Quinohemoprotein alcohol dehydrogenases: structure, function, and physiology

Quino(hemo)protein alcohol dehydrogenases (ADH) that have pyrroloquinoline quinone (PQQ) as the prosthetic group are classified into 3 groups, types I, II, and III. Type I ADH is a simple quinoprotein having PQQ as the only prosthetic group, while type II and type III ADHs are quinohemoprotein having heme c as well as PQQ in the catalytic polypeptide. Type II ADH is a soluble periplasmic enzyme and is widely distributed in Proteobacteria such as Pseudomonas, Ralstonia, Comamonas, etc. In contrast, type III ADH is a membrane-bound enzyme working on the periplasmic surface solely in acetic acid bacteria. It consists of three subunits that comprise a quinohemoprotein catalytic subunit, a triheme cytochrome c subunit, and a third subunit of unknown function. The catalytic subunits of all the quino(hemo)protein ADHs have a common structural motif, a quinoprotein-specific superbarrel domain, where PQQ is deeply embedded in the center. In addition, in the type II and type III ADHs this subunit contains a unique heme c domain. Various type II ADHs each have a unique substrate specificity, accepting a wide variety of alcohols, as is discussed on the basis of recent X-ray crystallographic analyses. Electron transfer within both type II and III ADHs is discussed in terms of the intramolecular reaction from PQQ to heme c and also from heme to heme, and in terms of the intermolecular reaction with azurin and ubiquinone, respectively. Unique physiological functions of both types of quinohemoprotein ADHs are also discussed.     

Natural alcohol exposure: is ethanol the main substrate for alcohol dehydrogenases in animals?

Alcohol dehydrogenase (ADH) activity is widely distributed in all phyla. In animals, three non-homologous NAD(P)(+)-dependent ADH protein families are reported. These arose independently throughout evolution and possess different structures and mechanisms of reaction: type I (medium-chain) ADHs are zinc-containing enzymes and comprise the most studied group in vertebrates; type II (short-chain) ADHs lack metal cofactor and have been extensively studied in Drosophila; and type III ADHs are iron-dependent/-activated enzymes that were initially identified only in microorganisms. The presence of these different ADHs in animals has been assumed to be a consequence of chronic exposure to ethanol. By far the most common natural source of ethanol is fermentation of fruit sugars by yeast, and available data support that this fruit trait evolved in concert with the characteristics of their frugivorous seed dispersers. Therefore, if the presence of ADHs in animals evolved as an adaptive response to dietary ethanol exposure, then it can be expected that the enzymogenesis of these enzymes began after the appearance of angiosperms with fleshy fruits, because substrate availability must precede enzyme selection. In this work, available evidence supporting this possibility is discussed. Phylogenetic analyses reveal that type II ADHs suffered several duplications, all of these restricted to flies (order Diptera). Induction of type II Adh by ethanol exposure, a positive correlation between ADH activity and ethanol resistance, and the fact that flies and type II Adh diversification occurred in concert with angiosperm diversification, strongly suggest that type II ADHs were recruited to allow larval flies to exploit new restricted niches with high ethanol content. In contrast, phyletic distribution of types I and III ADHs in animals showed that these appeared before angiosperms and land plants, independently of ethanol availability. Because these enzymes are not induced by ethanol exposure and possess a high affinity and/or catalytic efficiency for non-ethanol endogenous substrates, it can be concluded that the participation of types I and III ADHs in ethanol metabolism can be considered as incidental, and not adaptive.     

Three distinct quinoprotein alcohol dehydrogenases are expressed when Pseudomonas putida is grown on different alcohols.

