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J O Höög

Publications and source records attributed to J O Höög.

At least 19 recordsLinked to original sources
PubMed2002

Identification and characterisation of two allelic forms of human alcohol dehydrogenase 2.

The human alcohol dehydrogenase system is comprised of multiple forms that catalyse the oxidation/reduction of a large variety of alcohols and aldehydes. A transition that results in an Ile308Val substitution was identified in the human ADH2 gene by single-strand conformation polymorphism analysis. Screening a Swedish population revealed that Val308 was the most frequent allele (73%), and site-directed mutagenesis was used to obtain both allelozymes, which were expressed in Escherichia coli for characterisation. Thermostability was assayed by activity measurements and circular dichroism spectroscopy. The results showed that the 308Val substitution decreases protein stability, as compared to the Ile308 variant, an effect also demonstrated during prolonged storage. Ethanol, octanol, 12-hydroxydodecanoic acid and all-trans retinol were used as model substrates and, generally, slightly higher Km values were observed with Val at position 308. Finally, homology modelling, from mouse ADH2, further supported the decreased stability of the Val308 variant and located position 308 in the subunit interface of the molecule and in the vicinity of the active-site pocket entrance. In conclusion, the Ile308Val substitution represents a novel functional polymorphism within the human alcohol dehydrogenase gene cluster that may affect the metabolism of ethanol and other substrates.

Alcohol Dehydrogenase↗
PubMed2001

Micro-array chip analysis of carbonyl-metabolising enzymes in normal, immortalised and malignant human oral keratinocytes.

Enzymes involved in various protective and metabolic processes of carbonyl compounds were analysed utilising a micro-array method in a three-stage in vitro model for oral carcinogenesis involving cultured normal, immortalised and malignant human oral keratinocytes. A complete transcript profiling of identified carbonyl-metabolising enzymes belonging to the ADH, ALDH, SDR and AKR families is presented. Expression of 17 transcripts was detected in normal, 14 in immortalized and 19 in malignant keratinocytes of a total of 12,500 genes spotted on the micro-array chip. For the detected transcripts, about half were changed by cell transformation, and for the various enzyme families, differences in expression patterns were observed. The detected AKR transcripts displayed a conserved pattern of expression, indicating a requirement for the keratinocyte phenotype, while most of the detected SDRs displayed changed expression at the various stages of malignancy. The importance of multiple experiments in using a microarray technique for reliable results is underlined and, finally, the strength of the method in detecting co-expressed enzymes in metabolic pathways is exemplified by the detection of the formaldehyde-scavenging pathway enzymes and the polyol pathway enzymes.

Alcohol Dehydrogenase↗
PubMed2001

Functional polymorphism in the alcohol dehydrogenase 3 (ADH3) promoter.

The ADH3 gene encodes alcohol dehydrogenase 3 (ADH3)/glutathione-dependent formaldehyde dehydrogenase, the ancestral and most conserved form of alcohol dehydrogenase. ADH3 is expressed in all tissues examined and the enzyme is essential for formaldehyde scavenging. We have screened the promoter region including exon 1 and exons 5, 6 and 7 of the ADH3 gene for allelic variants. Using 80 samples of genomic DNA from Swedes as template, the various parts of the gene were PCR amplified and subsequently analyzed on single strand conformation polymorphism (SSCP) gels. No abnormal migration patterns could be detected by SSCP analysis of exons 5, 6 and 7 while for the promoter region, a large number of the samples displayed differences in SSCP gel migration patterns. Cloning and sequence analysis revealed four possible base pair exchanges in the promoter region. Two transitions were found at position -197 and -196, GG --> AA, one at position -79, G --> A and finally, close to the transcription start site, a fourth transition was found at position +9, C --> T. An allele specific PCR method was developed and allele frequencies were determined in three populations: Chinese, Spanish and Swedish. GG-197,-196 and AA-197,-196 alleles were common in all three populations, G-79 and A-79 were common in Swedes and Spaniards but only A-79 was found among Chinese. T+9 was the most rare allele with an allele frequency of 1.5% in Swedes. Finally, promoter activity assessments and electrophoretic mobility shift assays demonstrated that the C+9 --> T+9 exchange resulted in a significant transcriptional decrease in HeLa cells and a decreased binding of nuclear proteins. These base pair exchanges may have an effect on the expression of the enzyme and thereby influence the capacity of certain individuals to metabolize formaldehyde.

