Control of coenzyme binding to horse liver alcohol dehydrogenase.

The rate of association of NAD(+) with wild-type horse liver alcohol dehydrogenase (ADH) is maximal at pH values between pK values of about 7 and 9, and the rate of NADH association is maximal at a pH below a pK of 9. The catalytic zinc-bound water, His-51 (which interacts with the 2'- and 3'-hydroxyl groups of the nicotinamide ribose of the coenzyme in the proton relay system), and Lys-228 (which interacts with the adenosine 3'-hydroxyl group and the pyrophosphate of the coenzyme) may be responsible for the observed pK values. In this study, the Lys228Arg, His51Gln, and Lys228Arg/His51Gln (to isolate the effect of the catalytic zinc-bound water) mutations were used to test the roles of the residues in coenzyme binding. The steady state kinetic constants at pH 8 for the His51Gln enzyme are similar to those for wild-type ADH. The Lys228Arg and Lys228Arg/His51Gln substitutions decrease the affinity for the coenzymes up to 16-fold, probably due to altered interactions with the arginine at position 228. As determined by transient kinetics, the rate constant for association of NAD(+) with the mutated enzymes no longer decreases at high pH. The pH profile for the Lys228Arg enzyme retains the pK value near 7. The His51Gln and Lys228Arg/His51Gln substitutions significantly decrease the rate constants for NAD(+) association, and the pH dependencies show that these enzymes bind NAD(+) most rapidly at a pH above pK values of 8. 0 and 9.0, respectively. It appears that the pK of 7 in the wild-type enzyme is shifted up by the H51Q substitutions, and the resulting pH dependence is due to the deprotonation of the catalytic zinc-bound water. Kinetic simulations suggest that isomerization of the enzyme-NAD(+) complex is substantially altered by the mutations. In contrast, the pH dependencies for NADH association with His51Gln, Lys228Arg, and Lys228Arg/His51Gln enzymes were the same as for wild-type ADH, suggesting that the binding of NAD(+) and the binding of NADH are controlled differently.     

Substitution of arginine for histidine-47 in the coenzyme binding site of yeast alcohol dehydrogenase I

Molecular modeling of alcohol dehydrogenase suggests that His-47 in the yeast enzyme (His-44 in the protein sequence, corresponding to Arg-47 in the horse liver enzyme) binds the pyrophosphate of the NAD coenzyme. His-47 in the Saccharomyces cerevisiae isoenzyme I was substituted with an arginine by a directed mutation. Steady-state kinetic results at pH 7.3 and 30 degrees C of the mutant and wild-type enzymes were consistent with an ordered Bi-Bi mechanism. The substitution decreased dissociation constants by 4-fold for NAD+ and 2-fold for NADH while turnover numbers were decreased by 4-fold for ethanol oxidation and 6-fold for acetaldehyde reduction. The magnitudes of these effects are smaller than those found for the same mutation in the human liver beta enzyme, suggesting that other amino acid residues in the active site modulate the effects of the substitution. The pH dependencies of dissociation constants and other kinetic constants were similar in the two yeast enzymes. Thus, it appears that His-47 is not solely responsible for a pK value near 7 that controls activity and coenzyme binding rates in the wild-type enzyme. The small substrate deuterium isotope effect above pH 7 and the single exponential phase of NADH production during the transient oxidation of ethanol by the Arg-47 enzyme suggest that the mutation makes an isomerization of the enzyme-NAD+ complex limiting for turnover with ethanol.     

