A Novel, Non-redox-regulated NAD-dependent Malate Dehydrogenase from Chloroplasts of Arabidopsis thalianaL.

Abstract

We report a novel plastidic NAD-dependent malate dehydrogenase (EC 1.1.1.37), which is not redox-regulated in contrast to its NADP-specific counterpart (EC 1.1.1.82). Analysis of isoenzyme patterns revealed a single NAD-MDH associated with highly purified chloroplasts isolated from Arabidopsis and spinach. A cDNA clone encoding the novel enzyme was found in the Arabidopsis EST data base by sorting all putative clones for NAD-dependent malate dehydrogenase. A derived amino acid sequence is very similar to mitochondrial and peroxisomal NAD-MDHs within the region coding for the mature protein but possesses a 80-amino acid long N-terminal domain with typical characteristics of a chloroplast transit peptide. In vitro synthesized labeled precursor protein was imported into the stroma of spinach chloroplasts and processed to a mature enzyme subunit of 34 kDa. Expressed in Escherichia coli, the recombinant enzyme exhibited the same distinctive isoelectric point of 5.35 as the original enzyme from Arabidopsis chloroplasts. Northern analysis revealed that the protein is expressed in both autotrophic and heterotrophic tissues. The findings reported here indicate that the "malate valve" operates not only in the illuminated chloroplasts but also in dark chloroplasts and in heterotrophic plastids and is therefore a general mechanism to maintain the optimal ratio between ATP and reducing equivalents in plastids.

Mitochondrial Malate Dehydrogenase Lowers Leaf Respiration and Alters Photorespiration and Plant Growth in Arabidopsis

“…Two of these are targeted to peroxisomes, PMDH1 and PMDH2 (Pracharoenwattana et al, 2007;Eubel et al, 2008), and one to chloroplasts (Berkemeyer et al, 1998;Peltier et al, 2006); three have no apparent targeting sequences and are assumed to be cytosolic, and two have predicted mitochondrial targeting sequences and are the only MDH isoforms identified in purified mitochondrial samples (Heazlewood et al, 2004). The two putative mitochondrial enzymes will be referred to as mMDH1 (At1g53240) and mMDH2 (At3g15020).…”

Plastidial NAD-Dependent Malate Dehydrogenase Is Critical for Embryo Development and Heterotrophic Metabolism in Arabidopsis

“…Isoforms of MDH are present in various cell compartments (Gietl, 1992), and each isoform is specific to either cosubstrate NAD (NAD-MDH; EC 1.1.1.37) or NADP (NADP-MDH; EC 1.1.1.82). The Arabidopsis genome encodes eight putative NAD-MDH isoforms: two isoforms are peroxisomal MDH (PMDH; PMDH1 and PMDH2; Pracharoenwattana et al, 2007;Eubel et al, 2008), two isoforms are mitochondrial MDH (MMDH; MMDH1 and MMDH2; Millar et al, 2001;Lee et al, 2008;Tomaz et al, 2010), and one isoform is plastidial MDH (plastid-localized NAD-dependent MDH [pdNAD-MDH]; Berkemeyer et al, 1998). The remaining three isoforms have no detectable target sequence and are thought to be cytosolic MDH (CMDH; CMDH1, CMDH2, and CMDH3).…”

Computational Identification of 69 Retroposons in Arabidopsis

“…The best example was observed in the intronless chloroplast NADdependent malate dehydrogenase (At3g47520) that has two seven exonic paralogs (At1g53240 and At3g15020) that targeted to mitochondrion as well as two nine exonic paralogs (At2g22780 and At5g09660) that targeted to glyoxysome. Extensive analysis revealed that all of these three isoforms were derived from duplications of an ancient intronless mitochondrial isoform, and then only the chloroplast gene has kept intronless with a moderate divergence, occupying a different clade in the phylogenetic tree (Berkemeyer et al, 1998). Several tens of intronless genes were identified as ancient gene structures rather than retroposons and were not included in this work, such as 20S proteasome a-subunit C (At3g22110) and the histone H3-like protein family with seven members.…”