5' Untranslated Regions↗
PubMed2000

Human class V alcohol dehydrogenase (ADH5): A complex transcription unit generates C-terminal multiplicity.

The human ADH5 gene was reported to lack the last exon compared to other mammalian ADHs and consequently should be expressed as a truncated protein. Here we show with PCR amplification of 3'-cDNA ends that the ADH5 gene harbors the "missing" exon. Besides a cDNA identical to the published sequence, we found full-length transcripts that contained additional codons for eight amino acid residues. Northern blot analysis established the full-length variant as the major transcript with the strongest signal from adult liver. Sequence analysis of genomic DNA confirmed that the ADH5 gene displays composite internal/terminal exons, which can be differentially processed; i.e., 3'-end generation is a result of competition between polyadenylation and splicing.

Alcohol Dehydrogenase↗
PubMed2000

Crystal structures of mouse class II alcohol dehydrogenase reveal determinants of substrate specificity and catalytic efficiency.

The structure of mouse class II alcohol dehydrogenase (ADH2) has been determined in a binary complex with the coenzyme NADH and in a ternary complex with both NADH and the inhibitor N-cyclohexylformamide to 2.2 A and 2.1 A resolution, respectively. The ADH2 dimer is asymmetric in the crystal with different orientations of the catalytic domains relative to the coenzyme-binding domains in the two subunits, resulting in a slightly different closure of the active-site cleft. Both conformations are about half way between the open apo structure and the closed holo structure of horse ADH1, thus resembling that of ADH3. The semi-open conformation and structural differences around the active-site cleft contribute to a substantially different substrate-binding pocket architecture as compared to other classes of alcohol dehydrogenase, and provide the structural basis for recognition and selectivity of alcohols and quinones. The active-site cleft is more voluminous than that of ADH1 but not as open and funnel-shaped as that of ADH3. The loop with residues 296-301 from the coenzyme-binding domain is short, thus opening up the pocket towards the coenzyme. On the opposite side, the loop with residues 114-121 stretches out over the inter-domain cleft. A cavity is formed below this loop and adds an appendix to the substrate-binding pocket. Asp301 is positioned at the entrance of the pocket and may control the binding of omega-hydroxy fatty acids, which act as inhibitors rather than substrates. Mouse ADH2 is known as an inefficient ADH with a slow hydrogen-transfer step. By replacing Pro47 with His, the alcohol dehydrogenase activity is restored. Here, the structure of this P47H mutant was determined in complex with NADH to 2.5 A resolution. His47 is suitably positioned to act as a catalytic base in the deprotonation of the substrate. Moreover, in the more closed subunit, the coenzyme is allowed a position closer to the catalytic zinc. This is consistent with hydrogen transfer from an alcoholate intermediate where the Pro/His replacement focuses on the function of the enzyme.

Alcohol Dehydrogenase↗
PubMed2000

Studies on the interaction between ethanol and serotonin metabolism in rat, using deuterated ethanol and 4-methylpyrazole.

