Expression and characterization of monofunctional alcohol dehydrogenase enzymes in Clostridium thermocellum.

Clostridium thermocellum is a thermophilic anaerobic bacterium that could be used for cellulosic biofuel production due to its strong native ability to consume cellulose, however its ethanol production ability needs to be improved to enable commercial application. In our previous strain engineering work, we observed a spontaneous mutation in the native adhE gene that reduced ethanol production. Here we attempted to complement this mutation by heterologous expression of 18 different alcohol dehydrogenase (adh) genes. We were able to express all of them successfully in C. thermocellum. Surprisingly, however, none of them increased ethanol production, and several actually decreased it. Our findings contribute to understanding the correlation between C. thermocellum ethanol production and Adh enzyme cofactor preferences. The identification of a set of adh genes that can be successfully expressed in this organism provides a foundation for future investigations into how the properties of Adh enzymes affect ethanol production.

Fungal Alcohol Dehydrogenases: Physiological Function, Molecular Properties, Regulation of Their Production, and Biotechnological Potential.

Fungal alcohol dehydrogenases (ADHs) participate in growth under aerobic or anaerobic conditions, morphogenetic processes, and pathogenesis of diverse fungal genera. These processes are associated with metabolic operation routes related to alcohol, aldehyde, and acid production. The number of ADH enzymes, their metabolic roles, and their functions vary within fungal species. The most studied ADHs are associated with ethanol metabolism, either as fermentative enzymes involved in the production of this alcohol or as oxidative enzymes necessary for the use of ethanol as a carbon source; other enzymes participate in survival under microaerobic conditions. The fast generation of data using genome sequencing provides an excellent opportunity to determine a correlation between the number of ADHs and fungal lifestyle. Therefore, this review aims to summarize the latest knowledge about the importance of ADH enzymes in the physiology and metabolism of fungal cells, as well as their structure, regulation, evolutionary relationships, and biotechnological potential.

Signatures of Convergent Evolution and Natural Selection at the Alcohol Dehydrogenase Gene Region are Correlated with Agriculture in Ethnically Diverse Africans.

The alcohol dehydrogenase (ADH) family of genes encodes enzymes that catalyze the metabolism of ethanol into acetaldehyde. Nucleotide variation in ADH genes can affect the catalytic properties of these enzymes and is associated with a variety of traits, including alcoholism and cancer. Some ADH variants, including the ADH1B*48His (rs1229984) mutation in the ADH1B gene, reduce the risk of alcoholism and are under positive selection in multiple human populations. The advent of Neolithic agriculture and associated increase in fermented foods and beverages is hypothesized to have been a selective force acting on such variants. However, this hypothesis has not been tested in populations outside of Asia. Here, we use genome-wide selection scans to show that the ADH gene region is enriched for variants showing strong signals of positive selection in multiple Afroasiatic-speaking, agriculturalist populations from Ethiopia, and that this signal is unique among sub-Saharan Africans. We also observe strong selection signals at putatively functional variants in nearby lipid metabolism genes, which may influence evolutionary dynamics at the ADH region. Finally, we show that haplotypes carrying these selected variants were introduced into Northeast Africa from a West-Eurasian source within the last ∼2,000 years and experienced positive selection following admixture. These selection signals are not evident in nearby, genetically similar populations that practice hunting/gathering or pastoralist subsistence lifestyles, supporting the hypothesis that the emergence of agriculture shapes patterns of selection at ADH genes. Together, these results enhance our understanding of how adaptations to diverse environments and diets have influenced the African genomic landscape.

Ethanol Metabolism in the Liver, the Induction of Oxidant Stress, and the Antioxidant Defense System.

