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.     

A comparative study of the effect of pyrazole on the activity of enzymes in the alcohol/polyol dehydrogenase family]

A comparative study of the effect of pyrazol, an inhibitor of the coenzyme-binding site of alcohol dehydrogenases, on the activity of enzymes of the alcohol/polyol dehydrogenase group has been carried out. Commercial preparations of alcohol dehydrogenases from the cytoplasm of horse liver cells and yeast cells, as well as the enzyme from the cytoplasm of Trichosporon pullulans cells was completely inhibited by 1 mM pyrazol, while alcohol dehydrogenases from Candida utilis and Saccharomyces carlsbergensis were inhibited only by 25% and the enzymes from Saccharomyces cerevisiae and Torulopsis candida by 30 and 38%, respectively. The inhibition degree of alcohol dehydrogenases from the cytoplasm of liver cells of various mammals (bull, calf, rat, gopher) and birds (hen, pheasant, duck) varied from 12 to 42% in the presence of 1 mM pyrazol. The activity of sorbitol dehydrogenase from the liver cytoplasm of these mammals and birds changed neither in the presence of 1 mM pyrazol, nor in the case of a 15-fold increase of the inhibitor concentration. Possible structural differences in the coenzyme-binding site of the active center of the enzymes under study are discussed.     

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.     

Differences between alcohol dehydrogenases. Structural properties and evolutionary aspects.

Comparisons of the primary structures of yeast and horse liver alcohol dehydrogenases reveal that the enzymes are homologous but distantly related. The overall positional identity is 25% between common regions, and several deletions/insertions occur in either enzyme, the longest apparently corresponding to 21 residues, showing that the different subunit sizes are largely explained by internal differences. Variabilities in the structural similarities can be coupled with functional requirements but not directly with whole domains in the previously known tertiary structure of the horse protein. The two most similar regions of the enzymes affect active-site segments and the two most dissimilar regions seem to affect a loop structure without known function, and a segment participating in subunit interactions. The dissimilarities may probably be correlated with changes in zinc-binding properties and quaternary structures. The extra region corresponding to the large internal chain-length difference shows an apparent coincidence in sequence to a following segment of the horse enzyme, and additional elements of internal coincidences, or superficial similarities with other dehydrogenases, are noticed. These characteristics are not fully distinguishable from chance distributions but in view of the extensive species variations in alcohol dehydrogenases some evolutionary considerations may not be excluded, in which case a model relating all regions of these and associated enzymes to a common ancestor is shown to be compatible with all known observations.     

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.     

Different segment similarities in long-chain dehydrogenases

Long-chain dehydrogenases were scrutinized for common patterns. Overall molecular similarities are not discerned, in contrast to the situation for several short-chain and medium-chain dehydrogenases, but coenzyme-binding segments are discernible. Species variants of glucose-6-phosphate dehydrogenase reveal about 20% strictly conserved residues, grouped into three segments and supporting assignments of sites for coenzyme-binding and catalysis. Glycine is overrepresented among the residues conserved, typical of distantly related proteins. Two of the enzymes within the pentose phosphate pathway reveal a distant similarity of interest for further evaluation, between a C-terminal 178-residue segment of glucose-6-phosphate dehydrogenase and the N-terminal part of 6-phosphogluconate dehydrogenase.     

Selective carboxymethylation of cysteine-174 of the beta 2 beta 2 and beta 1 beta 1 human liver alcohol dehydrogenase isoenzymes by iodoacetate

The beta 1 beta 1 and beta 2 beta 2 human liver alcohol dehydrogenase isoenzymes differ by only one residue at the coenzyme-binding site; Arg-47 in beta 1 is replaced by His in the beta 2 subunit. Since Arg-47 is thought to facilitate the carboxymethylation of Cys-46 in horse liver alcohol dehydrogenase by binding halo acids in a Michaelis-Menten complex prior to inactivation, the specificity and kinetics of modification of the two human liver beta beta isoenzymes with iodoacetate were compared. Both of the beta beta isoenzymes were inactivated by treatment with iodo[14C]acetate, and one Cys per subunit was carboxymethylated. Cys-174, which is a ligand to the active-site zinc atom in horse liver alcohol dehydrogenase, was selectively carboxymethylated in each of the human beta beta isoenzymes; less than 15% of the iodo[14C]acetate incorporated into the enzyme appeared in Cys-46. Therefore, the three-dimensional structure of the basic amino acids in the anion-binding site of the human beta beta isoenzymes appears to be different from that of horse liver alcohol dehydrogenase. The kinetics of alkylation are consistent with the formation of a Michaelis-Menten complex before inactivation of the isoenzymes. The average Ki values for iodoacetate were 10 and 16 mM for beta 1 beta 1 and beta 2 beta 2, respectively, and maximal rate constants for inactivation were 0.22 and 0.17 min-1, respectively. From these data, it can be concluded that there is a relatively minor effect of the substitution of His for Arg at position 47 on the kinetics of inactivation.     