The metabolic interaction between ethanol and serotonin (5-hydroxytryptamine) via alcohol dehydrogenase (ADH; EC 1.1.1.1) was studied in tissue homogenates of Sprague-Dawley rats by following the transfer of deuterium from deuterated ethanol over endogenous NADH to 5-hydroxytryptophol (5HTOL). Homogenates of whole brain, lung, spleen, kidney, liver, stomach, jejunum, ileum, colon, and caecum were incubated in the presence of [2H2]ethanol and 5-hydroxyindole-3-acetaldehyde (5HIAL), and the [2H]5HTOL formed was identified and quantified using gas chromatography-mass spectrometry. ADH activity was most abundant in liver, kidney, and within the gastrointestinal tract. The highest incorporation of deuterium was obtained in homogenates of kidney, lung, and colon, whereas in brain, which contains very low ADH activity, no incorporation could be demonstrated. Addition of extra NAD+ (2.4 mM) increased the formation of [2H]5HTOL 2.6-fold in liver homogenates, but only 1.2-fold in kidney homogenates. 4-Methylpyrazole, a potent inhibitor of class I ADH, inhibited the 5HIAL reduction in homogenates of lung, kidney, jejunum, ileum, and colon, and caused a marked drop in 5HTOL oxidation in all tissues except stomach and spleen. These results demonstrate that in the rat a metabolic interaction between ethanol and serotonin via the ADH pathway may take place in several tissues besides the liver, which is the main tissue for ethanol detoxification.

Alcohol Dehydrogenase↗
PubMed2000

Expression of alcohol dehydrogenase 3 in tissue and cultured cells from human oral mucosa.

Because formaldehyde exposure has been shown to induce pathological changes in human oral mucosa, eg, micronuclei, the potential enzymatic defense by alcohol dehydrogenase 3 (ADH3)/glutathione-dependent formaldehyde dehydrogenase was characterized in oral tissue specimens and cell lines using RNA hybridization and immunological methods as well as enzyme activity measurements. ADH3 mRNA was expressed in basal and parabasal cell layers of oral epithelium, whereas the protein was detected throughout the cell layers. ADH3 mRNA and protein were further detected in homogenates of oral tissue and various oral cell cultures, including, normal, SV40T antigen-immortalized, and tumor keratinocyte lines. Inhibition of the growth of normal keratinocytes by maintenance at confluency significantly decreased the amount of ADH3 mRNA, a transcript with a determined half-life of 7 hours. In contrast, decay of ADH3 protein was not observed throughout a 4-day period in normal keratinocytes. In samples from both tissue and cells, the ADH3 protein content correlated to oxidizing activity for the ADH3-specific substrate S:-hydroxymethylglutathione. The composite analyses associates ADH3 mRNA primarily to proliferative keratinocytes where it exhibits a comparatively short half-life. In contrast, the ADH3 protein is extremely stable, and consequently is retained during the keratinocyte life span in oral mucosa. Finally, substantial capacity for formaldehyde detoxification is shown from quantitative assessments of alcohol- and aldehyde-oxidizing activities including K:(m) determinations, indicating that ADH3 is the major enzyme involved in formaldehyde oxidation in oral mucosa.

Aldehyde Oxidoreductases↗
PubMed2000

Human liver class I alcohol dehydrogenase gammagamma isozyme: the sole cytosolic 3beta-hydroxysteroid dehydrogenase of iso bile acids.

3beta-Hydroxy (iso) bile acids are formed during enterohepatic circulation from 3alpha-hydroxy bile acids and constitute normal compounds in plasma but are virtually absent in bile. Isoursodeoxycholic acid (isoUDCA) is a major metabolite of UDCA. In a recent study it was found that after administration of isoUDCA, UDCA became the major acid in bile. Thus, epimerization of the 3beta-hydroxy to a 3alpha-hydroxy group, catalyzed by 3beta-hydroxysteroid dehydrogenases (HSD) and 3-oxo-reductases must occur. The present study aims to characterize the human liver bile acid 3beta-HSD. Human liver cytosol and recombinant alcohol dehydrogenase (ADH) betabeta and gammagamma isozymes were subjected to native polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing. Activity staining with oxidized nicotinamide adenine dinucleotide (NAD(+)) or oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)) as cofactors and various iso bile acids as substrates was used to screen for 3beta-HSD activity. Reaction products were identified and quantified by gas chromotography/mass spectrometry (GC/MS). Computer-assisted substrate docking of isoUDCA to the active site of a 3-dimensional model of human class I gammagamma ADH was performed. ADH gammagamma isozyme was identified as the iso bile acid 3beta-HSD present in human liver cytosol, with NAD(+) as a cofactor. Values for k(cat)/K(m) were in the rank order isodeoxycholic acid (isoDCA), isochenodeoxycholic acid (isoCDCA), isoUDCA, and isolithocholic acid (isoLCA) (0.10, 0.09, 0.08, and 0. 05 min(-1) x micromol/L(-1), respectively). IsoUDCA fits as substrate to the 3-dimensional model of the active-site of ADH gammagamma. ADH gammagamma isozyme was defined as the only bile acid 3beta-HSD in human liver cytosol. Hydroxysteroid dehydrogenases are candidates for the binding and transport of 3alpha-hydroxy bile acids. We assume that ADH gammagamma isozyme is involved in cytosolic bile acid binding and transport processes as well.

