Thermodynamic behavior and conformational changes of alcohol oxidase from yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis>

We studied the influence of heating, ethanol, and sodium azide on the structural and conformational changes in the alcohol oxidase from yeast Hansenula polymorpha. An increase in fluorescence of the alcohol oxidase cofactor, flavine adenine dinucleotide, was revealed upon heating to 60°C while the enzymatic activity of alcohol oxidase was preserved. Differential scanning microcalorimetry revealed that ethanol stabilized the protein structure and increased the melting temperature. Based on the data of circular dichroism, we concluded that the percentage of helicity in the secondary structure of alcohol oxidase was not influenced by ethanol or sodium azide, but ethanol significantly modified the circular dichroism spectrum associated with the tertiary structure of alcohol oxidase.     

Reconstitution of the ethanol oxidase respiratory chain in membranes of quinoprotein alcohol dehydrogenase-deficient Gluconobacter suboxydans subsp. alpha strains.

The ethanol oxidase respiratory chain of Gluconobacter suboxydan was characterized by using G. suboxydans subsp. alpha, a variant species of G. suboxydans incapable of oxidizing ethanol. The membranes of G. suboxydans subsp. alpha exhibited neither alcohol dehydrogenase, ethanol oxidase, nor glucose-ferricyanide oxidoreductase activity. Furthermore, the respiratory chain of the organism exhibited an extremely diminished amount of cytochrome c and an increased sensitivity of the respiratory activity for cyanide or azide when compared with G. suboxydans. The first-subunit quinohemoprotein and the second-subunit cytochrome c of alcohol dehydrogenase complex in the membranes of G. suboxydans subsp. alpha were shown to be reduced and deficient, respectively, by using heme-staining and immunoblotting methods. Ethanol oxidase activity, lacking in G. suboxydans subsp. alpha, was entirely restored by reconstituting alcohol dehydrogenase purified from G. suboxydans to the membranes of G. suboxydans subsp. alpha; this also led to restoration of the cyanide or azide insensitivity and the glucose-ferricyanide oxidoreductase activity in the respiratory chain without affecting other respiratory activities such as glucose and sorbitol oxidases. Ethanol oxidase activity was also reconstituted with only the second-subunit cytochrome c of the enzyme complex. The results indicate that the second-subunit cytochrome c of the alcohol dehydrogenase complex is essential in ethanol oxidase respiratory chain and may be involved in the cyanide- or azide-insensitive respiratory chain bypass of G. suboxydans.     

beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: structure and activity in the presence of alcohols.

beta-Glycosidase from the extreme thermophilic archaeon Sulfolobus solfataricus is a tetrameric protein with a molecular mass of 240 kDa, stable in the presence of detergents, and with a maximal activity at temperatures above 95 degrees C. Understanding the structure-activity relationships of the enzyme under different conditions is of fundamental importance for both theoretical and applicative purposes. In this paper we report the effect of methanol, ethanol, 1-propanol, and 1-butanol on the activity of S. solfataricus beta-glycosidase expressed in Escherichia coli. The alcohols stimulated the enzyme activity, with 1-butanol producing its maximum effect at a lower concentration than the other alcohols. The structure of the enzyme was studied in the presence of 1-butanol by circular dichroism, and Fourier-transform infrared and fluorescence spectroscopies. Circular dichroism and steady-state fluorescence measurements revealed that at low temperatures the presence of the alcohol produced no significant changes in the tertiary structure of the enzyme. However, time-resolved fluorescence data showed that the alcohol modifies the protein microenvironment, leading to a more flexible enzyme structure, which is probably responsible for the enhanced enzymatic activity.     