A bacterial strain that can utilize several kinds of alcohols as its sole carbon and energy sources was isolated from soil and tentatively identified as Pseudomonas putida HK5. Three distinct dye-linked alcohol dehydrogenases (ADHs), each of which contained the prosthetic group pyrroloquinoline quinone (PQQ), were formed in the soluble fractions of this strain grown on different alcohols. ADH I was formed most abundantly in the cells grown on ethanol and was similar to the quinoprotein ADH reported for P. putida (H. Görisch and M. Rupp, Antonie Leeuwenhoek 56:35-45, 1989) except for its isoelectric point. The other two ADHs, ADH IIB and ADH IIG, were formed separately in the cells grown on 1-butanol and 1,2-propanediol, respectively. Both of these enzymes contained heme c in addition to PQQ and functioned as quinohemoprotein dehydrogenases. Potassium ferricyanide was an available electron acceptor for ADHs IIB and IIG but not for ADH I. The molecular weights were estimated to be 69,000 for ADH IIB and 72,000 for ADH IIG, and both enzymes were shown to be monomers. Antibodies raised against each of the purified ADHs could distinguish the ADHs from one another. Immunoblot analysis showed that ADH I was detected in cells grown on each alcohol tested, but ethanol was the most effective inducer. ADH IIB was formed in the cells grown on alcohols of medium chain length and also on 1,3-butanediol. Induction of ADH IIG was restricted to 1,2-propanediol or glycerol, of which the former alcohol was more effective. These results from immunoblot analysis correlated well with the substrate specificities of the respective enzymes. Thus, three distinct quinoprotein ADHs were shown to be synthesized by a single bacterium under different growth conditions.     

Purification and characterization of a primary-secondary alcohol dehydrogenase from two strains of Clostridium beijerinckii.

Two primary alcohols (1-butanol and ethanol) are major fermentation products of several clostridial species. In addition to these two alcohols, the secondary alcohol 2-propanol is produced to a concentration of about 100 mM by some strains of Clostridium beijerinckii. An alcohol dehydrogenase (ADH) has been purified to homogeneity from two strains (NRRL B593 and NESTE 255) of 2-propanol-producing C. beijerinckii. When exposed to air, the purified ADH was stable, whereas the partially purified ADH was inactivated. The ADHs from the two strains had similar structural and kinetic properties. Each had a native M(r) of between 90,000 and 100,000 and a subunit M(r) of between 38,000 and 40,000. The ADHs were NADP(H) dependent, but a low level of NAD(+)-linked activity was detected. They were equally active in reducing aldehydes and 2-ketones, but a much lower oxidizing activity was obtained with primary alcohols than with secondary alcohols. The kcat/Km value for the alcohol-forming reaction appears to be a function of the size of the larger alkyl substituent on the carbonyl group. ADH activities measured in the presence of both acetone and butyraldehyde did not exceed activities measured with either substrate present alone, indicating a common active site for both substrates. There was no similarity in the N-terminal amino acid sequence between that of the ADH and those of fungi and several other bacteria. However, the N-terminal sequence had 67% identity with those of two other anaerobes, Thermoanaerobium brockii and Methanobacterium palustre. Furthermore, conserved glycine and tryptophan residues are present in ADHs of these three anaerobic bacteria and ADHs of mammals and green plants.     

Characterization of a zinc-containing alcohol dehydrogenase with stereoselectivity from the hyperthermophilic archaeon Thermococcus guaymasensis

An alcohol dehydrogenase (ADH) from hyperthermophilic archaeon Thermococcus guaymasensis was purified to homogeneity and was found to be a homotetramer with a subunit size of 40 ± 1 kDa. The gene encoding the enzyme was cloned and sequenced; this gene had 1,095 bp, corresponding to 365 amino acids, and showed high sequence homology to zinc-containing ADHs and l-threonine dehydrogenases with binding motifs of catalytic zinc and NADP(+). Metal analyses revealed that this NADP(+)-dependent enzyme contained 0.9 ± 0.03 g-atoms of zinc per subunit. It was a primary-secondary ADH and exhibited a substrate preference for secondary alcohols and corresponding ketones. Particularly, the enzyme with unusual stereoselectivity catalyzed an anti-Prelog reduction of racemic (R/S)-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol. The optimal pH values for the oxidation and formation of alcohols were 10.5 and 7.5, respectively. Besides being hyperthermostable, the enzyme activity increased as the temperature was elevated up to 95°C. The enzyme was active in the presence of methanol up to 40% (vol/vol) in the assay mixture. The reduction of ketones underwent high efficiency by coupling with excess isopropanol to regenerate NADPH. The kinetic parameters of the enzyme showed that the apparent K(m) values and catalytic efficiency for NADPH were 40 times lower and 5 times higher than those for NADP(+), respectively. The physiological roles of the enzyme were proposed to be in the formation of alcohols such as ethanol or acetoin concomitant to the NADPH oxidation.     

