Avian alcohol dehydrogenase. Characterization of the quail enzyme, functional interpretations, and relationships to the different classes of mammalian alcohol dehydrogenase

The primary structure of the major quail liver alcohol dehydrogenase was determined. It is a long-chain, zinc-containing alcohol dehydrogenase of the type occurring also in mammals and hence allows judgement of the gene duplications giving rise to the classes of the human alcohol dehydrogenase system. The avian form is most closely related to the class I mammalian enzyme (72-75% residue identity), least related to class II (60% identity), and intermediately related to class III (64-65% identity). This pattern distinguishes the mammalian enzyme classes and separates classes I and II in particular. In addition to the generally larger similarities with class I, the avian enzyme exhibits certain residue patterns otherwise typical of the other classes, including an extra Trp residue, present in both class II and III but not in class I, with a corresponding increase in the UV absorbance. The avian enzyme further shows that a Gly residue at position 260 previously considered strictly conserved in alcohol dehydrogenases can be exchanged with Lys. However, zinc-binding residues, coenzyme-binding residues, and to a large extent substrate-binding residues are unchanged in the avian enzyme, suggesting its functional properties to be related to those of the class I mammalian alcohol dehydrogenases. In contrast, the areas of subunit interactions in the dimers differ substantially. These results show that (a) the vertebrate enzyme classes are of distant origin, (b) the submammalian enzyme exhibits partly mixed properties in relation to the classes, and (c) the three mammalian enzyme classes are not as equidistantly related as initially apparent but suggest origins from two sublevels.     

Characterization of Coturnix quail liver alcohol dehydrogenase enzymes

Livers from male or female Coturnix quail possess up to four electrophoretically distinct bands of alcohol dehydrogenase (ADH) activity. Three pyrazole-sensitive bands of enzymatic activity, designated ADH-1, ADH-2, and ADH-3, are cathodic at pH 8.2, and the fourth, ADH-An, is neutral to slightly anodic and insensitive to pyrazole. ADH-2 and ADH-3, and occasionally ADH-1, are present in livers from immature females. The predominant enzyme in immature male livers is ADH-3. At sexual maturity all three pyrazole-sensitive enzymes are present in livers from male birds, and livers from females possess predominantly ADH-3. ADH-2 and ADH-3, purified from female livers, are dimers of 80,000 daltons possessing 4 mol of Zn2+/mol of native protein. Both ADH-2 and ADH-3 were inhibited by 4-methylpyrazole with KI values of 430 and 335 nM, respectively. These values are similar to those of human class I isoenzymes. Neither enzyme oxidized methanol or ethylene glycol, which distinguished them from mammalian pyrazole-sensitive ADH isoenzymes. Both ADH-2 and ADH-3 showed specificity toward hydrophobic primary alcohols and were most active toward benzyl alcohol and n-octanol.     

Sequence and structural aspects of the functional diversification of plant alcohol dehydrogenases

The glycolytic proteins in plants are coded by small multigene families, which provide an interesting contrast to the high copy number of gene families studied to date. The alcohol dehydrogenase (Adh) genes encode glycolytic enzymes that have been characterized in some plant families. Although the amino acid sequences of zinc-containing long-chain ADHs are highly conserved, the metabolic function of this enzyme is variable. They also have different patterns of expression and are submitted to differences in nonsynonymous substitution rates between gene copies. It is possible that the Adh copies have been retained as a consequence of adaptative amino acid replacements which have conferred subtle changes in function. Phylogenetic analysis indicates that there have been a number of separate duplication events within angiosperms, and that genes labeled Adh1, Adh2 and Adh3 in different groups may not be homologous. Nonsynonymous/synonymous ratios yielded no signs of positive selection. However, the coefficients of functional divergence (theta) estimated between the Adh1 and Adh2 gene groups indicate statistically significant site-specific shift of evolutionary rates between them, as well as between those of different botanical families, suggesting that altered functional constraints may have taken place at some amino acid residues after their diversification. The theoretical three-dimensional structure of the alcohol dehydrogenase from Arabis blepharophylla was constructed and verified to be stereochemically valid.     

Action of shear on enzymes: studies with alcohol dehydrogenase.