Kinetics of native and modified liver alcohol dehydrogenase with coenzyme analogues: isomerization of enzyme-nicotinamide adenine dinucleotide complex

Coenzyme analogues with the adenosine ribose replaced with n-propyl, n-butyl, and n-pentyl groups; coenzyme analogues with the adenosine replaced with 3-(4-acetylanilino)propyl and 6-(4-acetylanilino)hexyl moieties; and nicotinamide mononucleotide, nicotinamide hypoxanthine dinucleotide, and 3-acetylpyridine adenine dinucleotide were used in steady-state kinetic studies with native and activated, amidinated enzymes. The Michaelis and inhibition constants increased up to 100-fold upon modification of coenzyme or enzyme. Turnover numbers with NAD+ and ethanol increased in some cases up to 10-fold due to increased rates of dissociation of enzyme-reduced coenzyme complexes. Rates of dissociation of oxidized coenzyme appeared to be mostly unaffected, but the values calculated (10-60 s-1) were significantly less than the turnover numbers with acetaldehyde and reduced coenzyme (20-900 s-1, at pH 8, 25 degrees C). Rates of association of coenzyme analogues also decreased up to 100-fold. When Lys-228 in the adenosine binding site was picolinimidylated, turnover numbers increased about 10-fold with NAD(H). Furthermore, the pH dependencies for association and dissociation of NAD+ and turnover number with NAD+ and ethanol showed the fastest rates above a pK value of 8.0. Turnover with NADH and acetaldehyde was fastest below a pK value of 8.1. These results can be explained by a mechanism in which isomerization of the enzyme-NAD+ complex (110 s-1) is partially rate limiting in turnover with NAD+ and ethanol (60 s-1) and is controlled by ionization of the hydrogen-bonded system that includes the water ligated to the catalytic zinc and the imidazole group of His-51.     

Drosophila lebanonensis alcohol dehydrogenase: pH dependence of the kinetic coefficients

The alcohol dehydrogenase (ADH) from Drosophila lebanonensis shows 82% positional identity to the alcohol dehydrogenases from Drosophila melanogaster. These insect ADHs belong to the short-chain dehydrogenase/reductase family which lack metal ions in their active site. In this family, it appears that the function of zinc in medium chain dehydrogenases has been replaced by three amino acids, Ser138, Tyr151 and Lys155. The present work on D. lebanonensis ADH has been performed in order to obtain information about reaction mechanism, and possible differences in topology and electrostatic properties in the vicinity of the catalytic residues in ADHs from various species of Drosophila. Thus the pH dependence of various kinetic coefficients has been studied. Both in the oxidation of alcohols and in the reduction of aldehydes, the reaction mechanism of D. lebanonensis ADH in the pH 6-10 region was consistent with a compulsory ordered pathway, with the coenzymes as the outer substrates. Over the entire pH region, the rate limiting step for the oxidation of secondary alcohols such as propan-2-ol was the release of the coenzyme product from the enzyme-NADH complex. In the oxidation of ethanol at least two steps were rate limiting, the hydride transfer step and the dissociation of NADH from the binary enzyme-NADH product complex. In the reduction of acetaldehyde, the rate limiting step was the dissociation of NAD+ from the binary enzyme-NAD+ product complex. The pH dependences of the kon velocity curves for the two coenzymes were the opposite of each other, i.e. kon increased for NAD+ and decreased for NADH with increasing pH. The two curves appeared complex and the kon velocity for the two coenzymes seemed to be regulated by several groups in the free enzyme. The kon velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD+ complex, with a pKa value of 7.1. The kon velocity for acetaldehyde was pH independent and showed that in the enzyme-NADH complex, the pKa value of the catalytic residue must be above 10. The koff velocity of NAD+ appeared to be partly regulated by the catalytic residue, and protonation resulted in an increased dissociation rate. The koff velocity for NADH and the hydride transfer step was pH independent. In D. lebanonensis ADH, the pKa value of the catalytic residue was 0.5 pH units lower than in the ADHS alleloenzyme from D. melanogaster. Thus it can be concluded that while most of the topology of the active site is mainly conserved in these two distantly related enzymes, the microenvironment and electrostatic properties around the catalytic residues differ.     

