Medium- and short-chain dehydrogenase/reductase gene and protein families : Dual functions of alcohol dehydrogenase 3: implications with focus on formaldehyde dehydrogenase and S-nitrosoglutathione reductase activities.

Alcohol dehydrogenase 3 (ADH3) is highly conserved, ubiquitously expressed in mammals and involved in essential cellular pathways. A large active site pocket entails special substrate specificities: shortchain alcohols are poor substrates, while medium-chain alcohols and particularly the glutathione adducts S-hydroxymethylglutathione (HMGSH) and S-nitrosoglutathione (GSNO) are efficiently converted under concomitant use of NAD(+)/NADH. By oxidation of HMGSH, the spontaneous glutathione adduct of formaldehyde, ADH3 is implicated in the detoxification of formaldehyde. Through the GSNO reductase activity, ADH3 can affect the transnitrosation equilibrium between GSNO and S-nitrosated proteins, arguing for an important role in NO homeostasis. Recent findings suggest that ADH3-mediated GSNO reduction and subsequent product formation responds to redox states in terms of NADH availability and glutathione levels. Finally, a dual function of ADH3 is discussed in view of its potential implications for asthma.

Serum-responsive expression of carbonyl-metabolizing enzymes in normal and transformed human buccal keratinocytes.

Gene expression of carbonyl-metabolizing enzymes (CMEs) was investigated in normal buccal keratinocytes (NBK) and the transformed buccal keratinocyte lines SVpgC2a and SqCC/Y1. Studies were performed at a serum concentration known to induce terminal squamous differentiation (TSD) in normal cells. Overall, 39 of 58 evaluated CMEs were found to be expressed at the transcript level. Together the transformed cell lines showed altered transcription of eight CME genes compared to NBK, substantiating earlier results. Serum increased transcript levels of ALDH1A3, DHRS3, HPGD and AKR1A1, and decreased those of ALDH4A1 in NBK; of these, the transformed, TSD-deficient cell lines partly retained regulation of ALDH1A3 and DHRS3. Activity measurements in crude cell lysates, including relevant enzymatic inhibitors, indicated significant capacity for CME-mediated xenobiotic metabolism among the cell lines, notably with an increase in serum-differentiated NBK. The results constitute the first evidence for differential CME gene expression and activity in non-differentiated and differentiated states of epithelial cells.

Energy-linked transhydrogenase. Characterization of a nucleotide-binding sequence in nicotinamide nucleotide transhydrogenase from beef heart.

Purified nicotinamide nucleotide transhydrogenase from beef heart was investigated with respect to labeling and subsequent sequence analysis of a nicotinamide nucleotide-binding site. A photo-activated azide derivative, 8-azidoadenosine 5'-monophosphate, was used as an active-site-directed photoaffinity label, which was shown to be specific for the NAD(H)-binding site in the dark. Light-activated incorporation of the label in transhydrogenase was accompanied by an inactivation, which approached 100% at the incorporation of about 1 mol label/mol transhydrogenase monomer. As expected from the assumed site-specificity of the label. NADH prevented both labeling and inactivation to some extent. However, NADPH also prevented labeling and inactivation marginally. The oxidized substrates NAD+ and NADP+ were inhibitory by themselves under these conditions, and the substrate analogs 5'-AMP and 2'-AMP were also poor protectors. The NAD(H)-site specificity of the azido compound was thus largely lost upon illumination and covalent modification. Radioactive labeling of transhydrogenase with 8-azido-[2-3H]-adenosine 5'-monophosphate followed by protease digestion, isolation of labeled peptides and amino-acid sequence analysis showed that Tyr 1006 in the sequence 1001-1027 close to the C-terminus was labeled. This sequence shows homologies with nucleotide-binding sequences in, e.g., F1-ATPase. On the basis of sequence homologies with other NAD(P)-dependent enzymes it is proposed that transhydrogenase contains 4 nucleotide-binding sites, of which 2 constitute the adenine nucleotide-binding domains of the catalytic sites for NAD(H) and NADP(H) close to the N- and C-terminals, respectively. Each of these domains has an additional vicinal nucleotide-binding sequence which may constitute a non-catalytic nucleotide-binding site or the nicotinamide nucleotide-binding domain of the catalytic site. The present results indicate that 8-azidoadenosine 5'-monophosphate is kinetically specific for the catalytic NAD(H)-binding site, but reacts covalently with Tyr 1006 of the putative non-catalytic site or nicotinamide nucleotide-binding domain formed by the 1001-1027 amino acid sequence of the catalytic NADP(H)-binding site. Interactions between the catalytic NAD(H) and NADP(H) binding sites, and the assumed non-catalytic sites, may be facilitated by a ligand-triggered formation of a narrow pocket, which normally allows an efficient hydride ion transfer between the natural substrates.

