Medium- and short-chain dehydrogenase/reductase gene and protein families

Zinc plays an important role in the structure and function of many enzymes, including alcohol dehydrogenases (ADHs) of the MDR type (mediumchain dehydrogenases/reductases). Active site zinc participates in catalytic events, and structural site zinc maintains structural stability. MDR-types of ADHs have both of these zinc sites but with some variation in ligands and spacing. The catalytic zinc sites involve three residues with different spacings from two separate protein segments, while the structural zinc sites involve four residues and cover a local segment of the protein chain (Cys97-Cys111 in horse liver class I ADH). This review summarizes properties of both ADH zinc sites, and relates them to zinc sites of proteins in general. In addition, it highlights a separate study of zinc binding peptide variants of the horse liver ADH structural zinc site. The results show that zinc coordination of the free peptide differs markedly from that of the enzyme (one His / three Cys versus four Cys), suggesting that the protein zinc site is in an energetically strained conformation relative to that of the peptide. This finding is a characteristic of an entatic state, implying a functional nature for this zinc site.     

Differential multiplicity of MDR alcohol dehydrogenases: enzyme genes in the human genome versus those in organisms initially studied

Screens were made for alcohol dehydrogenase (ADH) of the classical type (the MDR superfamily) in translations of human and other relevant genomes, corresponding to the organism types from which the enzyme was initially purified. Considerable multiplicities were detected in the dimeric enzymes from higher eukaryotes: seven forms in the human (plus three pseudogenes), all genes on chromosome 4, in the order class IV --> class Igamma --> class Ibeta --> class Ialpha --> class V --> class II --> class III, and eight forms in Arabidopsis thaliana (plus one pseudogene). These multiplicity patterns, and the species variability in the animal (human/mouse) and plant (Arabidopsis/pea) lines, suggest parallel but separate duplicatory events, giving rise to three families of dimeric MDR-ADH: class III, the animal non-class III, and the plant non-class III enzymes, with functions in formaldehyde elimination, in alcohol/aldehyde detoxication and in special pathways in higher eukaryotes. Multiplicity, although to a lesser extent, was also noted in tetrameric MDR-ADH, suggesting functional divergence between the dimeric and tetrameric enzymes. Combining these observations, at least five levels of divergence are reflected in the present ADH forms, corresponding to nodes at the SDR/MDR, the dimer/tetramer, the class III/non-class III, the class I/P, and the more recent class splits, each branch associated with separate functional patterns.     

Acetaldehyde oxidation inDrosophila null-mutants for alcohol dehydrogenase

Summary Aldehyde dehydrogenase (ALDH) activity is demonstrated in four strains ofD. melanogaster lacking active alcohol dehydrogenase (ADH-null mutants). In the four strains, ALDH activities are similar to those found in a wild strain. It is concluded that ADH-null flies are able to detoxify acetaldehyde. This finding is discussed in relation with the dual function of ADH proposed recently.This work was supported by a grant from the Medical Research Council of Canada (MRC 6920) to Dr F. Garcin.     

Medium- and short-chain dehydrogenase/reductase gene and protein families

The MDR superfamily with ~350-residue subunits contains the classical liver alcohol dehydrogenase (ADH), quinone reductase, leukotriene B4 dehydrogenase and many more forms. ADH is a dimeric zinc metalloprotein and occurs as five different classes in humans, resulting from gene duplications during vertebrate evolution, the first one traced to ~500 MYA (million years ago) from an ancestral formaldehyde dehydrogenase line. Like many duplications at that time, it correlates with enzymogenesis of new activities, contributing to conditions for emergence of vertebrate land life from osseous fish. The speed of changes correlates with function, as do differential evolutionary patterns in separate segments. Subsequent recognitions now define at least 40 human MDR members in the Uniprot database (corresponding to 25 genes when excluding close homologues), and in all species at least 10888 entries. Overall, variability is large, but like for many dehydrogenases, subdivided into constant and variable forms, corresponding to household and emerging enzyme activities, respectively. This review covers basic facts and describes eight large MDR families and nine smaller families. Combined, they have specific substrates in metabolic pathways, some with wide substrate specificity, and several with little known functions.     

