Identification and purification of a nicotinamide adenine dinucleotide-dependent secondary alcohol dehydrogenase from C1-utilizing microbes.
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Alcohol Oxidoreductases↗
Biomedical subjects
C T Hou
Publications and source records attributed to C T Hou.
Microbial oxidation of methane and methanol: purification and properties of a heme-containing aldehyde dehydrogenase from Methylomonas methylovora.
Procedures for the purification of an aldehyde dehydrogenase from extracts of the obligate methylotroph, Methylomonas methylovora are described. The purified enzyme is homogeneous as judged from polyacrylamide gel electrophoresis. In the presence of an artificial electron acceptor (phenazine methosulfate), the purified enzyme catalyzes the oxidation of straight chain aldehydes (C1--C10 tested), aromatic aldehydes (benzaldehyde, salicylaldehyde), glyoxylate, and glyceraldehyde. Biological electron acceptors such as NAD+, NADP+, FAD, FMN, pyridoxal phosphate, and cytochrome c cannot act as electron carriers. The activity of the enzyme is inhibited by sulfhydryl agents [p-chloromercuribenzoate, N-ethylmaleimide and 5,5-dithiobis (2-nitrobenzoic acid)], cuprous chloride, and ferrour nitrate. The molecular weight of the enzyme as estimated by gel filtration is approximately 45000 and the subunit size determined by sodium dodecyl sulfate-gel electrophoresis is approximately 23000. The purified enzyme is light brown and has an absorption peak at 410 nm. Reduction of enzyme with sodium dithionite or aldehyde substrate resulted in the appearance of peaks at 523 nm and 552nm. These results suggest that the enzyme is a hemoprotein. There was no evidence that flavins were present as prosthetic group. The amino acid composition of the enzyme is also presented.
Aldehyde Oxidoreductases↗
Microbial production of methyl ketones. Purification and properties of a secondary alcohol dehydrogenase from yeast.
Cell-free extracts derived from yeasts Candida utilis ATCC 26387, Hansenula polymorpha ATCC 26012, Pichia sp. NRRL-Y-11328 Torulopsis sp. strain A1 and Kloeckera sp. strain A2 catalyzed an NAD+-dependent oxidation of secondary alcohols (2-propanol, 2-butanol, 2-pentanol, 2-hexanol) to the corresponding methyl ketones (acetone, 2-butanone, 2-pentanone, 2-hexanone). We have purified a NAD+-specific secondary alcohol dehydrogenase from methanol-grown yeast, Pichia sp. The purified enzyme is homogenous as judged by polyacrylamide gel electrophoresis. The purified enzyme catalyzed the oxidation of secondary alcohols to the corresponding methyl ketones in the presence of NAD+ as an electron acceptor. Primary alcohols were not oxidized by the purified enzyme. The optimum pH for oxidation of secondary alcohols by the purified enzyme is 8.0. The molecular weight of the purified enzyme as determined by gel filtration is 98 000 and subunit size as determined by sodium dodecyl sulfate gel electrophoresis is 48 000. The activity of the purified secondary alcohol dehydrogenase was inhibited by sulfhydryl inhibitors and metal-binding agents.
Alcohol Oxidoreductases↗
Microbial oxidation of gaseous hydrocarbons: epoxidation of C2 to C4 n-alkenes by methylotrophic bacteria.
Over 20 new cultures of methane-utilizing microbes, including obligate (types I and III) and facultative methylotrophic bacteria were isolated. In addition to their ability to oxidize methane to methanol, resting cell-suspensions of three distinct types of methane-grown bacteria (Methylosinus trichosporium OB3b [type II, obligate]; Methylococcus capsulatus CRL M1 NRRL B-11219 [type I, obligate]; and Methylobacterium organophilum CRL-26 NRRL B-11222 [facultative]) oxidize C2 to C4 n-alkenes to their corresponding 1,2-epoxides. The product 1,2-epoxides are not further metabolized and accumulate extracellularly. Methanol-grown cells do not have either the epoxidation or the hydroxylation activities. Among the substrate gaseous alkenes, propylene is oxidized at the highest rate. Methane inhibits the epoxidation of propylene. The stoichiometry of the consumption of propylene and oxygen and the production of propylene oxide is 1:1:1. The optimal conditions for in vivo epoxidation are described. Results from inhibition studies indicate that the same monooxygenase system catalyzes both the hydroxylation and the epoxidation reactions. Both the hydroxylation and epoxidation activities are located in the cell-free particulate fraction precipitated between 10,000 and 40,000 x g centrifugation.
Alkenes↗
Microbial oxidation of gaseous hydrocarbons: production of methyl ketones from their corresponding secondary alcohols by methane- and methanol-grown microbes.