Estimation of rate dissociation constants involving ternary complexes in reactions catalysed by yeast alcohol dehydrogenase

Stopped-flow studies of oxidation of butan-1-ol and propan-2-ol by NAD(+) in the presence of Phenol Red and large concentrations of yeast alcohol dehydrogenase give no evidence for the participation of a group of pK(a) approx. 7.6 in alcohol binding. Such a group has been implicated in ethanol binding to horse liver alcohol dehydrogenase [Shore, Gutfreund, Brooks, Santiago & Santiago (1974) Biochemistry13, 4185-4190]. The present result supports previous findings based on steady-state kinetic studies with the yeast enzyme. Stopped-flow studies of the yeast alcohol dehydrogenase-catalysed reduction of acetaldehyde by NADH in the presence of ethanol as product inhibitor indicate that the rate-limiting step is NAD(+) release from the enzyme-NAD(+)-ethanol product complex. This finding permits calculation of K(3), the dissociation constant for ethanol from the enzyme-NAD(+)-ethanol complex, by using the product-inhibition data of Dickenson & Dickinson (1978) (Biochem. J.171, 613-627). The calculations show that K(3) varies very little with pH in the range 5.95-8.9, and this agrees with the findings of the stopped-flow experiments described above. Absorption and fluorescence measurements on mixtures of substrates and coenzymes in the presence of high concentrations of alcohol dehydrogenase have been used to estimate values for the ratio [enzyme-NADH-acetaldehyde]/ [enzyme-NAD(+)-ethanol] at equilibrium. The values obtained were in the range 0.11+/-0.04, and this value together with estimates of K(3) was used to provide estimates of values for rate constants and dissociation constants for steps within the catalytic mechanism.     

A study of the kinetics and mechanism of yeast alcohol dehydrogenase with a variety of substrates

1. The kinetics of oxidation of ethanol, propan-1-ol, butan-1-ol and propan-2-ol by NAD(+) and of reduction of acetaldehyde and butyraldehyde by NADH catalysed by yeast alcohol dehydrogenase were studied. 2. Results for the aldehyde-NADH reactions are consistent with a compulsory-order mechanism with the rate-limiting step being the dissociation of the product enzyme-NAD(+) complex. In contrast the results for the alcohol-NAD(+) reactions indicate that some dissociation of coenzyme from the active enzyme-NAD(+)-alcohol ternary complexes must occur and that the mechanism is not strictly compulsory-order. The rate-limiting step in ethanol oxidation is the dissociation of the product enzyme-NADH complex but with the other alcohols it is probably the catalytic interconversion of ternary complexes. 3. The rate constants describing the combination of NAD(+) and NADH with the enzyme and the dissociations of these coenzymes from binary complexes with the enzyme were measured.     

Kinetics of the reaction catalysed by rape alcohol dehydrogenase

The kinetics of the enzyme reaction of ethanol oxidation and acetaldehyde reduction catalysed by alcohol dehydrogenase (ADH) (EC 1.1.1.1) isolated from germinating rape seeds obeys the bi-bi ordered mechanism of Theorell and Chance. The enzyme reaction depends on the pH and temperature. The K_m values for the basic substrates have the lowest values around the pH optimum of the reaction. The enzyme is most stable at pH 6.5–7. The K_m values for ethanol and NAD increase with increasing temperature. The maximum rate of the ethanol oxidation satisfies the Arrhenius equation. The activation energy for the given temperature range is 40.11 kJ/mol. The rape ADH is denatured by heating above 60° but the enzyme-NAD complex is thermally more stable than the enzyme alone.     

The pH variation of steady-state kinetic parameters of site-specific Co(2+)-reconstituted liver alcohol dehydrogenase. A mechanistic probe for the assignment of metal-linked ionizations

