Extended superfamily of short alcohol-polyol-sugar dehydrogenases: structural similarities between glucose and ribitol dehydrogenases

The recently determined primary structure of glucose dehydrogenase from Bacillus megaterium was scanned by computerized comparisons for similarities with known polyol and alcohol dehydrogenases. The results revealed a highly significant similarity between this glucose dehydrogenase and ribitol dehydrogenase from Klebsiella aerogenes. Sixty-one positions of the 262 in glucose dehydrogenase are identical between these two proteins (23% identity), fitting into a homology alignment for the complete polypeptide chains. The extent of similarity is equivalent to that between other highly divergent but clearly related dehydrogenases (two zinc-containing alcohol dehydrogenases, 25% sorbitol and zinc-containing alcohol dehydrogenases, 25%; ribitol and non-zinc-containing alcohol dehydrogenases, 20%), and suggests an ancestral relationship between glucose and ribitol dehydrogenases from different bactera. The similarities fit into a previously suggested evolutionary scheme comprising short and long alcohol and polyol dehydrogenases, and greatly extend the former group to one composed of non-zinc-containing alcohol-polyol-glucose dehydrogenases.     

Extensive variations and basic features in the alcohol dehydrogenase-sorbitol dehydrogenase family

Structural comparisons of sorbitol dehydrogenase with zinc-containing 'long' alcohol dehydrogenases reveal distant but clear relationships. An alignment suggests 93 positional identities with horse liver alcohol dehydrogenase (25% of 374 positions) and 73 identities with yeast alcohol dehydrogenase (20%). Sorbitol dehydrogenase forms a link between these distantly related alcohol dehydrogenases and is in some regions more similar to one of them that they are to each other. 43 residues (11%) are common to all three enzymes and include a heavy over-representation of glycine (half of all glycine residues in sorbitol dehydrogenase), showing the importance of space restrictions in protein structures. Four regions are well conserved, two in each domain of horse liver alcohol dehydrogenase. They are two segments close to the active-site zinc atom of the catalytic domain, and two in the central beta-pleated sheet strands of the coenzyme-binding domain. These similarities demonstrate the general importance of internal and central building units in proteins. Large variations affect a region adjacent to the third protein ligand to the active-site zinc atom in horse liver alcohol dehydrogenase. Such changes at active sites of related enzymes are unusual. Other large differences concern the segment around the non-catalytic zinc atom of horse liver alcohol dehydrogenase; three of its four cysteine ligands are absent from sorbitol dehydrogenase. Three segments with several exchanges correspond to a continuous region with superficial areas, inter-domain contacts and inter-subunit interactions in the catalytic domain of alcohol dehydrogenase. They may correlate with the altered quaternary structure of sorbitol dehydrogenase. Regions corresponding to top and bottom beta-strands in the coenzyme-binding domain of the alcohol dehydrogenase are also little conserved. Within sorbitol dehydrogenase, a large segment shows an internal similarity. The two distantly related alcohol dehydrogenases and sorbitol dehydrogenase form a triplet of enzymes illustrating basic protein relationships. They are ancestrally close enough to establish similarities, yet sufficiently divergent to illustrate changes in all but fundamental properties.     

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.     

Sorbitol dehydrogenase is a zinc enzyme.

Evidence is given that tetrameric sorbitol dehydrogenase from sheep liver contains one zinc atom per subunit, most probably located at the active site, and no other specifically bound zinc or iron atom. In alcohol dehydrogenases that are structurally related to sorbitol dehydrogenase, more than one zinc atom per subunit can complicate investigations of zinc atom function. Therefore, sorbitol dehydrogenase will be particularly valuable for defining the precise roles of zinc in alcohol and polyol dehydrogenases, and for establishing correlations of structure and function with other important zinc-containing proteins.     

Molecular aspects of functional differences between alcohol and sorbitol dehydrogenases

The amino acid sequence of sheep liver sorbitol dehydrogenase has been fitted to the high-resolution model of the homologous horse liver alcohol dehydrogenase by computer graphics. This has allowed construction of a model of sorbitol dehydrogenase that provides explanations why sorbitol is not a substrate for alcohol dehydrogenase, why ethanol is not a substrate for sorbitol dehydrogenase, and what determines its specificity for polyols. An important feature of the model is that one of the ligands to the active site zinc atom is a glutamic acid residue instead of a cysteine residue, which is the corresponding ligand in the homologous alcohol dehydrogenases. This is one component of the structural change that can be related to the different substrate specificities, showing how altered enzymic activity might be brought about by structural changes of the kind that it is now possible to introduce by site-directed mutagenesis and recombinant DNA techniques.     