Cultures of methane- or methanol-utilizing microbes, including obligate (both types I and II) and facultative methylotrophic bacteria, obligate methanol utilizers, and methanol-grown yeasts were isolated from lake water of Warinanco Park, Linden, N.J., and lake and soil samples of Bayway Refinery, Linden, N.J. Resting-cell suspensions of these, and of other known C1-utilizing microbes, oxidized secondary alcohols to their corresponding methyl ketones. The product methyl ketones accumulated extracellularly. Succinate-grown cells of facultative methylotrophs did not oxidize secondary alcohols. Among the secondary alcohols, 2-butanol was oxidized at the highest rate. The optimal conditions for in vivo methyl ketone formation were compared among five different types of C1-utilizing microbes. Some enzymatic degradation of 2-butanone was observed. The product, 2-butanone, did not inhibit the oxidation of 2-butanol. The rate of the 2-butanone production was linear for the first 4 h of incubation for all five cultures tested. A yeast culture had the highest production rate. The optimum temperature for the production of 2-butanone was 35 degrees C for all the bacteria tested. The yeast culture had a higher temperature optimum (40 degrees C), and there was a reasonably high 2-butanone production rate even at 45 degrees C. Metal-chelating agents inhibit the production of 2-butanone, suggesting the involvement of metal(s) in the oxidation of secondary alcohols. Secondary alcohol dehydrogenase activity was found in the cell-free soluble extract of sonically disrupted cells. The cell-free system requires a cofactor, specifically nicotinamide adenine dinucleotide, for its activity. This is the first report of a nicotinamide adenine dinucleotide-dependent, secondary alcohol-specific enzyme.
Butanols↗
Oxidation of secondary alcohols to methyl ketones by yeasts.
Cell suspensions of yeasts, Candida utilis ATCC 26387, Hansenula polymorpha ATCC 26012, Pichia sp. NRRL-Y-11328, Torulopsis sp. strain A1, and Kloeckera sp. strain A2, grown on various C-1 compounds (methanol, methylamine, methylformate), ethanol, and propylamine catalyzed the oxidation of secondary alcohols to the corresponding methyl ketones. Thus, isopropanol, 2-butanol, 2-pentanol, and 2-hexanol were converted to acetone, 2-butanone, 2-pentanone, and 2-hexanone, respectively. Cell-free extracts derived from methanol-grown yeasts catalyzed an oxidized nicotinamide adenine dinucleotide-dependent oxidation of secondary alcohols to the corresponding methyl ketones, Primary alcohols were not oxidized. The effect of various environmental factors on the production of methyl ketones from secondary alcohols by methanol-grown Pichia sp. was investigated.
Alcohol Oxidoreductases↗
Microbial oxidation of gaseous hydrocarbons. II. Hydroxylation of alkanes and epoxidation of alkenes by cell-free particulate fractions of methane-utilizing bacteria.
Cell-free particulate fractions derived from methylotrophic bacteria catalyze the oxygen- and reduced nicotinamide adenine dinucleotide-dependent epoxidation of alkenes and hydroxylation of alkanes. Evidence presented indicates that the hydroxylation and epoxidation reactions are catalyzed by the same or a similar metal-containing monooxygenase.
Alkanes↗
Iron-binding ligands in the catalytic site of protocatechuate 3,4-dioxygenase.
The tryptophan fluorescence maximum for holoprotocatechuate 3,4-dioxygenase(holo PCD) is blue-shifted slightly (3 nm) from that of the apoenzyme. In the preparation of apoenzyme, increases in tryptophan fluorescence intensity coincided with decreases in enzyme activity and decreases in iron content. The tryptophan emission intensity of reconstituted enzyme having full enzyme activity was about 90% of that of the holoenzyme. Although apo PCD has similar molecular weight, amino acid content and essentially the same gross quaternary conformation as holo PCD, the absence of iron in apo PCD causes the changes in emission intensity of tryptophan. Findings indicate that some tryptophan residues may be (or may be near) the iron-binding ligands in the catalytic site of protocatechuate 3,4-dioxygenase.
Apoenzymes↗
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.
Alcohol Oxidoreductases↗
Microbial oxidation of methane and methanol: isolation of methane-utilizing bacteria and characterization of a facultative methane-utilizing isolate.
A methane-utilizing organism capable of growth both on methane and on more complex organic substrates as a sole source of carbon and energy, has been isolated and studied in detail. Suspensions of methane-grown cells of this organism oxidized C-1 compounds (methane, methanol, formaldehyde, formate); hydrocarbons (ethane, propane); primary alcohols (ethanol, propanol); primary aldehydes (acetaldehyde, propionaldehyde); alkenes (ethylene, propylene); dimethylether; and organic acids (acetate, malate, succinate, isocitrate). Suspensions of methanol-or succinate-grown cells did not oxidize methane, ethane, propane, ethylene, propylene, or dimethylether, suggesting that the enzymatic systems required for oxidation of these substrates are induced only during growth on methane. Extracts of methane-grown cells contained a particulate reduced nicotinamide adenine dinucleotide-dependent methane monooxygenase activity. Oxidation of methanol, formaldehyde, and primary alcohols was catalyzed by a phenazine methosulfate-linked, ammonium ion-requiring methanol dehydrogenase. Oxidation of primary aldehydes was catalyzed by a phenazine methosulfate-linked, ammonium ion-independent aldehyde dehydrogenase. Formate was oxidized by a nicotinamide adenine dinucleotide-specific formate dehydrogenase. Extracts of methane-grown, but not succinate-grown, cells contained the key enzymes of the serine pathway, hydroxypyruvate reductase and malate lyase, indicating that the enzymes of C-1 assimilation are induced only during growth on C-1 compounds. Glucose-6-phosphate dehydrogenase was induced during growth on glucose. Extracts of methane-grown cells contained low levels of enzymes of the tricarboxylic acid cycle, including alpha-keto glutarate dehydrogenase, relative to the levels found during growth on succinate.
