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Query: UNIPROT:P06889 (Mol)
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Drosophila alcohol dehydrogenase belongs to the short chain dehydrogenase/reductase (SDR) family which lack metal ions in their active site. In this family, it appears that the three amino acid residues, Ser138, Tyr151 and Lys155 have a similar function as the catalytic zinc in medium chain dehydrogenases. The present work has been performed in order to obtain information about the function of these residues. To obtain this goal, the pH and temperature dependence of various kinetic coefficients of the alcohol dehydrogenase from Drosophila lebanonensis was studied and three-dimensional models of the ternary enzyme-coenzyme-substrate complexes were created from the X-ray crystal coordinates of the D. lebanonensis ADH complexed with either NAD(+) or the NAD(+)-3-pentanone adduct. The kon velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD(+) complex, with a DeltaHion value of 74(+/-4) kJ/mol (18(+/-1) kcal/mol). Based on this result and the constructed three-dimensional models of the enzyme, the most likely candidate for this catalytic residue is Ser138. The present kinetic study indicates that the role of Lys155 is to lower the pKa values of both Tyr151 and Ser138 already in the free enzyme. In the binary enzyme-NAD(+) complex, the positive charge of the nicotinamide ring in the coenzyme further lowers the pKa values and generates a strong base in the two negatively charged residues Ser138 and Tyr151. With the OH group of an alcohol close to the Ser138 residue, an alcoholate anion is formed in the ternary enzyme NAD(+) alcohol transition state complex. In the catalytic triad, along with their effect on Ser138, both Lys155 and Tyr151 also appear to bind and orient the oxidized coenzyme.
J Mol Biol 1999 Nov 26
PMID:The catalytic triad in Drosophila alcohol dehydrogenase: pH, temperature and molecular modelling studies. 1061 Jul 83

The isoflavones daidzein, genistein, biochanin A and formononetin inhibit potently and preferentially the gamma-isozymes of mammalian alcohol dehydrogenase (gammagamma-ADH), the only ADH isozyme that catalyzes the oxidation of 3beta-hydroxysteroids. Based on these results, we proposed that these isoflavones might also act on other enzymes involved in 3beta-hydroxysteroid metabolism. Recently, we showed that they indeed are potent inhibitors of a bacterial beta-hydroxysteroid dehydrogenase (beta-HSD). To extend this finding to the mammalian systems, we hereby purified, characterized and studied the effects of isoflavones and structurally related compounds on, a bovine adrenal 3beta-hydroxysteroid dehydrogenase (3beta-HSD). This enzyme catalyzes the oxidation of 3beta-hydroxysteroids but not 3alpha-, 11beta- or 17beta-hydroxysteroids. The same enzyme also catalyzes 5-ene-4-ene isomerization, converting 5-pregnen 3, 20-dione to progesterone. The K(m) values of its dehydrogenase activity determined for a list of 3beta-hydroxysteroid substrates are similar (1 to 2 microM) and that of its isomerase activity, determined with 5-pregnen 3, 20-dione as a substrate, is 10 microM. The k(cat) value determined for its isomerase activity (18.2 min(-1)) is also higher than that for its dehydrogenase activity (1.4-2.4 min(-1)). A survey of more than 30 isoflavones and structurally related compounds revealed that daidzein, genistein, biochanin A and formononetin inhibit both the dehydrogenase and isomerase activity of this enzyme. Inhibition is potent and concentration dependent. IC(50) values determined for these compounds range from 0.4 to 11 microM, within the plasma and urine concentration ranges of daidzein and genistein of individuals on vegetarian diet or semi-vegetarian diet. These results suggest that dietary isoflavones may exert their biological effects by inhibiting the action of 3beta-HSD, a key enzyme of neurosteroid and/or steroid hormone biosynthesis.
J Steroid Biochem Mol Biol 1999 Dec 31
PMID:Bovine adrenal 3beta-hydroxysteroid dehydrogenase (E.C. 1.1.1. 145)/5-ene-4-ene isomerase (E.C. 5.3.3.1): characterization and its inhibition by isoflavones. 1070 8