Purification and characterization of alcohol oxidase from a genetically constructed over-producing strain of the methylotrophic yeast Hansenula polymorpha

Alcohol oxidase (AOX) has been purified 8-fold from a genetically constructed over-producing strain of the methylotrophic yeast Hansenula polymorpha C-105 (gcr1 catX) with impaired glucose-induced catabolite repression and completely devoid of catalase. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis and HPLC. Some physicochemical and biochemical properties of AOX were studied in detail: molecular weight (approximately 620 kD), isoelectric point (pI 6.1), and UV-VIS, circular dichroism (CD), and fluorescence spectra. The content of different secondary structure motifs of the enzyme has been calculated from the CD spectra using a computer program. It was found that the native protein contains about 50% alpha-helix, 25% beta-sheet, and about 20% random structures. The kinetic parameters for different substrates, such as methanol, ethanol, and formaldehyde, were measured using a Clark oxygen electrode. The rate of enzymatic oxidation of formaldehyde by alcohol oxidase from H. polymorpha is only twice lower compared to the best substrate of the enzyme, methanol.     

Interaction stabilizing tertiary structure of bacteriorhodopsin studied by denaturation experiments

The structural stability of bacteriorhodopsin was studied by denaturation experiments, using aliphatic alcohol as denaturants. The disappearance of a positive peak at 285 nm of the circular dichroism spectra, the change in the intrinsic fluorescence decay time, and the decrease of the regeneration activity bacteriorhodopsin indicated the denaturation of the tertiary structure of this protein at a methanol concentration of about 3 M. The circular dichroism band at 222 nm was unchanged by the denaturation. It was concluded that the alcohol-denatured state in water was similar to the molten globule state of soluble proteins, in which only the tertiary structure was destroyed. Solvent substitution from water to hexane did not cause denaturation of bacteriorhodopsin. However, further addition of alcohol destroyed the secondary as well as the tertiary structures. Comparing the alcohol effects on bacteriorhodopsin in water to that in hexane, the dominant interactions for the structure formation of this protein could be revealed: the hydrophobic interaction that arose from the structure of water is essential for the stability of membrane spanning helices, while the interaction which binds the helices is polar in nature. © 1995 Wiley-Liss, Inc.     

Chronic In Vivo Sodium Azide Infusion Induces Selective and Stable Inhibition of Cytochrome c Oxidase

Abstract: The effect of chronic subcutaneous infusion of sodium azide on the activity of mitochondrial respiratory chain enzymes was investigated in Sprague-Dawley rats. Treatment with ∼1 mg/kg/h sodium azide induced chronic, partial inhibition of cytochrome c oxidase, whereas the activities of respiratory complexes I and III were not significantly affected. The inhibition of cytochrome c oxidase was evident by 7 days after infusion began, and the effect was stable for at least 3 weeks. The selectivity of azide for cytochrome c oxidase is discussed in the context of other findings of azide effects on enzymes. The results of the present study indicate that the sodium azide infusion paradigm described here provides a useful tool for the evaluation of selective and stable cytochrome oxidase inhibition in vivo.     

Effects of some alcohols on the conformation of mitochondrial H+-ATPase complex and F1-ATPase from pig heart

The conformations of the H+-ATPase complex and F1-ATPase in low concentrations of methanol, ethanol, n-propanol, iso-propanol and t-butanol were studied by circular dichroism. For F1-ATPase, all but methanol first increased and then decreased the circular dichroism magnitude of helical bands as the alcohol concentration was increased. With ethanol, n-propanol, iso-propanol and t-butanol, the alpha-helix content reached a maximum at about 5% alcohol and began to decrease at 10%. The content of beta-sheet showed the opposite effect, reaching a minimum at 5% and increasing slightly at higher concentrations. None of the alcohols studied had a significant effect on the conformation of the H+-ATPase complex. This difference implies that the alcohols had a greater effect on free F1-ATPase than on the membrane-bound F1-ATPase. The hydrophobic protein F0 and the membrane lipids in the H+-ATPase complex may stabilize and protect F1 from the effects of the alcohols.     

Crystallization of alcohol oxidase from Pichia pastoris

Crystals of alcohol oxidase purified from Pichia pastoris were grown in microdialysis buttons in a solution of polyethylene glycol, sodium chloride and sodium azide. The crystals were stratified along the major axis and up to 3 mm in length. X-ray diffraction experiments indicated a space group of P2(1) and unit cell dimensions of a = 157.3 A, b = 171.5 A and c = 231.6 A. Crystals diffract to beyond 2.7 A and are suitable for X-ray structure analysis.     

