The conformation of adenosine diphosphoribose and 8-bromoadenosine diphosphoribose when bound to liver alcohol dehydrogenase.

8-Bromo-adenosine diphosphoribose (br8 ADP-Rib) and nicotinamide 8-bromoadenine dinucleotide (Nbr8AD+) which are analogues of the coenzyme NAD+, were prepared and their liver alcohol dehydrogenase complexes studied by crystallographic methods. Nbr8AD+ is active in alcohol dehydrogenase complexes studied by crystallographic methods. Nbr8AD+ is active in hydrogen transport and br8ADP-Rib is a coenzyme competitive inhibitor for the enzymes liver alcohol dehydrogenase and yeast alcohol dehydrogenase. X-ray data were obtained for the complex between liver alcohol dehydrogenase and br8ADP-Rib to 0.45 nm resolution and for the liver alcohol dehydrogenase-adenosine diphosphoribose complex to 0.29-nm resolution. The conformations of these analogues were determined from the X-ray data. It was found that ADP-Rib had a conformation very similar to the corresponding part of NAD+, when NAD+ is bound to lactate and malate dehydrogenase. br8ADP-Rib had the same anti conformation of the adenine ring with respect to the ribose as ADP-Rib and NAD+, in contrast to the syn conformation found in 8-bromo-adenosine. The overcrowding at the 8-position is relieved in br8ADP-Rib by having the ribose in the 2' endo condormation instead of the usual 3' endo as in ADP-Rib and NAD+.     

Synthesis and biological activity of N6-(N[(4-azido-3,5,6-trifluoro)-pyridin-2-yl]-2-aminoethyl)-adenosine 5'-monophosphate, a new AMP photoactivatable derivative. Covalent modification of horse liver alcohol dehydrogenase

N6-(N-[(4-Azido-3,5,6-trifluoro)pyridin-2-yl]-2-aminoethyl)- adenosine 5'-monophosphate has been synthesized and evidence presented for its structural assignment by ultraviolet and 19F-NMR spectroscopies. Its photolysis was shown to occur within 5 min. This AMP derivative behaves as a competitive inhibitor of NAD+ in horse-liver-alcohol-dehydrogenase-promoted oxidation of ethanol, with a Ki (0.95 mM) comparable to the Ki of AMP (1.9 mM). Moreover it is an activator of the enzyme when nicotinamide ribose is used as the oxidation cofactor. This activation is as good as that promoted by AMP or by the well known 8-azido-AMP. Upon photolysis of this new derivative in the presence of horse liver alcohol dehydrogenase, a covalent enzyme--analogue complex was isolated and assayed as a catalyst in the oxidation of ethanol using nicotinamide ribose as the cofactor. The reaction took place without complementation of AMP, indicating clearly that the AMP analogue is mainly covalently bound in the AMP-binding site, and that the linkage formed between the enzyme and the azido derivative has not dramatically altered the active site of the enzyme. A similar experiment with 8-azido-AMP produced a completely inactive complex.     

X-ray studies of the binding of Cibacron blue F3GA to liver alcohol dehydrogenase

The binding of Cibacron F3GA to orthorhombic crystals of liver alcohol dehydrogenase has been studied to 0.37-nm resolution. Similarities in the binding of this dye were found for rings B, C and D with the binding of the coenzyme NAD+. However, ring A of the dye and the nicotinamide ribose part of the coenzyme are quite differently bound to the enzyme.     

Temperature-dependent structure of the E x S complex of Bacillus stearothermophilus alcohol dehydrogenase