Yeast alcohol dehydrogenase (ADH) solutions (approximately 1 mg/ml, pH 7) were sheared in a coaxial cylindrical viscometer. This was fitted with a lid sealing the contents from the atmosphere and preventing evaporation. At 30 degrees C after a total of 5 hr intermittent shearing at 683 sec-1 no losses of activity were observed. No losses were found after 5 hr continuous shearing and in a no-shear control. At 40 degrees C and 683 sec-1 there were only small activity losses in 5 hr. Shearing at 3440 sec-1 no measurable losses of activity were found with a 1.03 mg/ml solution in 5 hr at 30 degrees C, a 1.03 mg/ml solution in 8 hr at 5 degrees C, and with a 3.89 mg/ml solution in 3 hr at 5 degrees C. In all these cases, however, a white precipitate formed that was not observed in zero shear control experiments. The sheared 3.89 mg/ml solution was clarified by centrifugation. It was shown that there were no ADH aggregates in the supernatant and that the precipitate was less than 2% of the original protein. At 30 degrees C under adverse pH conditions (pH 8.8) there was no significant difference in activity losses of an approximately 1 mg/ml solution sheared at 65 and 744 sec-1. An approximately 0.5 mg/ml ADH solution, pH 7, was agitated in a small reactor with no free air-liquid interface. Peak shear rates near the impeller were estimated to be about 9000 sec-1. Only a small decrease in specific activity was observed until over 15 hr total running at 5 degrees C.     

Comparative evolutionary analysis of chalcone synthase and alcohol dehydrogenase loci in Arabidopsis, Arabis, and related genera (Brassicaceae)

We analyzed sequence variation for chalcone synthase (CHS:) and alcohol dehydrogenase (ADH:) loci in 28 species in the genera ARABIDOPSIS: and ARABIS: and related taxa from tribe Arabideae. CHS: was single-copy in nearly all taxa examined, while ADH: duplications were found in several species. Phylogenies constructed from both loci confirmed that the closest relatives of Arabidopsis thaliana include Arabidopsis lyrata, Arabidopsis petraea, and Arabidopsis halleri (formerly in the genus CARDAMINOPSIS:). Slightly more distant are the North American n = 7 Arabis (BOECHERA:) species. The genus ARABIS: is polyphyletic-some unrelated species appear within this taxonomic classification, which has little phylogenetic meaning. Fossil pollen data were used to compute a synonymous substitution rate of 1.5 x 10 substitutions per site per year for both CHS: and Adh. Arabidopsis thaliana diverged from its nearest relatives about 5 MYA, and from Brassica roughly 24 MYA. Independent molecular and fossil data from several sources all provide similar estimates of evolutionary timescale in the Brassicaceae.     

Classification,origins, and patterns of diversification in New ZealandCarmichaelinae (Fabaceae)

Analysis of ITS sequences provides support for a clade that includes Carmichaelia, Clianthus, Montigena, and Swainsona. We provide a node-based definition and recommend that this clade be called Carmichaelinae. Results suggest that Carmichaelinae are derived from northern hemisphere Astragalinae. The clade has extensively radiated in Australia, and two independent lineages have diversified in New Zealand. The New Zealand lineages differ in species richness. One lineage consists of 24 species placed in Carmichaelia and Clianthus, while the other corresponds to the monotypic genus Montigena. The pattern of relationships inferred from ITS sequences suggests that the New Zealand radiation was recent and possibly accompanied episodes of mountain-building and glaciation.     

Intron-dependent evolution of the nucleotide-binding domains within alcohol dehydrogenase and related enzymes.

It has been suggested that the intron/exon structure of a gene corresponds to its evolutionary history. Accordingly, early in evolution DNA segments encoding short functional polypeptides may have been rearranged (exon-shuffling) to create full-length genes and RNA splicing may have been developed to remove intervening sequences (introns) in order to preserve translational reading frames. A conflicting viewpoint would be that introns were randomly inserted into previously uninterrupted genes after their initial evolutionary development. If so, the sites of introns would be unlikely to consistently reflect the domain structure of the protein. To address this question, the intron/exon structure of the gene encoding human alcohol dehydrogenase (ADH) was determined and compared to the gene structures for other ADHs and related proteins, all of which possess nucleotide-binding domains. Our results indicate that the introns in the nucleotide-binding domains of all the genes examined do indeed fall at positions which separate the short functional polypeptides (i.e. beta strands) which are believed to comprise this domain. We argue that our data is most easily explained by the hypothesis that introns were present in an ancestral nucleotide-binding domain which was later rearranged by exon-shuffling to form the various dehydrogenases and kinases which utilize such a domain.     

Expression and localization of human alcohol and aldehyde dehydrogenase enzymes in skin.