Molecular characterization of the recombinant iron-containing alcohol dehydrogenase from the hyperthermophilic Archaeon, Thermococcus strain ES1

The gene encoding a thermostable iron-containing alcohol dehydrogenase from Thermococcus Strain ES1 (ES1 ADH) was cloned, sequenced and expressed in Escherichia coli. The recombinant and native ES1 ADHs were purified using multistep column chromatography under anaerobic conditions. Both enzymes appeared to be homotetramers with a subunit size of 45 ± 1 kDa as revealed by SDS-PAGE, which was close to the calculated value (44.8 kDa). The recombinant ADH contained 1.0 ± 0.1 g-atom iron per subunit. Both enzymes were sensitive to oxygen with a half-life upon exposure to air of about 4 min. The recombinant enzyme exhibited a specific activity of 105 ± 2 U mg⁻¹, which was very similar to that of the native enzyme (110 ± 3 U mg⁻¹). The optimal pH-values for both enzymes for ethanol oxidation and acetaldehyde reduction were 10.4 and 7.0, respectively. Both enzymes also showed similar temperature-dependent activities, and catalyzed the oxidation of primary alcohols, but there was no activity towards methanol and secondary alcohols. Kinetic parameters of the enzymes showed lower K _m-values for acetaldehyde and NADPH and higher K _m-values for ethanol and NADP⁺. It is concluded that the gene encoding ES1 ADH was expressed successfully in E. coli. This is the first report of a fully active recombinant version of an iron-containing ADH from a hyperthermophile.     

Two zebrafish alcohol dehydrogenases share common ancestry with mammalian class I, II, IV, and V alcohol dehydrogenase genes but have distinct functional characteristics

Ethanol is teratogenic to many vertebrates. We are utilizing zebrafish as a model system to determine whether there is an association between ethanol metabolism and ethanol-mediated developmental toxicity. Here we report the isolation and characterization of two cDNAs encoding zebrafish alcohol dehydrogenases (ADHs). Phylogenetic analysis of these zebrafish ADHs indicates that they share a common ancestor with mammalian class I, II, IV, and V ADHs. The genes encoding these zebrafish ADHs have been named Adh8a and Adh8b by the nomenclature committee. Both genes were genetically mapped to chromosome 13. The 1450-bp Adh8a is 82, 73, 72, and 72% similar at the amino acid level to the Baltic cod ADH8 (previously named ADH1), the human ADH1B2, the mouse ADH1, and the rat ADH1, respectively. Also, the 1484-bp Adh8b is 77, 68, 67, and 66% similar at the amino acid level to the Baltic cod ADH8, the human ADH1B2, the mouse ADH1, and the rat ADH1, respectively. ADH8A and ADH8B share 86% amino acid similarity. To characterize the functional properties of ADH8A and ADH8B, recombinant proteins were purified from SF-9 insect cells. Kinetic studies demonstrate that ADH8A metabolizes ethanol, with a V(max) of 13.4 nmol/min/mg protein, whereas ADH8B does not metabolize ethanol. The ADH8A K(m) for ethanol as a substrate is 0.7 mm. 4-Methyl pyrazole, a classical competitive inhibitor of class I ADH, failed to inhibit ADH8A. ADH8B has the capacity to efficiently biotransform longer chain primary alcohols (>/=5 carbons) and S-hydroxymethlyglutathione, whereas ADH8A does not efficiently metabolize these substrates. Finally, mRNA expression studies indicate that both ADH8A and ADH8B mRNA are expressed during early development and in the adult brain, fin, gill, heart, kidney, muscle, and liver. Together these results indicate that class I-like ADH is conserved in zebrafish, albeit with mixed functional properties.     