The catalytic chemistry of the thermophilic Bacillus stearothermophilus alcohol dehydrogenase (HtADH) closely resembles that of mesophilic horse liver alcohol dehydrogenase (HLADH). Molecular dynamics (MD) simulations of the htADH x NAD+ x EtO- complex at 298, 323, and 348 K show that the structure of the ligated Zn2+...EtO- complex varies slightly with change in temperature. The MD-created Boltzmann distribution of htADH x NAD+ x EtO- structures establishes the formation of multiple states which increase in number with a decrease in temperature. The motions of the cofactor domain are highly correlated with the motions of NAD+ at the optimal growth temperature (348 K), with NAD+ being pushed toward the substrate by Val260. With a decrease in temperature, the motion together of the cofactor and substrate is reversed, and at 298 K, the nicotinamide ring of the cofactor moves away from the substrate. Both the distance between and the angle of approach of C4 of NAD+ and HD of EtO- become distorted from those of the reactive conformation. The percentages of ground state present as the reactive conformation at different temperatures are approximately correlated with the kcat for the htADH enzymatic reaction. The rate constant for the htADH x NAD+ x EtOH --> htADH x NAD+ x EtO- proton dissociation, which is mediated by Thr40-OH, becomes slower at lower temperatures. The time-dependent distance between EtO- and Thr40-OH reveals that the Thr40 hydroxyl group sways between the substrate and NAD+ ribose 2'-hydroxyl group at the optimal enzyme growth temperature, and this movement is effectively frozen out as the temperature decreases. The temperature dependence of active site conformations is due to the change in both long-range and short-range motions of the E x S complex.     

Active-site cobalt(II)-substituted horse liver alcohol dehydrogenase: characterization of intermediates in the oxidation and reduction processes as a function of pH

Substitution of Co(II) for the catalytic site Zn(II) of horse liver alcohol dehydrogenase (LADH) yields an active enzyme derivative, CoIIE, with characteristic Co(II) charge-transfer and d-d electronic transitions that are sensitive to the events which take place during catalysis [Koerber, S. C., MacGibbon, A. K. H., Dietrich, H., Zeppezauer, M., & Dunn, M. F. (1983) Biochemistry 22, 3424-3431]. In this study, UV-visible spectroscopy and rapid-scanning stopped-flow (RSSF) kinetic methods are used to detect and identify intermediates in the LADH catalytic mechanism. In the presence of the inhibitor isobutyramide, the pre-steady-state phase of alcohol (RCH2OH) oxidation at pH above 7 is characterized by the formation and decay of an intermediate with lambda max = 570, 640, and 672 nm for both aromatic and aliphatic alcohols (benzyl alcohol, p-nitrobenzyl alcohol, anisyl alcohol, ethanol, and methanol). By comparison with the spectrum of the stable ternary complex formed with oxidized nicotinamide adenine dinucleotide (NAD+) and 2,2',2'-trifluoroethoxide ion (TFE-), CoIIE(NAD+, TFE-), the intermediate which forms is proposed to be the alkoxide ion (RCH2O-) complex, CoIIE(NAD+, RCH2O-). The timing of reduced nicotinamide adenine dinucleotide (NADH) formation indicates that intermediate decay is limited by the interconversion of ternary complexes, i.e., CoIIE(NAD+, RCH2O-) in equilibrium CoIIE(NADH, RCHO). From competition experiments, we infer that, at pH values below 5, NAD+ and alcohol form a CoIIE(NAD+, RCH2OH) ternary complex. RSSF studies carried out as a function of pH indicate that the apparent pKa values for the ionization of alcohol within the ternary complex, i.e., CoIIE(NAD+, RCH2OH) in equilibrium CoIIE(NAD+, RCH2O-) + H+, fall in the range 5-7.5. Using pyrazole as the dead-end inhibitor, we find that the single-turnover time courses for the reduction of benzaldehyde, p-nitrobenzaldehyde, anisaldehyde, and acetaldehyde at pH above 7 all show evidence for the formation and decay of an intermediate. Via spectral comparisons with CoIIE-(NAD+, TFE-) and with the intermediate formed during alcohol oxidation, we identify the intermediate as the same CoIIE(NAD+, RCH2O-) ternary complex detected during alcohol oxidation.     