Class III human liver alcohol dehydrogenase: a novel structural type equidistantly related to the class I and class II enzymes

The primary structure of class III alcohol dehydrogenase (dimeric with chi subunits) from human liver has been determined by peptide analyses. The protein chain is a clearly distinct type of subunit distantly related to those of both human class I and class II alcohol dehydrogenases (with alpha, beta, gamma, and pi subunits, respectively). Disregarding a few gaps, residue differences in the chi protein chain with respect to beta 1 and pi occur at 139 and 140 positions, respectively. Compared to class I, the 373-residue chi structure has an extra residue, Cys after position 60, and two missing ones, the first two residues relative to class I, although the N-terminus is acetylated like that for those enzymes. The chi subunit contains two more tryptophan residues than the class I subunits, accounting for the increased absorbance at 280 nm. There are also four additional acidic and two fewer basic side chains than in the class I beta structure, compatible with the markedly different electrophoretic mobility of the class III enzyme. Residue differences between class III and the other classes occur with nearly equal frequency in the coenzyme-binding and catalytic domains. The similarity in the number of exchanges relative to that of the enzymes of the other two classes supports conclusions that the three classes of alcohol dehydrogenase reflect stages in the development of separate enzymes with distinct functional roles. In spite of the many exchanges, the residues critical to basic functional properties are either completely unchanged--all zinc ligands and space-restricted Gly residues--or partly unchanged--residues at the coenzyme-binding pocket.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active-site-specific zinc-depleted and reconstituted cobalt(II) human-liver alcohol dehydrogenase. Preparation, characterization and complexation with NADH and trans-4-(N,N-dimethylamino)-cinnamaldehyde

The active-site zinc atom of the beta 1 beta 1 isozyme of class I alcohol dehydrogenase (EC 1.1.1.1) from human liver was specifically removed by the chelating agent dipicolinic acid. From beta 1 gamma 1 and gamma 1 gamma 1 isozyme the active-site zinc is extracted much more slowly than from beta 1 beta 1 isozyme. Only partially active-site metal-depleted enzyme species were obtained from these isozymes. The active-site-specific reconstituted cobalt(II) derivative of the beta 1 beta 1 isozyme shows spectroscopic properties comparable to those of the active-site-specific reconstituted cobalt(II) horse liver alcohol dehydrogenase. The coenzyme-induced conformational change of the protein leads to a red shift of the d-d band from 648 nm to 673 nm. The chromophoric substrate trans-4-(N,N-dimethylamino)-cinnamaldehyde forms ternary complexes with NADH and the different isozymes, in close analogy to horse liver alcohol dehydrogenase. The differences in the active sites between beta 1 and gamma 1 subunits (threonine-48 instead of serine-48) or between zinc and cobalt(II) are reflected in the visible absorption spectra of the metal-bound chromophoric substrate.     

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.     

Pseudomonas 3 beta-hydroxysteroid dehydrogenase. Primary structure and relationships to other steroid dehydrogenases

