NAD-malic enzymes of Arabidopsis thaliana display distinct kinetic mechanisms that support differences in physiological control

TRONCONI, M. A.; GERRARD WHEELER, M. C.; MAURINO, V. G.; DRINCOVICH, M. F.; ANDREO, C. S.

BIOCHEMICAL JOURNAL

PORTLAND PRESS LTD

Año: 2010 vol. 430 p. 295 - 303

0264-6021

The Arabidopsis thaliana genome contains two genes encoding NAD-malic enzymes (NAD-ME1, At4G13560 and NAD-ME2, At4G00570). The encoded proteins are localized to mitochondria and assemble as homo- and heterodimers in vitro and in vivo. In this work, the kinetic mechanisms of NAD-ME1 and -2 homodimers and the heterodimer (NAD-MEH) were studied as an approach to understand the contribution of these enzymes to the plant physiology. Product inhibition and substrate analogue analyses indicated that NAD-ME2 follows a sequential ordered Bi-Ter mechanism, being NAD 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 in the first place. As a consequence of the kinetic mechanism, NAD-ME1 showed a partial inhibition by L-malate at low NAD concentrations. The analysis of a chimerical protein between NAD-ME 1 and -2, indicated that the first 176 amino acids are associated with the differences observed in the kinetic mechanisms of the enzymes. Furthermore, NAD-ME1, -2 and -H catalyze 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 under a metabolic condition in which mitochondrial NAD level would be low and L-malate level would be high, the activity of NAD-ME2 and/or -H would be preferred over that of NAD-ME1.