Cross-reactive monoclonal antibodies to alcohol dehydrogenases

Summary Three anti-horse liver alcohol dehydrogenase (HLADH) monoclonal antibodies are described. Two are specific for ADH and cross-react with class I and II enzymes from mouse, horse and Chinese hamster. They are specific for the native enzyme but do not inhibit enzyme activity except when combined at high concentration. The third antibody was isolated as a response to rabbit metallothionein. It binds metalloproteins and inhibits ADH activity.     

Alcohol dehydrogenase 2 is a major hepatic enzyme for human retinol metabolism

The metabolism of all-trans- and 9-cis-retinol/ retinaldehyde has been investigated with focus on the activities of human, mouse and rat alcohol dehydrogenase 2 (ADH2), an intriguing enzyme with apparently different functions in human and rodents. Kinetic constants were determined with an HPLC method and a structural approach was implemented by in silico substrate dockings. For human ADH2, the determined K_m values ranged from 0.05 to 0.3 μM and k_cat values from 2.3 to 17.6 min⁻¹, while the catalytic efficiency for 9-cis-retinol showed the highest value for any substrate. In contrast, poor activities were detected for the rodent enzymes. A mouse ADH2 mutant (ADH2Pro47His) was studied that resembles the human ADH2 setup. This mutation increased the retinoid activity up to 100-fold. The K_m values of human ADH2 are the lowest among all known human retinol dehydrogenases, which clearly support a role in hepatic retinol oxidation at physiological concentrations. Received 12 October 2006; received after revision 6 December 2006; accepted 8 January 2007     

Developmental changes of aldehyde dehydrogenase isozymes in human livers: mitochondrial ALDH2 isozyme is expressed in fetal livers

Previous reports suggested that the major cytosolic aldehyde dehydrogenase (ALDH1) was present in fetal and infant livers, but the major mitochondrial isozyme (ALDH2) was absent or severely diminished. Re-examination by means of starch gel electrophoresis followed by enzyme activity staining, and by means of dot blot immuno-hybridization of liver samples with known genotypes of the ALDH2 locus, indicated that both ALDH1 and ALDH2 genes are expressed in fetal and infant livers. In addition, ALDH4 isozyme was also observed. The results imply that a fetus with the 'usual' homozygous ALDH1(2)/ALDH1(2) genotype, but not one with the atypical ALDH1(2)/ALDH2(2) or ALDH2(2)/ALDH2(2), is capable of detoxifying acetaldehyde transferred from the mother.     

N-terminal acetylation in a third protein family of vertebrate alcohol dehydrogenase/retinal reductase found through a 'proteomics' approach in enzyme characterization

A recent finding of a novel class of retinol-active alcohol dehydrogenase (ADH) in frog prompted analysis of this activity in other vertebrate forms. Surprisingly, yet another and still more unrelated ADH was identified in chicken tissues. It was found to be a member of the aldo-keto reductase (AKR) enzyme family, not previously known as an ADH in vertebrates. Its terminal blocking group and the N-terminal segment, not assigned by protein and cDNA structure analysis, were determined by electrospray tandem mass spectrometry after protein isolation by two-dimensional gel electrophoresis. The N terminus is Acetyl-Ala- and the N-terminal segment contains two consecutive Asn residues. The results establish the new ADH enzyme of the AKR family and show the usefulness of combined gel separation and mass spectrometry in enzyme-characterization.     

Cross-reactive monoclonal antibodies to alcohol dehydrogenases

Three anti-horse liver alcohol dehydrogenase (HLADH) monoclonal antibodies are described. Two are specific for ADH and cross-react with class I and II enzymes from mouse, horse and Chinese hamster. They are specific for the native enzyme but do not inhibit enzyme activity except when combined at high concentration. The third antibody was isolated as a response to rabbit metallothionein. It binds metalloproteins and inhibits ADH activity.     