The 3 beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni commercially available was purified by an FPLC step and submitted to sequence determination by peptide analysis. The structure obtained reveals a 253-residue polypeptide chain, with an N-terminal, free alpha-amino group, and a low cysteine content. Comparisons with other hydroxysteroid dehydrogenases recently characterized reveal distant similarities with prokaryotic and, to some extent, also eukaryotic forms of separate specificities. Residue identities with a Streptomyces 20 beta-hydroxysteroid dehydrogenase are 35% and distributed over the entire molecule, whereas residue identities with the mammalian 17 beta-hydroxysteroid dehydrogenase only constitute 20%, and are essentially limited to the N-terminal and central parts, Nevertheless, all these enzymes exhibit a conserved tyrosine residue (position 151 in the present enzyme) noted as possibly having a functional role in some members of this protein family. Combined, the results establish the prokaryotic 3 beta-hydroxysteroid dehydrogenase as belonging to the family of short-chain alcohol dehydrogenases, reveal that the hydroxysteroid dehydrogenases are no more closely related than dehydrogenases with other enzyme activities within the family (e.g. glucose, ribitol, hydroxyprostaglandin dehydrogenases), show several of the mammalian hydroxysteroid dehydrogenases to have subunits of longer size with different patterns of similarity than those of the prokaryotic family members characterized, and define important segments of the coenzyme-binding region for this enzyme group.     

The detection of sorbitol dehydrogenase in the cytoplasm of liver cells in birds and its relation to alcohol dehydrogenase and glucose-6-phosphate dehydrogenase

Comparative studies have been made in the specific activity of sorbitol dehydrogenase, glucose-6-phosphate and alcohol dehydrogenases in the cytoplasm from the liver of wild and domestic ducks, hen and pheasant. High activity of all the three enzymes was found in ducks indicating the effective sorbitol (polyol) metabolism of glucose. The activity of glucose-6-phosphate dehydrogenase is an order lower as compared with the activity of sorbitol and alcohol dehydrogenases in the cytoplasm of hen liver. The same relationship was found for the activity of sorbitol dehydrogenase in the cytoplasm of pheasant liver.     

Alcohol dehydrogenase from the fruitfly Drosophila melanogaster. Inhibition studies of the alleloenzymes AdhS and AdhUF

Different metal binding inhibitors of horse liver alcohol dehydrogenase, similarly affect the Drosophila melanogaster AdhS and AdhUF alleloenzymes. However, binding is generally weaker and the experiments show that the alleloenzymes although not zinc metalloenzymes, behave to the metal binding reagents very much as if they were. The metal-directed, affinity-labelling, imidazole derivative BrImPpOH reversibly inhibits, but does not inactivate the alleolenzymes. This confirms there is no active site metal atom with cysteine as a metal ligand, as found in zinc alcohol dehydrogenases. Pyrazole is a strong ethanol-competitive inhibitor of AdhS and AdhUF alleloenzymes. Formation of the ternary enzyme-NAD-pyrazole complex gives an absorption increase between 295-330 nm. This enables an active site titration to be performed and the determination of epsilon (305 nm) of 15.8 . 10(3) M-1 . cm-1. Inhibition experiments with imidazole confirm that with secondary alcohols such as propan-2-ol, a Theorell-Chance mechanism predominates, but with ethanol and primary alcohols, interconversion of the ternary complexes is rate limiting. Salicylate is a coenzyme competitive inhibitor and KEI suggests that the coenzyme adenosine binding region is similar is Drosophila and horse liver alcohol dehydrogenase. Drosophila alcohol dehydrogenase is found not to form a ternary complex with NADH and isobutyramide. In this and other properties it is like carboxymethyl liver alcohol dehydrogenase. Both Drosophila and carboxymethyl alcohol dehydrogenase bind coenzyme in a similar manner to native horse liver alcohol dehydrogenase, but substrate binding differs between each. Inhibition by Cibacrone blue, indicates that amino acid 192 which is lysine in AdhS and threonine in AdhUF, is located in the coenzyme-binding region. Proteolytic activity present in preparations of alcohol dehydrogenase from D. melanogaster, is considered due to a metalloprotease, for which BrImPpOH is a potent inactivator.     

On the physiological significance of positive and negative cooperativity in enzymes.

Glyceraldehyde-3-phosphate dehydrogenases from mammalian muscle and yeast are known to display negative and positive cooperativity, respectively, in the binding of coenzyme, NAD. A possible physiological role of these coenzyme-binding anomalies is raised by comparing them to the coenzyme-binding properties of lactate dehydrogenase and alcohol dehydrogenase, the corresponding glycolytic NADH-reoxidizing enzymes in muscle and yeast. It is suggested that the observed coenzyme-binding relations reflect the evolutionary adaptation of enzymes to facilitate vectorial metabolite flow.     

