On plant alcohol dehydrogenases

We have found in a number of plants (lentil, lupine, bean, barley, oats, rye, wheat, cucumber, melon, flax, sunflower and rape) that varying amounts of ethanol are formed under natural anaerobiosis and, that in later growth periods these plants continue to react to anaerobiosis by formation of ethanol. When the testa has opened in germinating plants or, when plants are transferred from the anaerobic atmosphere to air, ethanol disappears. Plants contain alcohol dehydrogenases, the activity of which depends on the alcohol concentration in their tissue; the maximum concentration is reached during natural anaerobiosis, rising in the course of further growth when the plants are kept in a nitrogen atmosphere. Alcohol dehydrogenases of the plants studied are localised in the soluble cell fraction notsedimenting at 120 000 g, their pH optimum is in the weakly alkaline region and their Michaelis constants are equal in order of magnitude (10^?5 m). They are all inhibited in the same way by Zn²⁺, Cu²⁺, Hg²⁺, B₄ O ₇ ^2? ions, p-chloromercuric benzoate, iodoacetate, EDTA and phenantroline, which may be considered as evidence of the presence of ?SH groups. The specific activity of alcohol dehydrogenase preparations is higher in plants grown in light than in plants grown in the dark. The specific activity of plant alcohol dehydrogenases can be increased by precipitation with ammonium sulphate by at most one order of magnitude, while all the activity is lost by this purification process in the case of cereals. The following isoenzyme composition of ADH was found by means of electrophoresis on polyacrylamide: the enzyme from poas and sunflower, for example, is composed of three, that from wheat and oats six, the enzyme from maize and barley of five isoenzymes.     

Characteristics of alcohol/polyol dehydrogenases. The zinc-containing long-chain alcohol dehydrogenases

Sixteen characterized alcohol dehydrogenases and one sorbitol dehydrogenase have been aligned. The proteins represent two formally different enzyme activities (EC 1.1.1.1 and EC 1.1.1.14), three different types of molecule (dimeric alcohol dehydrogenase, tetrameric alcohol dehydrogenase, tetrameric sorbitol dehydrogenase), metalloproteins with different zinc contents (1 or 2 atoms per subunit), and polypeptide chains from different kingdoms and orders (mammals, higher plants, fungus, yeasts). Present comparisons utilizing all 17 forms reveal extensive variations in alcohol dehydrogenase, but with evolutionary changes that are of the same order in different branches and at different times. They emphasize the general importance of particular residues, suggesting related overall functional constraints in the molecules. The comparisons also define a few coincidences between intron positions in the genes and gap positions in the gene products. Only 22 residues are strictly conserved; half of these are Gly, and most of the remaining ones are Pro or acidic residues. No basic residue, no straight-chain hydrophobic residues, no aromatic residues, and essentially no branched-chain or polar neutral residues are invariable. Tentative consensus sequences were calculated, defining 13 additional residues likely to be typical of but not invariant among the alcohol dehydrogenases. These show a predominance of Val, charged residues, and Gly. Combined, the comparisons, which are particularly relevant to the data base for protein engineering, illustrate the requirements for functionally important binding interactions, and the extent of space restrictions in proteins with related overall conformations and functions.     

Quinone-dependent alcohol dehydrogenases and fad-dependent alcohol oxidases

This review considers quinone-dependent alcohol dehydrogenases and FAD-dependent alcohol oxidases, enzymes that are present in numerous methylotrophic eu- and prokaryotes and significantly differ in their primary and quaternary structure. The cofactors of the enzymes are bound to the protein polypeptide chain through ionic and hydrophobic interactions. Microorganisms containing these enzymes are described. Methods for purification of the enzymes, their physicochemical properties, and spatial structures are considered. The supposed mechanism of action and practical application of these enzymes as well as their producers are discussed.     