3-Hydroxysteroid Dehydrogenases↗
PubMed2000

Pharmacogenetics of the alcohol dehydrogenase system.

Alcohol dehydrogenase (ADH) constitutes a complex enzyme system with different forms and extensive multiplicity. A combination of constant and variable properties regarding function, multiplicity and structure of ADH is highlighted for the human system and extended to ADH forms in general. Future perspectives suggest continued studies in specific directions for distinction of metabolic, regulatory and pharmacogenetic roles of ADH.

Alcohol Dehydrogenase↗
PubMed1999

A novel subtype of class II alcohol dehydrogenase in rodents. Unique Pro(47) and Ser(182) modulates hydride transfer in the mouse enzyme.

Mice and rats were found to possess class II alcohol dehydrogenases with novel enzymatic and structural properties. A cDNA was isolated from mouse liver and the encoded alcohol dehydrogenase showed high identity (93.1%) with the rat class II alcohol dehydrogenase which stands in contrast to the pronounced overall variability of the class II line. The two heterologously expressed rodent class II enzymes exhibited over 100-fold lower catalytic efficiency (k(cat)/K(m)) for oxidation of alcohols as compared with other alcohol dehydrogenases and were not saturated with ethanol. Hydride transfer limited the rate of octanol oxidation as indicated by a deuterium isotope effect of 4.8. The mutation P47H improved hydride transfer and turnover rates were increased to the same level as for the human class II enzyme. Michaelis constants for alcohols and aldehydes were decreased while they were increased for the coenzyme. The rodent class II enzymes catalyzed reduction of p-benzoquinone with about the same maximal turnover as for the human form. This activity was not affected by the P47H mutation while a S182T mutation increased the K(m) value for benzoquinone 10-fold. omega-Hydroxy fatty acids were catalyzed extremely slow but functioned as potent inhibitors by binding to the enzyme-NAD(+) complex. All these data indicate that the mammalian class II alcohol dehydrogenase line is divided into two structurally and functionally distinct subgroups.

Alcohol Dehydrogenase↗
PubMed1999

Recommended nomenclature for the vertebrate alcohol dehydrogenase gene family.

The alcohol dehydrogenase (ADH) gene family encodes enzymes that metabolize a wide variety of substrates, including ethanol, retinol, other aliphatic alcohols, hydroxysteroids, and lipid peroxidation products. Studies on 19 vertebrate animals have identified ADH orthologs across several species, and this has now led to questions of how best to name ADH proteins and genes. Seven distinct classes of vertebrate ADH encoded by non-orthologous genes have been defined based upon sequence homology as well as unique catalytic properties or gene expression patterns. Each class of vertebrate ADH shares <70% sequence identity with other classes of ADH in the same species. Classes may be further divided into multiple closely related isoenzymes sharing >80% sequence identity such as the case for class I ADH where humans have three class I ADH genes, horses have two, and mice have only one. Presented here is a nomenclature that uses the widely accepted vertebrate ADH class system as its basis. It follows the guidelines of human and mouse gene nomenclature committees, which recommend coordinating names across species boundaries and eliminating Roman numerals and Greek symbols. We recommend that enzyme subunits be referred to by the symbol "ADH" (alcohol dehydrogenase) followed by an Arabic number denoting the class; i.e. ADH1 for class I ADH. For genes we recommend the italicized root symbol "ADH" for human and "Adh" for mouse, followed by the appropriate Arabic number for the class; i.e. ADH1 or Adh1 for class I ADH genes. For organisms where multiple species-specific isoenzymes exist within a class, we recommend adding a capital letter after the Arabic number; i.e. ADH1A, ADH1B, and ADH1C for human alpha, beta, and gamma class I ADHs, respectively. This nomenclature will accommodate newly discovered members of the vertebrate ADH family, and will facilitate functional and evolutionary studies.

