Mats Estonius

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.

Avian alcohol dehydrogenase: the chicken liver enzyme. Primary structure, cDNA-cloning, and relationships to other alcohol dehydrogenases.

The major ethanol-active form of chicken liver alcohol dehydrogenase was characterized. The primary structure was determined by peptide analysis and, to a large part, was also deduced by cDNA analysis of a near full-length cDNA clone. The latter was detected by screening of a chicken liver cDNA library with antibodies raised against the purified dehydrogenase. The structure shows that the avian enzyme exhibits characteristics of the complex mammalian alcohol dehydrogenase system, tracing its origin and divergence, and allowing functional correlations. The chicken protein analyzed proves to be a class I alcohol dehydrogenase, with 74% residue identity to gamma chains of the human enzyme, a Km for ethanol of 0.5 mM and a Ki for 4-methyl pyrazole of 2.5 microM. Relationships to the other two classes are non-identical; residue exchanges towards the human classes increase in the order I less than III less than II, and human/chicken differences are less than inter-class differences. Consequently, the origins of the classes are more distant than the avian/mammalian separation. They reflect duplicatory events separated in time, and the lines that lead to present-day classes I and II deviate early. Integrated with the data for the quail enzyme, the structure of the chicken protein shows that within the avian enzymes the degree of variation is comparable to that within the mammalian class I enzymes, which are more variable than the class III forms. The coenzyme-binding and substrate-binding residues of this chicken alcohol dehydrogenase are largely identical to those in the mammalian class I counterparts. However, the subunit-interacting areas are more variable and suggest some relationships of the avian enzyme with both class I and III mammalian forms. One of the residues, Gly260 (mammalian class I numbering system), previously considered characteristic of all alcohol dehydrogenases, is replaced by Gln.

Fast atom bombardment mass spectrometry and chemical analysis in determinations of acyl-blocked protein structures

Peptide generation and fast atom bombardment mass spectrometry in combination with conventional chemical analysis was used to identify the blocking group and establish the N‐terminal structure of six different proteins at the nanomole level. In this manner, the first terminal structures of three non‐mammalian alcohol dehydrogenases were determined, demonstrating the presence of N‐terminal acetylation in these piscine, amphibian, and avian enzymes. Similarly, two different yeast glucose‐6‐phosphate dehydrogenases and a minor variant of a human alcohol dehydrogenase were found to be acetylated. The exact end location of C‐terminal structures was also established. Together, the analyses permit the definition of terminal regions and blocking groups, thus facilitating the delineation of remaining structures.

Site-directed mutagenesis and enzyme properties of mammalian alcohol dehydrogenases correlated with their tissue distribution

Site-directed mutagenesis of mammalian alcohol dehydrogenases has helped to explain functional differences between enzymes within the protein family and traced these characteristics to specific amino acid residues. A threonine/serine exchange at position 48 in the human beta/gamma subunits can explain sensitivity to testosterone inhibition, as well as steroid dehydrogenase activity. It is possible to correlate the glutathione-dependent formaldehyde dehydrogenase activity of class III alcohol dehydrogenase with an arginine at position 115. Tissue distribution analysis of the three initially established classes of mammalian alcohol dehydrogenase show pronouncedly different patterns. Class I alcohol dehydrogenase is widespread but varies between the tissues, and exists in small amounts in the brain. The occurrence of class II is limited in contrast to the class III enzyme which is abundant in all tissues examined. The latter probably reflects the need for scavenging of formaldehyde in cytoprotection. Additional enzyme forms of mammalian alcohol dehydrogenase have been detected and have to be investigated further, together with the enzymes characterized earlier, regarding their physiological role in alcohol metabolism.