To identify ionizations of the active site metal-bound water in horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase; EC 1.1.1.1), the pH, solvent isotope, temperature, and anion dependences of the steady-state kinetic parameters kcat and kcat/KM have been evaluated under initial velocity conditions for the native and the active site-specific Co(2+)-reconstituted enzyme. In the oxidation of benzyl alcohol, a bell-shaped pattern of four prototropic equilibria was observed under conditions of saturating concentrations of NAD+. It is shown that the ionizations governing kcat (pK1 congruent to 6.7, pK2 congruent to 10.6) belong to the ternary enzyme-NAD(+)-alcohol complex, whereas the ionizations governing kcat/KM (pK1' congruent to 7.5, pK2' congruent to 8.9) belong to the binary enzyme-NAD+ complex. The ionizations pK1 and pK1' are not influenced by metal substitution and are ascribed to His-51 on the basis of experimental estimates of their associated enthalpies of ionization. On the other hand, pK2 and pK2' are significantly decreased (delta pKa congruent to 1.0) in the Co(2+)-enzyme and are attributed to the active site metal-bound water molecule. The shape of the pH profiles requires that the metal ion coordinates a neutral water molecule in the ternary enzyme-NAD(+)-alcohol complex under physiological conditions. The possible catalytic role of the water molecule within a pentacoordinate metal ion complex in the active site is discussed.     

Respiratory behaviour of a Zymomonas mobilis adhB::kan(r) mutant supports the hypothesis of two alcohol dehydrogenase isoenzymes catalysing opposite reactions

Perturbation of the aerobic steady-state in a chemostat culture of the ethanol-producing bacterium Zymomonas mobilis with a small pulse of ethanol causes a burst of ethanol oxidation, although the reactant ratio of the alcohol dehydrogenase (ADH) reaction ([NADH][acetaldehyde][H(+)])/([ethanol][NAD(+)]) remains above the K(eq) value. Simultaneous catalysis of ethanol synthesis and oxidation by the two ADH isoenzymes, residing in different redox microenvironments, has been proposed previously. In the present study, this hypothesis is verified by construction of an ADH-deficient strain and by demonstration that it lacks the oxidative burst in response to perturbation of its aerobic steady-state with ethanol.     

Dual coenzyme activities of high-Km aldehyde dehydrogenase from rat liver mitochondria

Various kinetic approaches were carried out to investigate kinetic attributes for the dual coenzyme activities of mitochondrial aldehyde dehydrogenase from rat liver. The enzyme catalyses NAD(+)- and NADP(+)-dependent oxidations of ethanal by an ordered bi-bi mechanism with NAD(P)+ as the first reactant bound and NAD(P)H as the last product released. The two coenzymes presumably interact with the kinetically identical site. NAD+ forms the dynamic binary complex with the enzyme, while the enzyme-NAD(P)H complex formation is associated with conformation change(s). A stopped-flow burst of NAD(P)H formation, followed by a slower steady-state turnover, suggests that either the deacylation or the release of NAD(P)H is rate limiting. Although NADP+ is reduced by a faster burst rate, NAD+ is slightly favored as the coenzyme by virtue of its marginally faster turnover rate.     

The catalytic triad in Drosophila alcohol dehydrogenase: pH, temperature and molecular modelling studies

Drosophila alcohol dehydrogenase belongs to the short chain dehydrogenase/reductase (SDR) family which lack metal ions in their active site. In this family, it appears that the three amino acid residues, Ser138, Tyr151 and Lys155 have a similar function as the catalytic zinc in medium chain dehydrogenases. The present work has been performed in order to obtain information about the function of these residues. To obtain this goal, the pH and temperature dependence of various kinetic coefficients of the alcohol dehydrogenase from Drosophila lebanonensis was studied and three-dimensional models of the ternary enzyme-coenzyme-substrate complexes were created from the X-ray crystal coordinates of the D. lebanonensis ADH complexed with either NAD(+) or the NAD(+)-3-pentanone adduct. The kon velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD(+) complex, with a DeltaHion value of 74(+/-4) kJ/mol (18(+/-1) kcal/mol). Based on this result and the constructed three-dimensional models of the enzyme, the most likely candidate for this catalytic residue is Ser138. The present kinetic study indicates that the role of Lys155 is to lower the pKa values of both Tyr151 and Ser138 already in the free enzyme. In the binary enzyme-NAD(+) complex, the positive charge of the nicotinamide ring in the coenzyme further lowers the pKa values and generates a strong base in the two negatively charged residues Ser138 and Tyr151. With the OH group of an alcohol close to the Ser138 residue, an alcoholate anion is formed in the ternary enzyme NAD(+) alcohol transition state complex. In the catalytic triad, along with their effect on Ser138, both Lys155 and Tyr151 also appear to bind and orient the oxidized coenzyme.     