Antibody-supported screening of alcohol dehydrogenases

Polyclonal antibodies raised against purified (R)-specific alcohol dehydrogenase of Lactobacillus kefir were used in Western blot analyses to search for structurally or immunologically related proteins. No immunochemical reactions were found with commercially available alcohol dehydrogenases (from yeast, horse liver and Thermoanaerobium brockii), but screening among the genus Lactobacillus revealed that each strain of a subgroup of Betabacterium gave positive results whereas strains of the other subgroups of Lactobacillus were found to be inactive. However, enzymatic assays with these antibody-positive strains showed, that besides L. kefir itself, only the strains of L. brevis possess alcohol dehydrogenase activity with acetophenone and NADPH as substrates.     

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.     

Human alcohol dehydrogenases and serotonin metabolism

Human liver alcohol dehydrogenases (ADH) may participate in serotonin (5-hydroxytryptamine) metabolism. Class I and II isozymes catalyze the oxidation of 5-hydroxytryptophol (5-HTOL) with kcat/Km values ranging from 10 to 100 mM-1 min-1 compared to 4-66 mM-1 min-1 for that of ethanol at pH 7.40, 25 degrees C. The product, 5-hydroxyindoleacetaldehyde, was purified as its semicarbazone and identified by mass spectrometry. Ethanol competitively inhibits 5-HTOL oxidation by beta 1 gamma 2 ADH with a Ki of 440 microM, a value similar to the Km of ethanol, 210 microM. The inhibition constants for 1,10-phenanthroline and 4-methylpyrazole are 20 microM and 80 nM respectively, essentially identical to those obtained with ethanol as substrate, 22 microM and 70 nM, respectively. The competition between ethanol and 5-HTOL for ADH can explain observations of ethanol induced changes in serotonin metabolism in vivo.     

Fluorometric assay for alcohol sulfotransferase

A sensitive fluorometric assay was developed for alcohol sulfotransferase (AST). This was the first continuous fluorometric assay reported for AST. It used 3'-phosphoadenosine 5'-phosphosulfate regenerated from 3-phosphoadenosine 5'-phosphate by a recombinant phenol sulfotransferase (PST) using 4-methylumbelliferyl sulfate as the sulfuryl group donor. The recombinant PST did not use the alcohol substrate under the designed condition, and the sensitivity for AST activity was found to be comparable to that of radioactive assay as reported in the literature. The change of fluorescence intensity of 4-methylumbelliferone corresponded directly to the amount of active AST and was sensitive enough to measure nanogram or picomole amounts of the enzyme activity. This fluorometric assay was used to determine the activities of AST as purified form and in crude extracts of pig liver, rat liver, and Escherichia coli. Some properties of human dehydroepiandrosterone sulfotransferase were determined by this method and were found to be comparable to published data. Under similar assay conditions, the contaminated activities of arylsulfatase in crude extracts were also determined. This method not only is useful for the routine and detailed kinetic study of this important class of enzymes but also has the potential for the development of a high-throughput procedure using microplate reader.     

Inhibition of alcohol dehydrogenases by thiol compounds

2-Mercaptoethanol is a strong inhibitor of LADH. The inhibitory effect is likely due to the binding of the SH group to the enzymatic zinc ion. Various thiol compounds do not inhibit YADH and it is suggested that the zinc atoms involved in the catalytic mechanism of LADH and YADH may have different structural arrangements and that these zinc atoms in YADH may not be blocked by thiol compounds. Thiol compounds also quench the enhanced fluorescence of LADH-NADH in a pH-dependent manner. At pH 9.2, the binding of coenzyme to LADH is replaced by 2-mercaptoethanol, whilst at pH 7.3, it further quenches the fluorescence of NADH-LADH. This quenching of fluorescence is likely attributed to a conformational change and energy transfer upon binding of 2-mercaptoethanol to the LADH-NADH complex. Complete reversal of the inhibitory effect of thiol compounds on LADH can be obtained by dialysis.     

