Aliphatic alcohol dehydrogenase from potato tubers

Potato tubers are shown to contain at least 3 alcohol dehydrogenases, one active with NAD and aliphatic alcohols, one active with NADP and terpene alcohols and one active with NADP and aromatic alcohols. The purification of the aliphatic alcohol dehydrogenase is described and its activity with a wide range of substrates is reported. On the basis of substrate specificity, the enzyme is shown to resemble yeast alcohol dehydrogenase rather than liver alcohol dehydrogenase. The enzyme shows high activity with and high affinity for ethanol, activity and affinity decline as the chain length is increased from ethanol to butanol, but a further increase in chain length leads to increased affinity for the alcohol. The physiological significance of the results is briefly discussed.     

A novel alcohol dehydrogenase present in Rhodomicrobium vannielii

Summary An alcohol dehydrogenase specific for NADP as coenzyme and with a pH optimum of 10.2 has been partially purified from the photosynthetic bacterium, Rhodomicrobium vannielii. With the exception of methan-1-ol, primary straight chain alcohols up to eight carbon atoms were active, highest rates being obtained with butan-1-ol. Substrate specificity was examined by both enzymic rate determination and Km value measurement. The alcohol dehydrogenase described was constitutive.     

Characterization of a Saccharomyces cerevisiae NADP(H)-dependent alcohol dehydrogenase (ADHVII), a member of the cinnamyl alcohol dehydrogenase family.

A new NADP(H)-dependent alcohol dehydrogenase (the YCR105W gene product, ADHVII) has been identified in Saccharomyces cerevisiae. The enzyme has been purified to homogeneity and found to be a homodimer of 40 kDa subunits and a pI of 6.2-6.4. ADHVII shows a broad substrate specificity similar to the recently characterized ADHVI (64% identity), although they show some differences in kinetic properties. ADHVI and ADHVII are the only members of the cinnamyl alcohol dehydrogenase family in yeast. Simultaneous deletion of ADH6 and ADH7 was not lethal for the yeast. Both enzymes could participate in the synthesis of fusel alcohols, ligninolysis and NADP(H) homeostasis.     

The microbial oxidation of methanol. The alcohol dehydrogenase of pseudomonas sp. M27

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.     

Characterization of cinnamyl alcohol dehydrogenase of Helicobacter pylori. An aldehyde dismutating enzyme

Cinnamyl alcohol dehydrogenases (CAD; 1.1.1.195) catalyse the reversible conversion of p-hydroxycinnamaldehydes to their corresponding alcohols, leading to the biosynthesis of lignin in plants. Outside of plants their role is less defined. The gene for cinnamyl alcohol dehydrogenase from Helicobacter pylori (HpCAD) was cloned in Escherichia coli and the recombinant enzyme characterized for substrate specificity. The enzyme is a monomer of 42.5 kDa found predominantly in the cytosol of the bacterium. It is specific for NADP(H) as cofactor and has a broad substrate specificity for alcohol and aldehyde substrates. Its substrate specificity is similar to the well-characterized plant enzymes. High substrate inhibition was observed and a mechanism of competitive inhibition proposed. The enzyme was found to be capable of catalysing the dismutation of benzaldehyde to benzyl alcohol and benzoic acid. This dismutation reaction has not been shown previously for this class of alcohol dehydrogenase and provides the bacterium with a means of reducing aldehyde concentration within the cell.     

Constitutive NADP-dependent alcohol dehydrogenase ofAcinetobacter sp. strain HO1-N

An NADP-dependent alcohol dehydrogenase was purified to homogeneity fromAcinetobacter sp. strain HO1-N. The enzyme appears to be a tetramer of sub-unit M_r 40,600, and it has kinetic and other properties almost identical to those of an enzyme previously isolated fromAcinetobacter calcoaceticus strain NCIB 8250. The alcohol dehydrogenases from both of these strains ofAcinetobacter oxidized primary alcohols. The highestk _cat(app) values were with alcohols containing from four to eight carbon atoms; there was activity up to tetradecan-l-ol, although it was a poor substrate, but there was not measurable activity with hexadecan-l-ol. The highest specificity constant was found with hexan-l-ol as substrate when the messurements were made in the absence of dioxan, and with decan-l-ol as substrate when assayed in the presence of dioxan. It seems unlikely that this enzyme is involved in the metabolism of wax esters or of long-chain alkanes.     

