The microbial oxidation of methanol. Purification and properties of the alcohol dehydrogenase of Pseudomonas sp. M27.
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ABSTRACT:
SUBMITTER: Anthony C
PROVIDER: S-EPMC1271237 | biostudies-other | 1967 Sep
REPOSITORIES: biostudies-other
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Project description:1. No primary hydrogen acceptor other than phenazine methosulphate has been found for the alcohol dehydrogenase from Pseudomonas sp. M27. 2. None of a wide range of vitamins or cofactors has any effect on the activity of the enzyme. 3. The enzyme is far less sensitive to metal-chelating agents and thiol reagents than are other alcohol dehydrogenases. 4. Methanol is oxidized at least as fast as other alcohols by this enzyme and its well-defined substrate specificity is different from that of other alcohol dehydrogenases. Only primary alcohols are oxidized; the general formula for an oxidizable substrate is R.CH(2).OH, where R may be H or [Formula: see text] 5. Whole organisms oxidize only those alcohols that are oxidized by the isolated enzyme.
| S-EPMC1207221 | biostudies-other
Project description:1. The purified alcohol dehydrogenase of Pseudomonas sp. M27, whose action is independent of nicotinamide nucleotides, has absorption peaks at 280mmu and at 350mmu with little or no absorption at or above 450mmu. 2. It does not fluoresce, but green-fluorescent material, diffusible on dialysis, is produced when the enzyme is treated with acid or alkali or when it is boiled. 3. Evidence is presented that the enzyme is not a flavoprotein. 4. Kinetic studies show a correlation between enzyme inactivation by acid, alkali or heat and liberation of the fluorescent material. 5. Some purification of the fluorescent material was achieved, but definite identification was not possible; the major component has a fluorescence maximum at about 460mmu with excitation maxima at about 260mmu and 365mmu. 6. Data are given (including absorption and fluorescence spectra) that support the suggestion that the prosthetic group of the enzyme is a pteridine derivative. 7. Possible mechanisms of action of the enzyme are discussed.
| S-EPMC1271238 | biostudies-other
Project description:At neutral pH, oxidation of CH(3)OH --> CH(2)O by an o-quinone requires general-base catalysis and the reaction is endothermic. The active-site -CO(2)(-) groups of Glu-171 and Asp-297 (Glu-171-CO(2)(-) and Asp-297-CO(2)(-)) have been considered as the required general base catalysts in the bacterial o-quinoprotein methanol dehydrogenase (MDH) reaction. Based on quantum mechanics/molecular mechanics (QM/MM) calculations, the free energy for MeOH reduction of o-PQQ when MeOH is hydrogen bonded to Glu-171-CO(2)(-) and the crystal water (Wat1) is hydrogen bonded to Asp-297-CO(2)(-) is DeltaG++ = 11.7 kcal/mol, which is comparable with the experimental value of 8.5 kcal/mol. The calculated DeltaG++ when MeOH is hydrogen bonded to Asp-297-CO(2)(-) is >50 kcal/mol. The Asp-297-CO(2)(-)...Wat1 complex is very stable. Molecular dynamics (MD) simulations on MDH.PQQ.Wat1 complex in TIP3P water for 5 ns does not result in interchange of Asp-297-CO(2)(-) bound Wat1 for a solvent water. Starting with Wat1 removed and MeOH hydrogen bonded to Asp-297-CO(2)(-), we find that MeOH returns to be hydrogen bonded to Glu-171-CO(2)(-) and Asp-297-CO(2)(-) coordinates to Ca(2+) during 3 ns simulation. The Asp-297-CO(2)(-)...Wat1 of reactant complex does play a crucial role in catalysis. By QM/MM calculation DeltaG++ = 1.1 kcal/mol for Asp-297-CO(2)(-) general-base catalysis of Wat1 hydration of the immediate CH(2)==O product --> CH(2)(OH)(2). By this means, the endothermic oxidation-reduction reaction is pulled such that the overall conversion of MeOH to CH(2)(OH)(2) is exothermic.
