Jan-Olov Höög

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.

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.

Mammalian alcohol dehydrogenase — Functional and structural implications

Mammalian alcohol dehydrogenase (ADH) constitutes a complex system with different forms and extensive multiplicity (ADH1-ADH6) that catalyze the oxidation and reduction of a wide variety of alcohols and aldehydes. The ADH1 enzymes, the classical liver forms, are involved in several metabolic pathways beside the oxidation of ethanol, e.g. norepinephrine, dopamine, serotonin and bile acid metabolism. This class is also able to further oxidize aldehydes into the corresponding carboxylic acids, i.e. dismutation. ADH2, can be divided into two subgroups, one group consisting of the human enzyme together with a rabbit form and another consisting of the rodent forms. The rodent enzymes almost lack ethanol-oxidizing capacity in contrast to the human form, indicating that rodents are poor model systems for human ethanol metabolism. ADH3 (identical to glutathione-dependent formaldehyde dehydrogenase) is clearly the ancestral ADH form and S-hydroxymethylglutathione is the main physiological substrate, but the enzyme can still oxidize ethanol at high concentrations. ADH4 is solely extrahepatically expressed and is probably involved in first pass metabolism of ethanol beside its role in retinol metabolism. The higher classes, ADH5 and ADH6, have been poorly investigated and their substrate repertoire is unknown. The entire ADH system can be seen as a general detoxifying system for alcohols and aldehydes without generating toxic radicals in contrast to the cytochrome P450 system.

Reduction of S-nitrosoglutathione by alcohol dehydrogenase 3 is facilitated by substrate alcohols via direct cofactor recycling and leads to GSH-controlled formation of glutathione transferase inhibitors.

GSNO (S-nitrosoglutathione) is emerging as a key regulator in NO signalling as it is in equilibrium with S-nitrosated proteins. Accordingly, it is of great interest to investigate GSNO metabolism in terms of competitive pathways and redox state. The present study explored ADH3 (alcohol dehydrogenase 3) in its dual function as GSNOR (GSNO reductase) and glutathione-dependent formaldehyde dehydrogenase. The glutathione adduct of formaldehyde, HMGSH (S-hydroxymethylglutathione), was oxidized with a k(cat)/K(m) value approx. 10 times the k(cat)/K(m) value of GSNO reduction, as determined by fluorescence spectroscopy. HMGSH oxidation in vitro was greatly accelerated in the presence of GSNO, which was concurrently reduced under cofactor recycling. Hence, considering the high cytosolic NAD(+)/NADH ratio, formaldehyde probably triggers ADH3-mediated GSNO reduction by enzyme-bound cofactor recycling and might result in a decrease in cellular S-NO (S-nitrosothiol) content in vivo. Formaldehyde exposure affected S-NO content in cultured cells with a trend towards decreased levels at concentrations of 1-5 mM, in agreement with the proposed mechanism. Product formation after GSNO reduction to the intermediate semimercaptal responded to GSH/GSNO ratios; ratios up to 2-fold allowed the spontaneous rearrangement to glutathione sulfinamide, whereas 5-fold excess of GSH favoured the interception of the intermediate to form glutathione disulfide. The sulfinamide and its hydrolysis product, glutathione sulfinic acid, inhibited GST (glutathione transferase) activity. Taken together, the findings of the present study provide indirect evidence for formaldehyde as a physiological trigger of GSNO depletion and show that GSNO reduction can result in the formation of GST inhibitors, which, however, is prevented under normal cellular redox conditions.

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 diners 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.

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.

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

Human gastric alcohol dehydrogenase (σσ‐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.

Species variations in cutaneous alcohol dehydrogenases and aldehyde dehydrogenases may impact on toxicological assessments of alcohols and aldehydes.

Alcohol dehydrogenase (ADH; EC. 1.1.1.1) and aldehyde dehydrogenase (ALDH; EC 1.2.1.3) play important roles in the metabolism of both endogenous and exogenous alcohols and aldehydes. The expression and localisation patterns of ADH (1-3) and ALDH (1-3) were investigated in the skin and liver of the mouse (BALB/c and CBA/ca), rat (F344) and guinea-pig (Dunkin-Hartley), using Western blot analysis and immunohistochemistry with class-specific antisera. ALDH2 expression and localisation was also determined in human skin, while ethanol oxidation, catalysed by ADH, was investigated in the mouse, guinea-pig and human skin cytosol. Western blot analysis revealed that ADH1, ADH3, ALDH1 and ALDH2 were expressed, constitutively, in the skin and liver of the mouse, rat and guinea-pig. ADH2 was not detected in the skin of any rodent species/strain, but was present in all rodent livers. ALDH3 was expressed, constitutively, in the skin of both strains of mouse and rat, but was not detected in guinea-pig skin and was absent in all livers. Immunohistochemistry showed similar patterns of expression for ADH and ALDH in both strains of mouse, rat, guinea-pig and human skin sections, with localisation predominantly in the epidermis, sebaceous glands and hair follicles. ADH activity (apparent V(max), nmoles/mg protein/min) was higher in liver (6.02-16.67) compared to skin (0.32-1.21) and lower in human skin (0.32-0.41) compared to mouse skin (1.07-1.21). The ADH inhibitor 4-methyl pyrazole (4-MP) reduced ethanol oxidation in the skin and liver in a concentration dependent manner: activity was reduced to approximately 30-40% and approximately 2-10% of the control activity, in the skin and liver, respectively, using 1 mM 4-MP. The class-specific expression of ADH and ALDH enzymes, in the skin and liver and their variation between species, may have toxicological significance, with respect to the metabolism of endogenous and xenobiotic alcohols and aldehydes.

Functionality of allelic variations in human alcohol dehydrogenase gene family: assessment of a functional window for protection against alcoholism

Alcohol dehydrogenase (ADH) catalyses the rate-determining reaction in ethanol metabolism. Genetic association studies of diverse ethnic groups have firmly demonstrated that the allelic variant ADH1B*2 significantly protects against alcoholism but that ADH1C*1, which is in linkage with ADH1B*2, produces a negligible protection. The influence of other potential candidate genes/alleles within the human ADH family, ADH1B*3 and ADH2, remains unclear or controversial. To address this question, functionalities of ADH1B3 and ADH2 were assessed at a physiological level of coenzyme and substrate range. Ethanol-oxidizing activities of recombinant ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2 and ADH2 were determined at pH 7.5 in the presence of 0.5 mm NAD with 2-50 mm ethanol. The activity differences between ADH1B2 and ADH1B1 were taken as a threshold for effective protection against alcoholism and those between ADH1C1 and ADH1C2 as a threshold for null protection. Over 2-50 mm ethanol, the activities of ADH1B3 were found 2.9-23-fold lower than those of ADH1B2, largely attributed to the Km effect (ADH1B2, 1.8 mm; ADH1B3, 61 mm). Strikingly, the ADH1B3 activity was only 84% that of ADH1B1 at a low ethanol concentration, 2 mm, but increased 10-fold at 50 mm. Corrected for relative expression levels of the enzyme in liver, the hepatic ADH2 activities were estimated to be 18-97% those of ADH1B1 over 2-50 mm ethanol and were 28-140% of the activity differences between ADH1C1 and ADH1C2. The assessment based on the proposed functional window for the human ADH gene family indicates that ADH1B*3 may show some degree of protection against alcoholism and that the ADH2 functional variants appear to be negligible for this protection.

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.