[
International C. elegans Meeting,
2001]
In my laboratory, we are overexpressing and biochemically characterizing C. elegans malate dehydrogenase (MDH), the last enzyme in the citric acid cycle. This enzyme, encoded by putative gene F20H11.3, appears to be the mitochondrial form of MDH, since it contains a mitochondrial import presequence. We have overexpressed and purified a version of this enzyme with the mitochondrial import presequence removed at the predicted cleavage site of the mitochondrial processing protease. The purified MDH enzyme has malate dehydrogenase activity that follows Michaelis-Menten kinetics when plotted versus oxaloacetate concentration, and the resulting Km is similar to the Km values for other MDH enzymes. Interestingly, the temperature dependence of the enzyme is a bell-shaped curve with a peak at approximately 30degC, suggesting that the enzyme is adapted to the growing temperature of C. elegans . We are grateful to Yuji Kohara for providing the cDNA clone of MDH.
[
International Worm Meeting,
2011]
Malate dehydrogenase (MDH) is the last enzyme in the citric acid cycle. MDH catalyzes the conversion of NAD+ and malate to NADH and oxaloacetate. Since the forward reaction is energetically unfavorable, the reverse reaction is usually studied. Eukaryotes have two versions of this enzyme, one that is imported into the mitochondria and one that remains in the cytoplasm. The cytoplasmic MDH in C. elegans (F46E10.10) was originally mis-classified as lactate dehydrogenase. Hold and Riddle (Mech. Aging Dev. 124, 779, 2003) realized that the amino acids in the active site were compatible with malate binding rather than lactate. We named this enzyme MDH-1 and renamed the mitochondrial enzyme (F20H11.3) MDH-2 so that the naming convention corresponded to that used for other eukaryotes. We overexpressed MDH-1 in E. coli and removed the chitin binding domain tag used for the purification. In kinetic assays with the purified enzyme, MDH-1 had malate dehydrogenase activity that followed Michaelis-Menten kinetics. For the reverse reaction, we determined that the KM for oxaloacetate was 37 mM, and the KM for NADH was 70 mM. Our gel filtration results indicated that MDH-1 was active as a dimer, which is similar to the quaternary structure of most MDH enzymes (Minarik, P. et al., Gen. Physiol. Biophys. 21, 257, 2002). We are in the process of purifying endogenous MDH-1 from worms to compare its activity to our recombinant enzyme.
[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
Malate dehydrogenase (MDH), the last enzyme in the citric acid cycle, is found in nearly all organisms, but most published work describing MDH enzyme kinetics involves prokaryotic proteins. MDH catalyzes the conversion of NAD + and malate to NADH and oxaloacetate. Since the forward reaction is energetically unfavorable, the reverse reaction is usually studied. We are biochemically characterizing overexpressed C. elegans malate dehydrogenase. This enzyme, called MDH-1 and encoded by gene F20H11.3, is the mitochondrial form of MDH since it contains a mitochondrial import presequence. Two different expression plasmids were constructed and tested in E. coli . The overexpressed MDH-1 N-terminus is at the predicted cleavage site for the mitochondrial processing protease. The purified MDH-1 enzyme had malate dehydrogenase activity that followed Michaelis-Menten kinetics when plotted versus oxaloacetate concentration. The K M value for oxaloacetate was 25 ?M, and this is similar to the K M values for other MDH enzymes. MDH-1 purified to apparent homogeneity had low specific activity, even when expressed with no large tags on either the N- or C-terminus. There is a 100-fold variation in published specific activity values for purified MDH enzymes from various sources, demonstrating a huge range of enzyme effectiveness. Our preliminary gel filtration results indicated that MDH-1 was active as a dimer, which is similar to the quaternary structure of most MDH enzymes (Minarik, P. et al. , Gen. Physiol. Biophys. 21, 257, 2002).