Three-dimensional structure of horse liver alcohol dehydrogenase at 2.4 Å resolution

Eklund, Hans; Nordström, B.; Zeppezauer, E.; Söderlund, Gustaf; Ohlsson, Ingrid; Boiwe, Torne; Söderberg, Bengt-Olof; Tapia, O.; Brändén, Carl‐Ivar; Åkeson, Åke

doi:10.1016/0022-2836(76)90072-3

Cited by 714 publications

(424 citation statements)

References 51 publications

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“…The new ADH-F sequence had Asp-224, which has been suggested to determine the specificity for NAD ϩ versus NADP ϩ as a coenzyme, and Thr-48, which is thought to form a hydrogen bond with the alcohol hydroxyl group bound to the catalytic zinc (19). Cysteines 98, 101, 104, and 112, which are responsible for binding the noncatalytic zinc (20), were conserved in the new ADH. However, the sequence of this ADH had less than 68% identity with any of the known ADH isozymes (Table I); hence, we conclude that ADH-F belongs to a separate class in the family of ADHs, class VII.…”

Section: Discussionmentioning

confidence: 99%

cDNA Sequence and Catalytic Properties of a Chick Embryo Alcohol Dehydrogenase That Oxidizes Retinol and 3β,5α-Hydroxysteroids

Kedishvili¹,

Gough²,

Chernoff

et al. 1997

Journal of Biological Chemistry

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This study was undertaken to identify the cytosolic 40-kDa zinc-containing alcohol dehydrogenases that oxidize all-trans-retinol and steroid alcohols in fetal tissues. Degenerate oligonucleotide primers were used to amplify by polymerase chain reaction 500-base pair fragments of alcohol dehydrogenase cDNAs from chick embryo limb buds and heart. cDNA fragments that encode an unknown putative alcohol dehydrogenase as well as the class III alcohol dehydrogenase were identified. The new cDNA hybridized with two messages of ϳ2 and 3 kilobase pairs in the adult chicken liver but not in the adult heart, muscle, testis, or brain. The corresponding complete cDNA clones with a total length of 1390 base pairs were isolated from a chicken liver gt11 cDNA library. The open reading frame encoded a 375-amino acid polypeptide that exhibited 67 and 68% sequence identity with chicken class I and III alcohol dehydrogenases, respectively, and had lower identity with mammalian class II (55-58%) and IV (62%) isozymes. Expression of the new cDNA in Escherichia coli yielded an active alcohol dehydrogenase (ADH-F) with subunit molecular mass of ϳ40 kDa. The specific activity of the recombinant enzyme, calculated from active site titration of NADH binding, was 3.4 min ؊1 for ethanol at pH 7.4 and 25°C. ADH-F was stereospecific for the 3␤,5␣-versus 3␤,5␤-hydroxysteroids. The K m value for ethanol at pH 7.4 was 17 mM compared with 56 M for all-transretinol and 31 M for epiandrosterone. Antiserum against ADH-F recognized corresponding protein in the chicken liver homogenate. We suggest that ADH-F represents a new class of alcohol dehydrogenase, class VII, based on its primary structure and catalytic properties.Cytosolic zinc-containing alcohol dehydrogenases (ADH) 1 with 40-kDa subunits are capable of oxidizing a variety of primary, secondary, and aliphatic alcohols and a limited number of cyclic alcohols (1). Six classes of dimeric ADH isozymes have been identified in mammals (1). Except for class I, all other classes of ADH have high K m values for ethanol and oxidize medium-chain and long-chain alcohols most effectively.Potential physiological substrates for ADH isozymes include retinoid and steroid alcohols. Human class IV is the most efficient retinol dehydrogenase, followed by class II and the class I ␣␣ ADH (2). Class IV and II ADH are not active with steroid alcohols, whereas class I isozymes oxidize both retinoid and steroid substrates with relatively low catalytic efficiency. Class I isozymes 2 exhibit stereospecificity toward alcohol substrates. For example, horse SS and human ␥␥ ADH oxidize 3␤-hydroxysteroids but not 3␣-hydroxysteroids (3). ADH isozymes vary in their tissue distribution; class IV ADH is expressed in the epithelial tissues of mammals and is present in the human stomach mucosa and esophagus (1), whereas class II is found in fetal and adult liver. Class I isozymes ␤

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Section: Discussionmentioning

confidence: 99%

cDNA Sequence and Catalytic Properties of a Chick Embryo Alcohol Dehydrogenase That Oxidizes Retinol and 3β,5α-Hydroxysteroids

Kedishvili¹,

Gough²,

Chernoff

et al. 1997

Journal of Biological Chemistry

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“…In the free enzyme, the tetracoordinate Zn2+ ion in the active site is ligated by the sulfhydryl groups of cysteine-46 and cysteine-174, the NE atom of histidine-67, and a water molecule (4). The coordination number of the active-site metal ion is considerably less well assigned for reaction intermediates.…”

mentioning

confidence: 99%

Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.

Makinen¹,

Yim²

1981

Proc. Natl. Acad. Sci. U.S.A.