Synergism between coenzyme and alcohol binding to liver alcohol dehydrogenase

Heterotropic cooperativity effects in the binding of alcohols and NAD+ or NADH to liver alcohol dehydrogenase have been examined by equilibrium measurements and stopped-flow kinetic studies. Equilibrium data are reported for benzyl alcohol, 2-chloroethanol, 2,2-dichloroethanol, and trifluoroethanol binding to free enzyme over the pH range 6-10. Binary-complex formation between enzyme and alcohols leads to inner-sphere coordination of the alcohol to catalytic zinc and shows a pH dependence reflecting the ionization states of zinc-bound water and the zinc-bound alcohol. The affinity of the binding protonation state of the enzyme for unionized alcohols increases approximately by a factor of 10 on complex formation between enzyme and NAD+ or NADH. The rate and kinetic cooperativity with coenzyme binding of the alcohol association step indicates that enzyme-bound alcohols participate in hydrogen bonding interactions which affect the rates of alcohol and coenzyme equilibration with the enzyme without providing any pronounced contribution to the net energetics of alcohol binding. The pKa values determined for alcohol deprotonation at the binary-complex level are linearly dependent on those of the free alcohols, and can be readily reconciled with the pKa values attributed to ionization of zinc-bound water. Alcohol coordination to catalytic zinc provides a major contribution to the pKa shift which ensures that the substrate is bound predominantly as an alcoholate ion in the catalytically productive ternary complex at physiological pH. The additional pKa shift contributed by NAD+ binding is less pronounced, but may be of particular mechanistic interest since it increases the acidity of zinc-bound alcohols relatively to that of zinc-bound water.     

Biochemical properties of alcohol dehydrogenase from Drosophila lebanonensis.

Purified Drosophila lebanonensis alcohol dehydrogenase (Adh) revealed one enzymically active zone in starch gel electrophoresis at pH 8.5. This zone was located on the cathode side of the origin. Incubation of D. lebanonensis Adh with NAD+ and acetone altered the electrophoretic pattern to more anodal migrating zones. D. lebanonensis Adh has an Mr of 56,000, a subunit of Mr of 28 000 and is a dimer with two active sites per enzyme molecule. This agrees with a polypeptide chain of 247 residues. Metal analysis by plasma emission spectroscopy indicated that this insect alcohol dehydrogenase is not a metalloenzyme. In studies of the substrate specificity and stereospecificity, D. lebanonensis Adh was more active with secondary than with primary alcohols. Both alkyl groups in the secondary alcohols interacted hydrophobically with the alcohol binding region of the active site. The catalytic centre activity for propan-2-ol was 7.4 s-1 and the maximum velocity of most secondary alcohols was approximately the same and indicative of rate-limiting enzyme-coenzyme dissociation. For primary alcohols the maximum velocity varied and was much lower than for secondary alcohols. The catalytic centre activity for ethanol was 2.4 s-1. With [2H6]ethanol a primary kinetic 2H isotope effect of 2.8 indicated that the interconversion of the ternary complexes was rate-limiting. Pyrazole was an ethanol-competitive inhibitor of the enzyme. The difference spectra of the enzyme-NAD+-pyrazole complex gave an absorption peak at 305 nm with epsilon 305 14.5 X 10(3) M-1 X cm-1. Concentrations and amounts of active enzyme can thus be determined. A kinetic rate assay to determine the concentration of enzyme active sites is also presented. This has been developed from active site concentrations established by titration at 305 nm of the enzyme and pyrazole with NAD+. In contrast with the amino acid composition, which indicated that D. lebanonensis Adh and the D. melanogaster alleloenzymes were not closely related, the enzymological studies showed that their active sites were similar although differing markedly from those of zinc alcohol dehydrogenases.     

Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD+

A new, acyclic NAD-analog, acycloNAD⁺ has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD⁺ with a redox potential of −324 mV and a 341 nm λ_max for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD⁺ by primary alcohols. With HLADH 1-butanol has the highest V_max at 49% that of β-NAD⁺. The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon–hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD⁺. In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD⁺ by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD⁺ has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases.     

