Microbial oxidation of methane and methanol: crystallization and properties of methanol dehydrogenase from Methylosinus sporium.
Obligate methylotrophs are divisible into two types on the basis of ultrastructural biochemical characteristics. Both groups possess a soluble phenazine methosulfate (PMS)-dependent methanol dehydrogenase. In addition, particulate PMS-dependent methanol dehydrogenase and PMS-independent methanol oxidase have been found in the type I membrane group. A procedure was developed for the crystallization of methanol dehydrogenase from the soluble fraction of the type II obligate methylotroph Methylosinus sporium. This is the first report of a crystalline methanol dehydrogenase from a methylotrophic bacterium. The crystallized enzyme is homogeneous as judged by ultracentrifugation and by acrylamide gel electrophoresis. In the presence of an electron acceptor (phenazine or phenazinium compound) and an activator (ammonium compound), the crystallized enzyme catalyzed the oxidation of primary alcohols and formaldehyde. Secondary, tertiary, and aromatic alcohols were not oxidized. The molecular weight of the enzyme as estimated by gel filtration is approximately 60,000, and as estimated by sedimentation equilibrium analysis it is 62,000. The sedimentation constant (S20,W) is 2.9. The subunit size determined by sodium dodecyl sulfate-gel electrophoresis is approximately 60,000. The amino acid composition and spectral properties of the enzyme are also presented. Antisera prepared against the crystalline enzyme are nonspecific, they cross-reacted and inhibited isofunctional enzymes from other obligate methylotrophic bacteria.
D000429:Alcohol Oxidoreductases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000643:Ammonium Chloride / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D000432:Methanol / Q000378:metabolism*; D008772:Methylococcaceae / Q000201:enzymology*; D008970:Molecular Weight; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013696:Temperature
D000596:Amino Acids; D004791:Enzyme Inhibitors; D000643:Ammonium Chloride; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor); D000432:Methanol