Yeast ADH Research Articles

Revealing ligand binding sites and quantifying subunit variants of noncovalent protein complexes in a single native top-down FTICR MS experiment.

"Native" mass spectrometry (MS) has been proven to be increasingly useful for structural biology studies of macromolecular assemblies. Using horse liver alcohol dehydrogenase (hADH) and yeast alcohol dehydrogenase (yADH) as examples, we demonstrate that rich information can be obtained in a single native top-down MS experiment using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Beyond measuring the molecular weights of the protein complexes, isotopic mass resolution was achieved for yeast ADH tetramer (147 kDa) with an average resolving power of 412,700 at m/z 5466 in absorption mode, and the mass reflects that each subunit binds to two zinc atoms. The N-terminal 89 amino acid residues were sequenced in a top-down electron capture dissociation (ECD) experiment, along with the identifications of the zinc binding site at Cys46 and a point mutation (V58T). With the combination of various activation/dissociation techniques, including ECD, in-source dissociation (ISD), collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD), 40% of the yADH sequence was derived directly from the native tetramer complex. For hADH, native top-down ECD-MS shows that both E and S subunits are present in the hADH sample, with a relative ratio of 4:1. Native top-down ISD of the hADH dimer shows that each subunit (E and S chains) binds not only to two zinc atoms, but also the NAD/NADH ligand, with a higher NAD/NADH binding preference for the S chain relative to the E chain. In total, 32% sequence coverage was achieved for both E and S chains.

Direct electron transfer from alcohol dehydrogenase

This study employed hydrodynamic cyclic voltammetry (HCV) with a glassy carbon (GC) rotating disk electrode (RDE), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) to investigate the direct electron transfer (DET) behavior of alcohol dehydrogenase (ADH, EC 1.1.1.1) embedded or otherwise in polymyxin (PM) and adsorbed concomitantly with cofactor NAD+ or NADH, or none on GC and covered with Nafion®. The hybrid GC/PM-ADH-NAD+/Nafion electrode thus constructed (and visualized by scanning electron microscopy (SEM)) persistently bioelectrocatalyzed ethanol oxidation and performed DET involving baker's yeast ADH, GC, and ethanol—a key finding in the present study—making this system a promising anode for use in biofuel cells. A rate constant (ks) of 0.82 s−1 was obtained for this electrode at a potential scan rate (ν) of 120 mV s−1. EIS experiments, particularly those conducted after higher potential HCV scans, allowed resistance to electron hopping (Reh) between redox centers in ADH and between these centers and ethanol molecules to be estimated as 84 kΩ lower for the GC/PM-ADH-NAD+/Nafion electrode during ethanol oxidation than for bare GC in the presence of ethanol. Nuclear magnetic resonance (NMR) unequivocally confirmed acetaldehyde production from ethanol oxidation by ADH DET.

Activity of yeast alcohol dehydrogenases on benzyl alcohols and benzaldehydes: Characterization of ADH1 from Saccharomyces carlsbergensis and transition state analysis

The substrate specificities of yeast alcohol dehydrogenases I and II from Saccharomyces cerevisiae ( SceADH1 and SceADH2) and Saccharomyces carlsbergensis ( ScbADH1) were studied. For this work, the gene for the S. carlsbergensis ADH1 was cloned, sequenced and expressed. The amino acid sequence of ScbADH1 differs at four positions as compared to SceADH1, including substitutions of two glutamine residues with glutamic acid residues, and has the same sequence as the commercial yeast enzyme, which apparently is prepared from S. carlsbergensis. The electrophoretic mobilities of ScbADH1, SceADH2 and commercial ADH are similar. The kinetics and specificities of ScbADH1 and SceADH1 acting on branched, long-chain and benzyl alcohols are very similar, but the catalytic efficiency of SceADH2 is about 10–100-fold higher on these substrates. A three-dimensional structure of SceADH1 shows that the substrate binding pocket has Met-270, whereas SceADH2 has Leu-270, which allows larger substrates to bind. The reduction of a series of p-substituted benzaldehydes catalyzed by SceADH2 is significantly enhanced by electron-withdrawing groups, whereas the oxidation of p-substituted aromatic alcohols may be only slightly affected by the substituents. The substituent effects on catalysis generally reflect the effects on the equilibrium constant for the reaction, where electron-withdrawing substituents favor alcohol. The results are consistent with a transition state that is electronically similar to the alcohol, supporting previous results obtained with commercial yeast ADH.