The liver metabolizes ethanol through three enzymatic pathways: alcohol dehydrogenase (ADH), cytochrome p450 (also called MEOS), and catalase. Alcohol dehydrogenase class I (ADH1) is considered the most important enzyme for the metabolism of ethanol, MEOS and catalase (CAT) are considered minor alternative pathways. However, contradicting experiments suggest that the non-ADH1 pathway may have a greater relevance for the metabolism of ethanol than previously thought. In some conditions, ethanol is predominately metabolized to acetaldehyde via cytochrome P450 family 2 (CYP2E1), which is involved in the generation of reactive oxygen species (ROS), mainly through electron leakage to oxygen to form the superoxide (O2•-) radical or in catalyzed lipid peroxidation. The CAT activity can also participate in the ethanol metabolism that produces ROS via ethanol directly reacting with the CAT-H2O2 complex, producing acetaldehyde and water and depending on the H2O2 availability, which is the rate-limiting component in ethanol peroxidation. We have shown that CAT actively participates in lactate-stimulated liver ethanol oxidation, where the addition of lactate generates H2O2, which is used by CAT to oxidize ethanol to acetaldehyde. Therefore, besides its known role as a catalytic antioxidant component, the primary role of CAT could be to function in the metabolism of xenobiotics in the liver.

Cometabolism of Ethanol in Azospirillum brasilense Sp7 Is Mediated by Fructose and Glycerol and Regulated Negatively by an Alternative Sigma Factor RpoH2.

Azospirillum brasilense is a plant growth-promoting rhizobacterium that is not known to utilize ethanol as a sole source of carbon for growth. This study shows that A. brasilense can cometabolize ethanol in medium containing fructose or glycerol as a carbon source and contribute to its growth. In minimal medium containing fructose or glycerol as a carbon source, supplementation of ethanol caused enhanced production of an alcohol dehydrogenase (ExaA) and an aldehyde dehydrogenase (AldA) in A. brasilense. However, this was not the case when malate was used as a carbon source. Inactivation of aldA in A. brasilense resulted in the loss of the AldA protein and its ethanol utilizing ability in fructose- or glycerol-supplemented medium. Furthermore, ethanol inhibited the growth of the aldA::Km mutant. The exaA::Km mutant also lost its ability to utilize ethanol in fructose-supplemented medium. However, in glycerol-supplemented medium, A. brasilense utilized ethanol due to the synthesis of a new paralog of alcohol dehydrogenase (ExaA1). The expression of exaA1 was induced by glycerol but not by fructose. Unlike exaA, expression of aldA and exaA1 were not dependent on σ54. Instead, they were negatively regulated by the RpoH2 sigma factor. Inactivation of rpoH2 in A. brasilense conferred the ability to use ethanol as a carbon source without or with malate, overcoming catabolite repression caused by malate. This is the first study showing the role of glycerol and fructose in facilitating cometabolism of ethanol by inducing the expression of ethanol-oxidizing enzymes and the role of RpoH2 in repressing them. IMPORTANCE This study unraveled a hidden ability of Azospirillum brasilense to utilize ethanol as a secondary source of carbon when fructose or glycerol were used as a primary growth substrate. It opens the possibility of studying the regulation of expression of the ethanol oxidation pathway for generating high yielding strains that can efficiently utilize ethanol. Such strains would be useful for economical production of secondary metabolites by A. brasilense in fermenters. The ability of A. brasilense to utilize ethanol might be beneficial to the host plant under the submerged growth conditions.

Optimizing the linker length for fusing an alcohol dehydrogenase with a cyclohexanone monooxygenase.

The use of enzymes in organic synthesis is highly appealing due their remarkably high chemo-, regio- and enantioselectivity. Nevertheless, for biosynthetic routes to be industrially useful, the enzymes must fulfill several requirements. Particularly, in case of cofactor-dependent enzymes self-sufficient systems are highly valuable. This can be achieved by fusing enzymes with complementary cofactor dependency. Such bifunctional enzymes are also relatively easy to handle, may enhance stability, and promote product intermediate channeling. However, usually the characteristics of the linker, fusing the target enzymes, are not thoroughly evaluated. A poor linker design can lead to detrimental effects on expression levels, enzyme stability and/or enzyme performance. In this chapter, the effect of the length of a glycine-rich linker was explored for the case study of ɛ-caprolactone synthesis through an alcohol dehydrogenase-cyclohexanone monooxygenase fusion system. The procedure includes cloning of linker variants, expression analysis, determination of thermostability and effect on activity and conversion levels of 15 variants of different linker sizes. The protocols can also be used for the creation of other protein-protein fusions.

Origin and Evolutionary Dynamics of the miR2119 and ADH1 Regulatory Module in Legumes.

