ELECTRON-MICROSCOPIC ANALYSIS AND BIOCHEMICAL-CHARACTERIZATION OF A NOVEL METHANOL DEHYDROGENASE FROM THE THERMOTOLERANT BACILLUS-SP C1

Methanol dehydrogenase from the thermotolerant Bacillus sp. C1 was studied by electron microscopy and image processing. Two main projections can be distinguished: one exhibits 5-fold symmetry and has a diameter of 15 nm, the other is rectangular with sides of 15 and 9 nm. Subsequent image processing showed that the 5-fold view possesses mirror symmetry. The rectangular views can be divided into two separate classes, one of which has 2-fold rotational symmetry. It is concluded that methanol dehydrogenase is a decameric molecule, and a tentative model is presented. The estimated molecular weight is 430,000, based on a subunit molecular weight of 43,000. The enzyme contains one zinc and one to two magnesium ions per subunit. N-terminal amino acid sequence analysis revealed substantial similarity with alcohol dehydrogenases from Saccharomyces cerevisiae, Zymomonas mobilis, Clostridium acetobutylicum, and Escherichia coli, which contain iron or zinc but no magnesium. In view of the aberrant structural and kinetic properties, it is proposed to distinguish the enzyme from common alcohol dehydrogenases (EC 1.1.1.1) by using the name NAD-dependent methanol dehydrogenase

Purification and characterization of an NAD+-linked formaldehyde dehydrogenase from the facultative RuMP cycle methylotroph Arthrobacter P1

When Arthrobacter P1 is grown on choline, betaine, dimethylglycine or sarcosine, an NAD+-dependent formaldehyde dehydrogenase is induced. This formaldehyde dehydrogenase has been purified using ammonium sulphate fractionation, anion exchange- and hydrophobic interaction chromatography. The molecular mass of the native enzyme was 115 kDa ± 10 kDa. Gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the molecular mass of the subunit was 56 kDa ± 3 kDa, which is consistent with a dimeric enzyme structure. After ammonium sulphate fractionation the partially purified enzyme required the addition of a reducing reagent in the assay mixture for maximum activity. The enzyme was highly specific for its substrates and the Km values were 0.10 and 0.80 mM for formaldehyde and NAD+, respectively. The enzyme was heat-stable at 50°C for at least 10 min and showed a broad pH optimum of 8.1 to 8.5. The addition of some metal-binding compounds and thiol reagents inhibited the enzyme activity