The activity of yeast ADH I and ADH II with long-chain alcohols and diols

The activities of yeast ADH I and ADH II towards long chain alcohols and diols were studied using rather unusual conditions (1.0 M Tris pH 8.75, approximately 0.3 mg/ml enzyme and [ S]< < < K m ) where the alcohols are oxidised quantitatively in a first-order manner. Plots of the apparent first-order rate constant versus primary alcohol chain length show double peaks with similar values for ethanol and 1-decanol and relatively low values for 1-butanol through to 1-octanol. With the α,ω diols only one peak of activity was observed with 1,14-tetradecanediol, the preferred substrate, being oxidised about the same rate as ethanol. Both enzymes were essentially inactive with short-chain diols (C 2–C 8). For all of these assays normalised rates with ADH II were about threefold faster than with ADH I.

Interaction of Alcohol Dehydrogenase with tert-Butylhydroperoxide: Stimulation of the Horse Liver and Inhibition of the Yeast Enzymes

Preincubation of horse liver alcohol dehydrogenase (HLADH) with the oxidative agent, tert-butyl hydroperoxide (tBOOH) results in a twofold stimulation of the ethanol dehydrogenase activity of this enzyme. This stimulation was dependent on tBOOH concentration up to 100 mM; above this concentration tBOOH did not further stimulate ethanol oxidation by HLADH. Active-site-directed reagents and classical ADH binary complexes were used to probe the possible mechanism of this activating effect. The rate and extent of stimulation by tBOOH is strongly reduced by binary complexes with NAD+ or NADH, whose pyrophosphate groups bind to Arg-47 and Arg-369. In contrast stimulation by tBOOH was not prevented by AMP or the sulfhydryl reagents dithiothreitol and glutathione, suggesting, respectively, a lack of role for Lys-228 and sulfhydryl group oxidation in the stimulation by tBOOH. In contrast to the liver enzyme, treatment of yeast ADH (YADH) with tBOOH irreversibly inhibited its ethanol dehydrogenase activity. Inhibition of YADH by tBOOH approximated first-order rate kinetics with respect to enzyme at fixed concentrations of tBOOH between 0.5 to 300 mM. Four –SH groups per molecule of YADH were modified by tBOOH, whereas only two –SH groups were modified in HLADH. The stimulation of HLADH by tBOOH is suggested to be due to destabilization of the catalytic Zn-coordination sphere and amino acids associated with coenzyme binding in the active site, while inactivation of YADH appears to be associated with –SH group oxidation by the peroxide.

New alcohol dehydrogenases for the synthesis of chiral compounds.

The enantioselective reduction of carbonyl groups is of interest for the production of various chiral compounds such as hydroxy acids, amino acids, hydroxy esters, or alcohols. Such products have high economic value and are most interesting as additives for food and feed or as building blocks for organic synthesis. Enzymatic reactions or biotransformations with whole cells (growing or resting) for this purpose are described. Although conversions with whole cells are advantageous with respect to saving expensive isolation of the desired enzymes, the products often lack high enantiomeric excess and the process results in low time-space-yield. For the synthesis of chiral alcohols, only lab-scale syntheses with commercially available alcohol dehydrogenases have been described yet. However, most of these enzymes are of limited use for technical applications because they lack substrate specificity, stability (yeast ADH) or enantioselectivity (Thermoanaerobium brockii ADH). Furthermore, all enzymes so far described are forming (S)-alcohols. Quite recently, we found and characterized several new bacterial alcohol dehydrogenases, which are suited for the preparation of chiral alcohols as well as for hydroxy esters in technical scale. Remarkably, of all these novel ADHs the (R)-specific enzymes were found in strains of the genus Lactobacillus. Meanwhile, these new enzymes were characterized extensively. Protein data (amino acid sequence, bound cations) confirm that these catalysts are novel enzymes. (R)-specific as well as (S)-specific ADHs accept a broad variety of ketones and ketoesters as substrates. The applicability of alcohol dehydrogenases for chiral syntheses as an example for the technical use of coenzyme-dependent enzymes is demonstrated and discussed in this contribution. In particular NAD-dependent enzymes coupled with the coenzyme regeneration by formate dehydrogenase proved to be economically feasible for the production of fine chemicals.