The AdhS alleloenzyme of alcohol dehydrogenase from Drosophila melanogaster. Variation of kinetic parameters with pH.

The variation with pH of the kinetic parameters for the alcohol and acetaldehyde reactions were studied for the alleloenzyme AdhS from Drosophila melanogaster. The variation of Ki (KEO,I) with pH for two ethanol-competitive inhibitors, pyrazole and 2,2,2-trifluoroethanol, was also studied. Both alcohol oxidation and acetaldehyde reduction follow a compulsory ordered pathway, with coenzyme binding first. The rate-limiting step for ethanol oxidation is complex and involves at least hydride transfer and dissociation of the enzyme-NADH complex (ER). In contrast with this, the rate-limiting step for the back reaction, i.e. the reduction of acetaldehyde, is dissociation of the enzyme-NAD+ complex (EO). A rate-limiting ER dissociation appears in the oxidation of the secondary alcohol propan-2-ol, whereas for the back reaction, i.e. acetone reduction, hydride transfer in the ternary complexes is rate-limiting. There is one group in the free enzyme, with a pK of approx. 8.0, that regulates the kon velocity for NADH, whereas for NAD+ several groups seem to be involved. A group in the enzyme is drastically perturbed by the formation of the binary EO complex. Protonation of this group with a pK of 7.6 in the EO complex resulted in weakened alcohol and inhibitor binding, in addition to an increased dissociation rate of NAD+ from the binary EO complex. Neither the binding of acetaldehyde nor the dissociation rate of NADH from the binary ER complex varied within the pH region studied.     

Crystal structure of the vertebrate NADP(H)-dependent alcohol dehydrogenase (ADH8)

The amphibian enzyme ADH8, previously named class IV-like, is the only known vertebrate alcohol dehydrogenase (ADH) with specificity towards NADP(H). The three-dimensional structures of ADH8 and of the binary complex ADH8-NADP(+) have been now determined and refined to resolutions of 2.2A and 1.8A, respectively. The coenzyme and substrate specificity of ADH8, that has 50-65% sequence identity with vertebrate NAD(H)-dependent ADHs, suggest a role in aldehyde reduction probably as a retinal reductase. The large volume of the substrate-binding pocket can explain both the high catalytic efficiency of ADH8 with retinoids and the high K(m) value for ethanol. Preference of NADP(H) appears to be achieved by the presence in ADH8 of the triad Gly223-Thr224-His225 and the recruitment of conserved Lys228, which define a binding pocket for the terminal phosphate group of the cofactor. NADP(H) binds to ADH8 in an extended conformation that superimposes well with the NAD(H) molecules found in NAD(H)-dependent ADH complexes. No additional reshaping of the dinucleotide-binding site is observed which explains why NAD(H) can also be used as a cofactor by ADH8. The structural features support the classification of ADH8 as an independent ADH class.     

The pH-dependent binding of NADH and subsequent enzyme isomerization of human liver beta 3 beta 3 alcohol dehydrogenase

