The three zinc-containing alcohol dehydrogenases from baker's yeast,Saccharomyces cerevisiae

This review is a summary of our current knowledge of the structure, function and mechanism of action of the three zinc-containing alcohol dehydrogenases, YADH-1, YADH-2 and YADH-3, in baker's yeast, Saccharomyces cerevisiae. The opening section deals with the substrate specificity of the enzymes, covering the steady-state kinetic data for its most known substrates. In the following sections, the kinetic mechanism for this enzyme is reported, along with the values of all rate constants in the mechanism. The complete primary structures of the three isoenzymes of YADH are given, and the model of the 3D structure of the active site is presented. All known artificial mutations in the primary structure of the YADH are covered in full and described in detail. Further, the chemical mechanism of action for YADH is presented along with the complement of steady-state and ligand-binding data supporting this mechanism. Finally, the bio-organic chemistry of the hydride-transfer reactions catalyzed by the enzyme is covered: this chemistry explains the narrow substrate specificity and the enantioselectivity of the yeast enzyme.     

Molecular evolution of the zinc-containing long-chain alcohol dehydrogenase genes

Phylogenetic relationships and rates of nucleotide substitution were studied for alcohol dehydrogenase (ADH) genes by using DNA sequences from mammals and plants. Mammalian ADH sequences include the three class I genes and a class II gene from humans and one gene each from baboon, rat, and mouse. Plant sequences include two ADH genes each from maize and rice, three genes from barley, and one gene each from wheat and two dicots, Arabidopsis and pea. Phylogenetic trees show that relationships among ADH genes are generally consistent with taxonomic relationships: mammalian and plant ADH genes are classified into two distinct groups; primate class I genes are clustered; and two dicot sequences are clustered separately from monocot sequences. Accelerated evolution has been detected among the duplicated ADH genes in plants, in which synonymous substitutions occurred more often within the coenzyme-binding domain than within the catalytic domains.     

NAD-linked, factor-dependent formaldehyde dehydrogenase or trimeric, zinc-containing, long-chain alcohol dehydrogenase from Amycolatopsis methanolica.

NAD-linked, factor-dependent formaldehyde dehydrogenase (FD-FA1DH) of the Gram-positive methylotrophic bacterium, Amycolatopsis methanolica, was purified to homogeneity. It is a trimeric enzyme with identical subunits (molecular mass 40 kDa) containing 6 atoms Zn/enzyme molecule. The factor is a heat-stable, low-molecular-mass compound, which showed retention on an Aminex HPX-87H column. Inactivation of the factor occurred during manipulation, but activity could be restored by incubation with dithiothreitol. The identity of the factor is still unknown. It could not be replaced by thiol compounds or cofactors known to be involved in metabolism of C1 compounds. Of the aldehydes tested, only formaldehyde was a substrate. However, the enzyme showed also activity with higher aliphatic alcohols and the presence of the factor was not required for this reaction. Methanol was not a substrate, but high concentrations of it could replace the factor in the conversion of formaldehyde. Presumably, a hemiacetal of formaldehyde is the genuine substrate, which, in the case of methanol, acts as a factor leading to methylformate as the product. This view is supported by the fact that formate could only be detected in the reaction mixture after acidification. Inhibition studies revealed that the enzyme contains a reactive thiol group, being protected by the binding of NAD against attack by heavy-metal ions and aldehydes. Studies on the effect of the order of addition of coenzyme and substrate suggested that optimal catalysis required NAD as the first binding component. Substrate specificity and the induction pattern clearly indicate a role of the enzyme in formaldehyde oxidation. However, since FD-FA1DH was also found in A. methanolica grown on n-butanol, but not on ethanol, it may have a role in the oxidation of higher aliphatic alcohols as well. FD-FA1DH and the factor from A. methanolica are very similar to a combination already described for Rhodococcus erythropolis [Eggeling, L. & Sahm, H. (1985) Eur. J. Biochem. 150, 129-134]. NAD-linked, glutathione-dependent formaldehyde dehydrogenase (GD-FA1DH) resembles FD-FA1DH in many respects. Since glutathione has so far not been detected in Gram-positive bacteria, FD-FA1DH could be the counterpart of this enzyme in Gram-positive bacteria. Alignment of the N-terminal sequence (31 residues) of FD-FA1DH with that of GD-FA1DH from rat liver indeed showed similarity (30% identical positions). However, comparable similarity was found with class I alcohol dehydrogenase from this organism and with cytosolic alcohol dehydrogenase from Saccharomyces cerevisiae, isozyme 1.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Similarities in active center geometries of zinc-containing enzymes, proteases and dehydrogenases.