Occurrence of a novel NADP(+)-linked alcohol dehydrogenase in Euglena gracilis

An NADP(+)-dependent alcohol dehydrogenase was found in Euglena gracilis Z grown on 1-hexanol, while it was detected at low activity in cells grown on ethanol or glucose as a carbon source, indicating that the enzyme is induced by the addition of 1-hexanol into the medium as a carbon source. This enzyme was extremely unstable, even at 4 degrees C, unless 20% ethylene glycol was added. The optimal pH was 8.8-9.0 for oxidation reaction. The apparent K(m) values for 1-hexanol and NADP(+) were found to be 6.79 mM and 46.7 microM for this enzyme, respectively. The substrate specificity of this enzyme was very different from that of already purified NAD(+)-specific ethanol dehydrogenase by showing the highest activity with 1-hexanol as a substrate, followed by 1-pentanol and 1-butanol, and there was very little activity with ethanol and 1-propanol. This enzyme was active towards the primary alcohols but not secondary alcohols. Accordingly, since the NADP(+)-specific enzyme was separated on DEAE cellulose column, Euglena was confirmed to contain a novel enzyme to be active towards middle and long-chain length of fatty alcohols.     

Characterization of two isozymes of coniferyl alcohol dehydrogenase from Streptomyces sp. NL15-2K

We purified two isozymes of coniferyl alcohol dehydrogenase (CADH I and II) to homogeneity from cell-free extracts of Streptomyces sp. NL15-2K. The apparent molecular masses of CADH I and II were determined to be 143 kDa and 151 kDa respectively by gel filtration, whereas their subunit molecular masses were determined to be 35,782.2 Da and 37,597.7 Da respectively by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Thus, it is probable that both isozymes are tetramers. The optimum pH and temperature for coniferyl alcohol dehydrogenase activity were pH 9.5 and 45 °C for CADH I and pH 8.5 and 40 °C for CADH II. CADH I oxidized various aromatic alcohols and allyl alcohol, and was most efficient on cinnamyl alcohol, whereas CADH II exhibited high substrate specificity for coniferyl alcohol, and showed no activity as to the other alcohols, except for cinnamyl alcohol and 3-(4-hydroxy-3-methoxyphenyl)-1-propanol. In the presence of NADH, CADH I and II reduced cinnamaldehyde and coniferyl aldehyde respectively to the corresponding alcohols.     

Purification and characterization of membrane-bound quinoprotein cyclic alcohol dehydrogenase from Gluconobacter frateurii CHM 9

A quinoprotein catalyzing oxidation of cyclic alcohols was found in the membrane fraction for the first time, after extensive screening among aerobic bacteria. Gluconobacter frateurii CHM 9 was finally selected in this study. The enzyme tentatively named membrane-bound cyclic alcohol dehydrogenase (MCAD) was found to occur specifically in the membrane fraction, and pyrroloquinoline quinone (PQQ) was functional as the primary coenzyme in the enzyme activity. MCAD catalyzed only oxidation reaction of cyclic alcohols irreversibly to corresponding ketones. Unlike already known cytosolic NAD(P)H-dependent alcohol-aldehyde or alcohol-ketone oxidoreductases, MCAD was unable to catalyze the reverse reaction of cyclic ketones or aldehydes to cyclic alcohols. MCAD was solubilized and purified from the membrane fraction of the organism to homogeneity. Differential solubilization to eliminate the predominant quinoprotein alcohol dehydrogenase (ADH), and the subsequent two steps of column chromatographies, brought MCAD to homogeneity. Purified MCAD had a molecular mass of 83 kDa by SDS-PAGE. Substrate specificity showed that MCAD was an enzyme oxidizing a wide variety of cyclic alcohols. Some minor enzyme activity was found with aliphatic secondary alcohols and sugar alcohols, but not primary alcohols, differentiating MCAD from quinoprotein ADH. NAD-dependent cytosolic cyclic alcohol dehydrogenase (CCAD) in the same organism was crystallized and its catalytic and physicochemical properties were characterized. Judging from the catalytic properties of CCAD, it was apparent that CCAD was distinct from MCAD in many respects and seemed to make no contributions to cyclic alcohol oxidation.     

Characterization of a nonhepatic alcohol dehydrogenase from rat hepatocellular carcinoma and stomach.