MicroRNAs are important regulators of gene expression in eukaryotes. Previously, we reported that in Phaseolus vulgaris, the precursor for miR2119 is located in the same gene as miR398a, conceiving a dicistronic MIR gene. Both miRNA precursors are transcribed and processed from a single transcript resulting in two mature microRNAs that regulate the mRNAs encoding ALCOHOL DEHYDROGENASE 1 (ADH1) and COPPER-ZINC SUPEROXIDE DISMUTASE 1 (CSD1). Genes for miR398 are distributed throughout the spermatophytes; however, miR2119 is only found in Leguminosae species, indicating its recent emergence. Here, we used public databases to explore the presence of the miR2119 sequence in several plant species. We found that miR2119 is present only in specific clades within the Papilionoideae subfamily, including important crops used for human consumption and forage. Within this subfamily, MIR2119 and MIR398a are found together as a single gene in the genomes of the Millettioids and Hologalegina. In contrast, in the Dalbergioids MIR2119 is located in a different locus from MIR398a, suggesting this as the ancestral genomic organization. To our knowledge, this is a unique example where two separate MIRNA genes have merged to generate a single polycistronic gene. Phylogenetic analysis of ADH1 gene sequences in the Papilionoideae subfamily revealed duplication events resulting in up to four ADH1 genes in certain species. Notably, the presence of MIR2119 correlates with the conservation of target sites in particular ADH1 genes in each clade. Our results suggest that post-transcriptional regulation of ADH1 genes by miR2119 has contributed to shaping the expansion and divergence of this gene family in the Papilionoideae. Future experimental work on ADH1 regulation by miR2119 in more legume species will help to further understand the evolutionary history of the ADH1 gene family and the relevance of miRNA regulation in this process.

Determining the Protective Activity of IDPs Under Partial Dehydration and Freeze-Thaw Conditions.

Unlike for structured proteins, the study of intrinsically disordered proteins (IDPs) requires selection of ad hoc assays and strategies to characterize their dynamic structure and function. Late embryogenesis abundant (LEA) proteins are important plant IDPs closely related to water-deficit stress response. Diverse hypothetical functions have been proposed for LEA proteins, such as membrane stabilizers during cold stress, oxidative regulators acting as ion metal binding molecules, and protein protectants during dehydration and cold/freezing conditions. Here we present two detailed protocols to characterize IDPs with potential protein/enzyme protection activity under partial dehydration and freeze-thaw treatments.

Germination temperatures affect the physiological quality of seeds of lettuce cultivars / Qualidade fisiológica de sementes de alface sob diferentes temperaturas na germinação

The objective of this study was to evaluate the physiological quality allied to biochemical quality of lettuce seeds by germination and enzymes expression at 20, 25, 30, 35, 40 and 42ºC. Germination speed index and percentage of germination were estimated. Isoenzyme expressions were assessed by alcohol dehydrogenase (ADH), malate dehydrogenase (MDH), catalase (CAT), esterase (EST), pyruvate decarboxylase (PDC) and glutamate oxaloacetate transferase (GOT). The experiment consisted of a completely randomized design in a factorial scheme 4x6, with four cultivars and six different temperatures, with four replications. The highest germination and vigor were observed for cv. 'Everglades' at 35°C, which proved that this cultivar is thermotolerant. Catalase can be considered a genetic marker for the identification ofthermotolerant lettuce cultivars. Cultivar 'Everglades' has potential to be used in lettuce breeding programs aimed at cultivars tolerant to high temperatures during germination.

O objetivo deste estudo foi avaliar a qualidade fisiológica e bioquímica de sementes de alface por meio da germinação e expressão de enzimas a 20, 25, 30, 35, 40 e 42ºC. As variáveis velocidade de germinação e o índice de velocidade de germinação foram estimadas. As expressões das enzimas alcool desidrogenase (ADH), malato desidrogenase (MDH), catalase (CAT), esterase (EST), piruvate descarboxilase (PDC) e glutamato oxaloacetato transferase (GOT) foram avaliadas. Para análise dos genótipos foi empregado o delineamento inteiramente casualizado em esquema fatorial 4x6, testando quatro cultivares e seis diferentes temperaturas, com quatro repetições. A maior germinação e vigor foram observadas para a cv. 'Everglades' a 35°C, o que prova que esta cultivar é termotolerante. A catalase pode ser considerada um marcador para a identificação de cultivares de alface termotolerantes. A cultivar 'Everglades' tem potential para uso em programas de melhoramento visando tolerância à alta temperatura durante a germinação.