Structures of human alcohol and aldehyde dehydrogenases

Human alcohol dehydrogenase is a dimeric zinc metalloenzyme for which forms of three classes, I, II and III, have been distinguished. Subunits hybridize within but not between classes. There are three types of subunit, alpha, beta, and gamma, in class I. The primary structures of all three forms have been established, as well as the overall properties and the effects of the amino acid substitutions between the various forms. Each subunit has 374 residues, of which 35 exhibit differences among the alpha, beta and gamma chains. Corresponding cDNA structures are also known, as are the genetic organization and details of the gene structures. Allelic variants occur at the beta and gamma loci. Corresponding amino acid substitutions have been characterized, and enzymatic differences between the allelic forms are explained by defined residue exchanges. The results also illustrate recent and repeated isozyme evolution, a subject where alcohol dehydrogenases exceptionally well offer detailed examples. Human aldehyde dehydrogenase occurs of two types, a mitochondrial and a cytosolic form. The enzymes are tetramers, do not contain functional metals, and have subunits which do not form inter-type hybrids. The primary structures have been determined, revealing a positional identity of 68% (in 500 residues) between the mitochondrial and cytosolic forms. The N-terminus is heterogeneous and is not blocked in the subunit of the mitochondrial enzyme, in contrast to that of the cytosolic enzyme or those of all the alcohol dehydrogenases (also cytosolic). A reactive cysteine residue at position 302 has been ascribed functional importance at or close to the active site, is conserved in the two aldehyde dehydrogenases, and is associated with the action of disulfiram on the enzyme. In Oriental populations, a mutant allelic variant of the mitochondrial protein with impaired enzyme function has also been characterized.     

Substituent effects in alkylated liver alcohol dehydrogenases

Alcohol dehydrogenase from horse liver was reductively alkylated with aldehydes having varied alkyl substituents. Kinetic studies of alkylated liver alcohol dehydrogenases which were modified in the absence and in the presence of NADH indicate that the alkylation of the specific lysine residues generally activates the enzyme by increasing Michaelis and inhibition constants for substrates and maximum velocities for the reactions. These kinetic parameters were analyzed in terms of electronic, steric, and hydrophobic effects of alkyl substituents. The hydrophilic character of the lysine residues is the most important factor which affects all kinetic parameters, particularly K_ia and V₂. In addition, the nucleophilic character of the lysine residues enhances the enzyme activity by increasing the maximum velocity of ethanol oxidation and the affinity of alcohol dehydrogenase for NADH and acetaldehyde. The steric interaction at the lysine residues favors the affinity of the enzyme for NADH and ethanol.     

Purification and comparative studies of alcohol dehydrogenases

Alcohol dehydrogenases from various animal and plant sources were purified by a common procedure which employed DEAE, Sephadex-G100 and affinity chromatographies. The procedure achieves an 80-130 fold purification for animal enzymes. However, only a 5-15 fold purification for plant enzymes was attained because of the instability of these enzymes. Purified alcohol dehydrogenases from animal and plant sources differ in coenzyme and substrate specificities. The enzymes from mammalian, avian and fish livers display aldehyde oxidizing and esterolytic activities in addition to alcohol oxidizing activity. However, the enzymes from plants and yeast show only the oxidative activity toward alcohols. Chemical modifications have been performed to identify amino acid residues which are essential to the oxidative and esterolytic activities of alcohol dehydrogenases.     

Developmental expression of alcohol dehydrogenases in maize

Alcohol dehydrogenase (ADH) in Zea mays exists in five distinct electrophoretic forms (isozymes), ADH-1, ADH-2, ADH-3, ADH-4, and ADH-T. The mode of inheritance of ADH-1 and ADH-2 has been previously reported; preliminary data suggest that ADH-3 is controlled by a different locus than ADH-2; no genetic analysis has yet been made for ADH-4 and ADH-T. Analyses at different stages of ontogenesis and of different organs have shown that the ADH isozyme pattern fluctuates qualitatively and quantitatively during the course of development and differentiation of the maize plant. ADH-T is controlled spatially and temporally in a very strict manner, being present only in extracts from the pericarp of 19- to 40-day-old kernels. ADH-3 and ADH-4 are present in the scutella of mature kernels and during early sporophytic development. ADH-1 and ADH-2 are the most common isozymes in all tissues examined, but ADH-1 is not found in endosperm of mature kernels or during germination. None of the isozymes have been found to be associated with any particulate cellular component at any stage of development. These findings are discussed with respect to differential gene expression, physiology, and cellular metabolism.     