Alcohol Dehydrogenase↗
PubMed1999

SDR and MDR: completed genome sequences show these protein families to be large, of old origin, and of complex nature.

Short-chain dehydrogenases/reductases (SDR) and medium-chain dehydrogenases/reductases (MDR) are protein families originally distinguished from characterisations of alcohol dehydrogenase of these two types. Screening of completed genome sequences now reveals that both these families are large, wide-spread and complex. In Escherichia coli alone, there are no fewer than 17 MDR forms, identified as open reading frames, considerably extending previously known MDR relationships in prokaryotes and including ethanol-active alcohol dehydrogenase. In entire databanks, 1056 SDR and 537 MDR forms are currently known, extending the multiplicity further. Complexity is also large, with several enzyme activity types, subgroups and evolutionary patterns. Repeated duplications can be traced for the alcohol dehydrogenases, with independent enzymogenesis of ethanol activity, showing a general importance of this enzyme activity.

Evolution, Molecular↗
PubMed1999

An ethanol-inducible MDR ethanol dehydrogenase/acetaldehyde reductase in Escherichia coli: structural and enzymatic relationships to the eukaryotic protein forms.

An ethanol-active medium-chain dehydrogenase/reductase (MDR) alcohol dehydrogenase was isolated and characterized from Escherichia coli. It is distinct from the fermentative alcohol dehydrogenase and the class III MDR alcohol dehydrogenase, both already known in E. coli. Instead, it is reminiscent of the MDR liver enzyme forms found in vertebrates and has a K(m) for ethanol of 0.7 mM, similar to that of the class I enzyme in humans, however, it has a very high k(cat), 4050 min(-1). It is also inhibited by pyrazole (K(i) = 0.2 microM) and 4-methylpyrazole (K(i)= 44 microM), but in a ratio that is the inverse of the inhibition of the human enzyme. The enzyme is even more efficient in the reverse direction of acetaldehyde reduction (K(m) = 30 microM and k(cat) = 9800 min(-1)), suggesting a physiological function like that seen for the fermentative non-MDR alcohol dehydrogenase. Growth parameters in complex media with and without ethanol show no difference. The structure corresponds to one of 12 new alcohol dehydrogenase homologs present as ORFs in the E. coli genome. Together with the previously known E. coli MDR forms (class III alcohol dehydrogenase, threonine dehydrogenase, zeta-crystallin, galactitol-1-phosphate dehydrogenase, sensor protein rspB) there is now known to be a minimum of 17 MDR enzymes coded for by the E. coli genome. The presence of this bacterial MDR ethanol dehydrogenase, with a structure compatible with an origin separate from that of yeast, plant and animal ethanol-active MDR forms, supports the view of repeated duplicatory origins of alcohol dehydrogenases and of functional convergence to ethanol/acetaldehyde activity. Furthermore, this enzyme is ethanol inducible in at least one E. coli strain, K12 TG1, with apparently maximal induction at an enthanol concentration of approximately 17 mM. Although present in several strains under different conditions, inducibility may constitute an explanation for the fairly late characterization of this E. coli gene product.

Alcohol Dehydrogenase↗
PubMed1999

Activities of human alcohol dehydrogenases in the metabolic pathways of ethanol and serotonin.