Effect of alcohols on the structure of caseins: circular dichroism studies of κ-casein A

The alcohols methanol, ethanol, propan-1-ol and 2,2,2-trifluoroethanol were added to aqueous solution of κ-casein A. Far u.v. circular dichroism spectroscopy revealed a greater proportion of -helix structure in the presence of alcohol; the effectiveness of the alcohol in causing this structural change increased in the order methanol to 2,2,2-trifluoroethanol. No alterations in secondary or tertiary structure were found in κ-casein A on reduction and S-carboxymethylation using near and far u.v.c.d. spectroscopy. The isolated macropeptide section of κ-casein was less sensitive to the addition of alcohols; it appeared that the linkage of macropeptide to the para-κ-casein section affected the case with which solvent induced conformational changes occurred. The conformational changes found in the presence of alcohols could not be correlated simply with the changes in the dielectric constant or with the alcohol-induced collapse of the hairy macropeptide layer of casein micelles or ther precipitation.     

Effects of surfactant, salt and solvent on the structure and activity of adenosine deaminase: molecular dynamic and spectrophotometric studies

Effects of sodium dodecyl sulfate, dodecyltrimethylammonium bromide, sodium chloride, sodium sulfate, methanol and ethanol, on the structure and activity of adenosine deaminase (ADA) were investigated by UV-Vis, circular dichroism spectrophotometry and molecular dynamics (MDs) studies. Relative activity, experimental and computational helix content, total accessible surface area (ASA) and exposed charged surface area (ECSA) were obtained. The relative activity of ADA in the absence and the presence of denaturants were compared with structural results. It was shown that an increase in the surface area and a decrease in the amount of helicity are associated with a decrease in the activity of ADA.     

Hepatic ethanol metabolism: Respective roles of alcohol dehydrogenase,the microsomal ethanol-oxidizing system,and catalase

The respective role of alcohol dehydrogenase, of the microsomal ethanol-oxidizing system, and of catalase in ethanol metabolism was assessed quantitatively in liver slices using various inhibitors and ethanol at a final concentration of 50 mm. Pyrazole (2 mm) virtually abolished cytosolic alcohol dehydrogenase activity but inhibited ethanol metabolism in liver slices by only 50–60%. The residual pyrazole-insensitive ethanol oxidation in liver slices remained unaffected by in vitro addition of the catalase inhibitor sodium azide (1 mm). At this concentration, sodium azide completely abolished catalatic activity of catalase in liver homogenate as well as peroxidatic activity of catalase in liver slices in the presence of dl-alanine. Similarly, in vivo administration of 3-amino-1,2,4-triazole, a compound which inhibits the activity of catalase but not that of the microsomal ethanol-oxidizing system, failed to decrease both the overall rates of ethanol oxidation and the activity of the pyrazole-insensitive pathway. Finally, butanol, a substrate and inhibitor of the microsomal ethanol-oxidizing system but not of catalase-H₂O₂, significantly decreased the pyrazole-insensitive ethanol metabolism in liver slices. These results indicate that alcohol dehydrogenase is responsible for half or more of ethanol metabolism by liver slices and that the microsomal ethanol-oxidizing system rather than catalase-H₂O₂ accounts for most if not all of the alcohol dehydrogenase-independent pathway.     

Active Transport of Alcohol in Corynebacterium acetophilum

The transport of alcohols was studied in Corynebacterium acetophilum, which was isolated as a strain growing well on acetate and ethanol. The transport of ethanol was found to be inducible by ethanol, n-propanol, n-butanol, and acetate, whereas transport of methanol occurred by noninducible passive diffusion. The entry of ethanol into the cells occurred against a concentration gradient and showed saturation kinetics with two K(m) values of 2.4 x 10(-5) M and 6.0 x 10(-5) M. Uptake of ethanol was inhibited by sodium azide, sodium cyanide, 2,4-dinitrophenol, and p-chloromercuribenzoate. The transport of ethanol was competitively inhibited by normal alcohols, but not by iso- or tert-alcohols. From these studies, we concluded that an inducible active alcohol transport system mediates the entry of ethanol, n-propanol, or n-butanol into the cells of C. acetophilum.     