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.

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.

Cloning and characterization of the human ADH4 gene.

Human alcohol dehydrogenase (ADH) constitutes a set of isozymes and enzymes with different tissue and substrate specificities. The subunits are coded for by at least five gene loci, ADH1-ADH5. We now report the cloning and analysis of the human ADH4 gene coding for the class-II ADH with pi-subunits. The gene spans a region of 21 kb and is divided into nine exons and eight introns. The arrangement is the same as for all analyzed mammalian class-I genes, but the region covered is 50% larger than that in the human class-I genes. The nucleotide (nt) sequences of the exons, exon/intron boundaries and 5'- and 3'-untranslated regions were determined. The transcription start point (tsp) of the ADH4 gene was defined by primer extension and localized to a position 61 nt upstream from the ATG start codon. A TATA box and a CAAT element were identified by homology to consensus sequences for tsp. No DNA structures homologous to the glucocorticoid-responsive elements (GRE) present in the ADH2 gene were found in the upstream region of the ADH4 gene, but two structures with a 70% identity to the GRE consensus sequence were found at nonhomologous locations. The difference and the overall low degree of identity, 41%, of the upstream regions suggest different regulatory mechanisms for the class-I and class-II genes.

Cloning and expression of the glutaredoxin (grx) gene of Escherichia coli.

Two DNA segments, together comprising 1147 bp and containing the glutaredoxin (GRX) gene, grx, from Escherichia coli K-12 were cloned and characterized in M13mp9. The gene was identified by hybridization with synthetic oligodeoxyribonucleotide probes corresponding to parts of the amino acid (aa) sequence of GRX. The sequence of 255 bp comprising the GRX structural gene gave a deduced as sequence identical to the directly determined one. The coding region is preceded by two possible ribosome-binding sites and three possible promoters with -10 and -35 regions as judged by homology to consensus sequences. The presence of a stable stem-loop structure, delta G = -17.0 kcal, followed by six thymine bases indicates that the transcription of the grx gene is Rho-independently terminated. An over-representation of rare codons in the grx gene, as compared to the genes for thioredoxin (TRX) and highly expressed proteins, is suggested as one possible explanation for the large difference in the synthesis between TRX and GRX in wild-type E. coli cells. GRX production was amplified at least 100 fold in strain JM103[pEMBL9ECG] over that in wild-type E. coli cells. The protein purified from the overproducing strain was identical in aa sequence with the previously analyzed GRX protein.

Cloning and characterization of a novel rat alcohol dehydrogenase of class II type.

A class II type alcohol dehydrogenase from rat liver was characterized at the cDNA level after screening cDNA libraries in combination with PCR amplification of the 5'-part. The open reading frame translates into a polypeptide of 376 amino acid residues, which show 73% positional identity to the human class II enzyme. This suggests that the class II enzyme is the most variable form of the mammalian alcohol dehydrogenases. A deletion is apparent corresponding to position 294 of the human enzyme and amino acid residues unique to the rat protein of those interacting with the coenzyme NAD+ are found at positions 47, 51, 178, and 271. Position 47 is occupied by Pro instead of Arg or His found in most mammalian alcohol dehydrogenases. This exchanged residue will not hydrogen bond to the pyrophosphate of the coenzyme and will change the local environment around position 47 to strictly hydrophobic.

Mitochondrial aldehyde dehydrogenase. Homology of putative targeting sequence to that of carbamyl phosphate synthetase I revealed by correlation of cDNA and protein data.