Alcohol Oxidoreductases↗
Immunological properties of protocatechuate 3, 4-dioxygenase isofunctional enzymes.
Antiserum prepared against protocatechuate 3, 4-dioxygenase from Pseudomonas aeruginosa forms precipitin bands without spurs with isofunctional enzymes from different strains of the fluorescent pseudomonads on immunodiffusion plates. Catalytic activity of the isofunctional enzymes was inhibited by an immunoglobulin fraction prepared against the enzyme from organisms of the same genus and not from different genera.
Cell-Free System↗
Inhibition of dimethyl ether and methane oxidation in Methylococcus capsulatus and Methylosinus trichosporium.
Metal-chelating or -binding agents inhibited the oxidation of dimethyl ether and methane, but not methanol, by cell suspensions of Methylococcus capsulatus and Methylosinus trichosporium. Evidence suggests that the involvement of metal-containing enzymatic systems in the initial step of oxidation of dimethyl ether and methane.
2,2'-Dipyridyl↗
Catechol 1,2-dioxygenase from Acinetobacter calcoaceticus: purification and properties.
Procedures for the purification of catechol 1,2-dioxygenase from extracts of Acinetobacter calcoaceticus strain ADP-96 are described. The purified enzyme was homogeneous as judged by ultracentrifugation and acrylamide gel electrophoresis. The enzyme contained 2 g-atoms of iron per mol of protein. The enzyme had a broad substrate specificity and catalyzed the oxidation of catechol, 4-methylcatechol, 3-methylcatechol, and 3-isopropyl catechol. The activity of the enzyme was inhibited by heavy metals, sulfhydryl inhibitors, and substrate analogues. The molecular weight of the enzyme was 85,000 as estimated by filtration on Bio-Gel agarose and 81,000 as estimated by sedimentation equilibrium analysis. The subunit size determined by sodium dodecyl sulfate-gel electrophoresis was 40,000. The amino terminal amino acid was methionine. The amino acid composition and spectral properties of 1,2-dioxygenase are also presented. Antisera prepared against the purified enzyme cross-reacted and inhibited enzyme activity in crude extracts from the other strain of A. calcoaceticus, but failed to cross-react and inhibit isofunctional enzyme from organisms of the genera Pseudomonas, Alcaligenes, and Nocardia.
Acinetobacter↗
Circular dichroism of holo- and apoprotocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa.
Circular dichroism studies have been carried out on both apo- and holoprotocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa, in the absence and presence of competitive inhibitors, protocatechualdehyde and 4-nitrocatechol. The apo- and holoenzyme showed identical spectra in the ultraviolet region between 200 and 250 nm (peptide back bone region), but the low intensity negative bands at 330 and 480 nm of the holoenzyme were completely absent in the apoenzyme. On the side chain region, the positive ellipticity peaks of the holoenzyme change into a lower intensity and broader band indicating the participation of aromatic amino acid residues in the primary binding of iron ion. Under anaerobic conditions, spectral changes were evident in the side chain region for the binary complexes of both the holo- and the apoenzyme with protocatechuate. The presence of iron in the holoenzyme results in an increase in positive ellipticity between 290 and 320 nm. Either with or without the iron, the enzyme protein binds protocatechuate and has a greater positive circular dichroism increase at 240-260 nm. CD difference spectra indicate that the modes of binding to form the binary complexes of holo- or apoenzyme with either substrates or competitive inhibitors are different. The bound iron ion stimulates binding. Spectral changes of the holoenzyme in the aromatic region were also observed in different pH environments of lower enzymatic activity. It is still not established whether these aromatic residues play an active or passive role in the binding of iron and/or substrates and inhibitors.
Apoenzymes↗
Circular dichroism of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa.
The circular dichroism spectrum of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa has been examined in the absence of the substrates, protocatechuic acid and O-2 and in the presence of the competitive inhibitors, protocatechualdehyde and p-hydroxybenzoic acid. The native enzyme has a low alpha-helical content (less than 1%) and exhibits several positive ellipticity bands between 250 and 300 nm (255,269,275 and 292 nm) and two, low intensity, negative bands at 330 and 480 nm. In the presence of protocatechuic acid and the absence of O-2, spectral changes are evident in the side chain and visible regions. There is a shift in the aromatic-region maximum from 275 to 267 and in the visible region from 330 to 348 and from 480 to 555 nm. No spectral changes are observed upon the removal or addition of only O-2. Different spectral changes in both the side chain and visible regions are observed in the enzyme with the two competitive inhibitors under either aerobic or anaerobic conditions. The spectral changes observed in the side chain region suggest the possible participation of aromatic residues in the binding process, but it is not yet established as to whether these residues play an active or passive role in binding and/or catalysis.
Aldehydes↗