Characterization of thermotolerant Acetobacter pasteurianus strains and their quinoprotein alcohol dehydrogenases

We isolated several thermotolerant Acetobacter species of which MSU10 strain, identified as Acetobacter pasteurianus, could grow well on agar plates at 41°C, tolerate to 1.5% acetic acid or 4% ethanol at 39°C, similarly seen with A. pasteurianus SKU1108 previously isolated. The MSU10 strain showed higher acetic acid productivity in a medium containing 6% ethanol at 37°C than SKU1108 while SKU1108 strain could accumulate more acetic acid in a medium supplemented with 4–5% ethanol at the same temperature. The fermentation ability at 37°C of these thermotolerant strains was superior to that of mesophilic A. pasteurianus IFO3191 strain having weak growth and very delayed acetic acid production at 37°C even at 4% ethanol. Alcohol dehydrogenases (ADHs) were purified from MSU10, SKU1108, and IFO3191 strains, and their properties were compared related to the thermotolerance. ADH of the thermotolerant strains had a little higher optimal temperature and heat stability than that of mesophilic IFO3191. More critically, ADHs from MSU10 and SKU1108 strains exhibited a higher resistance to ethanol and acetic acid than IFO3191 enzyme at elevated temperature. Furthermore, in this study, the ADH genes were cloned, and the amino acid sequences of ADH subunit I, subunit II, and subunit III were compared. The difference in the amino acid residues could be seen, seemingly related to the thermotolerance, between MSU10 or SKU1108 ADH and IFO 3191 ADH.     

Oxidation of methanol, ethylene glycol, and isopropanol with human alcohol dehydrogenases and the inhibition by ethanol and 4-methylpyrazole

Human alcohol dehydrogenases (ADHs) include multiple isozymes with broad substrate specificity and ethnic distinct allozymes. ADH catalyzes the rate-limiting step in metabolism of various primary and secondary aliphatic alcohols. The oxidation of common toxic alcohols, that is, methanol, ethylene glycol, and isopropanol by the human ADHs remains poorly understood. Kinetic studies were performed in 0.1M sodium phosphate buffer, at pH 7.5 and 25°C, containing 0.5 mM NAD(+) and varied concentrations of substrate. K(M) values for ethanol with recombinant human class I ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, and ADH1C2, and class II ADH2 and class IV ADH4 were determined to be in the range of 0.12-57 mM, for methanol to be 2.0-3500 mM, for ethylene glycol to be 4.3-2600mM, and for isopropanol to be 0.73-3400 mM. ADH1B3 appeared to be inactive toward ethylene glycol, and ADH2 and ADH4, inactive with methanol. The variations for V(max) for the toxic alcohols were much less than that of the K(M) across the ADH family. 4-Methylpyrazole (4MP) was a competitive inhibitor with respect to ethanol for ADH1A, ADH1B1, ADH1B2, ADH1C1 and ADH1C2, and a noncompetitive inhibitor for ADH1B3, ADH2 and ADH4, with the slope inhibition constants (K(is)) for the whole family being 0.062-960 μM and the intercept inhibition constants (K(ii)), 33-3000 μM. Computer simulation studies using inhibition equations in the presence of alternate substrate ethanol and of dead-end inhibitor 4MP with the determined corresponding kinetic parameters for ADH family, indicate that the oxidation of the toxic alcohols up to 50mM are largely inhibited by 20 mM ethanol or by 50 μM 4MP with some exceptions. The above findings provide an enzymological basis for clinical treatment of methanol and ethylene glycol poisoning by 4MP or ethanol with pharmacogenetic perspectives.     