Alcohol dehydrogenase (ADH; EC 1.1.1.1) and aldehyde dehydrogenase (ALDH; EC 1.2.1.3.) are important enzymes involved in the biotransformation of both alcohols and aldehydes. Today, six classes of ADH and twelve classes of ALDH have been defined in mammals. Here we report the detection and localisation of three classes of ADH and two classes of ALDH in human skin, using Western blot analysis and immunohistochemistry with class-specific antisera. Western blot analysis of human skin cytosol revealed that class I-III ADH and class 1 and class 3 ALDH enzymes are expressed, constitutively, in three different anatomical regions of human skin (foreskin, breast, abdomen). Densitometric analysis of the immunoreactive bands revealed differential constitutive expression of these enzymes in foreskin, breast, and abdomen skin. Immunohistochemistry showed the presence of class I ADH and class III ADH enzymes, predominantly in the epidermis with some localised expression in the dermal appendages of human skin. In comparison, staining for class II ADH was more faint in the epidermis with very little dermal expression. Class 1 ALDH and class 3 ALDH were predominantly localised to the epidermis with minimal, highly localised dermal appendageal expression. These cutaneous ADH and ALDH enzymes may play significant roles in the metabolism of endogenous or xenobiotic alcohols and aldehydes.     

Rat liver alcohol dehydrogenase of class III. Primary structure, functional consequences and relationships to other alcohol dehydrogenases

The amino acid sequence of alcohol dehydrogenase of class III from rat liver (the enzyme ADH-2) has been determined. This type of structure is quite different from those of both the class I and the class II alcohol dehydrogenases. The rat class III structure differs from the rat and human class I structures by 133-138 residues (exact value depending on species and isozyme type); and from that of human class II by 132 residues. In contrast, the rat/human species difference within the class III enzymes is only 21 residues. The protein was carboxymethylated with iodo[2(14)C]acetate, and cleaved with CNBr and proteolytic enzymes. Peptides purified by exclusion chromatography and reverse-phase high-performance liquid chromatography were analyzed by degradation with a gas-phase sequencer and with the manual 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate double-coupling method. The protein chain has 373 residues with a blocked N terminus. No evidence was obtained for heterogeneity. The rat ADH-2 enzyme of class III contains an insertion of Cys at position 60 in relation to the class I enzymes, while the latter alcohol dehydrogenase in rat (ADH-3) has another Cys insertion (at position 111) relative to ADH-2. The structure deduced explains the characteristic differences of the class III alcohol dehydrogenase in relation to the other classes of alcohol dehydrogenase, including a high absorbance, an anodic electrophoretic mobility and special kinetic properties. The main amino acid substitutions are found in the catalytic domain and in the subunit interacting segments of the coenzyme-binding domain, the latter explaining the lack of hybrid dimers between subunits of different classes. Several substitutions provide an enlarged and more hydrophilic substrate-binding pocket, which appears compatible with a higher water content in the pocket and hence could possibly explain the higher Km for all substrates as compared with the corresponding values for the class I enzymes. Finally the class III structure supports evolutionary relationships suggesting that the three classes constitute clearly separate enzymes within the group of mammalian zinc-containing alcohol dehydrogenases.     

经济林凋落叶浸提液对豆禾牧草生长的化感效应

摘 要【目的】化感作用是影响林下生草的主要因素之一,陕北地区红枣（Ziziphus jujuba）、核桃（Juglans regia）及苹果（Malus pumila）凋落叶的化感效应尚不明确。【方法】通过设置不同浓度红枣、核桃和苹果凋落叶浸提液（浓度为0.1,0.05,0.025,0.0125 g/mL）,进行室外紫花苜蓿（Medicago sativa）和多年生黑麦草（Lolium perenne）盆栽实验。【结果】（1）核桃和苹果浸提液处理,两种牧草的生物量显著降低,其中多年生黑麦草单株鲜重仅有0.45 g,同对照组相比紫花苜蓿仅有4.70 g。株高随浸提液浓度增加先上升后降低（P＜0.05）。（2）不同浸提液处理紫花苜蓿和多年生黑麦草的丙二醛含量高于对照组,不同类型浸提液处理2种草的生化指标均表现出显著差异（P＜0.05）。（3）核桃和苹果浸提液对紫花苜蓿和多年生黑麦草的根系性状有显著差异（P＜0.05）。【结论】两种牧草的生物量对不同浸提液的响应表现为“增加或降低”作用,红枣浸提液处理更适合多年生黑麦草生长,应充分考虑紫花苜蓿和多年生黑麦草与经济林之间的相互作用。     

Molecular phylogeny and diversification history of Prosopis (Fabaceae: Mimosoideae)