Lactate-stimulated ethanol oxidation: Revisiting an old hypothesis.

Liver slices from starved rats and incubated without other substrates oxidized ethanol at a rate of 4.1 µmols • h-1 • g-1. Addition of 10 mmols • L-1 lactate increased this rate 2-fold. 4-methylpyrazole (4-MP), an alcohol dehydrogenase (ADH) inhibitor, drastically decreased the rate of ethanol oxidation, but did not inhibit the stimulation due to lactate. In the same context, liver acetaldehyde production, as the main by-product of ethanol oxidation, appeared to be much less inhibited by 4-MP in the presence of lactate. Aminotriazole (a catalase inhibitor), however, completely inhibited the stimulation. Furthermore, 2-hydroxybut-3-ynoate, an alpha-hydroxy acid oxidase inhibitor, completely abolished the stimulated ethanol oxidation promoted by lactate. Moreover, to determine the origin of the H2O2 produced, we did liver subcellular fractionation and then analyzed their content in peroxisomes, mitochondria and catalase. We observed that cytoplasm and peroxisomes appears to be the main producers of H2O2, and that the acceleration of ethanol oxidation by lactate is completely dependent on catalase. In conclusion, the H2O2 necessary to boost the catalase-dependent oxidation of ethanol appears to come from cytoplasm and peroxisomes, and is produced by the enzyme lactate oxidase.

A dicistronic precursor encoding miR398 and the legume-specific miR2119 coregulates CSD1 and ADH1 mRNAs in response to water deficit.

Plant microRNAs are commonly encoded in transcripts containing a single microRNA precursor. Processing by DICER-LIKE 1 and associated factors results in the production of a small RNA, followed by its incorporation into an AGO-containing protein complex to guide silencing of an mRNA possessing a complementary target sequence. Certain microRNA loci contain more than one precursor stem-loop structure, thus encoding more than one microRNA in the same transcript. Here, we describe a unique case where the evolutionary conserved miR398a is encoded in the same transcript as the legume-specific miR2119. The dicistronic arrangement found in common bean was also observed in other legumes. In Phaseolus vulgaris, mature miR398 and miR2119 are repressed in response to water deficit, and we demonstrate that both are functional as they target the mRNAs for CSD1 and ADH1, respectively. Our results indicate that the repression of miR398 and miR2119 leads to coordinated up-regulation of CSD1 and ADH1 mRNAs in response to water deficit in common bean and possibly in other legumes. Furthermore, we show that miRNA directed CSD1 and ADH1 mRNAs up-regulation also occurs when common bean plants are exposed to flooding, suggesting that plant redox status and fermentation metabolism must be closely coordinated under different adverse conditions.

Associações entre polimorfismos genéticos da álcool: desidrogenase e o transtorno por uso de álcool / Associations between genetic polymorphisms of alcohol: dehydrogenase and alcohol use disorder

O transtorno por uso de álcool (TUA) é influenciado pela genética, principalmente na metabolização do etanol. Os genes da álcool desidrogenase (ADH1B/ADH1C), enzima que transforma o etanol, apresentam SNPs (single nucleotide polymorphisms) que resultam em isoenzimas com diferentes taxas catalíticas. Estudos demonstraram que os SNPs Arg48His, Arg370Cys, Arg272Gln e Ile350Val contribuem para o TUA. Este artigo revisou os estudos que investigaram SNPs em ADH1B (Arg48His/Arg370Cys) e ADH1C (Arg272Gln/Ile350Val), bem como avaliou as variações nas frequências alélicas desses genes e a influência no TUA nas diferentes populações no mundo. As frequências alélicas dos polimorfismos foram comparadas pelos testes qui-quadrado de Pearson e exato de Fisher (p < 0,05). O SNP Arg48His confere proteção para o TUA em euroamericanos, latino-americanos, europeus, brasileiros, asiáticos e australianos. O SNP Arg370Cys confere proteção para o TUA em afrodescendentes. Os SNPs Arg272Gln e Ile350Val predispõem o TUA principalmente em europeus. Os SNPs Arg48His, Arg370Cys e Arg272Gln/Ile350Val foram mais frequentes em amostras de leste-asiáticos (69,7%), africanos (19,1%) e europeus (40,5%), respectivamente (p < 0,01). Os diferentes alelos dos genes ADH1B/ADH1C devido a SNPs têm uma importante contribuição no TUA. As frequências desses alelos variam conforme a população, resultando em diferentes efeitos no TUA. (AU)