Reaction of 4-trans-(N,N-dimethylamino)cinnamaldehyde with the liver alcohol dehydrogenase-oxidized nicotinamide adenine dinucleotide complex

Evidence that horse liver alcohol dehydrogenase forms a ternary complex with 4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA) and oxidized nicotinamide adenine dinucleotide (NAD+) is presented. Formation of the complex is characterized by a 97-nm red shift of the free chromophore to 495 nm (epsilon 495 approximately 6.0 X 10(4) M-1 cm-1). This shift is larger than the 66-nm red shift of the E(NADH,-DACA) complex (lambda max = 464 nm) previously reported by Dunn and Hutchinson [Dunn, M.F., & Hutchison, J.S. (1973) Biochemistry 12, 4882-4892]. The large red shift of the E(NAD+,DACA) complex is due to the combined effects of coordination of the carbonyl oxygen of DACA to the active-site zinc ion and to the close proximity of the positively charged nicotinamide ring of NAD+. The stability of this complex is pH dependent and depends on a single apparent ionization with pKa = 7.6 +/- 0.3. The pH-independent dissociation constant for binding of DACA to E(NAD+) is 23 +/- 6 microM. The stoichiometry of DACA binding to the E(NAD+) complex is shown to be one per active site (two per enzyme molecule). Liver alcohol dehydrogenase is also shown to catalyze the NAD+-mediated oxidation of DACA to the corresponding carboxylic acid with a very slow turnover rate. The possibility that the observed E(NAD+,DACA) complex is an intermediate in the enzyme-catalyzed oxidation of DACA is discussed.     

Mechanism of nicotinamide inhibition and transglycosidation by Sir2 histone/protein deacetylases

Silent information regulator 2 (Sir2) enzymes catalyze NAD+-dependent protein/histone deacetylation, where the acetyl group from the lysine epsilon-amino group is transferred to the ADP-ribose moiety of NAD+, producing nicotinamide and the novel metabolite O-acetyl-ADP-ribose. Sir2 proteins have been shown to regulate gene silencing, metabolic enzymes, and life span. Recently, nicotinamide has been implicated as a direct negative regulator of cellular Sir2 function; however, the mechanism of nicotinamide inhibition was not established. Sir2 enzymes are multifunctional in that the deacetylase reaction involves the cleavage of the nicotinamide-ribosyl, cleavage of an amide bond, and transfer of the acetyl group ultimately to the 2'-ribose hydroxyl of ADP-ribose. Here we demonstrate that nicotinamide inhibition is the result of nicotinamide intercepting an ADP-ribosyl-enzyme-acetyl peptide intermediate with regeneration of NAD+ (transglycosidation). The cellular implications are discussed. A variety of 3-substituted pyridines was found to be substrates for enzyme-catalyzed transglycosidation. A Br?nsted plot of the data yielded a slope of +0.98, consistent with the development of a nearly full positive charge in the transition state, and with basicity of the attacking nucleophile as a strong predictor of reactivity. NAD+ analogues including beta-2'-deoxy-2'-fluororibo-NAD+ and a His-to-Ala mutant were used to probe the mechanism of nicotinamide-ribosyl cleavage and acetyl group transfer. We demonstrate that nicotinamide-ribosyl cleavage is distinct from acetyl group transfer to the 2'-OH ribose. The observed enzyme-catalyzed formation of a labile 1'-acetylated-ADP-fluororibose intermediate using beta-2'-deoxy-2'-fluororibo-NAD+ supports a mechanism where, after nicotinamide-ribosyl cleavage, the carbonyl oxygen of acetylated substrate attacks the C-1' ribose to form an initial iminium adduct.     

Insights into the sirtuin mechanism from ternary complexes containing NAD+ and acetylated peptide

Sirtuin proteins comprise a unique class of NAD+-dependent protein deacetylases. Although several structures of sirtuins have been determined, the mechanism by which NAD+ cleavage occurs has remained unclear. We report the structures of ternary complexes containing NAD+ and acetylated peptide bound to the bacterial sirtuin Sir2Tm and to a catalytic mutant (Sir2Tm(H116Y)). NAD+ in these structures binds in a conformation different from that seen in previous structures, exposing the alpha face of the nicotinamide ribose to the carbonyl oxygen of the acetyl lysine substrate. The NAD+ conformation is identical in both structures, suggesting that proper coenzyme orientation is not dependent on contacts with the catalytic histidine. We also present the structure of Sir2Tm(H116A) bound to deacteylated peptide and 3'-O-acetyl ADP ribose. Taken together, these structures suggest a mechanism for nicotinamide cleavage in which an invariant phenylalanine plays a central role in promoting formation of the O-alkylamidate reaction intermediate and preventing nicotinamide exchange.     