Alcohols and aldehydes in the metabolic pathways of ethanol and serotonin are substrates for alcohol dehydrogenases (ADH) of class I and II. In addition to the reversible alcohol oxidation/aldehyde reduction, these enzymes catalyse aldehyde oxidation. Class-I gammagamma ADH catalyses the dismutation of both acetaldehyde and 5-hydroxyindole-3-acetaldehyde (5-HIAL) into their corresponding alcohols and carboxylic acids. The turnover of acetaldehyde dismutation is high (kcat = 180 min-1) but saturation is reached first at high concentrations (Km = 30 mm) while dismutation of 5-HIAL is saturated at lower concentrations and is thereby more efficient (Km = 150 microm; kcat = 40 min-1). In a system where NAD+ is regenerated, the oxidation of 5-hydroxytryptophol to 5-hydroxyindole-3-acetic acid proceeds with concentration levels of the intermediary 5-HIAL expected for a two-step oxidation. Butanal and 5-HIAL oxidation is also observed for class-I ADH in the presence of NADH. The class-II enzyme is less efficient in aldehyde oxidation, and the ethanol-oxidation activity of this enzyme is competitively inhibited by acetate (Ki = 12 mm) and 5-hydroxyindole-3-acetic acid (Ki = 2 mm). Reduction of 5-HIAL is efficiently catalysed by class-I gammagamma ADH (kcat = 400 min-1; Km = 33 microm) in the presence of NADH. This indicates that the increased 5-hydroxytryptophol/5-hydroxyindole-3-acetic acid ratio observed after ethanol intake may be due to the increased NADH/NAD+ ratio on the class-I ADH.

Alcohol Dehydrogenase↗
PubMed1998

An attempt to transform class characteristics within the alcohol dehydrogenase family.

Human class I alcohol dehydrogenase was mutated at positions 57 and 115, exchanging for Asp and Arg respectively, in an attempt to introduce glutathione-dependent formaldehyde dehydrogenase characteristics. In addition, class III alcohol dehydrogenase, identical to glutathione-dependent formaldehyde dehydrogenase, was mutated at position 115, introducing Ser or Lys. The attempted class transformation was partly successful considering a higher affinity for 12-hydroxydodecanoate and a lower affinity for ethanol that was monitored for the class I mutant. However, the class I mutant displayed neither glutathione-dependent formaldehyde dehydrogenase activity nor fatty acid activation of alcohol oxidation. Interestingly, both class III mutants showed reduced activities for S-hydroxymethylglutathione and 12-hydroxydodecanoate through increased Km, values. Overall results show that it is not possible, by single point mutations, to completely transform enzyme characteristics between these two classes of alcohol dehydrogenase.

Alcohol Dehydrogenase↗
PubMed1998

Structural and functional divergence of class II alcohol dehydrogenase--cloning and characterisation of rabbit liver isoforms of the enzyme.

cDNAs coding for class II alcohol dehydrogenase were isolated from a rabbit-liver cDNA library. Deduced amino acid sequences show that isozymic forms of rabbit class II alcohol dehydrogenase exist, with a positional identity of 88.4%. A high variability in structure of class II alcohol dehydrogenase between the species is also reflected in function. The rabbit II-1 isozyme shows common characteristics with the human enzyme, but has a lower Km value for ethanol, 4.2 mM. The II-2 isozyme shows restriction for aliphatic alcohols longer than pentanol. For shorter alcohols the II-2 form has similar Km values as the II-1 isozyme, 5.5 mM for ethanol, but is a low activity variant with a 10-fold decrease in k(cat) values compared with II-1. Nevertheless, II-2 has a higher specificity for benzoquinone than II-1 due to a lower Km value, 80 microM compared with 1 mM, and is in this sense more like the human class II enzyme. In addition a rabbit class III alcohol dehydrogenase cDNA was isolated that encodes a typical class III enzyme/glutathione-dependent formaldehyde dehydrogenase. The finding of isozymic forms of class II alcohol dehydrogenase is in line with the evolution of the system of medium-chain alcohol dehydrogenases with different enzymes, different classes and different isozymes and further underline the complexity of the entire mammalian alcohol dehydrogenase system.

Alcohol Dehydrogenase↗