Development of the active site model for calcium-containing quinoprotein alcohol dehydrogenases

A new PQQ model compound [dimethyl 7-(1,4,7,10-tetraoxa-13-azacyclopentadec-13-yl)carbonyl-4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,9-dicarboxylate, 1], in which a 1-aza-15-crown-5 group is attached through an amide linkage at the 7-position, has been synthesized in order to develop an efficient model system of calcium-containing quinoprotein alcohol dehydrogenases. It has been found that Ca²⁺ binds to the quinone most strongly among the alkaline earth metal ions examined (Ca²⁺+>Sr²⁺+≫Ba²⁺+≫Mg²⁺) and the binding constant (K_M) for Ca²⁺ is as large as 2.1×10⁵ M⁻¹. Formation of the C-5 hemiacetal derivatives with ethanol is also investigated spectrophotometrically to show that the alcohol-addition to the quinone is enhanced in the presence of the metal ions. In this case, Ca²⁺ and Sr²⁺ show a similar efficiency that is several times larger than that of Ba²⁺. Addition of a strong base such as DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) into a MeCN solution containing the metal ion complex of 1 and ethanol leads to redox reactions to give the Ca²⁺ complex of 1H₂ (quinol form) and acetaldehyde. Kinetic studies on the redox reactions have been performed to gain insight into the mechanism of the alcohol-oxidation reaction catalyzed by the metal complexes of coenzyme PQQ.     

Cytosolic alcohol dehydrogenases from yeast Torulopsis candida]

A comparative study of cell cytosol alcohol dehydrogenase (ADH) from yeast Torulopsis candida IBFM-Y-127 grown on glucose and hexadecane which were the only source of carbon, was made. In both cases ADH had a pH optimum within the range of 7.0--10.0, when various normal primary alcohols (C2--C16) were used. The enzyme was active only in the presence of NAD, which cannot be substituted by NADP. The total activity of ADH decreased approximately 8-fold when the length of hydrocarbon radicals was changed from C2 up to C16. When the cells were grown on hexadecane, only ethyl, n-buthyl, n-amyl and n-hexyl alcohols were active as substrates. The dehydration rate of each alcohol was far lower than that for the cytosol of glucose-grown cells. In the latter case the enzyme activity also decreased with an increase in the alcohol radical from C2 to C6. In all cases studied methyl alcohol and cyclic (cinnamyl alcohol--C8) alcohol were not dehydrated at all. Disc-electrophoresis in polyacrylamide gel, involving gel colouration for the assay of enzyme activity showed that glucose--grown cell cytosol contained three forms of ADH. One of those forms was highly active when short--chain normal primary alcohols were used; this form may be probably regarded as "classical" ADH (EC 1.1.1.1). The two other forms caused intensive dehydration of long-chain alcohols (the best substrates were C7--C10 alcohols for one form and C10--C14 for the others). The two forms of ADH are probably isoenzymes of octanol dehydrogenase (EC 1.1.1.73). Cytosol of cells grown on n-alcane, had a reduced number of ADH forms. The data obtained are discussed in terms of the regulatory role of carbon and energy source (glucose or hexadecane) in the redistribution of alcohol dehydrogenases between structural components of cells (mitochondria) and cytosol.     