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The coordination environment of the catalytically active metal ion of horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) has been investigated by electron paramagnetic resonance (EPR) methods with use of the active-site-specific Co2+-reconstituted enzyme. The EPR absorption spectrum of the metal-substituted enzyme is characteristic of a rhombically distorted environment. The spectrum of the enzyme-NAD+ complex shows approximate axial symmetry of the metal ion site, indicating that binding of the coenzyme induces a structural alteration in the active-site region. This environment is not significantly altered further by binding of the competitive inhibitor pyrazole. To assign the coordination number of the active-site metal ion, the zero-field splitting was determined on the basis ofthe temperature dependence ofthe spin-lattice relaxation of the Co2+ ion. The zero-field splitting energies are '9 cm-' for the free Co2+-reconstituted enzyme and e46 and -47 cm-' for the enzyme-NAD+ and enzyme-NAD+-pyrazole complexes, respectively. On the basis of studies of structurally defined small molecule complexes, these values are compatible with a tetracoordinate metal ion in the active site of the free enzyme but a pentacoordinate metal ion in the binary enzyme-NAD+ complex and in the ternary enzyme-NAD+-inhibitor complex and, therefore, presumably also in the catalytically active ternary enzyme-NAD+-alcohol complex formed in the course of alcohol oxidation.The oxidation ofalcohol catalyzed by liver alcohol dehydrogenase (LADH; alcohol:NAD' oxidoreductase, EC 1.1.1.1) proceeds at the level of a ternary complex formed by the enzyme, the coenzyme NAD', and the substrate as the catalytically active intermediate of the reaction (1-3). In the free enzyme, the tetracoordinate Zn2+ ion in the active site is ligated by the sulfhydryl groups of cysteine-46 and cysteine-174, the NE atom of histidine-67, and a water molecule (4). The coordination number of the active-site metal ion is considerably less well assigned for reaction intermediates. The suggestion has been made on the basis of proton relaxation enhancement studies of enzyme-coenzyme-inhibitor complexes that substrates bind as outer sphere ligands only with no change in coordination number (5). Interpretations of the pH dependence of the kinetic parameters of the enzyme have invoked formation of both tetracoordinate (6, 7) and pentacoordinate (8, 9) intermediates as the catalytically active species. Difference Fourier syntheses of (alcohol) inhibitor substrate analogs bound to the active site of the enzyme-NAD' complex crystallized in the presence of 2-methyl-2,4-pentanediol suggest that the substrate displaces the metal-bound water molecule to form a tetracoordinate species only (10). However, the crystallizing reagent 2-methyl-2,4-pentanediol acts as substrate analog of the enzyme, resulting in a mixture of enzyme-NAD' and enzyme-NADH species in the crystal.* Under these conditions, addition of alcohol substrates may lead also to formation o...

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“…ADH class I and class IV have an activity in the first step of RA synthesis [16,17]. Crystal structures of ADH class I and class IV revealed a conserved active site that can accommodate long chain alcohols such as retinol [18][19][20]. ADH class I is expressed at high levels in many retinoid target tissues of embryos [15,21] and in adult epithelia, such as intestine, kidney, adrenal gland, testis, epididymis, uterus and ovary [15,[22][23][24][25].…”

Section: Discussionmentioning

confidence: 99%

Evidence for the Expression of Alcohol Dehydrogenase Class I Gene in Rat Uterus and Its Up-regulation by Progesterone

et al. 2008

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Abstract. The endometrium is one of the target tissues of the ovarian steroid hormones, estrogen and progesterone. In order to elucidate the mechanism of gene regulation in the endometrium, suppressive subtraction hybridization was performed to isolate the candidate genes controlled by progesterone in rat uterus. Alcohol dehydrogenase (ADH) class I gene was one of the candidate genes. Here we investigated the expression and regulation of ADH class I gene in rat uterus. The mRNA of ADH class I was detected in uterus by RT-PCR using specific primers. Using specific probe for ADH class I, in situ hybridization was performed to investigate localization in rat uterus. Positive signals were detected in the endometrial stromal cells of rat uterus by in situ hybridization and were not detected in endometrial epithelial cells and myometrium in rat uterus. Ovariectomized rats were treated with 17-β estradiol and progesterone and the uteri of these rats were used for Northern blot analysis and assay of the ADH activity. Northern blot analysis revealed that the expression of ADH class I mRNA in rat uteri was up-regulated approximately two-fold after progesterone treatment, but not estrogen. Likewise, ADH activity was approximately two-fold higher in progesterone-treated rat uteri compared with controls. This study demonstrated that ADH class I gene is progesterone-responsive in the uterus. This implies that progesterone might be involved with retinoic acid synthesis in the uterus, since ADH is the key enzyme for retinoic acid synthesis.

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Three-dimensional structure of horse liver alcohol dehydrogenase at 2.4 Å resolution

Cited by 714 publications

References 51 publications

cDNA Sequence and Catalytic Properties of a Chick Embryo Alcohol Dehydrogenase That Oxidizes Retinol and 3β,5α-Hydroxysteroids

cDNA Sequence and Catalytic Properties of a Chick Embryo Alcohol Dehydrogenase That Oxidizes Retinol and 3β,5α-Hydroxysteroids

Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.

Evidence for the Expression of Alcohol Dehydrogenase Class I Gene in Rat Uterus and Its Up-regulation by Progesterone

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