Alcohol oxidation by Candida guilliermondii yeasts grown on hexadecanol

A number of enzyme systems involved in the first steps of hexadecane oxidation can be induced by hexadecanol, an intermediate product of hexadecane degradation. It has also been found that, in Candida guilliermondii cells and in their mitochondrial fraction, an oxidase system is induced when the cells are grown on hexadecanol. This system is similar to that in cells grown on hexadecane; it oxidises higher alcohols at a high rate and is not inhibited by the inhibitors of the man phosphorylating respiration chain. The membrane-bound alcohol dehydrogenase and aldehyde dehydrogenase activities resistant to pyrazole, an inhibitor of cytosol ethanol dehydrogenase, are induced together with the oxidase activity when the cells are grown on hexadecanol as well as on hexadecane. The oxidation of higher alcohols by whole cells is entirely inhibited by azide although their oxidation by mitochondria is resistant to the action of azide; apparently, azide inhibits the transport of alcohols into the cell.     

Acetaldehyde-mediated hepatic lipid peroxidation: role of superoxide and ferritin

Evidence in alcoholics as well as in experimental models support the role of hepatic lipid peroxidation in the pathogenesis of alcohol-induced liver injury, but the mechanism of this injury is not fully delineated. Previous studies of the metabolism of ethanol by alcohol dehydrogenase revealed iron mobilization from ferritin that was markedly stimulated by superoxide radical generation by xanthine oxidase. Peroxidation of hepatic lipid membranes (assessed as malondialdehyde production) was studied during in vitro alcohol metabolism by alcohol dehydrogenase. Peroxidation was initiated by acetaldehyde-xanthine oxidase, stimulated by ferritin, and inhibited by superoxide dismutase or chelation or iron with desferrioxamine. In conclusion, lipid peroxidation may be initiated during the metabolism of ethanol by alcohol dehydrogenase by an iron-dependent acetaldehyde-xanthine oxidase mechanism.     

Circular dichroism studies on the effects of ethanol on the conformation of alpha1-acid glycoprotein, alpha1-antitrypsin, deoxyribonuclease, pepsinogen, soybean trypsin inhibitor and unfolded ribonucleases.

The effects of 25 to 75 volume-% ethanol on conformation of human serum alpha1-acid glycoprotein, human serum alpha1-antitrypsin, pancreatic deoxyribonuclease I, porcine pepsinogen, the "Kunitz" trypsin inhibitor from soybeans, and oxidized as well as reduced and S-carboxymethylated ribonucleases were tested by the circular dichroism (CD) probe. It was found that 25 volume-% ethanol had a slight effect, whereas 50--75 vol.-% alcohol significantly altered the conformation. The tertiary structure was perturbed and the polypeptide main chain was reorganized into new conformations of higher helix and beta-structure contents than in the native state. Comparison of the various proteins showed that the degree of reorganization depended chiefly on the cross-linking of the main chain by disulfide bridges. While the unfolded ribonucleases were refolded by 25 vol.-% ethanol into ordered conformations, the native ribonuclease and alpha1-antitrypsin was more sensitive to 25 vol.-% ethanol than the conformation of alpha1-acid glycoprotein, pepsinogen, and soybean trypsin inhibitor. Almost complete restoration of the native conformation was achieved by diluting the alcohol-containing solutions with water or by dialysis against water or buffer solutions. However, the renaturation depended on the time of contact with alcohol and on the temperature at which the alcohol-containing solutions were kept.     

