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Proc. Nati. Acad. Sci. USA Vol. 91, pp. 4980-4984, May 1994 Biochemistry Fundamental molecular differences between alcohol dehydrogenase classes (Drosophila octano dehydrogenase/class m alcohol dehydrogenase/mo ur patterns/zinc enyme famy) OLLE DANIELSSON*, SILVIA ATRIANt, TERESA LUQUEt, LARS HJELMQVIST*, ROSER GONZALEZ-DUARTEt, AND HANS J6RNVALL*f *Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden; tCenter for Biotechnology, Karolinska Institutet, S-141 86 Huddinge, Sweden; and tDepartment of Genetics, University of Barcelona, E-08071 Barcelona, Spain Communicated by Sune Bergstrom, January 18, 1994 ABSTRACT Two types of alcohol dehydrogenase in sepa- ary patterns, with class III being "constant" and class I rate protein families are the "medium-chain" zinc enzymes "variable" (10), result in a consistent picture of the enzyme (including the classical liver and yeast forms) and the "short- system and place the classes of medium-chain alcohol dehy- chain" enzymes (including the insect form). Although the drogenases as separate enzymes in the cellular metabolism. medium-chain family has been characterized in prokaryotes Similarly, another protein family, short-chain dehydroge- and many eukaryotes (fungi, plants, cephalopods, and verte- nases, has also evolved into a family comprising many brates), insects have seemed to possess only the short-chain different enzyme activities, including an alcohol dehydroge- enzyme. We have now also characterized a medium-chain nase (11). This form operates by means of a completely alcohol dehydrogenase in Drosophila. The enzyme is identical different catalytic mechanism and is related to mammalian to insect octanol dehydrogenase. It Is a typical class m alcohol prostaglandin dehydrogenases/carbonyl reductase (12). dehydrogenase, similar to the correspondin human form Thus far, this alcohol dehydrogenase has been found in (70% residue identity), with mostly the same residues involved insects, the Drosophila enzyme being recognized early to in substrate and coenzyme interactions. Changes that do occur differ from the zinc-containing alcohol dehydrogenases (13, are conservative, but Phe-Si is offunctional interest in relation 14). Its properties in various Drosophila species are well to decreased coenzyme binding and Increased overall activity. established (15). Extra residues versus the human enzyme near position 250 These two alcohol dehydrogenase types demonstrate that affect the coenzyme-binding domain. Enzymatic properties are ethanol dehydrogenase activity has evolved in different man- similar-i.e., very low activity toward ethanol (K. beyond ners, with many organisms now employing a medium-chain measurement) and high selectivity for formaldehyde/glu- enzyme, while others depend on a short-chain enzyme. The tathione (S-hydroxymethylgutathione; kt/Km = 160,000 medium-chain family has not been identified in insects, min'1 mM'1). Between the present class m1 and the ethanol- although it is of ancient origin and has been characterized in active class I enzymes, however, patterns of variability differ other eukaryotes and in prokaryotes. We now show that the greatly, highlighting fundamentally separate molecular prop- family is indeed present also in insects and that its major erties of these two alcohol with class representative is the typical class III type. In fact, the enzyme dehydrogenases, Im turns out to be identical to Drosophila octanol dehydroge- resembling enzymes in general and class I showing high nase, long known (16-20) but little studied. We have char- variation. The gene coding for the Drosophila class HI enzyme acterized the enzyme from Drosophila melanogaster enzy- produces an mRNA of about 1.36 kb that is present at all matically and structurally to prove its consistency with other developmental stages of the fly, compatible with the constitu- class III forms. We also have identified and sequenced the tive nature of the vertebrate enzyme. Taken together, the corresponding gene§ and detected its transcription product at results bridge a previously apparent gap in the distribution of all developmental stages. Thus, the family is now known to medium-chain alcohol dehydrogenases and establish a strictly be present essentially in all life forms, supporting the view conserved class m enzyme, consistent with an important role that medium-chain alcohol dehydrogenases are universal for this enzyme in cellular metabolism. factors in cellular defense mechanisms from prokaryotes to humans. In addition, we find fundamental differences be- The "classical" alcohol dehydrogenase is part of a wide- tween the class I and III