Purification and characterization of human liver sorbitol dehydrogenase

Sorbitol dehydrogenase from human liver was purified to homogeneity by affinity chromatography on immobilized triazine dyes, conventional cation-exchange chromatography, and high-performance liquid chromatography. The major form is a tetrameric, NAD-specific enzyme containing one zinc atom per subunit. Human liver sorbitol dehydrogenase oxidizes neither ethanol nor other primary alcohols. It catalyzes the oxidation of a secondary alcohol group of polyol substrates such as sorbitol, xylitol, or L-threitol. However, the substrate specificity of human liver sorbitol dehydrogenase is broader than that of the liver enzymes of other sources. The present report describes the stereospecific oxidation of (2R,3R)-2,3-butanediol, indicating a more general function of sorbitol dehydrogenase in the metabolism of secondary alcohols. Thus, the enzyme complements the substrate specificities covered by the three classes of human liver alcohol dehydrogenase.     

Sequence comparisons betweenAgrobacterium tumefaciens T-DNA-encoded octopine and nopaline dehydrogenases and other nucleotide-requiring enzymes: Structural and evolutionary implications

Summary The nucleotide sequences of the two T-DNA-encoded crown gall imino acid dehydrogenases octopine dehydrogenase and nopaline dehydrogenase were compared with each other and with the sequences of other dehydrogenases. A multistep strategy comprising computer sequence analysis and secondary- and antigenic-structure predictions was used. An alignment of octopine and nopaline dehydrogenase was obtained in which a 20-amino-acid N-terminal arm and six fairly long gaps in the C-terminal moiety were introduced. The aligned sequences have identities of 26% at the amino acid level and 38% at the nucleotide level. They appear to contain two domains. The N-terminal coenzyme-binding domains are similar to those of the well-characterized NAD(P) dehydrogenases. Conserved fragments were found in the C-terminal catalytic domains that likely contain essential residues for catalysis. Comparison of the sequences with those of two other 2-keto acid dehydrogenases, lactate and malate dehydrogenase, suggests that as in those enzymes, histidine, aspartic acid, and arginine residues are located at the octopine and nopaline dehydrogenase active sites. The crown gall enzymes could not be classified with any known family of dehydrogenases. Their evolutionary origin remains unknown. However, predictions concerning their internal organization may provide new insight into protein evolution.     

A synthetic peptide encompassing the binding site of the second zinc atom (the 'structural' zinc) of alcohol dehydrogenase.

A 23-residue peptide was synthesized that incorporates the loop which binds the structural zinc atom of mammalian alcohol dehydrogenases and contributes, in part, to subunit interactions in the native enzyme. Neither the amino acid composition nor the sequence of the peptide resemble those of zinc fingers. The reduced peptide stoichiometrically binds zinc or cobalt to form stable complexes with a dissociation constant of the peptide/CO2+ complex of 2.1 microM at pH 7.5. EDTA disrupts the complex. The absorption and magnetic circular dichroic spectra of the cobalt-peptide are indicative of a tetrahedral coordination geometry, and are similar to those of the cobalt-substituted structural site of horse and human (beta 1 beta 1) liver alcohol dehydrogenases. Consequently, the synthetic peptide can serve as a model for the metal-binding segment of alcohol dehydrogenase and for studies of fundamental problems concerning protein/metal interactions.     

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.     

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.     

Zinc environment in sheep liver sorbitol dehydrogenase

The extended X-ray absorption fine structure (EXAFS) associated with the zinc K-absorption edge has been recorded for sorbitol dehydrogenase. It is interpreted in terms of one cysteine sulfur among the ligands to the active site zinc atom. Simulations of the EXAFS based on the presence of two such sulfurs are less satisfactory, and comparison with the EXAFS of such systems points to the presence of only one sulfur ligand in sorbitol dehydrogenase. These results provide evidence that sorbitol dehydrogenase does not have the characteristic one water, one His, two Cys arrangement of ligands to the active site zinc found in the homologous alcohol dehydrogenases and are consistent with the one water, one His, one Cys, one Glu ligand arrangement of the proposed model of sorbitol dehydrogenase [Eklund, H., Horjales, E., J?rnvall, H., Br?ndén, C.-I., & Jeffery J. (1985) Biochemistry 24, 8005-8012]. Evidence for the correctness of the model is also evidence for validity of predictive techniques used in constructing the model, i.e., computer graphics fitting of the amino acid sequence to the crystallographically derived structure of a different but homologous protein.