Purification and properties of malate dehydrogenase from the extreme thermophileBacillus caldolyticus

Abstract

The enzyme malate dehydrogenase (EC 1.1.1.37) from an extreme thermophile B. Caldolyticus was purified to about 91% homogeneity. The molar mass of the enzyme was determined as 73 000 daltons and it is composed of two subunits, each with a molar mass of 37 000. Initial velocity studies with oxaloacetic acid and NADH as substrates at pH 8.1, over a range of temperatures, indicates that the enzyme operates via a sequential type mechanism. Van't Hoff plots of the kinetic parameters displayed sharp changes in slope at characteristic temperatures, whereas the Arrhenius plot exhibited no such breaks over the temperature interval investigated. The enzyme was found to be stable at 41 degrees C and lower temperatures. At 51 degrees C and 59 degrees C an almost immediate 20% reduction in activity was obtained, but no further inactivation occurred during the 60 min of incubation. At 59 degrees C the enzyme lost 50% of its initial activity in about 38 s. High concentration of NADH was observed to greatly stabilize the enzyme at that temperature. It is suggested that the slope changes in the Van't Hoff plots and the stability profiles at 51 degrees C and 59 degrees C are representative of a temperature induced conformational change in the enzyme.

Reassessment of the Transhydrogenase/Malate Shunt Pathway in Clostridium thermocellum ATCC 27405 through Kinetic Characterization of Malic Enzyme and Malate Dehydrogenase

“…The MDH from C. thermocellum was found to be thermostable, retaining over 50% activity after a 1-h incubation at 60°C. This is higher than what has been observed with the hyperthermophilic Bacillus candolyticus, which lost 50% of its activity after a 1-min incubation at 59°C, but similar to the values for Vulcanithermus medioatlanticus, which retained over 75% activity after 15 min of incubation at 60°C (30,32). The K m for OAA was significantly higher than for many characterized MDHs, such as the ones from V. medioatlanticus (0.048 mM), Corynebacterium glutamicum (0.057 mM), and Streptomyces aureofaciens (0.1 mM) (30,33,34 (33,38,39).…”

Malate Dehydrogenase from the Thermophilic Bacterium Vulcanithermus medioatlanticus

“…completely retains its activity after 10 min of incubation at temperatures up to 55°C [24]. Malate dehydrogenase from Bacillus candolyticus [25] lost 50% of its activity after 1 min of incubation at 59°C. Similar data on thermostability were obtained for MDH from Rhodospirillum rubrum [20] and Ch.…”

Synergistic ligand protection and intermediates in the denaturation of extremely thermophilic glutamine synthetase

“…for malate dehydrogenase (Kristjansson & Ponnamperuma, 1980), heavy meromyosin (Kimura et al, 1980), and pyridine nucleotide transhydrogenase (Voordouw et al, 1982). Light scattering and electron microscopy studies indicated that thermal denaturation of E-I, E-II, and BSGS did not produce detectable levels of hexamers, dimers, or monomers (Merkler, 1985).…”