Ethanol oxidation by the soluble fraction of a rat hepatoma was compared to that of the liver. Ethanol oxidation by the hepatoma was NAD⁺-dependent and sensitive to pyrazole, suggesting the presence of alcohol dehydrogenase. At low concentrations of ethanol (10.8 mm) the alcohol dehydrogenase activities of hepatoma and liver supernatant fractions were comparable. When the concentration of ethanol was raised to 108 mm, the activity of the liver enzyme decreased, whereas the activity in hepatoma supernatant fractions was strikingly elevated. m-Nitrobenzaldehyde-reducing activity was also conspicuously higher in hepatoma supernatant fractions. By contrast the ability to metabolize steroids and cyclohexanone was less than that in supernatant fractions of the liver.Electrophoresis of the liver supernatant fractions on ionagar at pH 7.0 revealed only one component that oxidized ethanol. On the other hand, hepatoma supernatant fractions contained two components with alcohol dehydrogenase activity; one with the same electrophoretic mobility as the liver enzyme, the other showing a slower rate of migration. The latter component, which is absent in the liver, is referred to as hepatoma alcohol dehydrogenase. By electrophoresis on starch gels at pH 8.5, it could be demonstrated that the liver and hepatoma enzymes moved in opposite directions.The liver and hepatoma enzymes differ in electrophoretic mobility, susceptibility to heat treatment, pH activity optimum and some catalytic properties. The substrate specificity of the hepatoma enzyme is narrower than that of liver alcohol dehydrogenase; cyclohexanone or 3β-hydroxysteroids of A/B cis configuration and the corresponding 3-ketones are not substrates for the hepatoma enzyme. The overall substrate specificity characteristics are, however, similar to those of the liver enzyme in that the effectiveness of substrates increases with an increase in chain length and introduction of unsaturation or an aromatic group. Both liver and hepatoma alcohol dehydrogenase cross-react with antibody to horse liver alcohol dehydrogenase EE. The Michaelis constant for ethanol with the hepatoma enzyme is 223 mm, compared to 0.3 mm for liver alcohol dehydrogenase; at 1.0 m ethanol the hepatoma enzyme is not fully saturated with substrate. The Michaelis constant for 2-hexene-1-ol is 0.3 mm, indicating that the hepatoma enzyme is better suited for dehydrogenation of longer chain alcohols. Stomach alcohol dehydrogenase has kinetic properties comparable to those of the hepatoma enzyme, as well as similar electrophoretic mobility. The hepatoma enzyme can be detected in the serum of rats bearing hepatomas.     

Isolation ofCaenorhabditis elegans mutants lacking alcohol dehydrogenase activity

Alcohol dehydrogenase (ADH) and the genes encoding this enzyme have been studied intensively in a broad range of organisms. Little, however, has been reported on ADH in the free-living nematodeCaenorhabiditis elegans. Extracts of wild-typeC. elegans contain ADH activity and display a single band of activity on a native polyacrylamide gel. Reaction rate for alcohol oxidation is more rapid with higher molecular weight alcohols as substrate than with ethanol. Primary alcohols are preferred to secondary alcohols.C. elegans is sensitive to allyl alcohol, a compound that has been used to select for ADH-null mutants of several organisms. Allyl alcohol-resistant mutant strains were selected from ethylmethanesulfonate (EMS)-mutagenized nematode populations. ADH activity was measured in extracts from eight of these strains and was found to be low or nondetectable. These results form a basis for molecular and genetic characterization of ADH expression inC. elegans.     

Rat liver alcohol dehydrogenase. Purification and properties

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.     

Subunit composition of alcohol dehydrogenase from Thea sinensis seeds and its substrate specificity for monoterpenes

Alcohol dehydrogenase (alcohol: NAD oxidoreductase, E.C. 1.1.1.1.) was purified from Thea sinensis seeds. Its M.W was 95000 and it was composed of two homogeneous subunits with MWs of 47000. The dissociation into subunits was caused by o-phenanthroline. Substrate specificity for monoterpene alcohols and aldehydes is discussed.     

