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

Abstract

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.

Comparative Transcriptome Atlases Reveal Altered Gene Expression Modules between Two Cleomaceae C3 and C4 Plant Species

“…The patterns of gene expression in T. hassleriana provide a good proxy for the ancestral C 3 expression pattern due to its phylogenetic proximity to G. gynandra (Inda et al, 2008;Cheng et al, 2013). Genes active in the C 4 cycle were recruited from previously existing metabolism (Matsuoka, 1995;Chollet et al, 1996;Streatfield et al, 1999;Wheeler et al, 2005;Tronconi et al, 2010). Expression patterns in T. hassleriana reflect known metabolism and expression; for instance, PPDK is expressed in seeds, stamens, and petals (Supplemental Figure 12B), which is similar to the expression domain reported by Chastain et al (2011).…”

Metabolic regulation of phytoplasma malic enzyme and phosphotransacetylase supports the use of malate as an energy source in these plant pathogens

“…The native molecular mass of AYWB-ME was calculated by size exclusion chromatography. The value obtained (76.0±9.9 kDa) indicated that AYWB-ME assembles as a dimer, consistent with other MEs that also assemble as dimers (Saigo et al, 2004) or tetramers through the dimerization of dimers (Xu et al, 1999 , which are typical conditions for NAD and NADP-ME activity measurements (Saigo et al, 2004;Tronconi et al, 2010) at the optimum pH (8.2). When NAD-dependent ME activity was assessed at varying NAD concentrations (from 0.01 to 2.5 mM), the data fitted to a sigmoid curve according to the Hill equation at low malate concentration (0.09 mM) and to a hyperbolic curve according to Michaelis-Menten equation at high malate concentration (30 mM) ( Table 1, Fig.…”

“…An absorption coefficient of 6.22 mM 21 cm 21 for NAD(P)H was used in the calculations. The kinetic parameters were calculated from triplicate experiments and subjected to nonlinear regression using the following equations (Detarsio et al, 2007;Tronconi et al, 2010):…”

Enzymatic Properties of Populus α- and β-NAD-ME Recombinant Proteins

“…Another study suggested that the β-subunit plays a regulatory role in NAD-ME activity [10]. However, recent studies showed that the separated recombinant proteins of Arabidopsis thaliana (AtNAD-ME1 and -2) showed NAD-ME activity and display distinct kinetic mechanisms [6,12]. …”

“…Plant α- and β-NAD-ME are encoded by single genes in the species analyzed so far. NAD-MEs assemble as heterodimers, heterotetramers or heterooctamers in some species, while it forms a homomer in others [6,12,14]. Some plant NAD-MEs are activated by fumarate and coenzyme A (CoA) [15,16] and are also potentially regulated via changes in aggregation state [7,11].…”