Tissue Distribution of Alcohol and Sorbitol Dehydrogenase mRNAs

Alcohol dehydrogenase (ADH) of class I is the principal enzyme in liver ethanol oxidation, and has been the subject of much research. It has been studied in many species and is a part of the enzyme system now constituting ADHs at large. The different mammalian ADHs can be divided into at least five classes according to structural properties (Pares et al., 1992). Class I is the classical liver ADH (Vallee and Bazzone, 1983), and class III ADH is the glutathione-dependent formaldehyde dehydrogenase (Koivusalo et al., 1989). ADH of class II shows a higher Km for ethanol than class I, and exhibits activity toward norepinephrine metabolites (Mardh et al., 1986), but is less studied than class I and class III ADH. Class IV is a stomach ADH characterized in rat and man (Pares et al., 1990; 1992; Moreno and Pares, 1991), and class V is a DNA-derived human structure recently reported (Yasunami et al., 1991).

Distinctive Class Relationships Within Vertebrate Alcohol Dehydrogenases

Mammalian alcohol dehydrogenases (ADH) constitute a well-studied enzyme system composed of sub-forms at different levels of multiplicity. The family has diverged into a number of different enzymes. At the next level (Fig. 1), fairly different forms (“classes”) of alcohol dehydrogenase, with distinct structural and enzymatic properties, occur. The subsequent level constitutes still more similar forms (“isozymes”) with gradual differences in properties and fewer residue exchanges.

Crystallizations of Novel Forms of Alcohol Dehydrogenase

Alcohol dehydrogenases in nature are derived from different protein families (cf Jornvall et al., 1993). The mammalian alcohol dehydrogenases constitute what presently appears to be six classes within a large family of medium-chain dehydrogenases and reductases, MDR, with at least seven characterized activity types (Persson et al., 1994). The classes of the mammalian alcohol dehydrogenases differ in structure (55–68% sequence identity), substrate pockets, subunit interactions, and other properties. Much of these aspects has been interpreted by comparisons and modelling studies based on crystallographic analyses of just two of the enzymes of one class, the class I horse (Eklund et al., 1976) and human (Hurley et al., 1991) enzymes, constituting the classical liver enzyme with ethanol activity. It is desirable to get direct crystallographic data on the conformations of more of the enzymes, especially since differences exist between the classes, class-hybrid properties have been found in early enzyme forms (of fish), and the two well-characterized classes, I and III, differ in internal variability patterns (Danielsson et al., 1994a). We have therefore crystallized five novel forms of these dehydrogenases, which should enable further structural characterizations and hence evaluation of the conclusions from modelling and from natural variants.

Mutations of human class III alcohol dehydrogenase.

The residues lining the active site pocket of class III alcohol dehydrogenase differ markedly from those of class I and other classes. Two charged residues in the vicinity of the catalytic site are characteristic of class III. One is Arg115, which is at the outer part of the substrate-binding cleft and is associated with major enzymatic characteristics of class III, i.e. fatty acid activation (Moulis et al., 1991; Holmquist et al., 1993) and glutathione-de-pendent formaldehyde dehydrogenase activity (Engeland et al., 1993). The other charged residue is Asp57, located in the middle part of the substrate pocket. In an attempt to study the relative importance of residues involved in differentiation of classes, we have by site-directed mutagenesis examined not only the function of Asp57, but also the roles of Tyr93 and Thr48 of class III (Estonius et al., 1994). The charge and polarity at positions 57 and 93 were altered by Asp57Leu and Tyr93Phe substitutions, respectively. As opposed to Asp in class III, Leu is found at position 57 in most class I alcohol dehydrogenases, and in contrast to Tyr in class III, Phe or Ala are found at position 93. Therefore, a combination of the Asp57Leu and Tyr93Phe exchanges in one mutant is of interest in order to see if a class I alcohol dehydrogenase substrate pocket, and class I enzymatic properties, can be mimicked by altering the active site structure in a direction toward those of class I enzymes. In addition, to check for differences in the oxidation of short-chain aliphatic alcohols contra S-hy-droxymethylglutathione (HMGSH), we have replaced Thr48 with Ala, a mutation deleterious to class I alcohol dehydrogenase activity (Hoog et al., 1992).