Medium-chain dehydrogenases/reductases (MDR). Family characterizations including genome comparisons and active site modeling.

Completed eukaryotic genomes were screened for medium-chain dehydrogenases/reductases (MDR). In the human genome, 23 MDR forms were found, a number that probably will increase, because the genome is not yet fully interpreted. Partial sequences already indicate that at least three further members exist. Within the MDR superfamily, at least eight families were distinguished. Three families are formed by dimeric alcohol dehydrogenases (ADH; originally detected in animals/plants), cinnamyl alcohol dehydrogenases (originally detected in plants) and tetrameric alcohol dehydrogenases (originally detected in yeast). Three further families are centred around forms initially detected as mitochondrial respiratory function proteins, acetyl-CoA reductases of fatty acid synthases, and leukotriene B4 dehydrogenases. The two remaining families with polyol dehydrogenases (originally detected as sorbitol dehydrogenase) and quinone reductases (originally detected as zeta-crystallin) are also distinct but with variable sequences. The most abundant families in the human genome are the dimeric ADH forms and the quinone oxidoreductases. The eukaryotic patterns are different from those of Escherichia coli. The different families were further evaluated by molecular modelling of their active sites as to geometry, hydrophobicity and volume of substrate-binding pockets. Finally, sequence patterns were derived that are diagnostic for the different families and can be used in genome annotations.     

Thermostable NAD(+)-dependent alcohol dehydrogenase from Sulfolobus solfataricus: gene and protein sequence determination and relationship to other alcohol dehydrogenases.

The NAD(+)-dependent alcohol dehydrogenase (EC 1.1.1.1) from the thermoacidophilic archaebacterium Sulfolobus solfataricus, DSM1617 strain (SSADH), has been purified and characterized. Its gene has been isolated by screening two S. Solfataricus genomic libraries using oligonucleotide probes. The encoding sequence consists of 1041 base pairs, and it shows a high preference for codons ending in T or A. The primary structure, determined by peptide and gene analysis, consists of 347 amino acid residues, yielding a molecular weight of 37,588. A level of identity of 24-25% was found with the amino acid sequences of horse liver, yeast, and Thermoanaerobium brockii alcohol dehydrogenases. The coenzyme-binding and catalytic and structural zinc-binding residues typical of eukaryotic alcohol dehydrogenases were found in SSADH with the difference that one out of the four structural zinc-binding Cys residues is substituted by Glu. The protein contains four zinc atoms per dimer, two of which are removed by chelating agents with a concomitant loss of structural stability.     

Structural relationship between the hexameric and tetrameric family of glutamate dehydrogenases.

The family of glutamate dehydrogenases include a group of hexameric oligomers with a subunit M(r) of around 50,000, which are closely related in amino acid sequence and a smaller group of tetrameric oligomers based on a much larger subunit with M(r) 115,000. Sequence comparisons have indicated a low level of similarity between the C-terminal portion of the tetrameric enzymes and a substantial region of the polypeptide chain for the more widespread hexameric glutamate dehydrogenases. In the light of the solution of the three-dimensional structure of the hexameric NAD(+)-linked glutamate dehydrogenase from Clostridium symbiosum, we have undertaken a detailed examination of the alignment of the sequence for the C-terminal domain of the tetrameric Neurospora crassa glutamate dehydrogenase against the sequence and the molecular structure of that from C. symbiosum. This analysis reveals that the residues conserved between these two families are clustered in the three-dimensional structure and points to a remarkably similar layout of the glutamate-binding site and the active-site pocket, though with some differences in the mode of recognition of the nucleotide cofactor.     

Electrophoretic analysis of substrate specificity of wheat alcohol dehydrogenases]

Electrophoresis in polyacrylamide gel slabs has been used to study the isoform composition and substrate specificity of alcohol dehydrogenases in the embryo and young seedlings of the diploid wheat Triticum monococcum L., the tetraploid T. dicoccon (Schrank) Schuebl and the hexaploid T. spelta L. Three alcohol dehydrogenases of different substrate specificity and developmental pattern were distinguished: a) the NAD-dependent alcohol dehydrogenase, catalyzing the oxidation of different primary and secondary aliphatic and aromatic alcohols, as well as certain compounds with several hydroxyl groups (tris, triethanolamin) and revealing, after electrophoresis, one major band in the diploid wheat and three bands in both polyploid wheats; b) the NADP-dependent aromatic alcohol dehydrogenase (substrate--cinnamic alcohol), revealing, after electrophoresis, one major fast moving band in the diploid wheat and two bands in polyploid wheats; c) an aromatic alcohol dehydrogenase (2-3 bands after electrophoreis) with no specificity to the cofactors (NAD or NADP).     