Alcohol use disorder (AUD) is influenced by genetics, especially in the metabolism of ethanol. The ethanol dehydrogenase genes (ADH1B/ADH1C), which convert ethanol, have single nucleotide polymorphisms (SNPs) that result in isoenzymes with different catalytic rates. Studies have shown that the Arg48His, Arg370Cys, Arg272Gln, and Ile350Val SNPs contribute to AUD. This article reviewed the studies that investigated SNPs in ADH1B (Arg48His/Arg370Cys) and ADH1C (Arg272Gln/Ile350Val) and evaluated variations in the allele frequencies of these genes and their influence on AUD in different populations worldwide. The allele frequencies of the polymorphisms were compared by Pearson's chi-square and Fisher's exact tests (p < 0.05). The Arg48His SNP provides protection against AUD in Euro-Americans, Latin Americans, Europeans, Brazilians, Asians, and Australians. The Arg370Cys SNP provides protection against AUD in Afro-descendants. The Arg272Gln and Ile350Val SNPs predispose to AUD mainly in Europeans. The Arg48His, Arg370Cys, and Arg272Gln/Ile350Val SNPs were more frequent in East Asians (69.7%), Africans (19.1%), and Europeans (40.5%), respectively (p < 0.01). The different alleles of the ADH1B/ADH1C genes due to SNPs make an important contribution to AUD. The frequencies of these alleles vary among different populations, resulting in different effects on AUD..(AU)

Polimorfismos en los genes de la enzima alcohol deshidrogenasa (ADH1) y la citocromo P450 2E1 (CYP2E1) en pacientes con cirrosis y carcinoma hepatocelular / Polymorphisms in alcohol dehydrogenase (ADH1) and cytochrome p450 2E1 (CYP2E1) genes in patients with cirrhosis and/or hepatocellular carcinoma

Introducción. Uno de los principales factores de riesgo del carcinoma hepatocelular es el consumo crónico de alcohol. En estudios en diferentes poblaciones, se sugiere que las variantes genéticas de las enzimas que participan en el metabolismo del alcohol, como la alcohol deshidrogenasa (ADH) y la citocromo P450 (CYP2E1), estarían asociadas con riesgo de enfermedades hepáticas terminales. Objetivo. Identificar y caracterizar las variantes alélicas de los genes ADH1B, ADH1C y CYP2E1 en pacientes colombianos con diagnóstico de cirrosis y carcinoma hepatocelular. Materiales y métodos. Se incluyeron muestras de pacientes atendidos entre el 2005 y el 2007, y entre el 2014 y el 2016, en la unidad de hepatología de un hospital de Medellín. La genotipificación de las muestras se hizo mediante reacción en cadena de la polimerasa (Polymerase Chain Reaction, PCR) con análisis de los polimorfismos en la longitud de los fragmentos de restricción (Restriction Fragment Length Polymorphism, RFLP). Los resultados se compararon con los de dos grupos de control y con lo reportado en la base de datos del 1000 Genomes Project. Resultados. Se recolectaron 97 muestras de pacientes con diagnóstico de cirrosis y carcinoma hepatocelular. Los dos factores de riesgo más frecuentes fueron el consumo crónico de alcohol (18,6 %) y las colangiopatías (17,5 %). Los genotipos más frecuentes en la población de estudio fueron el ADH1B*1/1 (82 %), el ADH1C*1/1 (59 %) y el CYP2E1*C/C (84 %). Conclusiones. En este primer estudio de los polimorfismos en pacientes colombianos con diagnóstico de cirrosis y carcinoma hepatocelular, los genotipos más frecuentes fueron el ADH1B*1/1, el ADH1C*1/1 y el CYP2E1*C/C. No se observaron diferencias estadísticamente significativas en la frecuencia de los genotipos entre los casos y los controles. Se requieren estudios adicionales en población colombiana para evaluar el riesgo de la enfermedad hepática terminal por consumo crónico de alcohol y la asociación con los polimorfismos.