Amperometric detection of ethanol with poly-( o-phenylenediamine)-modified enzyme electrodes

Poly-( o-phenylenediamine was electropolymerized on the surface of an alcohol dehydrogenase-nicotimanide adenine dinucleotide-modified carbon paste electrode to yield an electrochemical biosensor for ethanol. Efficient electrocatalytic oxidation, at low applied potentials, of the enzymatically produced NADH by the conducting film provided a current that was dependent on ethanol concentration. The influence of various experimental variables was explored to optimize the analytical performance. Linear responses for ethanol in the range 3 × 10 −8 − 3 × 10 −6 M and a response time of 20 s were obtained. The trend in sensitivity towards different alcohols is in agreement with the known biospecificity of yeast ADH. Ethanol sensor was applied to the analysis of different alcohol beverages, resulting in excellent accuracy and precision.

Purification and properties of alcohol dehydrogenase from a mutant strain of Candida guilliermondii deficient in one form of the enzyme

Alcohol dehydrogenase (ADH1) was purified from Candida guilliermondii strain B10-05 to homogeneity, using affinity chromatography on triazine dyes and gel filtration. The enzyme was tetrameric, with a subunit molecular weight of 38 000. The purified enzyme oxidized primary and secondary alcohols, although it preferred primary alcohols. Its activity toward secondary alcohols was better than those of other yeast ADH; however, the enzyme was less sensitive toward inhibitors. Kinetic studies indicated that C. guilliermondii ADH1 oxidized ethanol by an ordered bi–bi mechanism, with NAD as the first substrate fixed. Key words: Candida guilliermondii, alcohol dehydrogenase, ADH1, tetrameric.

On the oxidation of aldehydes by alcohol dehydrogenase of Drosophila melanogaster: Evidence for the gem-diol as the reacting substrate

Purified alcohol dehydrogenase from Drosophila melanogaster is able to catalyze the oxidation of aldehydes in addition to primary and secondary alcohols. Acetaldehyde, methoxy-acetaldehyde, chloro-acetaldehyde, and trichloro-acetaldehyde (chloral hydrate) are used here to show that the gem-diol structure is the reacting form of the aldehydes. The reaction rates depend primarily on the degree of hydration of the aldehydes and to a lesser extent on the resulting electronic state of the hydrated molecules. These results resemble the chemical oxidation of aldehydes. The V K m values for the oxidation of acetaldehyde hydrate and chloro-acetaldehyde hydrate are very similar to that for ethanol. The very high oxidation rates of secondary alcohols compared to primary alcohols by Drosophila ADH are explained as the result of the structural resemblance to hydrated aldehydes and a more favorable electronic state. This behavior is in contrast to mammalian and yeast ADH, which have a low affinity for secondary alcohols and aldehydes. The oxidation of aldehydes by ADH apparently enables D. melanogaster to live in alcoholic environments.

A New Means of Inducibly Inactivating a Cellular Protein

This paper presents a general means of eliminating the function of a single protein without relying on genetic alterations in its structure or level of synthesis. The strategy is based on the inducible cellular expression of neutralizing antibody to inactivate the protein selectively. The feasibility of this approach is illustrated by using alcohol dehydrogenase I (ADH I) in Saccharomyces cerevisiae as a model. Heavy- and light-chain cDNAs were isolated from a hybridoma secreting an antibody which neutralizes yeast ADH I. The cDNAs were characterized with respect to their length and identity, their signal sequences were removed, and synthetic translation initiation codons were joined to them. These truncated sequences were then inserted into an inducible expression vector and shown to be expressed as stable heavy and light chains, which assemble and bind antigen. The sequences were introduced into yeast mutants containing different levels of ADH activity, and evidence is provided that the antibodies produce limited neutralization of enzyme activity in vivo. In principle, the approach can be used for any cell type in which functional antibody can be inducibly expressed.

A new means of inducibly inactivating a cellular protein.

This paper presents a general means of eliminating the function of a single protein without relying on genetic alterations in its structure or level of synthesis. The strategy is based on the inducible cellular expression of neutralizing antibody to inactivate the protein selectively. The feasibility of this approach is illustrated by using alcohol dehydrogenase I (ADH I) in Saccharomyces cerevisiae as a model. Heavy- and light-chain cDNAs were isolated from a hybridoma secreting an antibody which neutralizes yeast ADH I. The cDNAs were characterized with respect to their length and identity, their signal sequences were removed, and synthetic translation initiation codons were joined to them. These truncated sequences were then inserted into an inducible expression vector and shown to be expressed as stable heavy and light chains, which assemble and bind antigen. The sequences were introduced into yeast mutants containing different levels of ADH activity, and evidence is provided that the antibodies produce limited neutralization of enzyme activity in vivo. In principle, the approach can be used for any cell type in which functional antibody can be inducibly expressed.