Antiquitin, a relatively unexplored member in the superfamily of aldehyde dehydrogenases with diversified physiological functions

Antiquitin is a member of the aldehyde dehydrogenase superfamily. Sequence analyses indicate that the protein is highly conserved from plants to animals. The plant antiquitins are generally believed to play a role in osmoregulation and/or detoxification. The physiological functions of animal antiquitins remain largely elusive, their involvement in a number of human diseases has been implicated. Received 28 February 2006; received after revision 13 July 2006; accepted 31 August 2006     

Selectivity determinants of the aldose and aldehyde reductase inhibitor-binding sites

Aldose reductase and aldehyde reductase belong to the aldo-keto reductase superfamily of enzymes whose members are responsible for a wide variety of biological functions. Aldose reductase has been identified as the first enzyme involved in the polyol pathway of glucose metabolism which converts glucose into sorbitol. Glucose over-utilization through the polyol pathway has been linked to tissue-based pathologies associated with diabetes complications, which make the development of a potent aldose reductase inhibitor an obvious and attractive strategy to prevent or delay the onset and progression of the complications. Structural studies of aldose reductase and the homologous aldehyde reductase in complex with inhibitor were carried out to explain the difference in the potency of enzyme inhibition. The aim of this review is to provide a comprehensive summary of previous studies to aid the development of aldose reductase inhibitors that may have less toxicity problems than the currently available ones. Received 4 December 2006; received after revision 12 February 2007; accepted 20 April 2007     

A common ancestor for a subunit in the mitochondrial proton-translocating NADH:ubiquinone oxidoreductase (complex I) and short-chain dehydrogenases/reductases

The proton-translocating NADH:ubiquinone oxidoreductase or complex I is located in the inner membranes of mitochondria, where it catalyzes the transfer of electrons from NADH to ubiquinone. Here we report that one of the subunits in complex I is homologous to short-chain dehydrogenases and reductases, a family of enzymes with diverse activities that include metabolizing steroids, prostaglandins and nucleotide sugars. We discovered that a subunit of complex I in human, cow, Neurospora crassa and Aquifex aeolius is homologous to nucleotide-sugar epimerases and hydroxysteroid dehydrogenases while seeking distant homologs of these enzymes with a hidden Markov model-based search of Genpept. This homology allows us to use information from the solved three-dimensional structures of nucleotide-sugar epimerases and hydroxysteroid dehydrogenases and our motif analysis of these enzymes to predict functional domains on their homologs in complex I. Received 26 November 1998; received after revision 12 January 1999; accepted 12 January 1999     

Structure and evolution of vertebrate aldehyde oxidases: from gene duplication to gene suppression

Aldehyde oxidases (AOXs) and xanthine dehydrogenases (XDHs) belong to the family of molybdo-flavoenzymes. Although AOXs are not identifiable in fungi, these enzymes are represented in certain protists and the majority of plants and vertebrates. The physiological functions and substrates of AOXs are unknown. Nevertheless, AOXs are major drug metabolizing enzymes, oxidizing a wide range of aromatic aldehydes and heterocyclic compounds of medical/toxicological importance. Using genome sequencing data, we predict the structures of AOX genes and pseudogenes, reconstructing their evolution. Fishes are the most primitive organisms with an AOX gene (AOXα), originating from the duplication of an ancestral XDH. Further evolution of fishes resulted in the duplication of AOXα into AOXβ and successive pseudogenization of AOXα. AOXβ is maintained in amphibians and it is the likely precursors of reptilian, avian, and mammalian AOX1. Amphibian AOXγ is a duplication of AOXβ and the likely ancestor of reptilian and avian AOX2, which, in turn, gave rise to mammalian AOX3L1. Subsequent gene duplications generated the two mammalian genes, AOX3 and AOX4. The evolution of mammalian AOX genes is dominated by pseudogenization and deletion events. Our analysis is relevant from a structural point of view, as it provides information on the residues characterizing the three domains of each mammalian AOX isoenzyme. We cloned the cDNAs encoding the AOX proteins of guinea pig and cynomolgus monkeys, two unique species as to the evolution of this enzyme family. We identify chimeric RNAs from the human AOX3 and AOX3L1 pseudogenes with potential to encode a novel microRNA.     