Affinity chromatography of mitochondrial nicotinamide nucleotide transhydrogenase

The binding of mitochondrial nicotinamide nucleotide transhydrogenase to NAD+ and NADP+ immobilized to agarose through different parts of the nicotinamide nucleotide molecule was investigated. NADP+ bound at the C8 atom in the adenine moiety proved to be the most efficient ligand whereas that bound at the C3 atom of the ribose moiety was relatively inefficient. NAD+ ligands were generally inactive independently of the site of attachment. Previous results suggest, however, that binding to immobilized NAD+ may be influenced by the detergent in which transhydrogenase is dispersed. Binding to neither ligand was affected by the presence of the second substrate.     

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.     

Participation of histidine-51 in catalysis by horse liver alcohol dehydrogenase

Histidine-51 in horse liver alcohol dehydrogenase (ADH) is part of a hydrogen-bonded system that appears to facilitate deprotonation of the hydroxyl group of water or alcohol ligated to the catalytic zinc. The contribution of His-51 to catalysis was studied by characterizing ADH with His-51 substituted with Gln (H51Q). The steady-state kinetic constants for ethanol oxidation and acetaldehyde reduction at pH 8 are similar for wild-type and H51Q enzymes. In contrast, the H51Q substitution significantly shifts the pH dependencies for steady-state and transient reactions and decreases by 11-fold the rate constant for the transient oxidation of ethanol at pH 8. Modest substrate deuterium isotope effects indicate that hydride transfer only partially limits the transient oxidation and turnover. Transient data show that the H51Q substitution significantly decreases the rate of isomerization of the enzyme-NAD(+) complex and becomes a limiting step for ethanol oxidation. Isomerization of the enzyme-NAD(+) complex is rate limiting for acetaldehyde reduction catalyzed by the wild-type enzyme, but release of alcohol is limiting for the H51Q enzyme. X-ray crystallography of doubly substituted His51Gln:Lys228Arg ADH complexed with NAD(+) and 2,3- or 2,4-difluorobenzyl alcohol shows that Gln-51 isosterically replaces histidine in interactions with the nicotinamide ribose of the coenzyme and that Arg-228 interacts with the adenosine monophosphate of the coenzyme without affecting the protein conformation. The difluorobenzyl alcohols bind in one conformation. His-51 participates in, but is not essential for, proton transfers in the mechanism.     

Molecular mechanics calculation of geometries of NAD+ derivatives, modified in the nicotinamide group, in a ternary complex with horse liver alcohol dehydrogenase

The geometry of seven NAD+ analogues bound to horse liver alcohol dehydrogenase (LADH) modified only in their nicotinamide group, have been studied using AMBER molecular mechanics energy-minimization procedures. Starting geometries were taken from X-ray crystallographic data for NAD+/Me2SO/LADH reported by Eklund and co-workers. In this study the NAD+ analogues were encaged by the constituent amino acids of the enzyme within a range of 0.6 nm from the initial NAD+/Me2SO/Zn2+ complex. The calculational method used is able to rationalize individual substituent effects and to evaluate the essential interactions between NAD+ analogue, enzyme, Me2SO and Zn2+ without the necessity of additional X-ray data. The results presented here demonstrate that the reactivity of NAD+ derivatives as reported in literature can be qualitatively related to the position of the pyridine moiety in the active site.     

An alcohol dehydrogenase ribozyme

We report an RNA molecule that exhibits activity analogous to that of alcohol dehydrogenase (ADH). Directed in vitro evolution was used to enrich nicotinamide adenine dinucleotide (NAD+)-dependent redox-active RNAs from a combinatorial pool. The most active ribozyme in the population forms a compact pseudoknotted structure and oxidizes an alcohol seven orders of magnitude faster than the estimated spontaneous rate. Moreover, this ADH RNA was coupled with a redox relay between NADH and flavin adenine dinucleotide to give a NAD+-regeneration system. Our demonstration of the redox ability of RNA adds support to an RNA-based metabolic system in ancient life.     