Immobilization of yeast ADH by adsorption onto polyaminomethylstyrene

Yeast alcohol dehydrogenase (EC 1.1.1.1) (Y-ADH) was immobilized by adsorption onto polyaminomethylstyrene (PAMS). The adsorption was rapid and yielded a product, the operational and storage stability of which depended on the initial content of enzyme and to a great extent on the composition of the adsorption buffer. The best results were obtained in the presence of 0.05 M phosphate solution, pH 8.8, containing 2 M sodium chloride. The amount of adsorbed ADH correlates with the position of anions in the Hofmeister series. After 1,000 bed volumes of the standard assay mixture have flowed through the column, there is no decrease in activity. It is possible to prepare adsorbates using crude yeast extracts as enzyme source. The stability of the immobilized enzyme is similar to that obtained with crystalline ADH preparations.

Partial complementation of the UV sensitivity of E. coli and yeast excision repair mutants by the cloned denV gene of bacteriophage T4.

The denV gene of bacteriophage T4 was reconstituted from two overlapping DNA fragments cloned in M13 vectors. The coding region of the intact gene was tailored into a series of plasmid vectors containing different promoters suitable for expression of the gene in E. coli and in yeast. Induction of the TAC promoter with IPTG resulted in overexpression of the gene, which was lethal to E. coli. Expression of the TACdenV gene in the absence of IPTG, or the use of the yeast GAL1 or ADH promoters resulted in partial complementation of the UV sensitivity of uvrA, uvrB, uvrC and recA mutants of E. coli and rad1, rad2, rad3, rad4 and rad10 mutants of S. cerevisiae. The extent of denV-mediated reactivation of excision-defective mutants was approximately equal to that of photoreactivation of such strains. Excision proficient E. coli cells transformed with a plasmid containing the denV gene were slightly more resistant to ultraviolet (UV) radiation than control cells without the denV gene. On the other hand, excision proficient yeast cells were slightly more sensitive to killing by UV radiation following transformation with a plasmid containing the denV gene. This effect was more pronounced in yeast mutants of the RAD52 epistasis group.

Molecular analysis of the alcohol dehydrogenase (Adh1) gene of maize.

A cDNA clone of maize Adh1 which contains the entire protein coding region of the gene has been constructed. The protein sequence predicted from the nucleotide sequence is in agreement with limited protein sequencing data for the ADH1 enzyme. An 11.5 kb genomic fragment containing the Adh1 gene has been isolated using the cDNA clone as a probe, and the gene region fully sequenced. The gene is interrupted by 9 introns, their junction sequences fitting the animal gene consensus sequence. Within the gene there is a triplication of a segment (104 bp) spanning an intron-exon junction. Presumptive promoter elements have been identified and are similar in nucleotide sequence and location, relative to the start of transcription, to those of other plant and animal genes. No recognizable poly(A+) addition signal is evident. Comparison of the nucleotide sequences of the cDNA (derived from an Adh1 -F allele) and genomic (derived from an Adh1 -S allele) clones has identified an amino acid difference consistent with the observed difference in electrophoretic mobility of the two enzymes. The maize ADH1 amino acid sequence is 50% homologous to that of horse liver ADH but is only 20% homologous to yeast ADH.

Alcohol dehydrogenase from leuconostoc mesenteroides: molecular properties in comparison with the yeast and horse liver enzymE

1. 1. Leuconostoc mesenteroides alcohol dehydrogenase is an acidic protein (pI =4.1) with a mol wt of 135,000 consisting of 4 identical subunits. 2. 2. It contains 2 Zn atoms and 5 cysteine residues per subunit. 3. 3. Thermal stability increases with increasing ionic strength. 4. 4. One NADPH molecule is bound per subunit. NADPH is bound about 10 times more strongly than NADH. Fluorescence spectra of bound coenzymes show characteristic blue shifts and intensity enhancements compared to those of free NAD(P)H. 5. 5. A comparison of molecular properties between horse liver-, yeast- and L. mesenteroides alcohol dehydrogenase shows the closer relationship of Leuconostoc ADH to yeast ADH.

