title: L-Malate dehydrogenase activity in the reductive arm of the incomplete citric acid cycle of Nitrosomonas europaea.
abstract: The autotrophic nitrifying bacterium Nitrosomonas europaea does not synthesize 2-oxoglutarate (α-ketoglutarate) dehydrogenase under aerobic conditions and so has an incomplete citric acid cycle. L-malate (S-malate) dehydrogenase (MDH) from N. europaea was predicted to show similarity to the NADP(+)-dependent enzymes from chloroplasts and was separated from the NAD(+)-dependent proteins from most other bacteria or mitochondria. MDH activity in a soluble fraction from N. europaea ATCC 19718 was measured spectrophotometrically and exhibited simple Michaelis-Menten kinetics. In the reductive direction, activity with NADH increased from pH 6.0 to 8.5 but activity with NADPH was consistently lower and decreased with pH. At pH 7.0, the K m for oxaloacetate was 20 μM; the K m for NADH was 22 μM but that for NADPH was at least 10 times higher. In the oxidative direction, activity with NAD(+) increased with pH but there was very little activity with NADP(+). At pH 7.0, the K m for L-malate was 5 mM and the K m for NAD(+) was 24 μM. The reductive activity was quite insensitive to inhibition by L-malate but the oxidative activity was very sensitive to oxaloacetate. MDH activity was not strongly activated or inhibited by glycolytic or citric acid cycle metabolites, adenine nucleotides, NaCl concentrations, or most metal ions, but increased with temperature up to about 55 °C. The reductive activity was consistently 10-20 times higher than the oxidative activity. These results indicate that the L-malate dehydrogenase in N. europaea is similar to other NAD(+)-dependent MDHs (EC 1.1.1.37) but physiologically adapted for its role in a reductive biosynthetic sequence.
authors.affiliation: School of Mathematical and Natural Sciences, Arizona State University at the West Campus, MC 2352, P.O. Box 37100, Phoenix, AZ, 85069-7100, USA, charles.deutch@asu.edu.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Deutch
mesh_terms: D002952:Citric Acid Cycle; D003067:Coenzymes / Q000378:metabolism; D004791:Enzyme Inhibitors; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008293:Malates / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D043370:Nitrosomonas europaea / Q000201:enzymology* / Q000378:metabolism*; D062907:Oxaloacetic Acid / Q000378:metabolism; D013053:Spectrophotometry
chemical_list: D003067:Coenzymes; D004791:Enzyme Inhibitors; D008293:Malates; D009243:NAD; D062907:Oxaloacetic Acid; D009249:NADP; C030298:malic acid; D008291:Malate Dehydrogenase