mechanisms of each NAD-ME in A. thaliana
4
5
6
7
Previous studies indicated that recombinant A. thaliana NAD-
ME1, -ME2 and -MEH behave differentially in terms of kinetic
and regulatory properties [17,18]. The results in the present paper
indicate that these enzymatic entities display different kinetic
mechanisms, the binding of L-malate to NAD-ME1 and -MEH in
the absence of NAD being one of the major differences compared
with NAD-ME2. Moreover, other differences in the kinetic
behaviour of NAD-ME1 and -MEH were observed (Figures 3 and
4), which imply a kinetically preferred pathway for the formation
of the ternary complex in NAD-ME1 (Figure 2B). The binding
of L-malate to NAD-ME1 and -MEH could be directly to the
active site or, alternatively, to an allosteric site that is absent in
NAD-ME2.
NAD-ME1 and -ME2 associate to generate a heterodimeric
enzyme (NAD-MEH) with different regulatory properties from
those shown by NAD-ME1 and -ME2 homodimers [18]. The
results of the present work suggest that, in NAD-MEH, each
subunit may contribute to the interaction with the substrate in
a different way. Essentially, the formation of the heterodimer
appears to result in a change of the kinetic mechanism of
NAD-ME2 to one similar to that shown by NAD-ME1. Another
possibility could be that, despite the observation that the NAD-
ME2 homodimer shows activity in vivo in the absence of
NAD-ME1, the NAD-ME2 subunit may play a regulatory role
rather than a catalytic one in the heterodimer, as was suggested
previously for the β subunit [16]. In either case, NAD-MEH is a
new enzymatic entity whose properties are not described by those
of the homodimers.
It is worth mentioning that the results obtained in this work are
based on the use of recombinant enzymes. However, the kinetic
parameters (Km and kcat values) and some regulatory properties
displayed by these recombinant forms [17,18] are similar to
those of characterized isofoms purified from several plant tissues
[19]. Thus the in vitro approach suggests that, under metabolic
conditions where the level of NAD is low and that of L-malate is
high, the activity of NAD-ME2 and/or -MEH would be preferred
over that of NAD-ME1. The different kinetic and regulatory
properties shown by each A. thaliana NAD-ME further support
the hypothesis of a specificity of function of each enzymatic entity
in plant metabolism. In this way, although these proteins act in
concert in most mature organs [17,18] they do not represent a case
of functional redundancy.
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(2002) Crystal structure of the malic enzyme from Ascaris suum complexed with
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AUTHOR CONTRIBUTION
19 Winning, B. M., Bourguignon, J. and Leaver, C. J. (1994) Plant mitochondrial
NAD-dependent malic enzyme. J. Biol. Chem. 269, 4780–4786
20 Douce, R. and Neuburger, M. (1989) The uniqueness of plant mitochondria. Annu. Rev.
Plant Physiol. Plant Mol. Biol. 40, 371–414
21 Palmer, J. M., Schwitzgue´bel, J. P. and Moller, I. M. (1982) Regulation of malate oxidation
in plant mitochondria. Biochem. J. 208, 703–711
Marcos Tronconi, Mariel Gerrard Wheeler and Mar´ıa Drincovich designed the concept
and experiments of this study. Marcos Tronconi prepared recombinant proteins and
was specifically involved in data acquisition. Marcos Tronconi, Mariel Gerrard Wheeler,
Vero´nica Maurino and Mar´ıa Drincovich were involved in the analysis and interpretation of
the results. Carlos Andreo, in collaboration with the other authors, drafted the manuscript.
22 Gerrard Wheeler, M. C., Arias, C. L., Tronconi, M. A., Maurino, V. G., Andreo, C. S. and
Drincovich, M. F. (2008) Arabidopsis thaliana NADP-malic enzyme isoforms: high degree
of identity but clearly distinct properties. Plant Mol. Biol. 67, 231–242
23 Maurino, V. G., Gerrard Wheeler, M. C., Andreo, C. S. and Drincovich, M. F. (2009)
Redundancy is sometimes seen only by the uncritical: does Arabidopsis need six malic
enzyme isoforms? Plant Sci. 176, 715–721
FUNDING
This work was supported by Agencia Nacional de Promocio´n Cient´ıfica y Tecnolo´gica
(ANPCyT), Argentina [grant number PICT 32233]; Consejo Nacional de Investigaciones
Cient´ıficas y Te´cnicas (CONICET), Argentina; and the Deutsche Forschungsgemeinschaft,
Germany.
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The Authors Journal compilation 2010 Biochemical Society