The spatial environment of the active centers for the four zinc-containing enzymes carbonic anhydrase, liver alcohol dehydrogenase, thermolysin and carboxypeptidase were compared and contrasted. The zinc is co-ordinated by three protein groups. In addition, a water molecule and substrate carbonyl may assume a fourth or fifth position. A group whose function is to abstract a proton from water during catalysis was found to have a constant spatial arrangement with respect to the zinc atom. The co-ordination sphere around the zinc is systematically distorted from a regular tetrahedral geometry with one specific ligand position being invariably occupied by a histidine residue. The orientation of the imidazole ring is moderately constant with respect to the Zn pyramid, a constraint possibly imposed by the adjacent substrate to permit its positioning suitable for catalysis.The comparison of carboxypeptidase and thermolysin was previously reported (Kester and Matthews, 1977a). The position of the water molecule as found in liver alcohol dehydrogenase when placed in thermolysin or carboxypeptidase would be consistent with a transient pentagonal Zn co-ordination during catalysis.Comparison of carboxypeptidase and carbonic anhydrase showed that the specificity pocket of carboxypeptidase superimposed onto a hydrophobic cavity of unknown function in carbonic anhydrase. The glycyl-l-tyrosine pseudo-substrate of carboxypeptidase fits well into the cavity, suggesting a probable binding site for esters in carbonic anhydrase. The excellent esterase activity of both these enzymes can thus be explained by a common binding mode and arrangement of catalytic groups.A comparison of trypsin and thermolysin demonstrates that, although their functional groups differ in character, the peptidase activity could be catalyzed in a similar manner. The proton-abstracting function of His57 in trypsin is generated by Glul43 acting on the Zn co-ordinated water, while the proton donor function of His57 in trypsin is generated by His231 in thermolysin.A comparison of liver alcohol dehydrogenase with other dehydrogenases suggests that His51 is not only a proton sink but also electronically provides an essential positive charge at crucial moments during catalysis. In contrast Arg 109 of lactafce dehydrogenase performs the same function by virtue of a conformational change. The superposition indicates that the zinc co-ordinated water oxygen has the proton acceptor function in liver alcohol dehydrogenase corresponding to the essential histidine groups in lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase.A compendium of the handedness of the catalytic configuration about the reactive atoms for ten different enzymes has been tabulated.     

Physiological function of alcohol dehydrogenases and long-chain (C(30)) fatty acids in alcohol tolerance of Thermoanaerobacter ethanolicus

A mutant strain (39E H8) of Thermoanaerobacter ethanolicus that displayed high (8% [vol/vol]) ethanol tolerance for growth was developed and characterized in comparison to the wild-type strain (39E), which lacks alcohol tolerance (<1.5% [vol/vol]). The mutant strain, unlike the wild type, lacked primary alcohol dehydrogenase and was able to increase the percentage of transmembrane fatty acids (i.e., long-chain C(30) fatty acids) in response to increasing levels of ethanol. The data support the hypothesis that primary alcohol dehydrogenase functions primarily in ethanol consumption, whereas secondary alcohol dehydrogenase functions in ethanol production. These results suggest that improved thermophilic ethanol fermentations at high alcohol levels can be developed by altering both cell membrane composition (e.g., increasing transmembrane fatty acids) and the metabolic machinery (e.g., altering primary alcohol dehydrogenase and lactate dehydrogenase activities).     

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.     

Biochemical differences between alcohol dehydrogenases of Drosophila melanogaster.

This paper describes substrate specificities, developmental changes in activity, pH profiles, and heat stabilities of isozymes produced by four Adh genotypes in D. melanogaster. No differences are found in the substrate specificities of isozymes from the different genotypes but studies of the other three properties reveal significant differences between the isozymes. Thus relatively low activities are found among extracts of AdhF Adhn2 larvae and among extracts of AdhF AdhF adults aged 44 days. Also AdhF AdhS and AdhS AdhS extracts have relatively high activities at pH 6-5, and AdhF Adhn2 extracts have relatively low activities at pH values above 10-0. Finally, extracts of AdhF AdhF and AdhF AdhS are more stable at 40 degrees C than are those of AdhS AdhS and AdhF Adhn2.     

The dye-linked alcohol dehydrogenase of Rhodopseudomonas acidophila. Comparison with dye-linked methanol dehydrogenases.