Introduction: One of the most important risk factors for hepatocellular carcinoma (HCC) is alcohol consumption: Studies in different populations suggest that the risk of liver disease could be associated with genetic variants of the enzymes involved in alcohol metabolism, such as alcohol dehydrogenase (ADH) and cytochrome P450 CYP2E1. Objective: To identify and characterize the allelic variants of ADH1B, ADH1C and CYP2E1 genes in Colombian patients with cirrhosis and/or HCC. Materials and methods: We included samples from patients attending the hepatology unit between 2005-2007 and 2014-2016 of a hospital in Medellin. Samples were genotyped using PCR-RFLP. We compared the results with two control groups and the 1000 Genomes Project database. Results: We collected 97 samples from patients with a diagnosis of cirrhosis and/or HCC. The two main risk factors were chronic alcohol consumption (18.6%) and cholangiopathies (17.5%). The most frequent genotypes in the study population were ADH1B*1/1 (82%), ADH1C*1/1 (59%), and CYP2E1*C/C (84%). Conclusions: This first study of polymorphisms in Colombian patients diagnosed with cirrhosis and/or HCC showed genotypes ADH1B*1/1, ADH1C*1/1 and CYP2E1*C/C as the most frequent. We found no significant differences in the genotype frequency between cases and controls. Further studies are necessary to explore the association between polymorphisms and the risk of end-stage liver disease from alcohol consumption.

Ethanol generation, oxidation and energy production in a cooperative bioelectrochemical system.

Integrating in situ biofuel production and energy conversion into a single system ensures the production of more robust networks as well as more renewable technologies. For this purpose, identifying and developing new biocatalysts is crucial. Herein, is reported a bioelectrochemical system consisting of alcohol dehydrogenase (ADH) and Saccharomyces cerevisiae, wherein both function cooperatively for ethanol production and its bioelectrochemical oxidation. Here, it is shown that it is possible to produce ethanol and use it as a biofuel in a tandem manner. The strategy is to employ flexible carbon fibres (FCF) electrode that could adsorb both the enzyme and the yeast cells. Glucose is used as a substrate for the yeast for the production of ethanol, while the enzyme is used to catalyse the oxidation of ethanol to acetaldehyde. Regarding the generation of reliable electricity based on electrochemical systems, the biosystem proposed in this study operates at a low temperature and ethanol production is proportional to the generated current. With further optimisation of electrode design, we envision the use of the cooperative biofuel cell for energy conversion and management of organic compounds.

Enhanced long-chain fatty alcohol oxidation by immobilization of alcohol dehydrogenase from S. cerevisiae.

This work reports on the oxidation of long-chain aliphatic alcohols catalyzed by a stabilized alcohol dehydrogenase from S. cerevisiae (yeast alcohol dehydrogenase (YADH)). In particular, the oxidation of the fatty alcohol tetracosanol (C24H50O) to yield lignoceric acid (C23H47COOH) was studied. The immobilization of YADH onto glyoxyl agarose supports crosslinked with a polymer (polyethylenimine) produced a highly stable catalyst (60-fold higher than the soluble enzyme at 40 °C). Aliphatic alcohols with different chain lengths (ranging from 2 to 24 carbons) were studied as substrates for YADH. The activity of YADH with aliphatic alcohols with a chain length higher than five carbon atoms is reported for the first time. The activities obtained with the immobilized YADH were all similar in magnitude, even with long-chain fatty alcohols such as docosanol and tetracosanol. As far as the oxidation of tetracosanol is concerned, the best values of reaction rate and substrate conversion were obtained at pH = 8.2 and T = 58 °C. At these conditions, the soluble enzyme inactivated rapidly, precluding its use in batch reaction. However, using the immobilized YADH, up to three sequential reaction batches were performed by recovering the catalyst after each batch. Several applications in the green oleochemical industry, e.g., for making plasticizers, lubricants, detergents, and personal care products, may benefit from having novel and stable biocatalysts able to oxidize long-chain fatty alcohols.