Malate dehydrogenases in phototrophic purple bacteria. Thermal stability, amino acid composition and immunological properties.

Purified malate dehydrogenases from four species of non-sulphur purple phototrophic bacteria were examined for their heat-stability, amino acid composition and antigenic relationships. Malate dehydrogenase from Rhodospirillum rubrum, Rhodobacter capsulatus and Rhodomicrobium vannielii (which are all tetrameric proteins) had an unusually high glycine content, but the enzyme from Rhodocyclus purpureus (which is a dimer) did not. R. rubrum malate dehydrogenase was extremely heat-stable relative to the other enzymes, withstanding 65 degrees C for over 1 h with no loss of activity. By contrast, malate dehydrogenase from R. vannielii lost activity above 35 degrees C, and that from R. capsulatus above 40 degrees C. Amino acid compositional relatedness and immunological studies indicated that tetrameric phototrophic-bacterial malate dehydrogenases were highly related to one another, but only distantly related to the tetrameric enzyme from Bacillus. This suggests that, despite differences in their thermal properties, the tetrameric malate dehydrogenases of non-sulphur purple bacteria constitute a distinct biochemical class of this catalyst.     

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.     

An enzymatic cycling method for nicotinamide-adenine dinucleotide with malic and alcohol dehydrogenases

A method for measuring nicotinamide-adenine dinucleotide by enzymatic cycling is described which uses malic and alcohol dehydrogenases (EC 1.1.1.37, and EC 1.1.1.1) for the enzyme couple. After cycling, malate is measured with either malic dehydrogenase or malic enzyme (EC 1.1.1.40). The method has a number of advantages compared to those previously described. The cycling rate is high (greater than 30 000/hr); blank values are low; the reaction is linear over a wide range of NAD concentrations; and the terminal indicator reaction requires only one step. In addition the system is well suited for double cycling. This was shown by measurements of NAD in nuclei and cytoplasm from single dorsal root ganglion cells (rabbit). The overall amplification in this case was about 1 000 000.     

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.     

The primary structure of alcohol dehydrogenase from Drosophila lebanonensis. Extensive variation within insect 'short-chain' alcohol dehydrogenase lacking zinc

Insect alcohol dehydrogenase is highly different from the well-known yeast and mammalian alcohol dehydrogenases. The enzyme from Drosophila lebanonensis has now been characterized by protein analysis and was found to have a 254-residue protein chain with an acetyl-blocked N-terminal Met. Comparisons with the structures of the enzyme from other species allows judgement of the extent of variability within the insect alcohol dehydrogenases. They have diverged to a considerable extent; two forms analyzed at the protein level differ at 18% of all residues, and all the known Drosophila alcohol dehydrogenase structures reveal differences at 72 positions. Some deviations, against a background similarity, in the extent of changes are noted among the parts corresponding to different exons. The structural variation within Drosophila is about as large as the one for the mammalian zinc-containing alcohol dehydrogenase. Consequently, the results illustrate Drosophila relationships and establish great variations also for group of alcohol dehydrogenases lacking zinc.     

Characteristics of mammalian class III alcohol dehydrogenases, an enzyme less variable than the traditional liver enzyme of class I

Class III alcohol dehydrogenase, whose activity toward ethanol is negligible, has defined, specific properties and is not just a "variant" of the class I protein, the traditional liver enzyme. The primary structure of the horse class III protein has now been determined, and this allows the comparison of alcohol dehydrogenases from human, horse, and rat for both classes III and I, providing identical triads for both these enzyme types. Many consistent differences between the classes separate the two forms as distinct enzymes with characteristic properties. The mammalian class III enzymes are much less variable in structure than the corresponding typical liver enzymes of class I: there are 35 versus 84 positional differences in these identical three-species sets. The class III and class I subunits contain four versus two tryptophan residues, respectively. This makes the differences in absorbance at 280 nm a characteristic property. There are also 4-6 fewer positive charges in the class III enzymes accounting for their electrophoretic differences. The substrate binding site of class III differs from that of class I by replacements at positions that form the hydrophobic barrel typical for this site. In class III, two to four of these positions contain residues with polar or even charged side chains (positions 57 and 93 in all species, plus positions 116 in the horse and 140 in the human and the horse), while corresponding intraclass variation is small. All these structural features correlate with functional characteristics and suggest that the enzyme classes serve different roles. In addition, the replacements between these triad sets illustrate further general properties of the two mammalian alcohol dehydrogenase classes.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Avian alcohol dehydrogenase. Characterization of the quail enzyme, functional interpretations, and relationships to the different classes of mammalian alcohol dehydrogenase