| S-EPMC3020142 | biostudies-literature
Project description:Desulfofundulus kuznetsovii is a thermophilic, spore-forming sulphate-reducing bacterium in the family Peptococcaceae. In this study, we describe a newly isolated strain of D. kuznetsovii, strain TPOSR, and compare its metabolism to the type strain D. kuznetsovii 17T. Both strains grow on a large variety of alcohols, such as methanol, ethanol and propane-diols, coupled to the reduction of sulphate. Strain 17T metabolizes methanol via two routes, one involving a cobalt-dependent methyl transferase and the other using a cobalt-independent alcohol dehydrogenase. However, strain TPOSR, which shares 97% average nucleotide identity with D. kuznetsovii strain 17T, lacks several genes from the methyl transferase operon found in strain 17T. The gene encoding the catalytically active methyl transferase subunit B is missing, indicating that strain TPOSR utilizes the alcohol dehydrogenase pathway exclusively. Both strains grew with methanol during cobalt starvation, but growth was impaired. Strain 17T was more sensitive to cobalt deficiency, due to the repression of its methyl transferase system. Our findings shed light on the metabolic diversity of D. kuznetsovii and their metabolic differences of encoding one or two routes for the conversion of methanol.
| S-EPMC10907483 | biostudies-literature
Project description:1. An improved procedure is reported for purification of the amine dehydrogenase from methylamine-grown Pseudomonas AM1 which yielded a product homogeneous by sedimentation and disc-electrophoretic analysis, with molecular weight of 133000. 2. The purified enzyme had absorption maxima at 280 and 430nm. On aging, a third peak appeared at 325nm, and the 430nm peak decreased in intensity. This spectrum was independent of pH. 3. Addition of 2.5mm-semicarbazide, phenylhydrazine, hydrazine or hydroxylamine produced modified spectra with maxima respectively at 400, 440, 395 and 425nm. 4. Aerobic addition of methylamine resulted in a bleaching of the 430nm peak and the appearance of a new one at 325nm. This spectral change was retained after removal of the methylamine by dialysis. The original spectrum could be restored on addition of phenazine methosulphate. 5. Addition of borohydride partially inactivated the enzyme and produced spectral changes similar to those observed with methylamine. Pre-treatment with methylamine prevented the inactivation by borohydride. The degree of inactivation could be increased by alternate phenazine methosulphate and borohydride treatments. 6. The addition of methylamine or borohydride each caused shifts in the fluorescence emission maximum from 348 to 380nm. 7. Lineweaver-Burk plots of reciprocal activity against reciprocal concentration of either of the substrates n-butylamine or phenazine methosulphate were consistent with a mechanism that involves interconversion of two free forms of the enzyme by the two substrates. 8. The enzyme, although spectrally modified, was not inactivated by dialysis against diethyldithiocarbamate, and contained about 0.27 g-atom of copper/mol, with small traces of cobalt, iron and zinc. 9. Conventional methods of resolution did not release the prosthetic group. Heat denaturation after treatment of the enzyme with methylamine liberated a yellow chromophore which did not reactivate resolved aspartate aminotransferase, and whose spectral, electrophoretic and fluorescence properties did not agree with any recognizable pyridoxal derivatives. 10. Despite the inconclusive results with the isolated chromophore, the observations on the enzyme suggest that it may contain a pyridoxal derivative bound as a Schiff's base which is converted into the pyridoxamine form on aerobic treatment with methylamine and reconverted into the pyridoxal form with phenazine methosulphate. 11. The copper detected is probably not involved in the enzyme mechanism, since most copper-chelating agents are not inhibitory, and since the enzyme does not react with oxygen.
| S-EPMC1177077 | biostudies-other
Project description:Alcohol dehydrogenase (EC 1.1.1.1) from the rat liver supernatant fraction has been purified 200-fold and partially characterized. The isolation procedure involved ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration. The purified enzyme behaved as a homogeneous preparation as evaluated by cellulose acetate and polyacrylamide-gel disc electrophoresis. Sulphoethyl-Sephadex chromatography and immunoelectrophoresis with rabbit antiserum indicated the presence of a minor component. Rat liver alcohol dehydrogenase appears to contain 4mol of zinc/mol, has an estimated molecular weight of 65000 and consists of two subunits of similar molecular weight. Heavy-metal ions, thiol-blocking reagents, urea at concentrations below 8m, low pH (5.5) and chelating agents deactivate the enzyme but do not dissociate it into subunits. Deactivated enzyme could not be reactivated. The enzyme is strictly specific for NAD(+) and has a broad specificity for alcohols, which are bound at a hydrophobic site. Inhibition occurred with the enzyme equilibrated with Zn(2+) at concentrations above 0.1mm.