Activation of a flavoprotein by proteolysis

Chymotryptic digestion of brain pyridoxine-5-P oxidase brings about a 4-fold enhancement of the catalytic power (Vmax/KM) using pyridoxine-5-P as substrate in the assay mixtures. The chymotrypsin-treated enzyme is less susceptible to inhibition by pyridoxal-5-P than the native enzyme. Fragments arising from limited proteolysis were separated by affinity chromatography using P-pyridoxal-Sepharose as supporting matrix. Catalytically active fractions, eluted by pyridoxine-5-P (5mM), displayed three bands when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular masses of the three protein bands are considerably lower than 28 kDa, the molecular mass of monomeric pyridoxine-5-P oxidase. Spectroscopic studies, absorption, fluorescence, and circular dichroism revealed that the microenvironment surrounding the cofactor flavin mononucleotide is not perturbed by limited proteolysis.     

Deflavination of flavo-oxidases by nucleophilic reagents

Using spectroscopic techniques we studied the effect of the nucleophilic reagents cyanide, cyanate and thiocyanate on three flavo-oxidases namely alcohol oxidase (AO), glucose oxidase (GOX) and D-amino acid oxidase (DAOX). All three ions, added at concentrations in the mM range, caused release of the flavin adenine dinucleotide (FAD) co-factors from the enzyme molecules. In the case of AO this was accompanied by significant conformational perturbations, which was not observed for GOX and DAOX. As suggested from fluorescence, absorption and circular dichroism spectral changes at least one phenolic hydroxyl group became ionized upon FAD release from AO and a new class of Trp residues, fluorescent only in apo-AO protein, was demasked.     

Purification and properties of alcohol oxidase from Poria contigua.

1. Alcohol oxidase (alcohol:oxygen oxidoreductase) was purified 22-fold from the brown rot fungus Poria contigua. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis, and by sedimentation in an ultracentrifuge. The molecular weight was calculated to be 610000 +/- 5000 from sedimentation equilibrium experiments. Electrophoresis in sodium dodecylsulfate gels and electron microscopic analysis indicate that the enzyme is an octamer composed of eight probably identical subunits, each having a molecular weight of 79 000. The enzyme contains eight mol FAD/mol as the prosthetic group. 2. This alcohol oxidase oxidizes not only methanol but also lower primary alcohols (C2-C4), 2-propin-1-ol and formaldehyde. The apparent Km value for methanol is 0.2 mM, and that for formaldehyde 6.1 mM. Sodium azide was found to be a competitive inhibitor with respect to methanol. 3. The enzyme from the fungus Poria contigua is immunologically different from the alcohol oxidase isolated from the methanol-utilizing yeast Candida boidinii. Furthermore antiserum raised against this enzyme did not cross-react with the alcohol oxidase from the white rot fungus Polyporus obtusus.     

Conformational studies on the gramicidin A transmembrane channel in lipid micelles and liposomes

The interaction of gramicidin A with lysolecithin micelles and with lecithin liposomes is demonstrated by circular dichroism to result in several metastable conformational states. A stable state can be obtained after extensive heating when the gramicidin A was added dry or in ethanol solution to the phospholipid dispersion but the stable state is readily obtained when gramicidin A is added in a trifluoroethanol solution. The circular dichroism of the stable conformational states is characterized by negative ellipticity below 205 nm and principally by a positive 220 nm band on which is superposed a weak 230 nm band (the latter likely arising from tryptophan side chains). The stable conformational state is considered to be that of the functional transmembrane channel primarily on the basis of extensive studies on its interaction with sodium ions.     

Conformational studies on the gramicidin A transmembrane channel in lipid micelles and liposomes

The interaction of gramicidin A with lysolecithin micelles and with lecithin liposomes is demonstrated by circular dichroism to result in several metastable conformational states. A stable state can be obtained after extensive heating when the gramicidin A was added dry or in ethanol solution to the phospholipid dispersion but the stable state is readily obtained when gramicidin A is added in a trifluoroethanol solution. The circular dichroism of the stable conformational state is characterized by negative ellipticity below 205 nm and principally by a positive 220 nm band on which is superposed a weak 230 nm band (the latter likely arising from tryptophan side chains). The stable conformational state is considered to be that of the functional transmembrane channel primarily on the basis of extensive studies on its interaction with sodium ions.