Human class I beta 3 beta 3 is one of the alcohol dehydrogenase dimers that catalyzes the reversible oxidation of ethanol. The beta 3 subunit has a Cys substitution for Arg-369 (beta 369C) in the coenzyme-binding site of the beta1 subunit. Kinetic studies have demonstrated that this natural mutation in the coenzyme-binding site decreases affinity for NAD+ and NADH. Structural studies suggest that the enzyme isomerizes from an open to closed form with coenzyme binding. However, the extent to which this isomerization limits catalysis is not known. In this study, stopped-flow kinetics were used from pH 6 to 9 with recombinant beta 369C to evaluate rate-limiting steps in coenzyme association and catalysis. Association rates of NADH approached an apparent zero-order rate with increasing NADH concentrations at pH 7.5 (42 +/- 1 s-1). This observation is consistent with an NADH-induced isomerization of the enzyme from an open to closed conformation. The pH dependence of apparent zero-order rate constants fit best a model in which a single ionization limits diminishing rates (pKa = 7.2 +/- 0.1), and coincided with Vmax values for acetaldehyde reduction. This indicates that NADH-induced isomerization to a closed conformation may be rate-limiting for acetaldehyde reduction. The pH dependence of equilibrium NADH-binding constants fits best a model in which a single ionization leads to a loss in NADH affinity (pKa = 8.1 +/- 0. 2). Rate constants for isomerization from a closed to open conformation were also calculated, and these values coincided with Vmax for ethanol oxidation above pH 7.5. This suggests that NADH-induced isomerization of beta 369C from a closed to open conformation is rate-limiting for ethanol oxidation above pH 7.5.     

Colloidal bismuth subcitrate and omeprazole inhibit alcohol dehydrogenase mediated acetaldehyde production by Helicobacter pylori

We have recently shown that Helicobacter pylori possesses marked alcohol dehydrogenase (ADH) activity and is capable - when incubated with an ethanol containing solution in vitro - of producing large amounts of acetaldehyde. In the present study we report that some drugs commonly used for the eradication of H. pylori and for the treatment of gastroduodenal diseases are potent ADH inhibitors and, consequently, effectively prevent bacterial oxidation of ethanol to acetaldehyde. Colloidal bismuth subcitrate (CBS), already at a concentration of 0.01 mM, inhibited H. pylori ADH by 93% at 0.5 M ethanol and decreased oxidation of 22 mM ethanol to acetaldehyde to 82% of control. At concentrations above 5 mM, CBS almost totally inhibited acetaldehyde formation. Omeprazole, a drug also known to suppress growth of H. pylori, also inhibited H. pylori ADH and suppressed bacterial acetaldehyde formation significantly to 69% of control at a drug concentration of 0.1 mM. By contrast, the H₂-receptor antagonists ranitidine and famotidine showed only modest effect on bacterial ADH and acetaldehyde production. We suggest that inhibition of bacterial ADH and a consequent suppression of acetaldehyde production from endogenous or exogenous ethanol may be a novel mechanism by which CBS and omeprazole exert their effect both on the growth of H. pylori as well as on H. pylori associated gastric injury.     

Expression and kinetic characterization of variants of human beta 1 beta 1 alcohol dehydrogenase containing substitutions at amino acid 47

Arg-47 of human beta 1 beta 1 alcohol dehydrogenase has been replaced with Lys, His, Gln, and Gly by site-directed mutagenesis. The mutated enzymes were expressed in Escherichia coli and purified to homogeneity. The recombinant enzymes with Arg and His at position 47 exhibit kinetic constants and stability which are similar to beta 1 beta 1 and beta 2 beta 2, respectively. The substitution of Lys, His, or Gln for Arg-47 resulted in active enzymes with lower affinity for coenzyme and higher Vmax values than beta 1 beta 1. The substitution of Gln at position 47 resulted in an enzyme with the highest Vmax for ethanol oxidation of any mammalian alcohol dehydrogenase. In this series of enzymes, the affinity for coenzyme decreases with decreasing pKa of the substituted amino acid side chains. The substitution of Gly at position 47 resulted in an enzyme with a Vmax that was one-half that of the low activity beta 1 beta 1 and coenzyme affinities that are lower than beta 1 beta 1, but are equal to or greater than the affinities exhibited by the His-47 or Gln-47 enzymes. Product inhibition studies indicated a change in mechanism from ordered Bi Bi for beta 1 beta 1 to rapid equilibrium random Bi Bi for the Gly-47 enzyme. The kinetic properties of the Gly-47 enzyme are substantially different from human liver alpha alpha which also has Gly at position 47.     