Purification and properties of F420- and NADP(+)-dependent alcohol dehydrogenases of Methanogenium liminatans and Methanobacterium palustre, specific for secondary alcohols

The F420-dependent alcohol dehydrogenase (ADH) of Methanogenium liminatans and the NADP(+)-dependent ADH of Methanobacterium palustre were purified to homogeneity. The native F420-dependent ADH of Mg. liminatans had a molecular mass of 150 kDa and consisted of four (presumably identical) subunits with a mass of 39 kDa. The temperature optimum was 42 degrees C, the optimum pH 6.0 and NaCl or KCl were inhibitory. The NADP(+)-dependent ADH of Mb. palustre had a molecular mass of 175 kDa and consisted also of four (presumably identical) subunits with a mass of 44 kDa. The temperature optimum was 60 degrees C, the optimum pH 8.0 and optimal activity was observed in the presence of 500 mM NaCl or KCl. The ADHs of both organisms catalysed the oxidation of various secondary and cyclic alcohols to the corresponding ketones and the reverse reaction. No primary alcohols were apparently oxidized. The NADP(+)-dependent ADH of Mb. palustre contained 4-8 mol atoms zinc/mol enzyme and was inhibited by low concentrations of iodoacetate and 4-hydroxymercuribenzoate, whereas the F420-dependent ADH of Mg. liminatans presumably contained no zinc ions and was inhibited by 1,10-phenanthroline or high concentrations (e.g. 100 microM) of 4-hydroxymercuribenzoate. Polyclonal antibodies against the NADP(+)-dependent ADH of Mb. palustre precipitated only the homologous ADH. A precipitation of the NADP(+)-dependent ADH of Methanocorpusculum parvum required a 10-fold higher antibody concentration, showing at least a distant relationship of both ADHs. Antibodies against the NADP(+)-dependent ADH of Mcp. parvum, however, formed precipitates with the homologous ADH of Mcp. parvum and with the NADP(+)-dependent ADH of Mb. palustre. They also formed precipitates with the ADH of Thermoanaerobium brockii, which is not related to methane bacteria. Antibodies against the F420-dependent ADH of Mg. liminatans reacted only with the homologous enzyme and did not form precipitates with NADP(+)-dependent ADHs. No immunological relation of the NADP(+)- or F420-dependent ADHs of methanogens with ADH of yeast or horse liver was found. In accordance with the immunological data, the N-terminal amino acid sequences of the NADP(+)-dependent ADHs of Mb. palustre and Mcp. parvum had a high degree of similarity, whereas the N-terminal amino acid sequence of the ADH of Mg. liminatans revealed no similarity with the two NADP(+)-dependent enzymes.     

Mammalian alcohol dehydrogenase of higher classes: analyses of human ADH5 and rat ADH6

Alcohol dehydrogenases (ADH) of classes V and VI, ADH5 and ADH6, have been defined in man and rodents, respectively. Sequence data have been obtained at cDNA and genomic levels, but limited data are available for functionality and substrate repertoire. The low positional identity (65%) between the two ADHs, place them into separate classes. We have shown that the ADH5 gene yields two differently processed mRNAs and harbors a gene organization identical to other mammalian ADHs. This is probably due to an alternative splicing in the eighth intron that results in a shorter message missing the ninth exon or a normal message with the expected number of codons. The isolated rat ADH6 cDNA was found to be fused to ADH2 at the 5′-end. The resulting main open reading frame translates into an N-terminally extended polypeptide. In vitro translation results in a polypeptide of about 42 kDa and further, protein was possible to express in COS cells as a fusion product with Green Fluorescent Protein. Both ADH5 and ADH6 show genes and gene products that are processed comparably to other mammalian ADHs and the deduced amino acid sequences indicate a lack of ethanol dehydrogenase activity that probably explains why no corresponding proteins have been isolated. The functionality of these ADHs is therefore still an enigma.     