The primary structure of the major quail liver alcohol dehydrogenase was determined. It is a long-chain, zinc-containing alcohol dehydrogenase of the type occurring also in mammals and hence allows judgement of the gene duplications giving rise to the classes of the human alcohol dehydrogenase system. The avian form is most closely related to the class I mammalian enzyme (72-75% residue identity), least related to class II (60% identity), and intermediately related to class III (64-65% identity). This pattern distinguishes the mammalian enzyme classes and separates classes I and II in particular. In addition to the generally larger similarities with class I, the avian enzyme exhibits certain residue patterns otherwise typical of the other classes, including an extra Trp residue, present in both class II and III but not in class I, with a corresponding increase in the UV absorbance. The avian enzyme further shows that a Gly residue at position 260 previously considered strictly conserved in alcohol dehydrogenases can be exchanged with Lys. However, zinc-binding residues, coenzyme-binding residues, and to a large extent substrate-binding residues are unchanged in the avian enzyme, suggesting its functional properties to be related to those of the class I mammalian alcohol dehydrogenases. In contrast, the areas of subunit interactions in the dimers differ substantially. These results show that (a) the vertebrate enzyme classes are of distant origin, (b) the submammalian enzyme exhibits partly mixed properties in relation to the classes, and (c) the three mammalian enzyme classes are not as equidistantly related as initially apparent but suggest origins from two sublevels.     

Rat liver alcohol dehydrogenase of class III. Primary structure, functional consequences and relationships to other alcohol dehydrogenases

The amino acid sequence of alcohol dehydrogenase of class III from rat liver (the enzyme ADH-2) has been determined. This type of structure is quite different from those of both the class I and the class II alcohol dehydrogenases. The rat class III structure differs from the rat and human class I structures by 133-138 residues (exact value depending on species and isozyme type); and from that of human class II by 132 residues. In contrast, the rat/human species difference within the class III enzymes is only 21 residues. The protein was carboxymethylated with iodo[2(14)C]acetate, and cleaved with CNBr and proteolytic enzymes. Peptides purified by exclusion chromatography and reverse-phase high-performance liquid chromatography were analyzed by degradation with a gas-phase sequencer and with the manual 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate double-coupling method. The protein chain has 373 residues with a blocked N terminus. No evidence was obtained for heterogeneity. The rat ADH-2 enzyme of class III contains an insertion of Cys at position 60 in relation to the class I enzymes, while the latter alcohol dehydrogenase in rat (ADH-3) has another Cys insertion (at position 111) relative to ADH-2. The structure deduced explains the characteristic differences of the class III alcohol dehydrogenase in relation to the other classes of alcohol dehydrogenase, including a high absorbance, an anodic electrophoretic mobility and special kinetic properties. The main amino acid substitutions are found in the catalytic domain and in the subunit interacting segments of the coenzyme-binding domain, the latter explaining the lack of hybrid dimers between subunits of different classes. Several substitutions provide an enlarged and more hydrophilic substrate-binding pocket, which appears compatible with a higher water content in the pocket and hence could possibly explain the higher Km for all substrates as compared with the corresponding values for the class I enzymes. Finally the class III structure supports evolutionary relationships suggesting that the three classes constitute clearly separate enzymes within the group of mammalian zinc-containing alcohol dehydrogenases.     

The possible occurrence of zinc in ribitol and sorbitol dehydrogenases from Mycobacterium sp. 279

The activity of polyhydric alcohol dehydrogenases in Mycobacterium sp. 279 was studied under limitation of zinc in the growth medium. It was found that the activity of ribitol and sorbitol dehydrogenases were markedly lowered and that of D-arabinitol dehydrogenase remained unchanged in the Zn2+-deficient cells. Other ions tested i.e., Co2+, Cu2+, Ni2+ and Mn2+ failed to substitute Zn2+ ions in their effect on the enzyme activities. The Zn2+-responsive enzymes were sensitive to the chelating agents (1,10-phenanthroline, 2,2'-dipyridyl), whereas D-arabinitol dehydrogenase was insensitive. The results indicate possible existence of a zinc component in the ribitol and sorbitol dehydrogenases from Mycobacterium sp. 279.     

Substrate specificity and stereospecificity of nicotinamide adenine dinucleotide-linked alcohol dehydrogenases from methanol-grown yeasts.

Nicotine adenine dinucleotide-linked primary alcohol dehydrogenase and a newly discovered secondary alcohol dehydrogenase coexist in most strains of methanol-grown yeasts. Alcohol dehydrogenases from methanol-grown yeasts oxidize (--)-2-butanol preferentially over its (+) enantiomorph. This is substantially different from alcohol dehydrogenases from bakers' yeast and horse liver.