Characteristics of short-chain alcohol dehydrogenases and related enzymes

Different short-chain dehydrogenases are distantly related, constituting a protein family now known from at least 20 separate enzymes characterized, but with extensive differences, especially in the C-terminal third of their sequences. Many of the first known members were prokaryotic, but recent additions include mammalian enzymes from placenta, liver and other tissues, including 15-hydroxyprostaglandin, 17 beta-hydroxysteroid and 11 beta-hydroxysteroid dehydrogenases. In addition, species variants, isozyme-like multiplicities and mutants have been reported for several of the structures. Alignments of the different enzymes reveal large homologous parts, with clustered similarities indicating regions of special functional/structural importance. Several of these derive from relationships within a common type of coenzyme-binding domain, but central-chain patterns of similarity go beyond this domain. Total residue identities between enzyme pairs are typically around 25%, but single forms deviate more or less (14-58%). Only six of the 250-odd residues are strictly conserved and seven more are conserved in all but single cases. Over one third of the conserved residues are glycine, showing the importance of conformational and spatial restrictions. Secondary structure predictions, residue distributions and hydrophilicity profiles outline a common, N-terminal coenzyme-binding domain similar to that of other dehydrogenases, and a C-terminal domain with unique segments and presumably individual functions in each case. Strictly conserved residues of possible functional interest are limited, essentially only three polar residues. Asp64, Tyr152 and Lys156 (in the numbering of Drosophila alcohol dehydrogenase), but no histidine or cysteine residue like in the completely different, classical medium-chain alcohol dehydrogenase family. Asp64 is in the suggested coenzyme-binding domain, whereas Tyr152 and Lys156 are close to the center of the protein chain, at a putative inter-domain, active-site segment. Consequently, the overall comparisons suggest the possibility of related mechanisms and domain properties for different members of the short-chain family.     

Structural insights into alcohol dehydrogenases catalyzing asymmetric reductions

Alcohol dehydrogenases are a group of oxidoreductases that specifically use NAD(P)⁺ or NAD(P)H as cofactors for electron acceptance or donation and catalyze interconversion between alcohols and corresponding carbonyl compounds. In addition to their physiological roles in metabolizing alcohols and aldehydes or ketones, alcohol dehydrogenases have received considerable attention with respect to their symmetry-breaking traits in catalyzing asymmetric reactions and have Accordingly, they have become widely applied in fine chemical synthesis, particularly in the production of chiral alcohols and hydroxyl compounds that are key elements in the synthesis of active pharmaceutical ingredients (API) employed in the pharmaceutical industry. The application of structural bioinformatics to the study of functional enzymes and recent scientific breakthroughs in modern molecular biotechnology provide us with an effective alternative to gain an understanding of the molecular mechanisms involved in asymmetric bioreactions and in overcoming the limitations of enzyme availability. In this review, we discuss molecular mechanisms underlying alcohol dehydrogenase-mediated asymmetric reactions, based on protein structure–function relationships from domain structure to functional active sites. The molecular principles of the catalytic machinery involving stereochemical recognition and molecular interaction are also addressed. In addition, the diversity of enzymatic functions and properties, for example, enantioselectivity, substrate specificity, cofactor dependence, metal requirement, and stability in terms of organic solvent tolerance and thermostability, are also discussed and based on a comparative analysis of high-resolution 3?D structures of representative alcohol dehydrogenases.     

Aerobic and anaerobic alcohol dehydrogenases in Escherichia coli

Abstract Mutants unable to use ethanol for carbon and energy were counterselected from an ethanolutilizing mutant of Escherichia coli K12 derepressed for alcohol dehydrogenase (ADH). Mutants of one class were devoid of ADH activity under anaerobic conditions but exhibited aerobic activities comparable to those of wild-type E. coli. Mutants of a second class exhibited ADH activity levels intermediate between those of the wild-type and derepressed parent. Immunological studies showed that mutants of the former class synthesized far less ADH protein than did the derepressed parent while mutants of the latter class synthesized about the same amount. The ADH mutations in both classes were located within the previously described adh region which contains the structural gene for the activity that is derepressed in the parent. An Eth⁻ adh-lac fusion mutant with an insertion in the structural gene was also isolated and characterized. It exhibited no ADH activity under anaerobic conditions and wild-type levels under aerobic conditions. These data are consistent with the existence in E. coli of distinct aerobic and anaerobic ADH enzymes and a derepression of the anaerobic but not the aerobic enzyme in the ethanol utilizing strain.