Purification and molecular properties of a Class II alcohol dehydrogenase (ADH-C2) from horse liver

An alcohol dehydrogenase (ADH) from horse liver was purified by ion-exchange and affinity chromatography. The enzyme (designated ADH-C2), is a dimer with a similar subunit size (47,300 mol. wt), as determined by SDS-polyacrylamide gel electrophoresis, to other mammalian ADHs. Zinc analyses and 1,10 phenanthroline inhibition studies indicated that each subunit contained 2 g atoms of zinc, with at least one involved catalytically. The enzyme exhibited similar kinetic properties to human pi-ADH and mouse ADH-C2, previously classified as class II ADHs [Vallee and Bazzone (1983) Isozymes, Vol. 8, pp. 219-244; Algar et al. (1983) Eur. J. Biochem. 137, 139-147] but differed in most respects from the extensively investigated horse Class I ADHs; EE, ES and SS. Horse ADH-C2 exhibited a Km value for ethanol of 42 mM and a broad substrate specificity, with Km values decreasing dramatically with an increase in chain length. The enzyme was much less sensitive to pyrazole inhibition (by at least 3 orders of magnitude) as compared with the Class I ADHs.     

Mammalian alcohol dehydrogenase of higher classes: analyses of human ADH5 and rat ADH6

Alcohol dehydrogenases (ADH) of classes V and VI, ADH5 and ADH6, have been defined in man and rodents, respectively. Sequence data have been obtained at cDNA and genomic levels, but limited data are available for functionality and substrate repertoire. The low positional identity (65%) between the two ADHs, place them into separate classes. We have shown that the ADH5 gene yields two differently processed mRNAs and harbors a gene organization identical to other mammalian ADHs. This is probably due to an alternative splicing in the eighth intron that results in a shorter message missing the ninth exon or a normal message with the expected number of codons. The isolated rat ADH6 cDNA was found to be fused to ADH2 at the 5'-end. The resulting main open reading frame translates into an N-terminally extended polypeptide. In vitro translation results in a polypeptide of about 42 kDa and further, protein was possible to express in COS cells as a fusion product with Green Fluorescent Protein. Both ADH5 and ADH6 show genes and gene products that are processed comparably to other mammalian ADHs and the deduced amino acid sequences indicate a lack of ethanol dehydrogenase activity that probably explains why no corresponding proteins have been isolated. The functionality of these ADHs is therefore still an enigma.     

Comparison of alcohol dehydrogenases from wild-type and mutant strain,JW200 Fe 4, of ‘Thermoanaerobacter ethanolicus’

Summary A mutant strain of Thermoanaerobacter ethanolicus (ATCC 31 550) designated JW200 Fe 4 contains primary and secondary alcohol dehydrogenases (ADHs). The primary ADH from JW000 Fe 4 was formed early in the growth cycle compared to the primary ADH form the wild-type strain (JW200 wt). The secondary ADH displayed 2.5-fold greater activity during the growth cycle of JW200 Fe 4 compared to the secondary ADH form JW200 wt. Both primary and secondary ADHs from JW200 Fe 4 were purified to homogeneity ADHs from JW200 Fe 4 were purified to homogeneity as determined by sodium dodecyl sulphate-gel electrophoresis. Relative molecular weight estimations indicated that both ADHs were tetrameric. Each ADH from JW200 Fe 4 contained approximately four Zn atoms per subunit and displayed Arrhenius plots similar to the ADHs from JW200 wt. The substrate specificity for the ADHs from JW200 Fe 4 was similar to that of the ADHs from JW200 wt. The secondary ADH oxidized 2-propanol at 51 times the rate of ethanol. Both ADHs from JW200 Fe 4 apparently reduce acetaldehyde to ethanol while only the secondary ADH from JW200 wt was suggested to contribute significantly to ethanol production.     