The genus Prosopis is an important member of arid and semiarid environments around the world. To study Prosopis diversification and evolution, a combined approach including molecular phylogeny, molecular dating, and character optimization analysis was applied. Phylogenetic relationships were inferred from five different molecular markers ( mat K- trn K, trn L- trn F, trn S- psb C, G3pdh, NIA). Taxon sampling involved a total of 30 Prosopis species that represented all Sections and Series and the complete geographical range of the genus. The results suggest that Prosopis is not a natural group. Molecular dating analysis indicates that the divergence between Section Strombocarpa and Section Algarobia plus Section Monilicarpa occurred in the Oligocene, contrasting with a much recent diversification (Late Miocene) within each of these groups. The diversification of the group formed by species of Series Chilenses, Pallidae, and Ruscifoliae is inferred to have started in the Pliocene, showing a high diversification rate. The moment of diversification within the major lineages of American species of Prosopis is coincident with the spreading of arid areas in the Americas, suggesting a climatic control for diversification of the group. Optimization of habitat parameters suggests an ancient occupation of arid environments by Prosopis species.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 93 , 621–640.     

Phylogeny, diversification rates and species boundaries of Mesoamerican firs (Abies, Pinaceae) in a genus-wide context

The genus Abies is distributed discontinuously in the temperate and subtropical montane forests of the northern hemisphere. In Mesoamerica (Mexico and northern Central America), modern firs originated from the divergence of isolated mountain populations of migrating North American taxa. However, the number of ancestral species, migratory waves and diversification speed of these taxa is unknown. Here, variation in repetitive (Pt30204, Pt63718, and Pt71936) and non-repetitive (rbcL, rps18-rpl20 and trnL-trnF) regions of the chloroplast genome was used to reconstruct the phylogenetic relationships of the Mesoamerican Abies in a genus-wide context. These phylogenies and two fossil-calibrated scenarios were further employed to estimate divergence dates and diversification rates within the genus, and to test the hypothesis that, as in many angiosperms, conifers may exhibit accelerated speciation rates in the subtropics. All phylogenies showed five main clusters that mostly agreed with the currently recognized sections of Abies and with the geographic distribution of species. The Mesoamerican taxa formed a single group with species from southwestern North America of sections Oiamel and Grandis. However, populations of the same species were not monophyletic within this group. Divergence of this whole group dated back to the late Paleocene and the early Miocene depending on the calibration used, which translated in very low diversification rates (r_0.0 = 0.026-0.054, r_0.9 = 0.009-0.019 sp/Ma). Such low rates were a constant along the entire genus, including both the subtropical and temperate taxa. An extended phylogeographic analysis on the Mesoamerican clade indicated that Abies flinckii and A. concolor were the most divergent taxa, while the remaining species (A. durangensis, A. guatemalensis, A. hickelii, A. religiosa and A. vejari) formed a single group. Altogether, these results show that divergence of Mesoamerican firs coincides with a model of environmental stasis and decreased extinction rate, being probably prompted by a series of range expansions and isolation-by-distance.     

A new approach for using cofactor dependent enzymes: example of alcohol dehydrogenase

The use of enzymes requiring a cofactor as substrate in organic synthesis is still a problem since the cofactors are expensive. This study deals with a new approach consisting of using fragments of NAD+. Three fragments of NAD(H) are examined. The activities of NMN+ and NMNH are greatly improved by the addition of adenosine in ethanol oxidation and in cyclohexanone reduction, respectively. Nicotinamide mononucleoside is not active in the ethanol oxidation but the addition of AMP promotes this reaction.     

Gene duplication,genome duplication,and the functional diversification of vertebrate globins

The functional diversification of the vertebrate globin gene superfamily provides an especially vivid illustration of the role of gene duplication and whole-genome duplication in promoting evolutionary innovation. For example, key globin proteins that evolved specialized functions in various aspects of oxidative metabolism and oxygen signaling pathways (hemoglobin [Hb], myoglobin [Mb], and cytoglobin [Cygb]) trace their origins to two whole-genome duplication events in the stem lineage of vertebrates. The retention of the proto-Hb and Mb genes in the ancestor of jawed vertebrates permitted a physiological division of labor between the oxygen-carrier function of Hb and the oxygen-storage function of Mb. In the Hb gene lineage, a subsequent tandem gene duplication gave rise to the proto α- and β-globin genes, which permitted the formation of multimeric Hbs composed of unlike subunits (α₂β₂). The evolution of this heteromeric quaternary structure was central to the emergence of Hb as a specialized oxygen-transport protein because it provided a mechanism for cooperative oxygen-binding and allosteric regulatory control. Subsequent rounds of duplication and divergence have produced diverse repertoires of α- and β-like globin genes that are ontogenetically regulated such that functionally distinct Hb isoforms are expressed during different stages of prenatal development and postnatal life. In the ancestor of jawless fishes, the proto Mb and Hb genes appear to have been secondarily lost, and the Cygb homolog evolved a specialized respiratory function in blood-oxygen transport. Phylogenetic and comparative genomic analyses of the vertebrate globin gene superfamily have revealed numerous instances in which paralogous globins have convergently evolved similar expression patterns and/or similar functional specializations in different organismal lineages.     

