Malate dehydrogenases from nitrifying bacteria : purification and properties

抄録

Malate dehydrogenases [EC 1.1.1.37, MDH] were purified as electrophoretically homogeneous proteins from Nitrosomonas sp. TK 794 (ammonia-oxidizing bacterium, Ns) and Nitrobacter agilis ATCC 14123 (nitrite-oxidizing bacterium, Nb). The molecular weights of the MDHs were 61.0 kDa (Ns-MDH) and 107.5 kDa (Nb-MDH), respectively, as indicated by gel filtration. Analysis by SDS-PAGE showed that these MDHs apparently consisted of two (Ns-MDH) and three (Nb-MDH) similar subunits with molecular weights of 35.5 and 35.9 kDa, respectively. The MDHs showed oxaloacetate-reducing activity with NAD^+. The K_m of the enzymes for oxaloacetate and NADH were estimated as 67 μM, 25 μM (Ns-MDH) and 74 μM, 53.6 μM (Nb-MDH), respectively. Optimal pH and temperature for the reaction were approximately 6.5-7.0, 60℃ (Ns-MDH) and 8.0-8.5, 45℃ (Nb-MDH), respectively. The enzymes were stable up to 60℃ (Ns-MDH) and 30℃ (Nb-MDH) and in the pH range between 5.5 and 9.5 (Ns-MDH) and 9.0 and 10.0 (Nb-MDH) after standing at 50℃ for 30 min. The Nb-MDH activity was strongly inhibited by N-ethylmaleimide and p-chloromercuribenzoate unlike the Ns-MDH activity. The N-terminal amino acid sequence of Nb-MDH was found to be ARNKIGLIGSGQIGGTLAHLIGLKELGNVVMFNIANGVPQ.