Developmental and environmental influences in the production of a single NAD-dependent fermentative alcohol dehydrogenase by the zygomycete Mucor rouxii

A soluble NAD-dependent alcohol dehydrogenase (ADH) activity was detected in mycelium and yeast cells of wild-type Mucor rouxii. In the mycelium of cells grown in the absence of oxygen, the enzyme activity was high, whereas in yeast cells, ADH activity was high regardless of the presence or absence of oxygen. The enzyme from aerobically or anaerobically grown mycelium or yeast cells exhibited a similar optimum pH for the oxidation of ethanol to acetaldehyde (∼pH 8.5) and for the reduction of acetaldehyde to ethanol (∼pH 7.5). Zymogram analysis conducted with cell-free extracts of the wild-type and an alcohol-dehydrogenase-deficient mutant strain indicated the existence of a single ADH enzyme that was independent of the developmental stage of dimorphism, the growth atmosphere, or the carbon source in the growth medium. Purified ADH from aerobically grown mycelium was found to be a tetramer consisting of subunits of 43 kDa. The enzyme oxidized primary and secondary alcohols, although much higher activity was displayed with primary alcohols. K _m values obtained for acetaldehyde, ethanol, NADH₂, and NAD⁺ indicated that physiologically the enzyme works mainly in the reduction of acetaldehyde to ethanol. Received: 11 March 1999 / Accepted: 14 July 1999     

Isolation of alcohol dehydrogenases from germinating seeds of pea,broad-bean,lentil and kidney-bean

The maximum of alcohol dehydrogenase (ADH) activity of germinating pea and broad-bean seeds sediments from 40 to 60% ammonium sulfate saturation, from lentil and kidney-bean seeds between 40 and 50%. This operation increased the specific activity of ADH preparations roughly tenfold. Chromatography on DEAE-eellulose and gel filtration increased the activity of the resulting preparation when compared with the initial preparation 178 times with pea, 334 times with broad-bean, 122 times with lentil and 77 times with kidney-bean. The ADHs resemble each other in coenzyme specificity: the reaction rate with NAD is one hundred times greater than with NADP. The substrate specificity is quite wide: besides ethanol, these enzymes oxidize 2-propene-l-ol (actually faster than ethanol), 2-butene-l-ol (at the rate of one half t h a t of ethanol) and butanol (even more slowly). In general, saturated alcohol analogues are oxidized more slowly than unsaturated ones. Methanol is a substrate for the enzym from pea only. The ADHs of the plants studied did not oxidize diols, sugar alcohols and cyclic alcohols. The enzyme from pea has the widest substrate specificity oxidizing isobutanol, phenylalcohol and mercaptoethanol. ADHs, which are widely encountered in plants, resemble each other to a certain degree — they have identical coenzymes, equal K_m values and equal values of the pH optimum, they differ in the purification process and in substrate specificity.     

Membrane-bound quinoprotein D-arabitol dehydrogenase of Gluconobacter suboxydans IFO 3257: a versatile enzyme for the oxidative fermentation of various ketoses

Solubilization of membrane-bound quinoprotein D-arabitol dehydrogenase (ARDH) was done successfully with the membrane fraction of Gluconobacter suboxydans IFO 3257. In enzyme solubilization and subsequent enzyme purification steps, special care was taken to purify ARDH as active as it was in the native membrane, after many disappointing trials. Selection of the best detergent, keeping ARDH as the holoenzyme by the addition of PQQ and Ca2+, and of a buffer system involving acetate buffer supplemented with Ca2+, were essential to treat the highly hydrophobic and thus labile enzyme. Purification of the enzyme was done by two steps of column chromatography on DEAE-Toyopearl and CM-Toyopearl in the presence of detergent and Ca2+. ARDH was homogenous and showed a single sedimentation peak in analytical ultracentrifugation. ARDH was dissociated into two different subunits upon SDS-PAGE with molecular masses of 82 kDa (subunit I) and 14 kDa (subunit II), forming a heterodimeric structure. ARDH was proven to be a quinoprotein by detecting a liberated PQQ from SDS-treated ARDH in HPLC chromatography. More preliminarily, an EDTA-treated membrane fraction lost the enzyme activity and ARDH activity was restored to the original level by the addition of PQQ and Ca2+. The most predominant unique character of ARDH, the substrate specificity, was highly versatile and many kinds of substrates were oxidized irreversibly by ARDH, not only pentitols but also other polyhydroxy alcohols including D-sorbitol, D-mannitol, glycerol, meso-erythritol, and 2,3-butanediol. ARDH may have its primary function in the oxidative fermentation of ketose production by acetic acid bacteria. ARDH contained no heme component, unlike the type II or type III quinoprotein alcohol dehydrogenase (ADH) and did not react with primary alcohols.     