Rabbit liver alcohol dehydrogenase: purification and properties

Alcohol dehydrogenase [EC 1.1.1.1] was purified to homogeneity from rabbit liver by water extraction, DEAE-cellulose treatment, affinity chromatography on 5'-AMP-Sepharose and gel filtration on Sephadex G-150 using dithiothreitol as a stabilizer. The purified enzyme has an estimated molecular weight of 72,000 and consists of two subunits with a molecular weight of about 36,000 each. The enzyme contains 4 g-atoms of zinc and 18 sulfhydryl groups per mol of protein and exhibits maximal activity at pH 10.8, with a second maximum at pH 7.5. The apparent Km values for ethanol and NAD+ are 0.45 mM and 53.19 microM, respectively, at pH 10.8 and 3.33 mM and 6.94 microM, respectively, at pH 7.5. The enzyme oxidizes ethanol most readily among the aliphatic alcohols studied and has very low substrate specificity for methanol. Among steroid alcohols, 5 beta-androstan-3 beta-ol-17-one serves as a substrate for the enzyme. Pyrazole and 4-methylpyrazole (which are well known alcohol dehydrogenase inhibitors), sulfhydryl reagents, heavy metal ions and metal-chelating agents inactivate the enzyme.     

Human alcohol dehydrogenase: dependence of secondary alcohol oxidation on the amino acids at positions 93 and 94.

The human liver alpha alpha and beta 1 beta 1 isoenzymes are straight-chain alcohol dehydrogenases with different efficiencies toward secondary alcohols. Two of the 24 amino acid substitutions in alpha alpha (A for F93 and I for T94) were made by site-directed mutagenesis of beta 1 beta 1 and the substrate specificity of beta 93A94I was examined. The Vmax/KM values of beta 93A94I for secondary alcohols (especially R enantiomers) are similar to that of alpha alpha and as much as 4000-fold greater than beta 1 beta 1, but the dependences of Vmax/KM on primary alcohol chain length are similar to beta 1 beta 1, but not alpha alpha. Thus, the substitutions of A for F93 and I for T94 in beta 1 beta 1 account for the increased efficiency towards secondary alcohols and stereoselectivity for enantiomeric alcohols, but not for the effects of chain length on the Vmax/KM for primary alcohols seen with alpha alpha.     

The involvement of alcohol dehydrogenase and aldehyde dehydrogenase in alcohol/aldehyde metabolism in Drosophila melanogaster

In this study we have examined the roles of alcohol dehydrogenase, aldehyde oxidase, and aldehyde dehydrogenase in the adaptation of Drosophila melanogaster to alcohol environments. Fifteen strains were characterized for genetic variation at the above loci by protein electrophoresis. Levels of in vitro enzyme activity were also determined. The strains examined showed considerable variation in enzyme activity for all three gene-enzyme systems. Each enzyme was also characterized for coenzyme requirements, effect of inhibitors, subcellular location, and tissue specific expression. A subset of the strains was chosen to assess the physiological role of each gene-enzyme system in alcohol and aldehyde metabolism. These strains were characterized for both the ability to utilize alcohols and aldehydes as carbon sources as well as the capacity to detoxify such substrates. The results of the above analyses demonstrate the importance of both alcohol dehydrogenase and aldehyde dehydrogenase in the in vivo metabolism of alcohols and aldehydes.     

Genetic and biochemical characterization of a short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

The gene encoding a short-chain alcohol dehydrogenase, AdhA, has been identified in the hyperthermophilic archaeon Pyrococcus furiosus, as part of an operon that encodes two glycosyl hydrolases, the beta-glucosidase CelB and the endoglucanase LamA. The adhA gene was functionally expressed in Escherichia coli, and AdhA was subsequently purified to homogeneity. The quaternary structure of AdhA is a dimer of identical 26-kDa subunits. AdhA is an NADPH-dependent oxidoreductase that converts alcohols to the corresponding aldehydes/ketones and vice versa, with a rather broad substrate specificity. Maximal specific activities were observed with 2-pentanol (46 U x mg(-1)) and pyruvaldehyde (32 U x mg(-1)) in the oxidative and reductive reaction, respectively. AdhA has an optimal activity at 90 degrees C, at which temperature it has a half life of 22.5 h. The expression of the adhA gene in P. furiosus was demonstrated by activity measurements and immunoblot analysis of cell extracts. A role of this novel type of archaeal alcohol dehydrogenase in carbohydrate fermentation is discussed.