A study of the oxidation of butan-1-ol and propan-2-ol by nicotinamide-adenine dinucleotide catalysed by yeast alcohol dehydrogenase.

1. The kinetics of oxidation of butan-1-ol and propan-2-ol by NAD+, catalysed by yeast alcohol dehydrogenase, were studied at 25 degrees C from pH 5.5 to 10, and at pH 7.05 from 14 degrees to 44 degrees C, 2. Under all conditions studied the results are consistent with a mechanism whereby some dissociation of coenzyme from the active enzyme-NAD+-alcohol ternary complexes occurs, and the mechanism is therefore not strictly compulsory order. 3. A primary 2H isotopic effect on the maximum rates of oxidation of [1-2H2]butan-1-ol and [2H7]propan-2-ol was found at 25 degrees C over the pH range 5.5-10. Further, in stopped-flow experiments at pH 7.05 and 25 degrees C, there was no transient formation of NADH in the oxidation of butan-1-ol and propan-2-ol. The principal rate-limiting step in the oxidation of dependence on pH of the maximum rates of oxidation of butan-1-ol and propan-2-ol is consisten with the possibility that histidine and cysteine residues may affect or control catalysis.     

Characterization of alcohol dehydrogenase 3 of the thermotolerant methylotrophic yeast Hansenula polymorpha

In this study, we identified and characterized mitochondrial alcohol dehydrogenase 3 from the thermotolerant methylotrophic yeast Hansenula polymorpha (HpADH3). The amino acid sequence of HpADH3 shares over 70% of its identity with the alcohol dehydrogenases of other yeasts and exhibits the highest similarity of 91% with the alcohol dehydrogenase 1 of H. polymorpha. However, unlike the cytosolic HpADH1, HpADH3 appears to be a mitochondrial enzyme, as a mitochondrial targeting extension exists at its N terminus. The recombinant HpADH3 overexpressed in Escherichia coli showed similar catalytic efficiencies for ethanol oxidation and acetaldehyde reduction. The HpADH3 displayed substrate specificities with clear preferences for medium chain length primary alcohols and acetaldehyde for an oxidation reaction and a reduction reaction, respectively. Although the H. polymorpha ADH3 gene was induced by ethanol in the culture medium, both an ADH isozyme pattern analysis and an ADH activity assay indicated that HpADH3 is not the major ADH in H. polymorpha DL-1. Moreover, HpADH3 deletion did not affect the cell growth on different carbon sources. However, when the HpADH3 mutant was complemented by an HpADH3 expression cassette fused to a strong constitutive promoter, the resulting strain produced a significantly increased amount of ethanol compared to the wild-type strain in a glucose medium. In contrast, in a xylose medium, the ethanol production was dramatically reduced in an HpADH3 overproduction strain compared to that in the wild-type strain. Taken together, our results suggest that the expression of HpADH3 would be an ideal engineering target to develop H. polymorpha as a substrate specific bioethanol production strain.     

Comparative study of plant alcohol dehydrogenases

Alcohol dehydrogenase was isolated both from monocotyledons and dicotyledons, some of them with proteins (bean, pea), others with lipids (rape, sunflower) and still others with sugars (rice) as reserve substances. Molecular weights of the isolated dehydrogenases ranged from 53 000 to 80 000. Plant alcohol dehydrogenases (ADH) catalyze the oxidation of ethanol as well as the reduction of acetaldehyde. pH optimum for the oxidation is in the alkaline region, for the reduction it is near neutrality. The Michaelis constants for ethanol oxidation are, with the exception of rice, higher than those for reduction of acetaldehyde. The specificity of plant ADH toward alcohols is relatively broad and only quantitatively different in the individual plants. Inhibitors of the ADH’s studied are oximes, amides and intermediates of sugar metabolism, such as malate, acetate or succinate. The degree of inhibition brought about by the inhibitors studied differs from plant to plant but the inhibition type is the same.