Opossum alcohol dehydrogenases: Sequences, structures, phylogeny and evolution: evidence for the tandem location of ADH genes on opossum chromosome 5

BLAT (BLAST-Like Alignment Tool) analyses and interrogations of the recently published opossum genome were undertaken using previously reported rat ADH amino acid sequences. Evidence is presented for six opossum ADH genes localized on chromosome 5 and organized in a comparable ADH gene cluster to that reported for human and rat ADH genes. The predicted amino acid sequences and secondary structures for the opossum ADH subunits and the intron-exon boundaries for opossum ADH genes showed a high degree of similarity with other mammalian ADHs, and four opossum ADH classes were identified, namely ADH1, ADH3, ADH6 and ADH4 (for which three genes were observed: ADH4A, ADH4B and ADH4C). Previous biochemical analyses of opossum ADHs have reported the tissue distribution and properties for these enzymes: ADH1, the major liver enzyme; ADH3, widely distributed in opossum tissues with similar kinetic properties to mammalian class 3 ADHs; and ADH4, for which several forms were localized in extrahepatic tissues, especially in the digestive system and in the eye. These ADHs are likely to perform similar functions to those reported for other mammalian ADHs in the metabolism of ingested and endogenous alcohols and aldehydes. Phylogenetic analyses examined opossum, human, rat, chicken and cod ADHs, and supported the proposed designation of opossum ADHs as class I (ADH1), class III (ADH3), class IV (ADH4A, ADH4B and ADH4C) and class VI (ADH6). Percentage substitution rates were examined for ADHs during vertebrate evolution which indicated that ADH3 is evolving at a much slower rate to that of the other ADH classes.     

Oxidation and reduction of 4-hydroxyalkenals catalyzed by isozymes of human alcohol dehydrogenase

4-Hydroxyalkenals, natural cytotoxic products of lipid peroxidation, are substrates for human alcohol dehydrogenases (ADH). Class I and II ADHs reduce aliphatic 4-hydroxyalkenals with chain lengths of from 5 to 15 carbons at pH 7 with kcat and Km values comparable to simple aliphatic aldehydes of the same chain length. Class II is particularly effective in the reduction with kcat values as high as 3300 min-1 for 4-hydroxyundecenal. Class III ADH is essentially inactive toward all of these substrates. The class I and II isozymes also catalyze the oxidation of the 4-hydroxy group at pH 10. However, during the reaction, an NAD(+)-dependent irreversible partial inactivation of the alpha beta 1 isozyme is observed which is attributed, with the aid of computer graphics modeling, to selective modification of the alpha subunit. Both ethanol and 1,10-phenanthroline, known to compete with conventional substrates, instantaneously, reversibly, and competitively inhibit 4-hydroxyalkenal reduction and oxidation, indicating that 4-hydroxyalkenals bind at the same site as do conventional substates. The fact that the class II enzyme pi pi-ADH so far is found only in the liver and that the 4-hydroxyalkenals are the best substrates known for this isozyme suggest that it may play a significant role in cellular defenses in the conversion of the cytotoxic aldehydes to the less reactive 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.