1. A dye-linked alcohol dehydrogenase was purified 20-fold from extracts of Rhodopseudomonas acidophila 10050 grown anaerobically in the light on methanol/HCO3-. 2. The enzyme resembled many previously reported methanol dehydrogenases from other methylotrophic organisms in coupling to phenazine methosulphate, requiring ammonia as an activator, possessing a pH optimum of 9 and a mol.wt. of approx. 116000. In many other respects the enzyme showed singular properties. 3. The stability of the enzyme under various conditions of temperature and pH was studied. 4. Primary aliphatic amines containing up to nine carbon atoms (the longest tested) were better activators than ammonia. 5. A wide range of primary alcohols and aldehydes served as substrates, with apparent Km values ranging from 57 mM for methanol to 6 micron for ethanol. 6. O2 was an inhibitor competitive with respect to the alcohol substrate. In the presence of O2, apparent Km values of 145 mM were recorded for methanol. 6. Cyanide and alphaalpha'-bipyridine were inhibitors competitive with respect to the amine activator. 7. The properties of the enzyme from Rhodopseudomonas acidophila are compared with those of similar enzymes from other organisms, and implications of the salient differences are discussed.     

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.     

Short-chain dehydrogenases. Proteolysis and chemical modification of prokaryotic 3 alpha/20 beta-hydroxysteroid, insect alcohol and human 15-hydroxyprostaglandin dehydrogenases.

Prokaryotic 3 alpha/20 beta-hydroxysteroid dehydrogenase exhibits one segment sensitive to proteolysis with Glu-C protease and trypsin (cleaving after Glu192 and Arg196, respectively). Cleavage is associated with dehydrogenase inactivation; the presence of NADH offers almost complete protection and substrate (cortisone) gives some protection. Distantly related insect alcohol dehydrogenase is more resistant to proteolysis, but cleavage in a corresponding segment is detectable with Asp-N protease (cleaving before Asp198), while a second site (at Glu243) is sensitive to cleavage with both Glu-C and Asp-N proteases. Combined, the results suggest the presence of limited regions especially sensitive to proteolysis and the possibility of some association between the enzyme active site and the sensitive site(s). Modification of the hydroxysteroid dehydrogenase with tetranitromethane is paralleled by enzyme inactivation. With a 10-fold excess of reagent, labeling corresponds to 1.2 nmol Tyr/nmol protein chain and is recovered largely in Tyr152, with lesser amounts in Tyr251. Tetranitromethane also rapidly inhibits the other two dehydrogenases, but they contain Cys residues, preventing direct correlation with Tyr modification. Together, the proteolysis and chemical modifications highlight three segments of short-chain dehydrogenase subunits, one mid-chain, containing Tyr152 of the steroid dehydrogenase (similar numbers in the other enzymes), strictly conserved and apparently close to the enzyme active site, the other around position 195, sensitive to proteolysis and affected by coenzyme binding, while the third is close to the C-terminus.     

Double-ternary complex affinity chromatography: preparation of alcohol dehydrogenases.

A general affinity chromatographic method for alcohol dehydrogenase purification has been developed by employing immobilized 4-substituted pyrazole derivatives that isolate the enzyme through formation of a specific ternary complex. Sepharose 4B is activated with 300 mg of cyanogen bromide/ml of packed gel and coupled to 4-[3-(N-6-aminocaproyl)aminopropyl]pyrazole. From crude liver extracts in 50 mM phosphate-0.37 mM nicotinamide adenine dinucleotide, pH 7.5, alcohol dehydrogenase is optimally bound at a capacity of 4-5 mg of enzyme/ml of gel. Addition of ethanol, propanol, or butanol, 500 mM, results in the formation of a second ternary complex, which allows the elution of bound enzyme in high yield and purity. This double-ternary complex affinity chromatography has been applied successfully to human, horse, rat, and rabbit liver extracts to isolate the respective homogeneous alcohol dehydrogenases.     

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.     

The alcohol dehydrogenases of Saccharomyces cerevisiae: a comprehensive review

Alcohol dehydrogenases (ADHs) constitute a large family of enzymes responsible for the reversible oxidation of alcohols to aldehydes with the concomitant reduction of NAD(+) or NADP(+). These enzymes have been identified not only in yeasts, but also in several other eukaryotes and even prokaryotes. The ADHs of Saccharomyces cerevisiae have been studied intensively for over half a century. With the ever-evolving techniques available for scientific analysis and since the completion of the Yeast Genome Project, a vast amount of new information has been generated during the past 10 years. This review attempts to provide a brief summary of the wealth of knowledge gained from earlier studies as well as more recent work. Relevant aspects regarding the primary and secondary structure, kinetic characteristics, function and molecular regulation of the ADHs in S. cerevisiae are discussed in detail. A brief outlook also contemplates possible future research opportunities.