Bull semen nicotinamide adenine dinucleotide nucleosidase. VII. Nonenzymatic basis of apparent transglycosidation with histamine

The reaction between NAD and histamine in the presence of purified bull semen nicotinamide adenine dinucleotide nucleosidase (NADase) was studied with respect to the rate of disappearance of the nicotinamide ribosidic linkage of NAD and the rate of the loss of one orcinol-positive ribose of NAD. It was observed that in the presence of this enzyme, 50% of the ribosidic linkage was hydrolyzed prior to any change in orcinol-positive ribose. A nonenzymatic reaction of the product of hydrolysis, adenosine diphosphoribose with histamine was observed to result in the loss of one orcinol-positive ribose. Similar nonenzymatic reactions of histamine were observed with ribose and ribose-5-phosphate. The data suggest that the bull semen NADase does not catalyze a transglycosidation reaction between NAD and histamine as had been claimed previously.     

An aspartate residue in yeast alcohol dehydrogenase I determines the specificity for coenzyme

In the three-dimensional structures of enzymes that bind NAD or FAD, there is an acidic residue that interacts with the 2'- and 3'-hydroxyl groups of the adenosine ribose of the coenzyme. The size and charge of the carboxylate might repel the binding of the 2'-phosphate group of NADP and explain the specificity for NAD. In the NAD-dependent alcohol dehydrogenases, Asp-223 (horse liver alcohol dehydrogenase sequence) appears to have this role. The homologous residue in yeast alcohol dehydrogenase I (residue 201 in the protein sequence) was substituted with Gly, and the D223G enzyme was expressed in yeast, purified, and characterized. The wild-type enzyme is specific for NAD. In contrast, the D223G enzyme bound and reduced NAD+ and NADP+ equally well, but, relative to wild-type enzyme, the dissociation constant for NAD+ was increased 17-fold, and the reactivity (V/K) on ethanol was decreased to 1%. Even though catalytic efficiency was reduced, yeast expressing the altered or wild-type enzyme grew at comparable rates, suggesting that equilibration of NAD and NADP pools is not lethal. Asp-223 participates in binding NAD and in excluding NADP, but it is not the only residue important for determining specificity for coenzyme.     

Classical Raman spectroscopic studies of NADH and NAD+ bound to liver alcohol dehydrogenase by difference techniques

We report the Raman spectra of reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD+, respectively) and adenosine 5'-diphosphate ribose (ADPR) when bound to the coenzyme site of liver alcohol dehydrogenase (LADH). The bound NADH spectrum is calculated by taking the classical Raman difference spectrum of the binary complex, LADH/NADH, with that of LADH. We have investigated how the bound NADH spectrum is affected when the ternary complexes with inhibitors are formed with dimethyl sulfoxide (Me2SO) or isobutyramide (IBA), i.e., LADH/NADH/Me2SO or LADH/NADH/IBA. Similarly, the difference spectra of LADH/NAD+/pyrazole or LADH/ADPR with LADH are calculated. The magnitude of these difference spectra is on the order of a few percent of the protein Raman spectrum. We report and discuss the experimental configuration and control procedures we use in reliably calculating such small difference signals. These sensitive difference techniques could be applied to a large number of problems where the classical Raman spectrum of a "small" molecule, like adenine, bound to the active site of a protein is of interest. The spectrum of bound ADPR allows an assignment of the bands of the bound NADH and NAD+ spectra to normal coordinates located primarily on either the nicotinamide or the adenine moiety. By comparing the spectra of the bound coenzymes with model compound data and through the use of deuterated compounds, we confirm and characterize how the adenine moiety is involved in coenzyme binding and discuss the validity of the suggestion that the adenine ring is protonated upon binding. The nicotinamide moiety of NADH shows significant molecular changes upon binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid residues in the nicotinamide binding site contribute to catalysis by horse liver alcohol dehydrogenase