Equilibria and Substrate Specificity Studies with Alcohol Dehydrogenases

Abstract While secondary alcohols and NAD+, and ketones and NADH react much slowly than the ethanol–acetaldehyde system in the presence of yeast and liver alcohol dehydrogenases, by starting near equilibrium concentrations, it has been shown that expected thermodynamic equilibria are obtained. With liver ADH, cycloalkanols are remarkably reactive, while with yeast ADH those with six or more carbon atoms in the ring are unreactive. Horse liver ADH shows stereospecificity for 2-octanol, but no stereospecificity for 2-butanol. On the other hand, yeast ADH shows the same stereoselectivity for both alcohols. In the presence of yeast ADH, R(−)-isomers of 2-butanol, 2-octanol and 1-methoxy-2-propanol are not reactive, comparing to the big reactivity of S(+)-isomers of these alcohols, but R(−)-isomer of propylene glycol is also slightly reactive as S(+)-isomer. Further, the reactivity of secondary alcohols with NAD+ in the presence of liver ADH is following, 2-octanol&amp;gt;2-butanol&amp;gt;2-propanol. These results suggest that horse liver ADH is much less sterically hindered at its active site than yeast ADH and that not only the steric factor but also hydrophobic bonding between the end alkyl group of alcohol and the hydrophobic region of enzyme might be another important factor to determine stereospecificity and reactivity of enzyme.

A technique for the isolation of yeast alcohol dehydrogenase mutants with altered substrate specificity

Electrophoretically distinguishable mutants of yeast alcohol dehydrogenase with specifically altered kinetics have been obtained using a modification of the allyl alcohol selection technique of Megnet. The modification depends on the fact that mutant cells producing no detectable ADH or only the ADH sequestered inside the mitochondria cannot become petite. Allyl alcohol is oxidized by yeast ADH to the poisonous compound acrolein, so petite cells possessing a cytoplasmic ADH will die unless the structural gene is altered to permit the enzyme to distinguish between ethanol and allyl alcohol. The independent nuclear control of yeast mitochondrial ADH, suspected by other workers, is demonstrated. The fact that strains carrying this ADH cannot become petite provides direct genetic evidence that mitochondrial and cytoplasmic NAD pools are separate in Saccharomyces cerevisiae.

Methanol activity of alcohol dehydrogenases from human liver, horse liver, and yeast

Formaldehyde and NADH are formed from methanol and NAD in the reaction catalyzed by horse liver ADH and yeast ADH. In this reaction the above enzymes resemble closely human liver ADH, which has been known for some time to catalyze methanol dehydrogenation. The formation of formaldehyde from melhanol has been followed by chromatography of 2:4-DNPH derivatives of the reaction products and quantitation by the highly specific chromotropic acid method. Kinetic results have shown that turnover numbers × sec −1 × active site −1 are very similar for all three enzymes. The specificity of all three enzymes with respect to methanol is therefore identical. Human liver ADH, however, has the lowest K m for methanol, and on this basis only may be considered to be the best catalyst of this reaction.

Immunologische analyse des alterungsprozesses von kristalliner hefe-alkohol-dehydrogenase

The decrease of enzymatic activity of crystalline yeast-ADH caused by aging was tested with immuno-electrophoresis (Grabar), double-diffusion (Ouchterlony), quantitative immunological methods (techniques of Mancini, Laurell, and Clarke besides the quantitative precipitation) and the use of the density-gradient-ultracentrifugation. Others authors like Bühner [4] described the dissociation of aging native yeast ADH into a subunit slower sedimenting than the native enzyme; we could confirm this. In addition, we found that this protein without enzymatic activity has an SH-titer of about 4 moles cysteine/150.000 g (native ADH of our crystalline preparations has about 20 moles cystein/150.000 g) and its antigenic determinants are completely different to those of the native ADH-molecule. Immunization of rabbits with freshly isolated, crystalline ADH-preparations as well as with purchased, crystalline ADH-charges in incomplete Freund's adjuvant results in immunsera containing non-cross-reacting anti-ADH-antibodies and anti-ADH-subunit-antibodies besides six to eight further antibody-populations against contaminating proteins in the injected enzyme-charges. Comparing the sedimentation-diagrams with the patterns of the quantitative one- or two-dimensional immunoelectrophoresis by Laurell and Clarke of different aged samples of an ADH-preparation we found the same results: the decrease of the enzymatic active ADH-peak caused by aging is accompanied by an increase of the antigenic ADH-subunit.

Specific determination of hydroxy groups with yeast ADH.

Specific determination of hydroxy groups with yeast ADH.