Comparison of existing protein and cDNA data for human liver mitochondrial aldehyde dehydrogenase reveals deviations in two segments. They are shown to correspond exactly to localized frameshifts in the cDNA data, which are likely to have three reading errors. After correction of the cDNA frameshifts, a deduced amino acid sequence corresponds exactly to the data established at the protein level. In addition, extension of the shifted frame into the cDNA corresponding to the mitochondrial leader sequence allows reinterpretation of that sequence. The new leader sequence is consistent with characteristics of such segments of other mitochondrial protein pro-forms. Furthermore, the sequence displays a homology, when centered around the cleavage site, with the leader sequence of rat liver carbamyl phosphate synthetase I, suggesting a novel similarity between mitochondrial targeting sequences of two different enzymes.

Alcohol dehydrogenase in human tissues: localisation of transcripts coding for five classes of the enzyme.

Tissue distribution of the five identified classes of human alcohol dehydrogenase was studied by assessment of mRNA levels in 23 adult and four fetal tissues. Alcohol dehydrogenase of class I was found in most tissues, brain and placenta excluded, but expression levels among tissues differed widely. The distribution pattern of class III transcripts was consistent with those of housekeeping enzymes while, in contrast, class IV transcripts were found only in stomach. Transcripts of multiple length were detected for most classes and were due to different gene products arising through the use of different poly-A signals or transcription from different gene loci. Both class II and class V showed a pattern of liver-enriched expression. However, low mRNA levels were detected also in stomach, pancreas and small intestine for class II, and in fetal kidney and small intestine for class V. Significantly higher levels of class V transcripts were present in fetal liver when compared with levels in adult liver, which suggests that human class V is a predominantly fetal alcohol dehydrogenase.

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.

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.

Aldehyde dismutase activity of human liver alcohol dehydrogenase.

Human alcohol dehydrogenases of class I and class II but not class III catalyse NAD+-dependent aldehyde oxidation in addition to the NADH-dependent aldehyde reduction. The two reactions are coupled, i.e. the enzymes display dismutase activity. Dismutase activity of recombinantly expressed human class I isozymes beta1beta1 and gamma2gamma2, class II and class III alcohol dehydrogenases was assayed with butanal as substrate by gas chromatographic-mass spectrometric quantitations of butanol and butyric acid. The class I gamma2gamma2 isozyme showed a pronounced dismutase activity with a high kcat, 1300 min(-1), and a moderate Km, 1.2 mM. The class I beta1beta1 isozyme and the class II alcohol dehydrogenase showed moderate catalytic efficiencies for dismutase activity with lower kcat values, 60-75 min(-1). 4-Methylpyrazole, a potent class I ADH inhibitor, inhibited the class I dismutation completely, but cyanamide, an inhibitor of mitochondrial aldehyde dehydrogenase, did not affect the dismutation. The dismutase reaction might be important for metabolism of aldehydes during inhibition or lack of mitochondrial aldehyde dehydrogenase activity.

Class IV mammalian alcohol dehydrogenase. Structural data of the rat stomach enzyme reveal a new class well separated from those already characterized.

The stomach form of alcohol dehydrogenase has been structurally evaluated by peptide analysis covering six separate regions of the rat enzyme. Overall, this new structure differs widely (32-40% residue differences) from the structures of three classes of alcohol dehydrogenase characterized before from the same species. Consequently, this novel enzyme constitutes a true fourth class of mammalian alcohol dehydrogenase. In particular, differences are extensive also towards class II, although enzymatic and physicochemical properties initially suggested overall similarities with class II. The new structure establishes the presence of one further alcohol dehydrogenase mammalian gene, extends the enzyme family derived from repeated gene duplications, and confirms tissue-specific expressions.

Isolation, characterization and structure of subtilisin from a thermostable Bacillus subtilis isolate.

A serine protease has been isolated and characterized from Bacillus subtilis, strain RT-5 (a thermostable soil isolate from the Tharparkar desert of Pakistan) able to grow at 55 degrees C. The primary structure was established by a combination of protein and DNA-sequence analyses. The amino-acid sequence, inhibition pattern and solubility properties identify the enzyme as a subtilisin. It has 43 amino-acid replacements toward subtilisin BPN' and as much as 83 replacements toward another subtilisin, confirming that strain variabilities are extensive between different subtilisin forms. However, the structure is identical to one of unknown functional properties deduced from DNA and is closely related to mesentericopeptidase but that homologue is not thermostable. From comparisons with that form and with subtilisin BPN', it is concluded that replacements of Ala --> Ser at positions 85 and 89, Ser --> Ala at position 88 and Asp or Ser --> Asn at position 259 may promote thermostability.