Model for the structure of formaldehyde dehydrogenase based on alcohol dehydrogenase

The three-dimensional structure of rat liver formaldehyde dehydrogenase (FALDH), previously known as class III alcohol dehydrogenase, was constructed using computer graphics and computer programs developed for model building. The construction is based on horse liver alcohol dehydrogenase (EE-ADH), whose structure has been elucidated by X-ray crystallography. The high sequence homology between the two enzymes makes knowledge-based modelling feasible in this case. The model shows a remarkable similarity to horse liver alcohol dehydrogenase especially in the NAD-binding domain. Certain mutations, and the one insertion in FALDH compared to EE-ADH in particular, have cause important changes in the substrate binding site, and thus aliphatic alcohols have been replaced by hemi-thioacetals as favourable substrates.     

Interaction of alcohol dehydrogenase with tert-butylhydroperoxide: stimulation of the horse liver and inhibition of the yeast enzymes

Preincubation of horse liver alcohol dehydrogenase (HLADH) with the oxidative agent, tert-butyl hydroperoxide (tBOOH) results in a twofold stimulation of the ethanol dehydrogenase activity of this enzyme. This stimulation was dependent on tBOOH concentration up to 100 mM; above this concentration tBOOH did not further stimulate ethanol oxidation by HLADH. Active-site-directed reagents and classical ADH binary complexes were used to probe the possible mechanism of this activating effect. The rate and extent of stimulation by tBOOH is strongly reduced by binary complexes with NAD(+) or NADH, whose pyrophosphate groups bind to Arg-47 and Arg-369. In contrast stimulation by tBOOH was not prevented by AMP or the sulfhydryl reagents dithiothreitol and glutathione, suggesting, respectively, a lack of role for Lys-228 and sulfhydryl group oxidation in the stimulation by tBOOH. In contrast to the liver enzyme, treatment of yeast ADH (YADH) with tBOOH irreversibly inhibited its ethanol dehydrogenase activity. Inhibition of YADH by tBOOH approximated first-order rate kinetics with respect to enzyme at fixed concentrations of tBOOH between 0.5 to 300 mM. Four -SH groups per molecule of YADH were modified by tBOOH, whereas only two -SH groups were modified in HLADH. The stimulation of HLADH by tBOOH is suggested to be due to destabilization of the catalytic Zn-coordination sphere and amino acids associated with coenzyme binding in the active site, while inactivation of YADH appears to be associated with -SH group oxidation by the peroxide.     

Phylogeny, historical biogeography, and patterns of diversification for Pinus (Pinaceae): phylogenetic tests of fossil-based hypotheses

Pines comprise one of the largest coniferous genera, are distributed throughout the Northern Hemisphere, and have an abundant fossil record. Distributions of fossils have been used to derive a three-step hypothesis of early pine evolution, which postulates a Mesozoic origin for the genus, east-west expansions across Laurasia, and retraction into Eocene refugia. Here, we present phylogenetic tests of this hypothesis using chloroplast sequence data from four loci for 83 pine species. We used the fossil-based hypothesis to derive null expectations concerning monophyly of taxonomic groups, dates of cladogenesis, and patterns of diversification. Phylogenetic analyses using several algorithms subsequently provided rigorous tests of these expectations. Our inferred phylogenies illustrated broad congruence with taxonomic groups, but highlighted consistent problems within subgenus Strobus. Estimated minimum dates of divergence derived from relaxed clock methods were largely consistent with the fossil record and yielded a date for the ingroup node of Pinus of 128+/-4 mya, depending upon the calibration used for subgenus Pinus. Ancestral area reconstructions showed Pinus to have most likely originated in Eurasia. Major clades differed in biogeographic patterns, but were consistent with the fossil-based hypothesis. We found weak support, however, for a change in diversification rate in the Eocene as interpretations of fossil distributions would have predicted.