enzymes, defining separate prop- spread system ofzinc-containing enzymes (1). In mammalian erties of these related proteins. tissues, at least six classes ofthis enzyme occur. They differ considerably and represent stages between separate enzymes and ordinary isozymes. Class I is the well-known liver MATERIALS AND METHODS enzyme with ethanol dehydrogenase activity (2), class III is Protein. D. melanogaster whole flies were bred and har- identical with glutathione-dependent formaldehyde dehydro- vested as described (21). After homogenization, centrifuga- genase (3), class IV is a form preferentially expressed in tion, isoelectric focusing, and activity staining with 1 mM stomach (4, 5), while classes II, V, and VI, although little octanol or 33 mM ethanol at pH 10 and 1 mM glutathione/1 studied, are known also to exhibit distinct properties (6, 7, mM formaldehyde at pH 8, alcohol dehydrogenase activity of 44). The class origins have been traced to gene duplications the medium-chain type was detected and purified by utilizing early in vertebrate evolution [the I/III duplication (8)] or ion-exchange chromatography on DEAE-Sepharose, affinity during that evolution [the IV/I duplication (5)], with emerg- chromatography on AMP-Sepharose, and afast protein liquid ing activities toward ethanol (9); class III corresponds to an chromatography (FPLC) step on Mono Q as described for the ancestral form. These properties and the different evolution- class III enzyme from other sources (9). Short-chain alcohol dehydrogenase activity was also monitored (isopropanol at The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U07641). 4980 Downloaded by guest on September 29, 2021 Biochemistry: Danielsson et al. Proc. NatL Acad. Sci. USA 91 (1994) 4981 pH 8.6), pooled after the ion-exchange chromatography step, A : iB and purified to homogeneity on Blue-Sepharose (22). The strain used contained the adhF allele yielding the rapidly migrating form of the short-chain enzyme. 1,-f. Structural Analysis. The pure protein was carboxymethy- t~~~~~.a 40 Z lated by treatment with 14C-labeled iodoacetate and digested in separate batches with proteolytic enzymes (8). Peptide digests were fractionated by reverse-phase HPLC, and all fragments obtained were submitted to structural analysis. Amino acid compositions were determined with a Pharmacia LKB Alpha FIG. 1. Gel electrophoretic patterns of homogenates (A) and plus analyzer after acid hydrolysis for 24 hr at purified alcohol dehydrogenases (B and C) of D. melanogaster. (A) 1100C with 6 M HCl/0.5% phenol, and sequence degradations Isoelectric focusing under native conditions and activity staining were carried out with MilliGen Prosequencers 6600 and 6625 with formaldehyde/glutathione (lane 1), octanol (lane 2), isopropanol utilizing arylamine coupling for membrane attachments or (lane 3), and ethanol (lane 4). (B) Isoelectric focusing as inA, but with with an Applied Biosystems 470 sequencer with an on-line protein staining ofthe purified proteins, octanol dehydrogenase (lane 120A analyzer. 5), and short-chain dehydrogenase (lane 6). (C) Isoelectric focusing PCR Amplfication and Northern Analysis. Peptide struc- after SDS/polyacrylamide gel electrophoresis and protein staining of tures determined were utilized for construction oftwo 23-mer standard proteins (lane 7; molecular masses 94, 67, 43, 30, 20.1, and degenerate oligonucleotide probes (corresponding to amino 14.4 kDa from top to bottom) and pure octanol dehydrogenase (lane 8). In lanes 3, 4, and 6, a minor more-acidic form is acid residues 92-99 and 261-268), which served as primers for visible, representing one ofinterconvertible forms in flies homozygous at the PCR amplification with genomic DNA of several Drosophila ADH locus (35). species. Total cellular RNA was isolated by the guanidinium isothiocyanate method (23) from larval, pupal, and adult D. melanogaster; separated by 1.2% agarose/formaldehyde gel raphy on DEAE-Sepharose, AMP-Sepharose, and Mono Q FPLC column in a protocol similar to that for other class Ill electrophoresis; and transferred to a nylon membrane (Am- ersham) for hybridization at high stringency (42TC in 50% alcohol dehydrogenases (9). All octanol dehydrogenase ac- formamide) (24). A control rehybridization was performed tivity was monitored and coincided either with that of the with a D. melanogaster actin gene probe. Autoradiographs glutathione-dependent formaldehyde dehydrogenase through were measured with an UltroScan XL (Pharmacia LKB) all purification steps or with the short-chain alcohol dehy- enhanced laser densitometer. drogenase, which was also purified. The
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