Molecular evolution and functional divergence of alcohol dehydrogenases in animals, fungi and plants

Abstract Alcohol dehydrogenases belong to the large superfamily of medium-chain dehydrogenases/reductases, which occur throughout the biological world and are involved with many important metabolic routes. We considered the phylogeny of 190 ADH sequences of animals, fungi, and plants. Non-class III Caenorhabditis elegans ADHs were seen closely related to tetrameric fungal ADHs. ADH3 forms a sister group to amphibian, reptilian, avian and mammalian non-class III ADHs. In fishes, two main forms are identified: ADH1 and ADH3, whereas in amphibians there is a new ADH form (ADH8). ADH2 is found in Mammalia and Aves, and they formed a monophyletic group. Additionally, mammalian ADH4 seems to result from an ADH1 duplication, while in Fungi, ADH formed clusters based on types and genera. The plant ADH isoforms constitute a basal clade in relation to ADHs from animals. We identified amino acid residues responsible for functional divergence between ADH types in fungi, mammals, and fishes. In mammals, these differences occur mainly between ADH1/ADH4 and ADH3/ADH5, whereas functional divergence occurred in fungi between ADH1/ADH5, ADH5/ADH4, and ADH5/ADH3. In fishes, the forms also seem to be functionally divergent. The ADH family expansion exemplifies a neofunctionalization process where reiterative duplication events are related to new activities.

Analysis of the Association of Nonsynonymous Polymorphisms in ADH Genes with Hazardous Drinking in HIV-1-Positive Individuals.

BACKGROUND: Hazardous drinking (HD) is a serious health problem in people infected with human immunodeficiency virus type 1 (HIV-1). Single nucleotide polymorphisms (SNPs) in alcohol dehydrogenase (ADH) genes have been associated with HD in different populations, but there were no data about this in HIV-1-positive individuals. This study investigated the association of 4 nonsynonymous SNPs in ADH genes (Arg48His and Arg370Cys in ADH1B gene; Arg272Gln and Ile350Val in ADH1C gene) with HD in people living with HIV-1. METHODS: This case-control study included 365 HIV-1-positive individuals (121 with HD and 244 without HD). Sociodemographic variables were collected with a standardized individual questionnaire. HD (score ≥8) and binge drinking (BD) (drinks on the same occasion ≥5) were detected with the Alcohol Use Disorders Identification Test. The 4 SNPs were genotyped by the polymerase chain reaction-restriction fragment length polymorphism method. Adjusted odds ratios (aORs) and 95% confidence intervals (CIs) were calculated using logistic regression analysis. The Bonferroni correction was used (considering the 4 SNPs studied). RESULTS: There were no significant differences in the frequencies of Arg370Cys, Arg272Gln, and Ile350Val polymorphisms between HD cases and controls. Otherwise, Arg/His genotype (rs1229984) in ADH1B gene showed a protective effect against HD (aOR = 0.36; 95% CI: 0.14 to 0.90) and BD (aOR = 0.49; 95% CI: 0.21 to 0.95). Nevertheless, these differences were no longer significant after Bonferroni correction. CONCLUSIONS: The results of this study suggest a possible effect of the Arg48His genotype on the protection against HD in HIV-1-positive individuals.

Regulation of a Glycerol-Induced Quinoprotein Alcohol Dehydrogenase by σ54 and a LuxR-Type Regulator in Azospirillum brasilense Sp7.