| S-EPMC1178266 | biostudies-other
Project description:1. A dye-linked methanol dehydrogenase, resembling many others from a variety of methylotrophic bacteria, was purified to homogeneity from extracts of methanol-grown Methylophilus methylotrophus. 2. The enzyme was very stable in the presence of methanol; in the absence of methanol it had a half-life of 1-2 days at 4 degrees C. 3. The value of A1% 1cm,280 was 17.5. 4. The enzyme retained bound methanol after passage through Sephadex G-25. This tightly-bound methanol slowly exchanged with free [14C]-methanol from a value of 0.27 mol of [14C]methanol/mol of enzyme after 48 h incubation at 4 degrees C to a limiting value of approx. 2.5 mol of [14C]methanol/mol of enzyme after 3 weeks incubation at 4 degrees C. 5. One mol of this enzyme reduced 89.4 mol of 2,6-dichlorophenol-indophenol (via phenazine methosulphate) in the absence of any additional methanol in the assay mixture. The source of the electrons involved in this reduction is not known.
| S-EPMC1163357 | biostudies-other
Project description:Methyl formate synthase, which catalyzes methyl formate formation during the growth of methylotrophic yeasts, was purified to homogeneity from methanol-grown Candida boidinii and Pichia methanolica cells. Both purified enzymes were tetrameric, with identical subunits with molecular masses of 42 to 45 kDa, containing two atoms of zinc per subunit. The enzymes catalyze NAD(+)-linked dehydrogenation of the hydroxyl group of the hemiacetal adduct [CH2(OH)OCH3] of methanol and formaldehyde, leading to the formation of a stoichiometric amount of methyl formate. Although neither methanol nor formaldehyde alone acted as a substrate for the enzymes, they showed simple NAD(+)-linked alcohol dehydrogenase activity toward aliphatic long-chain alcohols such as octanol, showing that they belong to the class III alcohol dehydrogenase family. The methyl formate synthase activity of C. boidinii was found in the mitochondrial fraction in subcellular fractionation experiments, suggesting that methyl formate synthase is a homolog of Saccharomyces cerevisiae Adh3p. These results indicate that formaldehyde could be oxidized in a glutathione-independent manner by methyl formate synthase in methylotrophic yeasts. The significance of methyl formate synthase in both formaldehyde resistance and energy metabolism is also discussed.
| S-EPMC168467 | biostudies-other
Project description:Most plant-produced monoterpenes can be degraded by soil microorganisms. Borneol is a plant terpene that is widely used in traditional Chinese medicine. Neither microbial borneol dehydrogenase (BDH) nor a microbial borneol degradation pathway has been reported previously. One borneol-degrading strain, Pseudomonas sp. strain TCU-HL1, was isolated by our group. Its genome was sequenced and annotated. The genome of TCU-HL1 consists of a 6.2-Mbp circular chromosome and one circular plasmid, pTHL1 (12.6 kbp). Our results suggest that borneol is first converted into camphor by BDH in TCU-HL1 and is further decomposed through a camphor degradation pathway. The recombinant BDH was produced in the form of inclusion bodies. The apparent Km values of refolded recombinant BDH for (+)-borneol and (-)-borneol were 0.20 ± 0.01 and 0.16 ± 0.01 mM, respectively, and the kcat values for (+)-borneol and (-)-borneol were 0.75 ± 0.01 and 0.53 ± 0.01 s-1, respectively. Two plant BDH genes have been reported previously. The kcat and kcat/Km values of lavender BDH are about 1,800-fold and 500-fold lower, respectively, than those of TCU-HL1 BDH.ImportanceThe degradation of borneol in a soil microorganism through a camphor degradation pathway is reported in this study. We also report a microbial borneol dehydrogenase. The kcat and kcat/Km values of lavender BDH are about 1,800-fold and 500-fold lower, respectively, than those of TCU-HL1 BDH. The indigenous borneol- and camphor-degrading strain isolated, Pseudomonas sp. strain TCU-HL1, reminds us of the time 100 years ago when Taiwan was the major producer of natural camphor in the world.
| S-EPMC5066354 | biostudies-literature
Project description:Methanol dehydrogenases isolated from bacteria belonging to different classes of methylotrophs contain the same prosthetic group. A procedure for its purification from whole cells is given. The reduced and oxidized form of the enzyme from Hyphomicrobium X and those of the isolated group are compared and it is concluded that the latter indeed functions in the enzyme. Further evidence is presented that the prosthetic group is not a pterine or lumazine derivative, but a water-soluble nitrogen-containing quinone.
| S-EPMC1162511 | biostudies-other