Microbial oxidation of methane and methanol: crystallization of methanol dehydrogenase and properties of holo- and apomethanol dehydrogenase from Methylomonas methanica.
Procedures are described for the purification and crystallization of methanol dehydrogenase from the soluble fraction of the type I obligate methylotroph Methylomonas methanica strain S1. The crystallized enzyme is homogeneous as judged by acrylamide gel electrophoresis and ultracentrifugation. The enzyme had a high pH optimum (9.5) and required ammonium salt as an activator. In the presence of phenazine methosulfate as an electron acceptor, the enzyme catalyzed the oxidation of primary alcohols and formaldehyde. Secondary, tertiary, and aromatic alcohols were not oxidized. The molecular weight as well as subunit size of methanol dehydrogenase was 60,000, indicating that it is monomeric. The sedimentation constant (s(20,w)) was 3.1S. The amino acid composition of the crystallized enzyme is also presented. Antisera prepared against the crystalline enzyme were nonspecific; they cross-reacted with and inhibited the isofunctional enzyme from other obligate methylotrophic bacteria. The crystalline methanol dehydrogenase had an absorption peak at 350 nm in the visible region and weak fluorescence peaks at 440 and 470 nm due to the presence of a pteridine derivative as the prosthetic group. A procedure was developed for the preparation of apo-methanol dehydrogenase. The molecular weights, sedimentation constants, electrophoretic mobilities, and immunological properties of apo- and holo-methanol dehydrogenases are identical. Apo-methanol dehydrogenase lacked the absorption peak at 350 nm and the fluorescence peaks at 440 and 470 nm and was catalytically inactive. All attempts to reconstitute an active enzyme from apo-methanol dehydrogenase, using various pteridine derivatives, were unsuccessful.
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D005779:Immunodiffusion; D008697:Methane / Q000378:metabolism*; D000432:Methanol / Q000378:metabolism*; D008772:Methylococcaceae / Q000201:enzymology*; D008970:Molecular Weight; D013379:Substrate Specificity
D000429:Alcohol Oxidoreductases; D008697:Methane; D000432:Methanol