Two hammerhead ribozymes flanked by Giardia lamblia alcohol dehydrogenase E (ADHE) antisense RNA fragments, ARzS and ARzL, were designed, synthesized and found capable of cleaving an ADHE mRNA fragment at the anticipated position in vitro. The ribozymes were then electroporated into Giardia trophozoites and expressed via the giardiavirus-mediated RNA expression system. Expression of the ribozyme with two short antisense arms, ARzS, was stabilized under puromycin selection and demonstrated a 33% reduction in ADHE mRNA and 25% decrease in NAD+-dependent ADH activity in the transfectants. Expression of ARzL, the ribozyme with two long antisense arms, cannot be enriched under puromycin without killing the transfected cells, probably due to excessive depletion of ADHE. Without the drug selection, however, transient expression of ARzL 20-40 h after electroporation resulted in an 83.7% loss of ADHE mRNA and an 84.5% reduction in ADH activity in the transfected cells. When the ribozyme moiety was removed from ARzL, the latter retained some of its in vivo activity of lowering ADHE mRNA and ADH activity, suggesting that inhibition of ADHE gene expression in Giardia can be accomplished by the antisense RNA alone, albeit less efficiently. The ADHE deficient transfectant demonstrated relatively poorer anaerobic growth but grew more vigorously than the wild type under aerobic conditions, suggesting that the role of ADHE in providing NAD+ through anaerobic reduction of acetyl-CoA to ethanol could be replaced by a yet unidentified aerobic enzyme(s) in Giardia. The close association consistently observed between the levels of ADHE mRNA and ADH activity in transfected Giardia cells suggests that ADHE could be the only functional alcohol dehydrogenase in Giaradia. One other Giardia gene encoding a putative Class III ADH, GIADH3, was identified and cloned, but no Class III ADH activity could be detected in Giardia by the conventional enzyme assays. This gene is thus probably unexpressed in Giardia trophozoite.
Mol Biochem Parasitol 2000 Jun
PMID:Role of alcohol dehydrogenase E (ADHE) in the energy metabolism of Giardia lamblia. 1092 54

Jingwei (jgw) is the first gene found to be of sufficiently recent origin in Drosophila to offer insights into the origin of a gene. While its chimerical gene structure was partially resolved as including a retrosequence of alcohol dehydrogenase (ADH:), the structure of its non-ADH: parental gene, the donor of the N-terminal domain of jgw, is unclear. We characterized this non-ADH: parental locus, yellow emperor (ymp), by cloning it, mapping it onto the polytene chromosomes, sequencing the entire locus, and examining its expression patterns in Drosophila melanogaster. We show that ymp is located in the 96-E region; the N-terminal domain of ymp has donated the non-ADH: portion of jgw via a duplication. The similar 5' portions of the gene and its regulatory sequences give rise to similar testis-specific expression patterns in ymp and jgw in Drosophila teissieri. Furthermore, between-species comparison of ymp revealed purifying selection in the protein sequence, suggesting a functional constraint in ymp. While the structure of ymp provides clear information for the molecular origin of the new gene jgw, it unexpectedly casts a new light on the concept of genes. We found, for the first time, that the single locus of the ymp gene encompasses three major molecular mechanisms determining structure of eukaryotic genes: (1) the 5' exons of ymp are involved in an exon-shuffling event that has created the portion recruited by jgw; (2) using alternative cleavage sites and alternative splicing sites, the 3' exon groups of ymp produce two proteins with nonhomologous C-terminal domains, both exclusively in the testis; and (3) in the opposite strand of the third intron of ymp is an essential gene, musashi (msi), which encodes an RNA-binding protein. The composite gene structure of ymp manifests the complexity of the gene concept, which should be considered in genomic research, e.g., gene finding.
Mol Biol Evol 2000 Sep
PMID:The origin of the Jingwei gene and the complex modular structure of its parental gene, yellow emperor, in Drosophila melanogaster. 1095 46

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.
J Mol Biol 2000 Sep 15
PMID:Crystal structures of mouse class II alcohol dehydrogenase reveal determinants of substrate specificity and catalytic efficiency. 1097 Jul 44

Scarce bibliographical data exists on the enzymes in Lepidosiren paradoxa and analysis of several enzymes was considered worthy of investigation. Distribution of ADH, ALP, FBALD, GAPDH, G3PDH, G6PDH, GPI, LDH, MDH, and PGM was identified in ten tissues (retina, heart, muscle, liver, kidney, lung, gut, gills, brain, and ovary) of the South American lungfish and compared with patterns previously described in other vertebrates. Compared with earlier results differences in the number of loci expressed were observed for ADH, G3PDH, GPI, and MDH. The number of loci expressed and/or in tissue specificity of several enzymes (ADH, FBALD, GAPDH, G3PDH, G6PDH and PGM) were found to be similar to those of other vertebrates. Differences were detected in ALP due to the absence of an intestinal-specific form typical of fish, amphibians, reptiles and birds; further differences were observed in GPI and MDH due to their tissue expression. The differences in LDH involve the LDH-A4 isozyme which was most common in tissues. Overall, comparison with other vertebrates reveals that in L. paradoxa the tissue-restricted expressions of some enzymes are similar, while others have retained an ancestral pattern and exhibit a more widespread tissue expression of genes.
Comp Biochem Physiol B Biochem Mol Biol 2000 Aug
PMID:Isozyme distribution of ten enzymes and their loci in South American lungfish, Lepidosiren paradoxa (Osteichthyes, Dipnoi). 1102 62