Class IV alcohol dehydrogenase (the gastric enzyme). Structural analysis of human sigma sigma-ADH reveals class IV to be variable and confirms the presence of a fifth mammalian alcohol dehydrogenase class.

Human gastric alcohol dehydrogenase (sigma sigma-ADH) was submitted to peptide analysis at picomole scale. A total of 72 positions were determined in the protein chain, providing information on three aspects of alcohol dehydrogenase structures in general. First, the data establish the presence of a unique class of the enzyme, now confirmed as class IV, expressed in gastric tissue and separate from another novel class, now termed class V. Second, the class IV gastric enzyme has active site relationships compatible with an ethanol-active, zinc-containing alcohol dehydrogenase. Third, this enzyme class is of the variable type, like that for the 'variable', classical liver alcohol dehydrogenase of class I, and in contrast to that for the 'constant' class III enzyme. Known human alcohol dehydrogenase structures now prove the presence of at least seven human genes for the enzyme and nine for the whole protein family.

Reptilian alcohol dehydrogenase. Heterogeneity relevant to class multiplicity of the mammalian enzyme.

Liver alcohol dehydrogenase of the ethanol-active type ('class I enzyme') from the lizard, Uromastix hardwickii, was purified and screened for relationships with other vertebrate forms of the enzyme. Two different acetylated N-termini (acetyl-Gly and acetyl-Ser) and further positional differences already in the N-terminal segments establish the presence of two types of protein chain. The multiplicity is different from that hitherto detected within vertebrate class I alcohol dehydrogenase isozymes but typical of that which would be expected for subunits of different classes. In particular, relationships to class II or to class II-related forms appear likely. This may indicate yet further vertebrate alcohol dehydrogenase multiplicity or discovery of a class II non-mammalian enzyme. The results give prospects of defining gene duplications corresponding to more than one alcohol dehydrogenase class split to at an early vertebrate stage.

Alcohol dehydrogenase of class I: kiwi liver enzyme, parallel evolution in separate vertebrate lines, and correlation with 12S rRNA patterns.

Alcohol dehydrogenase class I from kiwi liver has been purified, analyzed, and compared with that of other alcohol dehydrogenases. The results show that several avian and mammalian forms of the enzyme exhibit parallel evolutionary patterns in two independent lineages of a single protein, establishing a pattern in common. Furthermore, the data correlate the enzyme evolutionary pattern with that of 12S rRNA. Biologically, the patterns complement those on ratite and other avian relationships. Functionally, the enzyme has a low Km with ethanol and a branched-chain residue at position 141, like the mammalian enzymes but in contrast to the other characterized ratite enzyme (with Ala-141 and a higher Km). This pattern of natural variability suggests a frequent but not fully complete correlation between a large residue size at position 141 and tight ethanol binding.

cDNA clones coding for the beta-subunit of human liver alcohol dehydrogenase have differently sized 3'-non-coding regions.

Three different size classes of cDNA clones coding for the beta 1-subunit of human alcohol dehydrogenase (ADH) were characterized from a human liver cDNA library. Clones were identified by hybridization with synthetic oligodeoxyribonucleotides. A total of 2530 nucleotides were determined, covering an ADH-coding region of 1122 nucleotides, a preceding 72-nucleotide segment and 3 types of 3'-non-coding region. The coding nucleotide sequence is in full agreement with the amino acid sequence of the beta 1-subunit. Of 8 clones identified, 6 had a short, 213-nucleotide 3'-non-coding region; 1 an intermediate, 590-nucleotide 3'-region; and 1 a long, 1330-nucleotide 3'-region. In addition, 2 unused polyadenylation signals were found. These results suggest that human liver beta-ADH mRNAs occur in several size classes, and that in addition to the consensus sequence AATAAA further signals are important for 3'-end formation.

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.