Ethanol metabolizing system inDrosophila melanogaster: subcellular distribution of some main enzymes

Summary The subcellular distribution of some enzymes which play a part in ethanol metabolism have been determined by differential centrifugation of homogenates of adultD. melanogaster flies of various genotypes. Aldehyde dehydrogenase, recently discovered inD. melanogaster, is present in the five genotypes studied. It has been found however to be, in vitro at least, most active in a strain lacking both alcohol dehydrogenase and aldehyde oxidase.     

Nicotinoprotein (NAD+ -containing) alcohol dehydrogenase: structural relationships and functional interpretations

The primary structure of nicotinoprotein alcohol dehydrogenase (ADH) from Amycolatopsis methanolica was determined and used for modelling against known ADH structures, and for evaluation of the coenzyme binding. The results establish the medium-chain dehydrogenase/reductase nature of the nicotinoprotein ADH. Its subunit model and that of the human class Ibeta ADH subunit structure are similar, with mean a carbon deviations of 0.95 A, but they differ in seven loops. Nicotinoprotein ADH occupies a phylogenetic position intermediate between the dimeric and tetrameric ADH families. Two of the differing loops are important for coenzyme binding in the nicotinoprotein model, where one (with a Thr271Arg exchange towards the traditional enzyme) may suggest a slight rotation of the coenzyme adenine ring in the nicotinoprotein, and the other, with an Asn288 insertion, may suggest an extra hydrogen bond to its nicotinamide ribose, favouring stronger binding of the coenzyme. Combined with previous data, this suggests differences in the details of the tight coenzyme binding in different nicotinoproteins, but a common mode for this binding by loop differences.     

Decreased levels of complex III core protein 1 and complex V beta chain in brains from patients with Alzheimer's disease and Down syndrome

Ubiquinol:cytochrome c oxidoreductase (complex III) and ATP synthase (complex V) are important enzymes in the mitochondrial electron transport chain. Defects in mitochondrial respiratory enzymes have been reported for several neurodegenerative diseases. In this study, we applied the proteomic approach to investigate protein levels of complex III core protein and complex V beta chain in brain regions of Alzheimer's disease (AD) and Down syndrome (DS) patients. Complex III core protein 1 was significantly reduced in the temporal cortex of AD patients. Complex V beta chain was significantly reduced in the frontal cortex of DS patients. We conclude that decreased mitochondrial respiratory enzymes could contribute to the impairment of energy metabolism observed in DS. These decreases could also cause the generation of reactive oxygen species and neuronal cell death (apoptosis) in DS as well as AD.     

Class III adenylyl cyclases: molecular mechanisms of catalysis and regulation

Class III adenylyl cyclases are the most abundant type of cyclic AMP-producing enzymes. The adjustment of the cellular levels of this second messenger is achieved by a variety of regulatory mechanisms which couple signals to adenylyl cyclase activity. Because of the divergent nature of stimuli which impinge on these enzymes, highly individualized class III adenylyl cyclases have evolved in metazoans, eukaryotic unicells and bacteria. Regulation usually exploits the dimeric structure of the catalyst, whose active centres form at the dimer interface. The fold of the catalytic domains and the basic catalytic mechanisms are similar in all class III adenylyl cyclases, and substrate binding generally closes the active site by an induced-fit mechanism. Regulatory inputs can result in dramatic rearrangements of the catalytic domains within the dimer, which often are based on rotational movements. Received 13 February 2006; received after revision 16 March 2006; accepted 20 April 2006