Amino acid residues Thr-178, Val-203, and Val-292, which interact with the nicotinamide ring of the coenzyme bound to alcohol dehydrogenase (ADH), may facilitate hydride transfer and hydrogen tunneling by orientation and dynamic effects. The T178S, T178V, V203A, V292A, V292S, and V292T substitutions significantly alter the steady state and transient kinetics of the enzyme. The V292A, V292S, and V292T enzymes have decreased affinity for coenzyme (NAD+ by 30-50-fold and NADH by 35-75-fold) as compared to the wild-type enzyme. The substitutions in the nicotinamide binding site decrease the rate constant of hydride transfer for benzyl alcohol oxidation by 3-fold (for V292T ADH) to 16-fold (for V203A ADH). The modest effects suggest that catalysis does not depend critically on individual residues and that several residues in the nicotinamide binding site contribute to catalysis. The structures of the V292T ADH-NAD+-pyrazole and wild-type ADH-NAD+-4-iodopyrazole ternary complexes are very similar. Only subtle changes in the V292T enzyme cause the large changes in coenzyme binding and the small change in hydride transfer. In these complexes, one pyrazole nitrogen binds to the catalytic zinc, and the other nitrogen forms a partial covalent bond with C4 of the nicotinamide ring, which adopts a boat conformation that is postulated to be relevant for hydride transfer. The results provide an experimental basis for evaluating the contributions of dynamics to hydride transfer.     

Raman spectroscopy of oxidized and reduced nicotinamide adenine dinucleotides

We have measured the Raman spectra of oxidized nicotinamide adenine dinucleotide, NAD+, and its reduced form, NADH, as well as a series of fragments and analogues of NAD+ and NADH. In addition, we have studied the effects of pH as well as deuteration of the exchangeable protons on the Raman spectra of these molecules. In comparing the positions and intensities of the peaks in the fragment and analogue spectra with those of NADH and NAD+, we find that it is useful to consider these large molecules as consisting of component parts, namely, adenine, two ribose groups, two phosphate groups, and nicotinamide, for the purpose of assigning their spectral features. The Raman bands of NADH and NAD+ are found generally to arise from molecular motions in one or another of these molecular moieties, although some peaks are not quite so easily identified in this way. This type of assignment is the first step in a detailed understanding of the Raman spectra of NAD+ and NADH. This is needed to understand the binding properties of NADH and NAD+ acting as coenzymes with the NAD-linked dehydrogenases as deduced recently by using Raman spectroscopy.     

The interaction of borate and sulfite with pyridine nucleotides.

The kinetics and equilibria of the borate interaction at ribose with NAD+ and NMN+ have been measured using as a chromophoric probe the perturbation effect borate has on the addition of sulfite to the 4 position of the nicotinamide ring. NAD+ and NMN+ have more favorable borate association constants than do their corresponding sulfite addition complexes. The rate of interaction of the ribose moiety with borate at low borate buffer concentration is dependent on the concentration of both borate and boric acid. At high borate concentration the rate becomes independent of borate concentration, indicating the existence of a two-step process for the interaction of NAD-sulfite with borate with a change of rate-determining step from the interaction of the ribose hydroxyl group with borate at low borate to an elimination of sulfite at high borate concentration. A linear free energy relationship with a slope of 0.94 describes an increased reactivity of the nucleotide for sulfite as the affinity of the nucleotide for sulfite increases.     

The absence of quinoprotein alcohol dehydrogenase in Acinetobacter calcoaceticus

It is shown that the unusual NAD(P)+-independent quinoprotein alcohol dehydrogenase, said previously to be responsible for oxidation of ethanol during growth of Acinetobacter calcoaceticus LMD 79.39, was in fact isolated from an unidentified organism which contained cytochrome c and which has now been lost. Several genuine strains of A. calcoaceticus do not contain cytochrome c nor do they contain a quinoprotein alcohol dehydrogenase. The enzyme responsible for ethanol oxidation in these bacteria is an inducible NAD+-linked alcohol dehydrogenase.