Azospirillum brasilense Sp7 uses glycerol as a carbon source for growth and nitrogen fixation. When grown in medium containing glycerol as a source of carbon, it upregulates the expression of a protein which was identified as quinoprotein alcohol dehydrogenase (ExaA). Inactivation of exaA adversely affects the growth of A. brasilense on glycerol. A determination of the transcription start site of exaA revealed an RpoN-dependent -12/-24 promoter consensus. The expression of an exaA::lacZ fusion was induced maximally by glycerol and was dependent on σ54 Bioinformatic analysis of the sequence flanking the -12/-24 promoter revealed a 17-bp sequence motif with a dyad symmetry of 6 nucleotides upstream of the promoter, the disruption of which caused a drastic reduction in promoter activity. The electrophoretic mobility of a DNA fragment containing the 17-bp sequence motif was retarded by purified EraR, a LuxR-type transcription regulator that is transcribed divergently from exaA EraR also showed a positive interaction with RpoN in two-hybrid and pulldown assays.IMPORTANCE Quinoprotein alcohol dehydrogenase (ExaA) plays an important role in the catabolism of alcohols in bacteria. Although exaA expression is thought to be regulated by a two-component system consisting of EraS and EraR, the mechanism of regulation was not known. This study shows the details of the regulation of expression of the exaA gene in A. brasilense We have shown here that exaA of A. brasilense is maximally induced by glycerol and harbors a σ54-dependent promoter. The response regulator EraR binds to an inverted repeat located upstream of the exaA promoter. This study shows that a LuxR-type response regulator (EraR) binds upstream of the exaA gene and physically interacts with σ54 The unique feature of this regulation is that EraR is a LuxR-type transcription regulator that lacks the GAFTGA motif, a characteristic feature of the enhancer binding proteins that are known to interact with σ54 in other bacteria.

Evaluation of single and stack membraneless enzymatic fuel cells based on ethanol in simulated body fluids.

The purpose of this work is to evaluate single and double-cell membraneless microfluidic fuel cells (MMFCs) that operate in the presence of simulated body fluids SBF, human serum and blood enriched with ethanol as fuels. The study was performed using the alcohol dehydrogenase enzyme immobilised by covalent binding through an array composed of carbon Toray paper as support and a layer of poly(methylene blue)/tetrabutylammonium bromide/Nafion and glutaraldehyde (3D bioanode electrode). The single MMFC was tested in a hybrid microfluidic fuel cell using Pt/C as the cathode. A cell voltage of 1.035V and power density of 3.154mWcm-2 were observed, which is the highest performance reported to date. The stability and durability were tested through chronoamperometry and polarisation/performance curves obtained at different days, which demonstrated a slow decrease in the power density on day 10 (14%) and day 20 (26%). Additionally, the cell was tested for ethanol oxidation in simulated body fluid (SBF) with ionic composition similar to human blood plasma. Those tests resulted in 0.93V of cell voltage and a power density close to 1.237mWcm-2. The double cell MMFC (Stack) was tested using serum and human blood enriched with ethanol. The stack operated with blood in a serial connection showed an excellent cell performance (0.716mWcm-2), demonstrating the feasibility of employing human blood as energy source.

Taxonomic status of two morphotypes of Coryphaena hippurus (Perciformes: Coryphaenidae)

Two Coryphaena hippurus morphotypes (dourado and palombeta) are found along the Brazilian coast and are considered by Rio de Janeiro’s fisherman and fishmongers as two different species. Furthermore, these morphotypes are commercialized under different values and suffer different fishing pressure. Therefore, a definition of their taxonomic status is an important economic and biological matter. In order to investigate this problem, allozyme electrophoresis method was undertaken for seventeen loci on 117 individuals of C. hippurus sampled at Cabo Frio/RJ (Brazil). The data indicate homogeneity between the morphotypes gene pools. Nevertheless, differences were found for genetic variation among dourado and palombeta, especially due to alcohol dehydrogenase locus. Natural selection hypothesis is discussed in explaining these findings.(AU)

Dois morfotipos de Coryphaena hippurus (dourado e palombeta) encontrados ao longo da costa brasileira são considerados espécies diferentes por pescadores e mercadores das regiões de desembarque do estado do Rio de Janeiro. Além disso, esses morfotipos são comercializados por valores diferentes e sofrem diferentes pressões de pesca. Desta forma, a definição do status taxonômico desses morfotipos é importante, tanto em termos econômicos quanto biológicos. A fim de investigar esse problema foi utilizado o método de eletroforese de aloenzimas com a amostragem de dezessete loci para 117 indivíduos dos dois morfotipos de C. hippurus obtidos em desembarques pesqueiros na região de Cabo Frio/RJ (Brasil). Os dados indicaram uma homogeneidade entre os conjuntos gênicos dos morfotipos. A despeito disso, diferenças entre os conjuntos gênicos de dourado e palombeta foram encontradas, devido, especialmente, ao locus álcool desidrogenase. A hipótese de seleção natural é discutida como possível explicação para esses resultados.(AU)