Alterations in gene expression during early stages of dormancy release in grapevine buds were analyzed to facilitate the identification of gene products that may mediate the signal transduction of a dormancy-release signal, or derepression of meristematic activity. In the present report we describe the identification of GDBRPK, a transcript for an SNF-like protein kinase that is up-regulated upon chemical induction of dormancy release by hydrogen cyanamide (HC). Since SNF and SNF-like protein kinases are known as sensors of stress signals, we hypothesize that GDBRPK may be involved in the perception of a stress signal induced by HC. We also describe a simultaneous and remarkable induction of both PDC and ADH transcripts that was observed shortly after HC application, and was of a transient nature. These data may imply that HC application leads to a transient respiratory stress, which likely results in a temporary increase in the AMP/ATP ratio. Since AMP is known as a stress signal that is sensed by SNF-like kinases, we suggest that the SNF-like GDBRPK could serve as the sensor of this signal.
Plant Mol Biol 2000 Jul
PMID:The transduction of the signal for grape bud dormancy breaking induced by hydrogen cyanamide may involve the SNF-like protein kinase GDBRPK. 1105

We report the cloning and structural characterization of two Adh loci of the olive fruit fly, Bactrocera oleae. Each of the two genes, named Adh1 and Adh2, consists of three exons and two introns for a total length of 1981 and 988 nucleotides, respectively. Their deduced amino acid sequences of 257 and 258 residues exhibit a 77% identity and display the characteristics of the insect ADH enzymes, which belong to the short-chain dehydrogenases/reductases family. The Adh genes of B. oleae are compared to the two genes of the Mediterranean fly, Ceratitis capitata, the only other species of the Tephritidae family in which the Adh genes have been studied. On the basis of amino acid divergence the four genes form two clusters each containing one gene from each species, as expected if there was one duplication event before speciation. On the basis of nucleotide sequence the four sequences form two clusters each containing the two sequences from the same species, as expected if there was a separate duplication event in each species. To help decide between the two alternatives, we compared at both the amino acid and DNA level the Adh genes of five Drosophila species that are known to carry two such genes and observed that, with only one exception at the amino acid level, conspecific loci cluster together. We conclude that the information we have at present does not allow a firm choice between the hypothesis of a single duplication event that occurred before the split of Bactrocera and Ceratitis from their common ancestor and the hypothesis of two independent duplication events, one in each of the two genera.
J Mol Evol 2001 Jan
PMID:Characterization of two alcohol dehydrogenase (Adh) loci from the olive fruit fly, Bactrocera (Dacus) oleae and implications for Adh duplication in dipteran insects. 1113 92

To investigate the level and pattern of DNA polymorphism in the noncoding regulatory region in the plant nuclear genome, 2.4 kb of nucleotide sequence of the 5' upstream region of ADH: was determined for 14 ecotypes of Arabidopsis thaliana and five accessions of Arabis gemmifera. Using this data set and previously determined ADH: sequence data, DNA variation was analyzed in a 4.4-kb region of the locus. Two divergent sequence types detected in the transcriptional unit of ADH: were not present in the 5' region of the ADH: gene in A. thaliana. Nucleotide diversity of the entire 5' region was estimated to be 0.0040, which is lower than that in the transcriptional unit. The level of variation was not uniform. There were peaks of variations in a approximately 400-bp region where cis-regulatory elements for ADH: expression were clustered and in exon 4. In interspecific comparison with A. gemmifera, lower divergence was observed in the 5' flanking region than in the exons. High peaks of divergence in the 400-bp regulatory region and exon 4 were also detected, although there were many other peaks. These results indicate that regions of functional importance have a high level of polymorphism and divergence in the ADH: locus of these genera. The possibility of balancing selection in the ADH: gene of these plants is discussed.
Mol Biol Evol 2001 Feb
PMID:DNA variation in the 5' upstream region of the Adh locus of the wild plants Arabidopsis thaliana and Arabis gemmifera. 1115 75

Low levels of genetic diversity and divergence at nuclear loci have previously been observed for cycloidea and fil1-like genes within and between several Antirrhinum species, and divergence at these loci is also low between species in genera at different levels of relatedness in the former family Scrophulariaceae (Digitalis and Verbascum). The low divergence values are surprising, because (based on the sequences of chloroplast loci) the Scrophulariaceae are thought to be polyphyletic, with two anciently diverged clades, and the species we compared belonged to the two different clades. Here, we extend our studies of sequence divergence to more nuclear genes: fil2, far, globosa, and ADH: Detailed studies revealed that in Antirrhinum these genes belong to gene families. Low levels of divergence between Antirrhinum and Verbascum were observed for four of the loci studied, fil2-1, fil2-2, far-L, and globosa, similar to our previous observations. We discuss hypotheses to explain these low synonymous divergence values. For Adh, no cases of very similar sequences were found, but, rather, our sequences from the three different genera (Antirrhinum, Digitalis, and Verbascum) were all very diverged. Repeated gene duplication and loss of elements in the Adh gene family is likely in these lineages, making it impossible to determine orthology of the Adh genes.
Mol Biol Evol 2001 Oct
PMID:Low rates of silent substitution in nuclear genes of two distantly related Scrophulariaceae (Antirrhinum and Verbascum). 1155 99


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