Purification and properties of methanol dehydrogenase from Methylocystis sp. GB 25.
Methanol dehydrogenase (MDH) from Methylocystis sp. GB 25, which belongs to the group II of methanotrophic bacteria, is able to catalyse the oxidation of methanol to formate directly. The enzyme was purified 20-fold by a 5 step procedure to electrophoretic homogeneity. After cell disruption by French press, about 95% of MDH-activity was found in the soluble fraction. The relative molecular mass of the native enzyme has been estimated to be 122 kDa by gel filtration and 115 kDa by the method of Hedrick and Smith (1968). It seems to be composed of two identical subunits with a relative molecular mass of 62 kDa (estimated by SDS gel electrophoresis). The isoelectric point was found to be about 8.3. The amino terminal sequence shows a strong similarity to the alpha-chain of MDH from the facultative methylotrophic bacterium Methylobacterium extorquens AM1. PQQ, the probable prosthetic group of MDH, could be detected in the supernatant of the culture by using the apoenzyme of a membrane-bound glucose dehydrogenase from Pseudomonas aeruginosa but not absolutely in the absorption spectra of the enzyme after DEAE-chromatography. The purified MDH has an optimum activity at pH 9.0 and at 45 degrees C. MDH of Methylocystis sp. GB 25 oxidises only primary alcohols from methanol to heptanol and aldehydes from formaldehyde to propionaldehyde and the glutaraldehyde, respectively. The estimated Km-values show no dependence upon the chain length of substrates.
Grosse
S
S
Institut für Biochemie, Fakultät für Biowissenschaften, Pharmazie und Psychologie, Universität Leipzig, Germany.
Wendlandt
K D
KD
Kleber
H P
HP
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017421:Sequence Analysis; D013379:Substrate Specificity; D013696:Temperature
D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor)