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Sample records for aldehyde dehydrogenase enzymes

  1. Non-P450 aldehyde oxidizing enzymes: the aldehyde dehydrogenase superfamily

    PubMed Central

    Marchitti, Satori A; Brocker, Chad; Stagos, Dimitrios; Vasiliou, Vasilis

    2009-01-01

    Background Aldehydes are highly reactive molecules. While several non-P450 enzyme systems participate in their metabolism, one of the most important is the aldehyde dehydrogenase (ALDH) superfamily, composed of NAD(P)+-dependent enzymes that catalyze aldehyde oxidation. Objective This article presents a review of what is currently known about each member of the human ALDH superfamily including the pathophysiological significance of these enzymes. Methods Relevant literature involving all members of the human ALDH family was extensively reviewed, with the primary focus on recent and novel findings. Conclusion To date, 19 ALDH genes have been identified in the human genome and mutations in these genes and subsequent inborn errors in aldehyde metabolism are the molecular basis of several diseases, including Sjögren-Larsson syndrome, type II hyperprolinemia, γ-hydroxybutyric aciduria and pyridoxine-dependent seizures. ALDH enzymes also play important roles in embryogenesis and development, neurotransmission, oxidative stress and cancer. Finally, ALDH enzymes display multiple catalytic and non-catalytic functions including ester hydrolysis, antioxidant properties, xenobiotic bioactivation and UV light absorption. PMID:18611112

  2. A specific affinity reagent to distinguish aldehyde dehydrogenases and oxidases. Enzymes catalyzing aldehyde oxidation in an adult moth

    SciTech Connect

    Tasayco, M.L.; Prestwich, G.D. )

    1990-02-25

    Aldehyde dehydrogenase (ALDH) and oxidase (AO) enzymes from the tissue extracts of male and female tobacco budworm moth (Heliothis virescens) were identified after electrophoretic protein separation. AO activity was visualized using formazan- or horseradish peroxidase-mediated staining coupled to the AO-catalyzed oxidation of benzaldehyde. A set of six soluble AO enzymes with isoelectric points from pI 4.6 to 5.3 were detected primarily in the antennal extracts. Partially purified antennal AO enzymes also oxidized both (Z)-9-tetradecenal and (Z)-11-hexadecenal, the two major pheromone components of this moth. ALDH activity was detected using a tritium-labeled affinity reagent based on a known irreversible inhibitor of this enzyme. This labeled vinyl ketone, (3H)(Z)-1,11-hexadecadien-3-one, was synthesized and used to covalently modify the soluble ALDH enzymes from tissue extracts. Molecular subunits of potential ALDH enzymes were visualized in the fluorescence autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated proteins of the antenna, head, and leg tissues. Covalent modification of these protein subunits decreased specifically in the presence of excess pheromone aldehyde or benzaldehyde. Labeled vinyl ketones are thus novel tools for the identification of molecular subunits of ALDH enzymes.

  3. Evaluation of alcohol dehydrogenase and aldehyde dehydrogenase enzymes as bi-enzymatic anodes in a membraneless ethanol microfluidic fuel cell

    NASA Astrophysics Data System (ADS)

    Galindo-de-la-Rosa, J.; Arjona, N.; Arriaga, L. G.; Ledesma-García, J.; Guerra-Balcázar, M.

    2015-12-01

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldH) enzymes were immobilized by covalent binding and used as the anode in a bi-enzymatic membraneless ethanol hybrid microfluidic fuel cell. The purpose of using both enzymes was to optimize the ethanol electro-oxidation reaction (EOR) by using ADH toward its direct oxidation and AldH for the oxidation of aldehydes as by-products of the EOR. For this reason, three enzymatic bioanode configurations were evaluated according with the location of enzymes: combined, vertical and horizontally separated. In the combined configuration, a current density of 16.3 mA cm-2, a voltage of 1.14 V and a power density of 7.02 mW cm-2 were obtained. When enzymes were separately placed in a horizontal and vertical position the ocp drops to 0.94 V and to 0.68 V, respectively. The current density also falls to values of 13.63 and 5.05 mA cm-2. The decrease of cell performance of bioanodes with separated enzymes compared with the combined bioanode was of 31.7% and 86.87% for the horizontal and the vertical array.

  4. Molecular cloning of a plant betaine-aldehyde dehydrogenase, an enzyme implicated in adaptation to salinity and drought.

    PubMed

    Weretilnyk, E A; Hanson, A D

    1990-04-01

    Many plants, as well as other organisms, accumulate betaine (N,N,N-trimethylglycine) as a nontoxic or protective osmolyte under saline or dry conditions. In plants, the last step in betaine synthesis is catalyzed by betaine-aldehyde dehydrogenase (BADH, EC 1.2.1.8), a nuclear-encoded chloroplastic enzyme. A cDNA clone for BADH (1812 base pairs) was selected from a lambda gt10 cDNA library derived from leaves of salt-stressed spinach (Spinacia oleracea L.). The library was screened with oligonucleotide probes corresponding to amino acid sequences of two peptides prepared from purified BADH. The authenticity of the clone was confirmed by nucleotide sequence analysis; this analysis demonstrated the presence of a 1491-base-pair open reading frame that contained sequences encoding 12 peptide fragments of BADH. The clone hybridized to a 1.9-kilobase mRNA from spinach leaves; this mRNA was more abundant in salt-stressed plants, consistent with the known salt induction of BADH activity. The amino acid sequence deduced from the BADH cDNA sequence showed substantial similarities to those for nonspecific aldehyde dehydrogenases (EC 1.2.1.3 and EC 1.2.1.5) from several sources, including absolute conservation of a decapeptide in the probable active site. Comparison of deduced and determined amino acid sequences indicated that the transit peptide may comprise only 7 or 8 residues, which is atypically short for precursors to stromal proteins. PMID:2320587

  5. Betaine aldehyde dehydrogenase in sorghum.

    PubMed Central

    Wood, A J; Saneoka, H; Rhodes, D; Joly, R J; Goldsbrough, P B

    1996-01-01

    The ability to synthesize and accumulate glycine betaine is wide-spread among angiosperms and is thought to contribute to salt and drought tolerance. In plants glycine betaine is synthesized by the two-step oxidation of choline via the intermediate betaine aldehyde, catalyzed by choline monooxygenase and betaine aldehyde dehydrogenase (BADH). Two sorghum (Sorghum bicolor) cDNA clones, BADH1 and BADH15, putatively encoding betaine aldehyde dehydrogenase were isolated and characterized. BADH1 is a truncated cDNA of 1391 bp. BADH15 is a full-length cDNA clone, 1812 bp in length, predicted to encode a protein of 53.6 kD. The predicted amino acid sequences of BADH1 and BADH15 share significant homology with other plant BADHs. The effects of water deficit on BADH mRNA expression, leaf water relations, and glycine betaine accumulation were investigated in leaves of preflowering sorghum plants. BADH1 and BADH15 mRNA were both induced by water deficit and their expression coincided with the observed glycine betaine accumulation. During the course of 17 d, the leaf water potential in stressed sorghum plants reached -2.3 MPa. In response to water deficit, glycine betaine levels increased 26-fold and proline levels increased 108-fold. In severely stressed plants, proline accounted for > 60% of the total free amino acid pool. Accumulation of these compatible solutes significantly contributed to osmotic potential and allowed a maximal osmotic adjustment of 0.405 MPa. PMID:8934627

  6. Betaine aldehyde dehydrogenase isozymes of spinach

    SciTech Connect

    Hanson, A.D.; Weretilnyk, E.A.; Weigel, P.

    1986-04-01

    Betaine is synthesized in spinach chloroplasts via the pathway Choline ..-->.. Betaine Aldehyde ..-->.. Betaine; the second step is catalyzed by betaine aldehyde dehydrogenase (BADH). The subcellular distribution of BADH was determined in leaf protoplast lysates; BADH isozymes were separated by 6-9% native PAGE. The chloroplast stromal fraction contains a single BADH isozyme (number1) that accounts for > 80% of the total protoplast activity; the extrachloroplastic fraction has a minor isozyme (number2) which migrates more slowly than number1. Both isozymes appear specific for betaine aldehyde, are more active with NAD than NADP, and show a ca. 3-fold activity increase in salinized leaves. The phenotype of a natural variant of isozyme number1 suggests that the enzyme is a dimer.

  7. cDNA cloning and analysis of betaine aldehyde dehydrogenase, a salt inducible enzyme in sugar beet

    SciTech Connect

    McCue, K.F.; Hanson, A.D. )

    1990-05-01

    Betaine accumulates and serves as a compatible osmolyte in some plants subjected to drought or salinity stress. The last enzyme in the betaine biosynthetic pathway is betaine aldehyde dehydrogenase (BADH). The activity of BADH increases in response to increasing salinity levels. This increase in activity corresponds to an increase in protein detectable by immunoblotting, and to an increase in the translatable BADH mRNA. BADH was cloned from a cDNA library constructed in {lambda}gt10 using poly(A){sup +} RNA from sugar beets salinized to 500 mM NaCl. cDNAs were size selected (>1kb) before ligation into the vector, and the library was screened with a spinach BADH cDNA probe. Three nearly full length clones obtained were confirmed as BADH by their nucleotide and deduced amino acid homology to spinach BADH. Clones averaged 1.8 kb and contained open reading frames of 500 amino acids at 80% identity with spinach BADH. RNA gel blot analysis of poly(A){sup +} RNA indicated that salinization to 500 mM NaCl resulted in a 5-fold increase of BADH mRNA level.

  8. Structural shifts of aldehyde dehydrogenase enzymes were instrumental for the early evolution of retinoid-dependent axial patterning in metazoans.

    PubMed

    Sobreira, Tiago J P; Marlétaz, Ferdinand; Simões-Costa, Marcos; Schechtman, Deborah; Pereira, Alexandre C; Brunet, Frédéric; Sweeney, Sarah; Pani, Ariel; Aronowicz, Jochanan; Lowe, Christopher J; Davidson, Bradley; Laudet, Vincent; Bronner, Marianne; de Oliveira, Paulo S L; Schubert, Michael; Xavier-Neto, José

    2011-01-01

    Aldehyde dehydrogenases (ALDHs) catabolize toxic aldehydes and process the vitamin A-derived retinaldehyde into retinoic acid (RA), a small diffusible molecule and a pivotal chordate morphogen. In this study, we combine phylogenetic, structural, genomic, and developmental gene expression analyses to examine the evolutionary origins of ALDH substrate preference. Structural modeling reveals that processing of small aldehydes, such as acetaldehyde, by ALDH2, versus large aldehydes, including retinaldehyde, by ALDH1A is associated with small versus large substrate entry channels (SECs), respectively. Moreover, we show that metazoan ALDH1s and ALDH2s are members of a single ALDH1/2 clade and that during evolution, eukaryote ALDH1/2s often switched between large and small SECs after gene duplication, transforming constricted channels into wide opened ones and vice versa. Ancestral sequence reconstructions suggest that during the evolutionary emergence of RA signaling, the ancestral, narrow-channeled metazoan ALDH1/2 gave rise to large ALDH1 channels capable of accommodating bulky aldehydes, such as retinaldehyde, supporting the view that retinoid-dependent signaling arose from ancestral cellular detoxification mechanisms. Our analyses also indicate that, on a more restricted evolutionary scale, ALDH1 duplicates from invertebrate chordates (amphioxus and ascidian tunicates) underwent switches to smaller and narrower SECs. When combined with alterations in gene expression, these switches led to neofunctionalization from ALDH1-like roles in embryonic patterning to systemic, ALDH2-like roles, suggesting functional shifts from signaling to detoxification. PMID:21169504

  9. Structural shifts of aldehyde dehydrogenase enzymes were instrumental for the early evolution of retinoid-dependent axial patterning in metazoans

    PubMed Central

    Sobreira, Tiago J. P.; Marlétaz, Ferdinand; Simões-Costa, Marcos; Schechtman, Deborah; Pereira, Alexandre C.; Brunet, Frédéric; Sweeney, Sarah; Pani, Ariel; Aronowicz, Jochanan; Lowe, Christopher J.; Davidson, Bradley; Laudet, Vincent; Bronner, Marianne; de Oliveira, Paulo S. L.; Schubert, Michael; Xavier-Neto, José

    2011-01-01

    Aldehyde dehydrogenases (ALDHs) catabolize toxic aldehydes and process the vitamin A-derived retinaldehyde into retinoic acid (RA), a small diffusible molecule and a pivotal chordate morphogen. In this study, we combine phylogenetic, structural, genomic, and developmental gene expression analyses to examine the evolutionary origins of ALDH substrate preference. Structural modeling reveals that processing of small aldehydes, such as acetaldehyde, by ALDH2, versus large aldehydes, including retinaldehyde, by ALDH1A is associated with small versus large substrate entry channels (SECs), respectively. Moreover, we show that metazoan ALDH1s and ALDH2s are members of a single ALDH1/2 clade and that during evolution, eukaryote ALDH1/2s often switched between large and small SECs after gene duplication, transforming constricted channels into wide opened ones and vice versa. Ancestral sequence reconstructions suggest that during the evolutionary emergence of RA signaling, the ancestral, narrow-channeled metazoan ALDH1/2 gave rise to large ALDH1 channels capable of accommodating bulky aldehydes, such as retinaldehyde, supporting the view that retinoid-dependent signaling arose from ancestral cellular detoxification mechanisms. Our analyses also indicate that, on a more restricted evolutionary scale, ALDH1 duplicates from invertebrate chordates (amphioxus and ascidian tunicates) underwent switches to smaller and narrower SECs. When combined with alterations in gene expression, these switches led to neofunctionalization from ALDH1-like roles in embryonic patterning to systemic, ALDH2-like roles, suggesting functional shifts from signaling to detoxification. PMID:21169504

  10. The Oxidative Fermentation of Ethanol in Gluconacetobacter diazotrophicus Is a Two-Step Pathway Catalyzed by a Single Enzyme: Alcohol-Aldehyde Dehydrogenase (ADHa)

    PubMed Central

    Gómez-Manzo, Saúl; Escamilla, José E.; González-Valdez, Abigail; López-Velázquez, Gabriel; Vanoye-Carlo, América; Marcial-Quino, Jaime; de la Mora-de la Mora, Ignacio; Garcia-Torres, Itzhel; Enríquez-Flores, Sergio; Contreras-Zentella, Martha Lucinda; Arreguín-Espinosa, Roberto; Kroneck, Peter M. H.; Sosa-Torres, Martha Elena

    2015-01-01

    Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2–C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde. PMID:25574602

  11. The oxidative fermentation of ethanol in Gluconacetobacter diazotrophicus is a two-step pathway catalyzed by a single enzyme: alcohol-aldehyde Dehydrogenase (ADHa).

    PubMed

    Gómez-Manzo, Saúl; Escamilla, José E; González-Valdez, Abigail; López-Velázquez, Gabriel; Vanoye-Carlo, América; Marcial-Quino, Jaime; de la Mora-de la Mora, Ignacio; Garcia-Torres, Itzhel; Enríquez-Flores, Sergio; Contreras-Zentella, Martha Lucinda; Arreguín-Espinosa, Roberto; Kroneck, Peter M H; Sosa-Torres, Martha Elena

    2015-01-01

    Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2-C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde. PMID:25574602

  12. SAXS fingerprints of aldehyde dehydrogenase oligomers.

    PubMed

    Tanner, John J

    2015-12-01

    Enzymes of the aldehyde dehydrogenase (ALDH) superfamily catalyze the nicotinamide adenine dinucleotide-dependent oxidation of aldehydes to carboxylic acids. ALDHs are important in detoxification of aldehydes, amino acid metabolism, embryogenesis and development, neurotransmission, oxidative stress, and cancer. Mutations in genes encoding ALDHs cause metabolic disorders, including alcohol flush reaction (ALDH2), Sjögren-Larsson syndrome (ALDH3A2), hyperprolinemia type II (ALDH4A1), γ-hydroxybutyric aciduria (ALDH5A1), methylmalonic aciduria (ALDH6A1), pyridoxine dependent epilepsy (ALDH7A1), and hyperammonemia (ALDH18A1). We previously reported crystal structures and small-angle X-ray scattering (SAXS) analyses of ALDHs exhibiting dimeric, tetrameric, and hexameric oligomeric states (Luo et al., Biochemistry 54 (2015) 5513-5522; Luo et al., J. Mol. Biol. 425 (2013) 3106-3120). Herein I provide the SAXS curves, radii of gyration, and distance distribution functions for the three types of ALDH oligomer. The SAXS curves and associated analysis provide diagnostic fingerprints that allow rapid identification of the type of ALDH oligomer that is present in solution. The data sets provided here serve as a benchmark for characterizing oligomerization of ALDHs. PMID:26693506

  13. Targeting Aldehyde Dehydrogenase 2: New Therapeutic Opportunities

    PubMed Central

    Chen, Che-Hong; Ferreira, Julio Cesar Batista; Gross, Eric R.; Mochly-Rosen, Daria

    2014-01-01

    A family of detoxifying enzymes called aldehyde dehydrogenases (ALDHs) has been a subject of recent interest, as its role in detoxifying aldehydes that accumulate through metabolism and to which we are exposed from the environment has been elucidated. Although the human genome has 19 ALDH genes, one ALDH emerges as a particularly important enzyme in a variety of human pathologies. This ALDH, ALDH2, is located in the mitochondrial matrix with much known about its role in ethanol metabolism. Less known is a new body of research to be discussed in this review, suggesting that ALDH2 dysfunction may contribute to a variety of human diseases including cardiovascular diseases, diabetes, neurodegenerative diseases, stroke, and cancer. Recent studies suggest that ALDH2 dysfunction is also associated with Fanconi anemia, pain, osteoporosis, and the process of aging. Furthermore, an ALDH2 inactivating mutation (termed ALDH2*2) is the most common single point mutation in humans, and epidemiological studies suggest a correlation between this inactivating mutation and increased propensity for common human pathologies. These data together with studies in animal models and the use of new pharmacological tools that activate ALDH2 depict a new picture related to ALDH2 as a critical health-promoting enzyme. PMID:24382882

  14. ALDEHYDE DEHYDROGENASES EXPRESSION DURING POSTNATAL DEVELOPMENT: LIVER VS. LUNG

    EPA Science Inventory

    Aldehydes are highly reactive molecules present in the environment, and can be produced during biotransformation of xenobiotics. Although the lung can be a major target for aldehyde toxicity, development of aldehyde dehydrogenases (ALDHs), which detoxify aldehydes, in lung has be...

  15. Exploring the evolutionary route of the acquisition of betaine aldehyde dehydrogenase activity by plant ALDH10 enzymes: implications for the synthesis of the osmoprotectant glycine betaine

    PubMed Central

    2014-01-01

    Background Plant ALDH10 enzymes are aminoaldehyde dehydrogenases (AMADHs) that oxidize different ω-amino or trimethylammonium aldehydes, but only some of them have betaine aldehyde dehydrogenase (BADH) activity and produce the osmoprotectant glycine betaine (GB). The latter enzymes possess alanine or cysteine at position 441 (numbering of the spinach enzyme, SoBADH), while those ALDH10s that cannot oxidize betaine aldehyde (BAL) have isoleucine at this position. Only the plants that contain A441- or C441-type ALDH10 isoenzymes accumulate GB in response to osmotic stress. In this work we explored the evolutionary history of the acquisition of BAL specificity by plant ALDH10s. Results We performed extensive phylogenetic analyses and constructed and characterized, kinetically and structurally, four SoBADH variants that simulate the parsimonious intermediates in the evolutionary pathway from I441-type to A441- or C441-type enzymes. All mutants had a correct folding, average thermal stabilities and similar activity with aminopropionaldehyde, but whereas A441S and A441T exhibited significant activity with BAL, A441V and A441F did not. The kinetics of the mutants were consistent with their predicted structural features obtained by modeling, and confirmed the importance of position 441 for BAL specificity. The acquisition of BADH activity could have happened through any of these intermediates without detriment of the original function or protein stability. Phylogenetic studies showed that this event occurred independently several times during angiosperms evolution when an ALDH10 gene duplicate changed the critical Ile residue for Ala or Cys in two consecutive single mutations. ALDH10 isoenzymes frequently group in two clades within a plant family: one includes peroxisomal I441-type, the other peroxisomal and non-peroxisomal I441-, A441- or C441-type. Interestingly, high GB-accumulators plants have non-peroxisomal A441- or C441-type isoenzymes, while low-GB accumulators

  16. A coniferyl aldehyde dehydrogenase gene from Pseudomonas sp. strain HR199 enhances the conversion of coniferyl aldehyde by Saccharomyces cerevisiae.

    PubMed

    Adeboye, Peter Temitope; Olsson, Lisbeth; Bettiga, Maurizio

    2016-07-01

    The conversion of coniferyl aldehyde to cinnamic acids by Saccharomyces cerevisiae under aerobic growth conditions was previously observed. Bacteria such as Pseudomonas have been shown to harbor specialized enzymes for converting coniferyl aldehyde but no comparable enzymes have been identified in S. cerevisiae. CALDH from Pseudomonas was expressed in S. cerevisiae. An acetaldehyde dehydrogenase (Ald5) was also hypothesized to be actively involved in the conversion of coniferyl aldehyde under aerobic growth conditions in S. cerevisiae. In a second S. cerevisiae strain, the acetaldehyde dehydrogenase (ALD5) was deleted. A prototrophic control strain was also engineered. The engineered S. cerevisiae strains were cultivated in the presence of 1.1mM coniferyl aldehyde under aerobic condition in bioreactors. The results confirmed that expression of CALDH increased endogenous conversion of coniferyl aldehyde in S. cerevisiae and ALD5 is actively involved with the conversion of coniferyl aldehyde in S. cerevisiae. PMID:27070284

  17. Aldehyde dehydrogenase protein superfamily in maize.

    PubMed

    Zhou, Mei-Liang; Zhang, Qian; Zhou, Ming; Qi, Lei-Peng; Yang, Xiong-Bang; Zhang, Kai-Xuan; Pang, Jun-Feng; Zhu, Xue-Mei; Shao, Ji-Rong; Tang, Yi-Xiong; Wu, Yan-Min

    2012-11-01

    Maize (Zea mays ssp. mays L.) is an important model organism for fundamental research in the agro-biotechnology field. Aldehydes were generated in response to a suite of environmental stresses that perturb metabolism including salinity, dehydration, desiccation, and cold and heat shock. Many biologically important aldehydes are metabolized by the superfamily of NAD(P)(+)-dependent aldehyde dehydrogenases. Here, starting from the database of Z. mays, we identified 28 aldehyde dehydrogenase (ALDH) genes and 48 transcripts by the in silico cloning method using the ALDH-conserved domain amino acid sequence of Arabidopsis and rice as a probe. Phylogenetic analysis shows that all 28 members of the ALDH gene families were classified to ten distinct subfamilies. Microarray data and quantitative real-time PCR analysis reveal that ZmALDH9, ZmALDH13, and ZmALDH17 genes involve the function of drought stress, acid tolerance, and pathogens infection. These results suggested that these three ZmALDH genes might be potentially useful in maize genetic improvement. PMID:22983498

  18. Some properties of aldehyde dehydrogenase from sheep liver mitochondria.

    PubMed Central

    Hart, G J; Dickinson, F M

    1977-01-01

    Aldehyde dehydrogenase from sheep liver mitochondria was purified to homogeneity as judged by electrophoresis on polyacrylamide gels, and by sedimentation-equilibrium experiments in the analytical ultracentrifuge. The enzyme has a molecular weight of 198000 and a subunit size of 48000, indicating that the molecule is a tetramer. Fluorescence and spectrophotometric titrations indicate that each subunit can bind 1 molecule of NADH. Enzymic activity is completely blocked by reaction of 4mol of 5,5'-dithiobis-(2-nitrobenzoate)/mol of enzyme. Excess of disulfiram or iodoacetamide decreases activity to only 50% of the control value, and only two thiol groups per molecule are apparently modified by these reagents. PMID:194582

  19. Drosophila melanogaster alcohol dehydrogenase: mechanism of aldehyde oxidation and dismutation.

    PubMed

    Winberg, J O; McKinley-McKee, J S

    1998-02-01

    Drosophila alcohol dehydrogenase (Adh) catalyses the oxidation of both alcohols and aldehydes. In the latter case, the oxidation is followed by a reduction of the aldehyde, i.e. a dismutation reaction. At high pH, dismutation is accompanied by a small release of NADH, which is not observed at neutral pH. Previously it has been emphasized that kinetic coefficients obtained by measuring the increase in A340, i.e. the release of NADH at high pH is not a direct measure of the aldehyde oxidation reaction and these values cannot be compared with those for alcohol dehydrogenation. In this article we demonstrate that this is not entirely true, and that the coefficients phiB and phiAB, where B is the aldehyde and A is NAD+, are the same for a dismutation reaction and a simple aldehyde dehydrogenase reaction. Thus the substrate specificity of the aldehyde oxidation reaction can be determined by simply measuring the NADH release. The coefficients for oxidation and dehydrogenation reactions (phi0d and phiAd respectively) are complex and involve the constants for the dismutation reaction. However, dead-end inhibitors can be used to determine the quantitative contribution of the kinetic constants for the aldehyde oxidation and reduction pathways to the phi0d and phiAd coefficients. The combination of dead-end and product inhibitors can be used to determine the reaction mechanism for the aldehyde oxidation pathway. Previously, we showed that with Drosophila Adh, the interconversion between alcohols and aldehydes followed a strictly compulsory ordered pathway, although aldehydes and ketones formed binary complexes with the enzyme. This raised the question regarding the reaction mechanism for the oxidation of aldehydes, i.e. whether a random ordered pathway was followed. In the present work, the mechanism for the oxidation of different aldehydes and the accompanying dismutation reaction with the slow alleloenzyme (AdhS) from Drosophila melanogaster has been studied. To obtain

  20. Drosophila melanogaster alcohol dehydrogenase: mechanism of aldehyde oxidation and dismutation.

    PubMed Central

    Winberg, J O; McKinley-McKee, J S

    1998-01-01

    Drosophila alcohol dehydrogenase (Adh) catalyses the oxidation of both alcohols and aldehydes. In the latter case, the oxidation is followed by a reduction of the aldehyde, i.e. a dismutation reaction. At high pH, dismutation is accompanied by a small release of NADH, which is not observed at neutral pH. Previously it has been emphasized that kinetic coefficients obtained by measuring the increase in A340, i.e. the release of NADH at high pH is not a direct measure of the aldehyde oxidation reaction and these values cannot be compared with those for alcohol dehydrogenation. In this article we demonstrate that this is not entirely true, and that the coefficients phiB and phiAB, where B is the aldehyde and A is NAD+, are the same for a dismutation reaction and a simple aldehyde dehydrogenase reaction. Thus the substrate specificity of the aldehyde oxidation reaction can be determined by simply measuring the NADH release. The coefficients for oxidation and dehydrogenation reactions (phi0d and phiAd respectively) are complex and involve the constants for the dismutation reaction. However, dead-end inhibitors can be used to determine the quantitative contribution of the kinetic constants for the aldehyde oxidation and reduction pathways to the phi0d and phiAd coefficients. The combination of dead-end and product inhibitors can be used to determine the reaction mechanism for the aldehyde oxidation pathway. Previously, we showed that with Drosophila Adh, the interconversion between alcohols and aldehydes followed a strictly compulsory ordered pathway, although aldehydes and ketones formed binary complexes with the enzyme. This raised the question regarding the reaction mechanism for the oxidation of aldehydes, i.e. whether a random ordered pathway was followed. In the present work, the mechanism for the oxidation of different aldehydes and the accompanying dismutation reaction with the slow alleloenzyme (AdhS) from Drosophila melanogaster has been studied. To obtain

  1. Reversible, partial inactivation of plant betaine aldehyde dehydrogenase by betaine aldehyde: mechanism and possible physiological implications.

    PubMed

    Zárate-Romero, Andrés; Murillo-Melo, Darío S; Mújica-Jiménez, Carlos; Montiel, Carmina; Muñoz-Clares, Rosario A

    2016-04-01

    In plants, the last step in the biosynthesis of the osmoprotectant glycine betaine (GB) is the NAD(+)-dependent oxidation of betaine aldehyde (BAL) catalysed by some aldehyde dehydrogenase (ALDH) 10 enzymes that exhibit betaine aldehyde dehydrogenase (BADH) activity. Given the irreversibility of the reaction, the short-term regulation of these enzymes is of great physiological relevance to avoid adverse decreases in the NAD(+):NADH ratio. In the present study, we report that the Spinacia oleracea BADH (SoBADH) is reversibly and partially inactivated by BAL in the absence of NAD(+)in a time- and concentration-dependent mode. Crystallographic evidence indicates that the non-essential Cys(450)(SoBADH numbering) forms a thiohemiacetal with BAL, totally blocking the productive binding of the aldehyde. It is of interest that, in contrast to Cys(450), the catalytic cysteine (Cys(291)) did not react with BAL in the absence of NAD(+) The trimethylammonium group of BAL binds in the same position in the inactivating or productive modes. Accordingly, BAL does not inactivate the C(450)SSoBADH mutant and the degree of inactivation of the A(441)I and A(441)C mutants corresponds to their very different abilities to bind the trimethylammonium group. Cys(450)and the neighbouring residues that participate in stabilizing the thiohemiacetal are strictly conserved in plant ALDH10 enzymes with proven or predicted BADH activity, suggesting that inactivation by BAL is their common feature. Under osmotic stress conditions, this novel partial and reversible covalent regulatory mechanism may contribute to preventing NAD(+)exhaustion, while still permitting the synthesis of high amounts of GB and avoiding the accumulation of the toxic BAL. PMID:26792760

  2. Aldehyde dehydrogenases: From eye crystallins to metabolic disease and cancer stem cells

    PubMed Central

    Vasiliou, Vasilis; Thompson, David C.; Smith, Clay; Fujita, Mayumi; Chen, Ying

    2014-01-01

    The aldehyde dehydrogenase (ALDH) superfamily is composed of nicotinamide adenine dinucleotide (phosphate) (NAD(P)+)-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. To date, 24 ALDH gene families have been identified in the eukaryotic genome. In addition to aldehyde metabolizing capacity, ALDHs have additional catalytic (e.g. esterase and reductase) and non-catalytic activities. The latter include functioning as structural elements in the eye (crystallins) and as binding molecules to endobiotics and xenobiotics. Mutations in human ALDH genes and subsequent inborn errors in aldehyde metabolism are the molecular basis of several diseases. Most recently ALDH polymorphisms have been associated with gout and osteoporosis. Aldehyde dehydrogenase enzymes also play important roles in embryogenesis and development, neurotransmission, oxidative stress and cancer. This article serves as a comprehensive review of the current state of knowledge regarding the ALDH superfamily and the contribution of ALDHs to various physiological and pathophysiological processes. PMID:23159885

  3. Aldehyde dehydrogenase inhibitors from the mushroom Clitocybe clavipes.

    PubMed

    Kawagishi, Hirokazu; Miyazawa, Toshiyuki; Kume, Hiroko; Arimoto, Yasushi; Inakuma, Takahiro

    2002-11-01

    Five fatty acid derivatives including three novel compounds were isolated from the mushroom Clitocybe clavipe. Their structures were elucidated by spectral analyses. These compounds inhibited aldehyde dehydrogenase in vitro. PMID:12444711

  4. Human liver aldehyde dehydrogenase: coenzyme binding

    SciTech Connect

    Kosley, L.L.; Pietruszko, R.

    1987-05-01

    The binding of (U-/sup 14/C) NAD to mitochondrial (E2) and cytoplasmin(E1) aldehyde dehydrogenase was measured by gel filtration and sedimentation techniques. The binding data for NAD and (E1) yielded linear Scatchard plots giving a dissociation constant of 25 (+/- 8) uM and the stoichiometry of 2 mol of NAD bound per mol of E1. The binding data for NAD and (E2) gave nonlinear Scatchard plots. The binding of NADH to E2 was measured via fluorescence enhancement; this could not be done with E1 because there was no signal. The dissociation constant for E2 by this technique was 0.7 (+/- 0.4) uM and stoichiometry of 1.0 was obtained. The binding of (U-/sup 14/C) NADH to (E1) and (E2) was also measured by the sedimentation technique. The binding data for (E1) and NADH gave linear Scatchard plots giving a dissociation constant of 13 (+/- 6) uM and the stoichiometry of 2.0. The binding data for NADH to (E2) gave nonlinear Scatchard plots. With (E1), the dissociation constants for both NAD and NADH are similar to those determined kinetically, but the stoichiometry is only half of that found by stopped flow technique. With (E2) the dissociation constant by fluorometric procedure was 2 orders of magnitude less than that from catalytic reaction.

  5. Aldehyde dehydrogenases in cellular responses to oxidative/electrophilic stress.

    PubMed

    Singh, Surendra; Brocker, Chad; Koppaka, Vindhya; Chen, Ying; Jackson, Brian C; Matsumoto, Akiko; Thompson, David C; Vasiliou, Vasilis

    2013-03-01

    Reactive oxygen species (ROS) are continuously generated within living systems and the inability to manage ROS load leads to elevated oxidative stress and cell damage. Oxidative stress is coupled to the oxidative degradation of lipid membranes, also known as lipid peroxidation. This process generates over 200 types of aldehydes, many of which are highly reactive and toxic. Aldehyde dehydrogenases (ALDHs) metabolize endogenous and exogenous aldehydes and thereby mitigate oxidative/electrophilic stress in prokaryotic and eukaryotic organisms. ALDHs are found throughout the evolutionary gamut, from single-celled organisms to complex multicellular species. Not surprisingly, many ALDHs in evolutionarily distant, and seemingly unrelated, species perform similar functions, including protection against a variety of environmental stressors such as dehydration and ultraviolet radiation. The ability to act as an "aldehyde scavenger" during lipid peroxidation is another ostensibly universal ALDH function found across species. Upregulation of ALDHs is a stress response in bacteria (environmental and chemical stress), plants (dehydration, salinity, and oxidative stress), yeast (ethanol exposure and oxidative stress), Caenorhabditis elegans (lipid peroxidation), and mammals (oxidative stress and lipid peroxidation). Recent studies have also identified ALDH activity as an important feature of cancer stem cells. In these cells, ALDH expression helps abrogate oxidative stress and imparts resistance against chemotherapeutic agents such as oxazaphosphorine, taxane, and platinum drugs. The ALDH superfamily represents a fundamentally important class of enzymes that contributes significantly to the management of electrophilic/oxidative stress within living systems. Mutations in various ALDHs are associated with a variety of pathological conditions in humans, highlighting the fundamental importance of these enzymes in physiological and pathological processes. PMID:23195683

  6. Chemical modification of aldehyde dehydrogenase by a vinyl ketone analogue of an insect pheromone.

    PubMed

    Blatter, E E; Tasayco, M L; Prestwich, G; Pietruszko, R

    1990-12-01

    A major component of the sex pheromone from the tobacco budworm moth Heliothis virescens is a C16 straight-chain aldehyde with a single unsaturation at the eleventh position. The sex pheromones are inactivated when metabolized to their corresponding acids by insect aldehyde dehydrogenase. During this investigation it was demonstrated that the C16 aldehyde is a good substrate for human aldehyde dehydrogenase (EC 1.2.1.3) isoenzymes E1 and E2 with Km and Kcat. values at pH 7.0 of 2 microM and 0.4 mumol of NADH/min per mg and of 0.6 microM and 0.24 mumol of NADH/min per mg respectively. A vinyl ketone analogue of the pheromone inhibited insect pheromone metabolism; it also inactivated human aldehyde dehydrogenase. Total inactivation of both isoenzymes was achieved at stoichiometric (equal or less than the subunit number) concentrations of vinyl ketone, incorporating 2.1-2.6 molecules/molecule of enzyme. Substrate protection was observed in the presence of the parent aldehyde and 5'-AMP. Peptide maps of tryptic digests of the E2 isoenzyme modified with 3H-labelled vinyl ketone showed that incorporation occurred into a single peptide peak. The labelled peptide of E2 isoenzyme was further purified on h.p.l.c. and sequenced. The label was incorporated into cysteine-302 in the primary structure of E2 isoenzyme, thus indicating that cysteine-302 is located in the aldehyde substrate area of the active site of aldehyde dehydrogenase. Affinity labelling of aldehyde dehydrogenase with vinyl ketones may prove to be of general utility in biochemical studies of these enzymes. PMID:2268265

  7. Aldehyde dehydrogenase 1A1 in stem cells and cancer

    PubMed Central

    Tomita, Hiroyuki; Tanaka, Kaori; Tanaka, Takuji; Hara, Akira

    2016-01-01

    The human genome contains 19 putatively functional aldehyde dehydrogenase (ALDH) genes, which encode enzymes critical for detoxification of endogenous and exogenous aldehyde substrates through NAD(P)+-dependent oxidation. ALDH1 has three main isotypes, ALDH1A1, ALDH1A2, and ALDH1A3, and is a marker of normal tissue stem cells (SC) and cancer stem cells (CSC), where it is involved in self-renewal, differentiation and self-protection. Experiments with murine and human cells indicate that ALDH1 activity, predominantly attributed to isotype ALDH1A1, is tissue- and cancer-specific. High ALDH1 activity and ALDH1A1 overexpression are associated with poor cancer prognosis, though high ALDH1 and ALDH1A1 levels do not always correlate with highly malignant phenotypes and poor clinical outcome. In cancer therapy, ALDH1A1 provides a useful therapeutic CSC target in tissue types that normally do not express high levels of ALDH1A1, including breast, lung, esophagus, colon and stomach. Here we review the functions and mechanisms of ALDH1A1, the key ALDH isozyme linked to SC populations and an important contributor to CSC function in cancers, and we outline its potential in future anticancer strategies. PMID:26783961

  8. Fluorescence lifetime analysis and effect of magnesium ions on binding of NADH to human aldehyde dehydrogenase 1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aldehyde dehydrogenase 1 (ALDH1) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions influence ALDH1 activity in part by increasing NADH binding affinity to the enzyme thus reducing activity. By using time-resolved fluorescence spectroscopy, we have resolved t...

  9. Targeting Aldehyde Dehydrogenase: a Potential Approach for Cell labeling

    PubMed Central

    Vaidyanathan, Ganesan; Song, Haijing; Affleck, Donna; McDougald, Darryl L.; Storms, Robert W.; Zalutksy, Michael R.; Chin, Bennett B.

    2009-01-01

    Introduction To advance the science and clinical application of stem cell therapy, the availability of a highly sensitive, quantitative, and translational method for tracking stem cells would be invaluable. Because hematopoetic stem cells express high levels of the cytosolic enzyme aldehyde dehydrogenase-1A1 (ALDH1), we sought to develop an agent that is specific to ALDH1 and thus to cells expressing the enzyme. Such an agent might be also helpful in identifying tumors that are resistant to cyclophosphomide chemotherapy because ALDH1 is known to be responsible for this resistance. Methods We developed schemes for the synthesis of two 3radioiodinated aldehdyes—N-formylmethyl-5-[*I]iodopyridine-3-carboxamide ([*I]FMIC) and 4-diethylamino-3-[*I]iodobenzaldehyde ([*I]DEIBA)—at no-carrier-added levels from their respective tin precursors. These agents were evaluated using pure ALDH1 and tumor cells that expressed the enzyme. Results The average radiochemical yields for the synthesis [125I]FMIC and [125I]DEIBA were 70 ± 5% and 47 ± 14%, respectively. ALDH1 converted both compounds to respective acids suggesting their suitability as ALDH1 imaging agents. Although ability of ALDH1 within the cells to oxidize one of these substrates was shown, specific uptake in ALDH-expressing tumor cells could not be demonstrated. Conclusion To pursue this approach for ALDH1 imaging, radiolabeled aldehydes need to be designed such that, in addition to being good substrates for ALDH1, the cognate products should be sufficiently polar so as to be retained within the cells. PMID:19875048

  10. Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells

    PubMed Central

    Jean, Elise; Laoudj-Chenivesse, Dalila; Notarnicola, Cécile; Rouger, Karl; Serratrice, Nicolas; Bonnieu, Anne; Gay, Stéphanie; Bacou, Francis; Duret, Cédric; Carnac, Gilles

    2011-01-01

    Abstract Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties, we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant-derived cells were incubated with ALDEFLUOR, a fluorescent substrate for ALDH, and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast-CD56+ fraction in those cells, but, we also observed heterogeneity of ALDH activity levels within CD56-purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly, ALDH activity and Aldh1a1 expression levels were very low in mouse, rat, rabbit and non-human primate myoblasts. Using different approaches, from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde, an inhibitor of class I ALDH, to cell fractionation by flow cytometry using the ALDEFLUOR assay, we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H2O2)-induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity, as a purification strategy, could allow non-toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage. PMID:19840193

  11. DEVELOPMENTAL EXPRESSION OF ALDEHYDE DEHYDROGENASE IN RAT: A COMPARISON OF LIVER AND LUNG DEVELOPMENT

    EPA Science Inventory

    Metabolism is one of the major determinants for age-related susceptibility changes to chemicals. Aldehydes are highly reactive molecules present in the environment and can be produced during biotransformation of xenobiotics. Aldehyde dehydrogenases (ALDH) are important in aldehyd...

  12. Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus

    PubMed Central

    Halavaty, Andrei S.; Rich, Rebecca L.; Chen, Chao; Joo, Jeong Chan; Minasov, George; Dubrovska, Ievgeniia; Winsor, James R.; Myszka, David G.; Duban, Mark; Shuvalova, Ludmilla; Yakunin, Alexander F.; Anderson, Wayne F.

    2015-01-01

    When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL (SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NAD+) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD+, NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme. PMID:25945581

  13. Aldehyde dehydrogenase (ALDH) in Alzheimer's and Parkinson's disease.

    PubMed

    Grünblatt, Edna; Riederer, Peter

    2016-02-01

    Evidence suggests that aldehyde dehydrogenase (ALDH; E.C. 1.2.1.3) gene, protein expression and activity are substantially decreased in the substantia nigra of patients with Parkinson's disease (PD). This holds especially true for cytosolic ALDH1A1, while mitochondrial ALDH2 is increased in the putamen of PD. Similarly, in Alzheimer's disease (AD) several studies in genetic, transcriptomic, protein and animal models suggest ALDH involvement in the neurodegeneration processes. Such data are in line with findings of increased toxic aldehydes, like for example malondialdehyde, nonenal, 3,4-dihydroxyphenylacetaldehyde and others. Genetic, transcriptomic and protein alterations may contribute to such data. Also in vitro and in vivo experimental work points to an important role of ALDH in the pathology of neurodegenerative disorders. Aims at investigating dysfunctions of aldehyde detoxification are suitable to define genetic/molecular targets for new therapeutic strategies balancing amine metabolism in devastating disorders like PD and probably also AD. PMID:25298080

  14. Crystal structure of the NADP+-dependent aldehyde dehydrogenase from Vibrio harveyi: structural implications for cofactor specificity and affinity.

    PubMed Central

    Ahvazi, B; Coulombe, R; Delarge, M; Vedadi, M; Zhang, L; Meighen, E; Vrielink, A

    2000-01-01

    Aldehyde dehydrogenase from the bioluminescent bacterium, Vibrio harveyi, catalyses the oxidation of long-chain aliphatic aldehydes to acids. The enzyme is unique compared with other forms of aldehyde dehydrogenase in that it exhibits a very high specificity and affinity for the cofactor NADP(+). Structural studies of this enzyme and comparisons with other forms of aldehyde dehydrogenase provide the basis for understanding the molecular features that dictate these unique properties and will enhance our understanding of the mechanism of catalysis for this class of enzyme. The X-ray structure of aldehyde dehydrogenase from V. harveyi has been solved to 2.5-A resolution as a partial complex with the cofactor NADP(+) and to 2. 1-A resolution as a fully bound 'holo' complex. The cofactor preference exhibited by different forms of the enzyme is predominantly determined by the electrostatic environment surrounding the 2'-hydroxy or the 2'-phosphate groups of the adenosine ribose moiety of NAD(+) or NADP(+), respectively. In the NADP(+)-dependent structures the presence of a threonine and a lysine contribute to the cofactor specificity. In the V. harveyi enzyme an arginine residue (Arg-210) contributes to the high cofactor affinity through a pi stacking interaction with the adenine ring system of the cofactor. Further differences between the V. harveyi enzyme and other aldehyde dehydrogenases are seen in the active site, in particular a histidine residue which is structurally conserved with phosphorylating glyceraldehyde-3-phosphate dehydrogenase. This may suggest an alternative mechanism for activation of the reactive cysteine residue for nucleophilic attack. PMID:10903148

  15. Fatty aldehyde dehydrogenases in Acinetobacter sp. strain HO1-N: role in hexadecane and hexadecanol metabolism

    SciTech Connect

    Singer, M.E.; Finnerty, W.R.

    1985-12-01

    The role of fatty aldehyde dehydrogenases (FALDHs) in hexadecane and hexadecanol metabolism was studied in Acinetobacter sp. strain HO1-N. Two distinct FALDHs were demonstrated in Acinetobacter sp. strain HO1-N: (i) a membrane-bound, NADP-dependent FALDH activity induced 5-, 15-, and 9 fold by growth on hexadecanol, dodecyl aldehyde, and hexadecane, respectively, and (ii) a constitutive, NAD-dependent, membrane-localized FALDH. Dodecyl aldehyde-negative mutants were isolated and grouped into two phenotypic classes based on growth: class 1 mutants were hexadecane and hexadecanol negative and class 2 mutants were hexadecane and hexadecanol positive. Specific activity of NADP-dependent FALDH in Ald21 (class 1 mutant) was 85% lower than that of wild-type FALDH, while the specific activity of Ald24 (class 2 mutant) was 55% greater than that of wild-type FALDH. Ald21R, a dodecyl aldehyde-positive revertant able to grow on hexadecane, hexadecanol, and dodecyl aldehyde, exhibited a 100% increase in the specific activity of the NADP-dependent FALDH. This study provides genetic and physiological evidence for the role of fatty aldehyde as an essential metabolic intermediate and NADP-dependent FALDH as a key enzyme in the dissimilation of hexadecane, hexadecanol, and dodecyl aldehyde in Acinetobacter sp. strain HO1-N.

  16. The use of tomato aminoaldehyde dehydrogenase 1 for the detection of aldehydes in fruit distillates.

    PubMed

    Frömmel, Jan; Tarkowski, Petr; Kopečný, David; Šebela, Marek

    2016-09-25

    Plant NAD(+)-dependent aminoaldehyde dehydrogenases (AMADHs, EC 1.2.1.19) belong to the family 10 of aldehyde dehydrogenases. They participate in the metabolism of polyamines or osmoprotectants. The enzymes are characterized by their broad substrate specificity covering ω-aminoaldehydes, aliphatic and aromatic aldehydes as well as nitrogen-containing heterocyclic aldehydes. The isoenzyme 1 from tomato (Solanum lycopersicum; SlAMADH1) oxidizes aliphatic aldehydes very efficiently and converts also furfural, its derivatives or benzaldehyde, which are present at low concentrations in alcoholic distillates such as fruit brandy. In this work, SlAMADH1 was examined as a bioanalytical tool for their detection. These aldehydes arise from fermentation processes or thermal degradation of sugars and their presence is related to health complications after consumption including nausea, emesis, sweating, decrease in blood pressure, hangover headache, among others. Sixteen samples of slivovitz (plum brandy) from local producers in Moravia, Czech Republic, were analyzed for their aldehyde content using a spectrophotometric activity assay with SlAMADH1. In all cases, there were oxidative responses observed when monitoring NADH production in the enzymatic reaction. Aldehydes in the distillate samples were also subjected to a standard determination using reversed-phase HPLC with spectrophotometric and tandem mass spectrometric detection after a derivatization with 2,4-dinitrophenylhydrazine. Results obtained by both methods were found to correlate well for a majority of the analyzed samples. The possible applicability of SlAMADH1 for the evaluation of aldehyde content in food and beverages has now been demonstrated. PMID:26703808

  17. Discovery of a novel class of covalent inhibitor for aldehyde dehydrogenases

    SciTech Connect

    Khanna, Mary; Chen, Che-Hong; Kimble-Hill, Ann; Parajuli, Bibek; Perez-Miller, Samantha; Baskaran, Sulochanadevi; Kim, Jeewon; Dria, Karl; Vasiliou, Vasilis; Mochly-Rosen, Daria; Hurley, Thomas D.

    2012-10-23

    Human aldehyde dehydrogenases (ALDHs) comprise a family of 17 homologous enzymes that metabolize different biogenic and exogenic aldehydes. To date, there are relatively few general ALDH inhibitors that can be used to probe the contribution of this class of enzymes to particular metabolic pathways. Here, we report the discovery of a general class of ALDH inhibitors with a common mechanism of action. The combined data from kinetic studies, mass spectrometric measurements, and crystallographic analyses demonstrate that these inhibitors undergo an enzyme-mediated {beta}-elimination reaction generating a vinyl ketone intermediate that covalently modifies the active site cysteine residue present in these enzymes. The studies described here can provide the basis for rational approach to design ALDH isoenzyme-specific inhibitors as research tools and perhaps as drugs, to address diseases such as cancer where increased ALDH activity is associated with a cellular phenotype.

  18. Modulation of ethanol stress tolerance by aldehyde dehydrogenase in the mycorrhizal fungus Tricholoma vaccinum.

    PubMed

    Asiimwe, Theodore; Krause, Katrin; Schlunk, Ines; Kothe, Erika

    2012-08-01

    We report the first mycorrhizal fungal aldehyde dehydrogenase gene, ald1, which was isolated from the basidiomycete Tricholoma vaccinum. The gene, encoding a protein Ald1 of 502 amino acids, is up-regulated in ectomycorrhiza. Phylogenetic analyses using 53 specific fungal aldehyde dehydrogenases from all major phyla in the kingdom of fungi including Ald1 and two partial sequences of T. vaccinum were performed to get an insight in the evolution of the aldehyde dehydrogenase family. By using competitive and real-time RT-PCR, ald1 is up-regulated in response to alcohol and aldehyde-related stress. Furthermore, heterologous expression of ald1 in Escherichia coli and subsequent in vitro enzyme activity assay demonstrated the oxidation of propionaldehyde and butyraldehyde with different kinetics using either NAD(+) or NADP(+) as cofactors. In addition, overexpression of ald1 in T. vaccinum after Agrobacterium tumefaciens-mediated transformation increased ethanol stress tolerance. These results demonstrate the ability of Ald1 to circumvent ethanol stress, a critical function in mycorrhizal habitats. PMID:22159964

  19. Aldehydic load and aldehyde dehydrogenase 2 profile during the progression of post-myocardial infarction cardiomyopathy: benefits of Alda-1

    PubMed Central

    Gomes, Katia M.S.; Bechara, Luiz R.G.; Lima, Vanessa M.; Ribeiro, Márcio A.C.; Campos, Juliane C.; Dourado, Paulo M.; Kowaltowski, Alicia J.; Mochly-Rosen, Daria; Ferreira, Julio C.B.

    2015-01-01

    Background/Objectives We previously demonstrated that reducing cardiac aldehydic load by aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme responsible for metabolizing the major lipid peroxidation product, protects against acute ischemia/reperfusion injury and chronic heart failure. However, time-dependent changes in ALDH2 profile, aldehydic load and mitochondrial bioenergetics during progression of post-myocardial infarction (post-MI) cardiomyopathy is unknown and should be established to determine the optimal time window for drug treatment. Methods Here we characterized cardiac ALDH2 activity and expression, lipid peroxidation, 4-hydroxy-2-nonenal (4-HNE) adduct formation, glutathione pool and mitochondrial energy metabolism and H2O2 release during the 4 weeks after permanent left anterior descending (LAD) coronary artery occlusion in rats. Results We observed a sustained disruption of cardiac mitochondrial function during the progression of post-MI cardiomyopathy, characterized by >50% reduced mitochondrial respiratory control ratios and up to 2 fold increase in H2O2 release. Mitochondrial dysfunction was accompanied by accumulation of cardiac and circulating lipid peroxides and 4-HNE protein adducts and down-regulation of electron transport chain complexes I and V. Moreover, increased aldehydic load was associated with a 90% reduction in cardiac ALDH2 activity and increased glutathione pool. Further supporting an ALDH2 mechanism, sustained Alda-1 treatment (starting 24hrs after permanent LAD occlusion surgery) prevented aldehydic overload, mitochondrial dysfunction and improved ventricular function in post-MI cardiomyopathy rats. Conclusion Taken together, our findings demonstrate a disrupted mitochondrial metabolism along with an insufficient cardiac ALDH2-mediated aldehyde clearance during the progression of ventricular dysfunction, suggesting a potential therapeutic value of ALDH2 activators during the progression of post-myocardial infarction

  20. Targeting Aldehyde Dehydrogenase Cancer Stem Cells in Ovarian Cancer

    PubMed Central

    Landen, Charles N.; Goodman, Blake; Katre, Ashwini A.; Steg, Adam D.; Nick, Alpa M.; Stone, Rebecca L.; Miller, Lance D.; Mejia, Pablo Vivas; Jennings, Nicolas B.; Gershenson, David M.; Bast, Robert C.; Coleman, Robert L.; Lopez-Berestein, Gabriel; Sood, Anil K.

    2010-01-01

    Aldehyde dehydrogenase-1A1 (ALDH1A1) expression characterizes a subpopulation of cells with tumor initiating or cancer stem cell properties in several malignancies. Our goal was to characterize the phenotype of ALDH1A1-positive ovarian cancer cells and examine the biological effects of ALDH1A1 gene silencing. In our analysis of multiple ovarian cancer cell lines, we found that ALDH1A1 expression and activity was significantly higher in taxane and platinum-resistant cell lines. In patient samples, 72.9% of ovarian cancers had ALDH1A1 expression, in whom the percent of ALDH1A1-positive cells correlated negatively with progression-free survival (6.05 v 13.81 months, p<0.035). Subpopulations of A2780cp20 cells with ALDH1A1 activity were isolated for orthotopic tumor initiating studies, where tumorigenicity was approximately 50-fold higher with ALDH1A1-positive cells. Interestingly, tumors derived from ALDH1A1-positive cells gave rise to both ALDH1A1-positive and ALDH1A1-negative populations, but ALDH1A1-negative cells could not generate ALDH1A1-positive cells. In an in vivo orthotopic mouse model of ovarian cancer, ALDH1A1 silencing using nanoliposomal siRNA sensitized both taxane- and platinum-resistant cell lines to chemotherapy, significantly reducing tumor growth in mice compared to chemotherapy alone (a 74–90% reduction, p<0.015). These data demonstrate that the ALDH1A1 subpopulation is associated with chemoresistance and outcome in ovarian cancer patients, and targeting ALDH1A1 sensitizes resistant cells to chemotherapy. ALDH1A1-positive cells have enhanced, but not absolute, tumorigenicity, but do have differentiation capacity lacking in ALDH1A1-negative cells. This enzyme may be important for identification and targeting of chemoresistant cell populations in ovarian cancer. PMID:20889728

  1. Bifunctional aldehyde/alcohol dehydrogenase (ADHE) in chlorophyte algal mitochondria.

    PubMed

    Atteia, Ariane; van Lis, Robert; Mendoza-Hernández, Guillermo; Henze, Katrin; Martin, William; Riveros-Rosas, Hector; González-Halphen, Diego

    2003-09-01

    Protein profiles of mitochondria isolated from the heterotrophic chlorophyte Polytomella sp. grown on ethanol at pH 6.0 and pH 3.7 were analyzed by Blue Native and denaturing polyacrylamide gel electrophoresis. Steady-state levels of oxidative phosphorylation complexes were influenced by external pH. Levels of an abundant, soluble, mitochondrial protein of 85 kDa and its corresponding mRNA increased at pH 6.0 relative to pH 3.7. N-terminal and internal sequencing of the 85 kDa mitochondrial protein together with the corresponding cDNA identified it as a bifunctional aldehyde/alcohol dehydrogenase (ADHE) with strong similarity to homologues from eubacteria and amitochondriate protists. A mitochondrial targeting sequence of 27 amino acids precedes the N-terminus of the mature mitochondrial protein. A gene encoding an ADHE homologue was also identified in the genome of Chlamydomonas reinhardtii, a photosynthetic relative of Polytomella. ADHE reveals a complex picture of sequence similarity among homologues. The lack of ADHE from archaebacteria indicates a eubacterial origin for the eukaryotic enzyme. Among eukaryotes, ADHE has hitherto been characteristic of anaerobes since it is essential to cytosolic energy metabolism of amitochondriate protists such as Giardia intestinalis and Entamoeba histolytica. Its abundance and expression pattern suggest an important role for ADHE in mitochondrial metabolism of Polytomella under the conditions studied. The current data are compatible with the view that Polytomella ADHE could be involved either in ethanol production or assimilation, or both, depending upon environmental conditions. Presence of ADHE in an oxygen-respiring algal mitochondrion and co-expression at ambient oxygen levels with respiratory chain components is unexpected with respect to the view that eukaryotes acquired ADHE genes specifically as an adaptation to an anaerobic lifestyle. PMID:14756315

  2. Vascular Bioactivation of Nitroglycerin by Aldehyde Dehydrogenase-2

    PubMed Central

    Lang, Barbara S.; Gorren, Antonius C. F.; Oberdorfer, Gustav; Wenzl, M. Verena; Furdui, Cristina M.; Poole, Leslie B.; Mayer, Bernd; Gruber, Karl

    2012-01-01

    Aldehyde dehydrogenase-2 (ALDH2) catalyzes the bioactivation of nitroglycerin (glyceryl trinitrate, GTN) in blood vessels, resulting in vasodilation by nitric oxide (NO) or a related species. Because the mechanism of this reaction is still unclear we determined the three-dimensional structures of wild-type (WT) ALDH2 and of a triple mutant of the protein that exhibits low denitration activity (E268Q/C301S/C303S) in complex with GTN. The structure of the triple mutant showed that GTN binds to the active site via polar contacts to the oxyanion hole and to residues 268 and 301 as well as by van der Waals interactions to hydrophobic residues of the catalytic pocket. The structure of the GTN-soaked wild-type protein revealed a thionitrate adduct to Cys-302 as the first reaction intermediate, which was also found by mass spectrometry (MS) experiments. In addition, the MS data identified sulfinic acid as the irreversibly inactivated enzyme species. Assuming that the structures of the triple mutant and wild-type ALDH2 reflect binding of GTN to the catalytic site and the first reaction step, respectively, superposition of the two structures indicates that denitration of GTN is initiated by nucleophilic attack of Cys-302 at one of the terminal nitrate groups, resulting in formation of the observed thionitrate intermediate and release of 1,2-glyceryl dinitrate. Our results shed light on the molecular mechanism of the GTN denitration reaction and provide useful information on the structural requirements for high affinity binding of organic nitrates to the catalytic site of ALDH2. PMID:22988236

  3. Crystallization and preliminary X-ray analysis of aldehyde dehydrogenase from Vibrio harveyi.

    PubMed Central

    Croteau, N.; Vedadi, M.; Delarge, M.; Meighen, E.; Abu-Abed, M.; Howell, P. L.; Vrielink, A.

    1996-01-01

    Aldehyde dehydrogenase from Vibrio harveyi catalyzes the oxidation of long-chain aliphatic aldehydes to acids. The enzyme is unique among the family of aldehyde dehydrogenases in that it exhibits much higher specificity for the cofactor NADP+ than for NAD+. The sequence of this form of the enzyme varies significantly from the NAD+ dependent forms, suggesting differences in the three-dimensional structure that may be correlated to cofactor specificity. Crystals of the enzyme have been grown both in the presence and absence of NADP+ using the hanging drop vapor diffusion technique. In order to improve crystal size and quality, iterative seeding techniques were employed. The crystals belong to space group P2(1), with unit cell dimensions a = 79.4 A, b = 131.1 A, c = 92.2 A, and beta = 92.4 degrees. Freezing the crystal to 100 K has enabled a complete set of data to be collected using a rotating anode source (lambda = 1.5418 A). The crystals diffract to a minimum d-spacing of 2.6 A resolution. Based on density calculations, two homodimers of molecular weight 110 kDa are estimated to be present in the asymmetric unit. Self-rotation functions show the presence of 3 noncrystallographic twofold symmetry axes. PMID:8897616

  4. NAD(P)-Dependent Aldehyde Dehydrogenases Induced during Growth of Ralstonia eutropha Strain Bo on Tetrahydrofurfuryl Alcohol

    PubMed Central

    Schräder, Thomas; Zarnt, Grit; Andreesen, Jan R.

    2001-01-01

    Different aldehyde dehydrogenases (AlDHs) were formed during growth of Ralstonia eutropha Bo on tetrahydrofurfuryl alcohol (THFA). One of these enzymes, AlDH 4, was purified and characterized as a homodimer containing no prosthetic groups, showing a strong substrate inhibition, and having an N-terminal sequence similar to those of various NAD(P)-dependent AlDHs. The conversion rate of THFA by the quinohemoprotein THFA dehydrogenase was increased by AlDH 4. PMID:11717302

  5. Characterization of a periplasmic quinoprotein from Sphingomonas wittichii that functions as aldehyde dehydrogenase.

    PubMed

    Zeiser, Jessica; Mühlenbeck, Larissa Helen; Schweiger, Paul; Deppenmeier, Uwe

    2014-03-01

    The α-proteobacterium Sphingomonas wittichii RW1 is known for its ability to degrade dioxins and related toxic substances. Bioinformatic analysis of the genome indicated that this organism may contain the largest number of pyrroloquinoline quinone-dependent dehydrogenases of any bacteria sequenced so far. Sequence analysis also showed that one of these genes (swit_4395) encodes an enzyme that belongs to the class of periplasmic glucose dehydrogenases. This gene was fused to a pelB signal sequence and a strep-tag coding region at the 5' and 3' ends, respectively. The fusion product was cloned into the broad-host range expression vector pBBR1p264-Streplong and the corresponding protein was heterologously produced in Escherichia coli, purified via Strep-Tactin affinity chromatography, and characterized. The protein Swit_4395 had a subunit mass of 39.3 kDa and formed active homooctamers and homododecamers. The enzyme showed the highest activities with short- and medium-chain aldehydes (chain length C1-C6) and ketoaldehydes, such as methylglyoxal and phenylglyoxal. Butyraldehyde was the best substrate, with V max and apparent K M values of 3,970 U/mg protein and 12.3 mM, respectively. Pyrroloquinoline quinone was detected using UV-Vis spectroscopy and was found to be a prosthetic group of the purified enzyme. Therefore, Swit_4395 was identified as a pyrroloquinoline quinone-dependent aldehyde dehydrogenase. The enzyme could be purified from the native host when the expression vector was introduced into S. wittichii RW1, indicating homologous protein production. Overproduction of Swit_4395 in S. wittichii RW1 dramatically increased the tolerance of the bacterium toward butyraldehyde and thus might contribute to the detoxification of toxic aldehydes. PMID:23828599

  6. Insight into Coenzyme A cofactor binding and the mechanism of acyl-transfer in an acylating aldehyde dehydrogenase from Clostridium phytofermentans

    PubMed Central

    Tuck, Laura R.; Altenbach, Kirsten; Ang, Thiau Fu; Crawshaw, Adam D.; Campopiano, Dominic J.; Clarke, David J.; Marles-Wright, Jon

    2016-01-01

    The breakdown of fucose and rhamnose released from plant cell walls by the cellulolytic soil bacterium Clostridium phytofermentans produces toxic aldehyde intermediates. To enable growth on these carbon sources, the pathway for the breakdown of fucose and rhamnose is encapsulated within a bacterial microcompartment (BMC). These proteinaceous organelles sequester the toxic aldehyde intermediates and allow the efficient action of acylating aldehyde dehydrogenase enzymes to produce an acyl-CoA that is ultimately used in substrate-level phosphorylation to produce ATP. Here we analyse the kinetics of the aldehyde dehydrogenase enzyme from the fucose/rhamnose utilisation BMC with different short-chain fatty aldehydes and show that it has activity against substrates with up to six carbon atoms, with optimal activity against propionaldehyde. We have also determined the X-ray crystal structure of this enzyme in complex with CoA and show that the adenine nucleotide of this cofactor is bound in a distinct pocket to the same group in NAD+. This work is the first report of the structure of CoA bound to an aldehyde dehydrogenase enzyme and our crystallographic model provides important insight into the differences within the active site that distinguish the acylating from non-acylating aldehyde dehydrogenase enzymes. PMID:26899032

  7. Insight into Coenzyme A cofactor binding and the mechanism of acyl-transfer in an acylating aldehyde dehydrogenase from Clostridium phytofermentans.

    PubMed

    Tuck, Laura R; Altenbach, Kirsten; Ang, Thiau Fu; Crawshaw, Adam D; Campopiano, Dominic J; Clarke, David J; Marles-Wright, Jon

    2016-01-01

    The breakdown of fucose and rhamnose released from plant cell walls by the cellulolytic soil bacterium Clostridium phytofermentans produces toxic aldehyde intermediates. To enable growth on these carbon sources, the pathway for the breakdown of fucose and rhamnose is encapsulated within a bacterial microcompartment (BMC). These proteinaceous organelles sequester the toxic aldehyde intermediates and allow the efficient action of acylating aldehyde dehydrogenase enzymes to produce an acyl-CoA that is ultimately used in substrate-level phosphorylation to produce ATP. Here we analyse the kinetics of the aldehyde dehydrogenase enzyme from the fucose/rhamnose utilisation BMC with different short-chain fatty aldehydes and show that it has activity against substrates with up to six carbon atoms, with optimal activity against propionaldehyde. We have also determined the X-ray crystal structure of this enzyme in complex with CoA and show that the adenine nucleotide of this cofactor is bound in a distinct pocket to the same group in NAD(+). This work is the first report of the structure of CoA bound to an aldehyde dehydrogenase enzyme and our crystallographic model provides important insight into the differences within the active site that distinguish the acylating from non-acylating aldehyde dehydrogenase enzymes. PMID:26899032

  8. [Characterization of aldehyde dehydrogenase gene fragment from mung bean Vigna radiata using the polymerase chain reaction].

    PubMed

    Ponomarev, A G; Bubiakina, V V; Tatarinova, T D; Zelenin, S M

    1998-01-01

    Two degenerate oligonucleotide sequence primers and polymerase chain reactions on total DNA have been utilized to clone on 651--bp gene fragment coding the central part of amino acid sequence of an earlier unknown aldehyde dehydrogenase (ALDH) from mung bean. The deduced partial amino acid sequence for this aldehyde dehydrogenase shows about 65% sequence identity to ALDHs of Vibrio cholerae Rhodococcus sp., Alcaligenes eutrophus and about 45% sequence identity to mammalian ALDHs 1 and 2, ALDHs of Aspergillus niger and A, nidulans, the betain aldehyde dehydrogenase from spinach. Alignment of the mung bean aldehyde dehydrogenase partial amino acid sequence with the sequence of 16 NAD(P)(+)-dependent aldehyde dehydrogenases has demonstrated that all strictly conserved amino acid residues and all three conservative regions are identical. PMID:9778740

  9. Aldehyde dehydrogenase (ALDH) superfamily in plants: gene nomenclature and comparative genomics.

    PubMed

    Brocker, Chad; Vasiliou, Melpomene; Carpenter, Sarah; Carpenter, Christopher; Zhang, Yucheng; Wang, Xiping; Kotchoni, Simeon O; Wood, Andrew J; Kirch, Hans-Hubert; Kopečný, David; Nebert, Daniel W; Vasiliou, Vasilis

    2013-01-01

    In recent years, there has been a significant increase in the number of completely sequenced plant genomes. The comparison of fully sequenced genomes allows for identification of new gene family members, as well as comprehensive analysis of gene family evolution. The aldehyde dehydrogenase (ALDH) gene superfamily comprises a group of enzymes involved in the NAD(+)- or NADP(+)-dependent conversion of various aldehydes to their corresponding carboxylic acids. ALDH enzymes are involved in processing many aldehydes that serve as biogenic intermediates in a wide range of metabolic pathways. In addition, many of these enzymes function as 'aldehyde scavengers' by removing reactive aldehydes generated during the oxidative degradation of lipid membranes, also known as lipid peroxidation. Plants and animals share many ALDH families, and many genes are highly conserved between these two evolutionarily distinct groups. Conversely, both plants and animals also contain unique ALDH genes and families. Herein we carried out genome-wide identification of ALDH genes in a number of plant species-including Arabidopsis thaliana (thale crest), Chlamydomonas reinhardtii (unicellular algae), Oryza sativa (rice), Physcomitrella patens (moss), Vitis vinifera (grapevine) and Zea mays (maize). These data were then combined with previous analysis of Populus trichocarpa (poplar tree), Selaginella moellindorffii (gemmiferous spikemoss), Sorghum bicolor (sorghum) and Volvox carteri (colonial algae) for a comprehensive evolutionary comparison of the plant ALDH superfamily. As a result, newly identified genes can be more easily analyzed and gene names can be assigned according to current nomenclature guidelines; our goal is to clarify previously confusing and conflicting names and classifications that might confound results and prevent accurate comparisons between studies. PMID:23007552

  10. Rice Aldehyde Dehydrogenase7 Is Needed for Seed Maturation and Viability1[W][OA

    PubMed Central

    Shin, Jun-Hye; Kim, Sung-Ryul; An, Gynheung

    2009-01-01

    Aldehyde dehydrogenases (ALDHs) catalyze the irreversible oxidation of a wide range of reactive aldehydes to their corresponding carboxylic acids. Although the proteins have been studied from various organisms and at different growth stages, their roles in seed development have not been well elucidated. We obtained T-DNA insertional mutants in OsALDH7, which is remarkably inducible by oxidative and abiotic stresses. Interestingly, endosperms from the osaldh7 null mutants accumulated brown pigments during desiccation and storage. Extracts from the mutant seeds showed a maximum absorbance peak at 360 nm, the wavelength that melanoidin absorbs. Under UV light, those extracts also exhibited much stronger fluorescence than the wild type, suggesting that the pigments are melanoidin. These pigments started to accumulate in the late seed developmental stage, the time when OsALDH7 expression began to increase significantly. Purified OsALDH7 protein showed enzyme activities to malondialdehyde, acetaldehyde, and glyceraldehyde. These results suggest that OsALDH7 is involved in removing various aldehydes formed by oxidative stress during seed desiccation. The mutant seeds were more sensitive to our accelerated aging treatment and accumulated more malondialdehyde than the wild type. These data imply that OsALDH7 plays an important role in maintaining seed viability by detoxifying the aldehydes generated by lipid peroxidation. PMID:19052152

  11. Aldehyde dehydrogenase-2 regulates nociception in rodent models of acute inflammatory pain

    PubMed Central

    Zambelli, Vanessa O.; Gross, Eric R.; Chen, Che-Hong; Gutierrez, Vanessa P.; Cury, Yara; Mochly-Rosen, Daria

    2014-01-01

    Exogenous aldehydes can cause pain in animal models, suggesting that aldehyde dehydrogenase 2 (ALDH2), which metabolizes many aldehydes, may regulate nociception. To test this hypothesis, we generated a knock-in mouse with an inactivating point mutation in ALDH2 (ALDH2*2), which is also present in human ALDH2 of ~540 million East Asians. The ALDH2*1/*2 heterozygotic mice exhibited a larger response to painful stimuli than their wild-type littermates, and this heightened nociception was inhibited by an ALDH2-selective activator (Alda-1). No effect on inflammation per se was observed. Using a rat model, we then showed that nociception tightly correlated with ALDH activity (R2=0.90) and that reduced nociception was associated with less early growth response protein 1 (EGR1) in the spinal cord and less reactive aldehyde accumulation at the insult site (including acetaldehyde and 4-hydroxynonenal). Further, acetaldehyde and formalin-induced nociceptive behavior was greater in the ALDH2*1/*2 mice than wild-type mice. Finally, Alda-1 treatment was also beneficial when given even after the inflammatory agent was administered. Our data in rodent models suggest that the mitochondrial enzyme ALDH2 regulates nociception and could serve as a molecular target for pain control, with ALDH2 activators, such as Alda-1, as potential non-narcotic cardiac-safe analgesics. Furthermore, our results suggest a possible genetic basis for East Asians’ apparent lower pain tolerance. PMID:25163478

  12. Alcohol and aldehyde dehydrogenase polymorphisms in Chinese and Indian populations.

    PubMed

    Tan, Ene-Choo; Lim, Leslie; Leong, Jern-Yi; Lim, Jing-Yan; Lee, Arthur; Yang, Jun; Tan, Chay-Hoon; Winslow, Munidasa

    2010-01-01

    The association between two functional polymorphisms in alcohol dehydrogenase (ADH2/ADH1B) and aldehyde dehydrogenase (ALDH2) genes and alcohol dependence was examined in 182 Chinese and Indian patients undergoing treatment for alcohol dependence and 184 screened control subjects from Singapore. All subjects were screened by the Alcohol Use Disorders Identification Test (AUDIT). Patients were also administered the Severity of Alcohol Dependence Questionnaire (SADQ). Polymorphisms were genotyped by allele-specific polymerase chain reaction and selected genotypes confirmed by DNA sequencing or restriction fragment length polymorphism. Our results showed that frequencies of ADH1B*2 and ALDH2*2 were higher in controls compared to alcohol-dependent subjects for both Chinese and Indians. Frequencies of these two alleles were also higher in the 104 Chinese controls compared to the 80 Indian controls. None of the eight Chinese who were homozygous for both protective alleles was alcohol dependent. The higher frequencies of the protective alleles could explain the lower rate of alcohol dependence in Chinese. PMID:20025435

  13. Oxidation of fatty aldehydes to fatty acids by Escherichia coli cells expressing the Vibrio harveyi fatty aldehyde dehydrogenase (FALDH).

    PubMed

    Buchhaupt, Markus; Guder, Jan; Sporleder, Fenja; Paetzold, Melanie; Schrader, Jens

    2013-03-01

    Fatty acids represent an important renewable feedstock for the chemical industry. To enable biotechnological one carbon truncations of fatty acids, the enzymes α-dioxygenase and fatty aldehyde dehydrogenase (FALDH) have to be combined in a two-step process. We expressed an FALDH from V. harveyi in E. coli and characterized its substrate spectrum with a focus on the number and position of double bonds in the fatty aldehyde molecules. Synthesis of the expected fatty acid products was proven by analysis of whole cell biotransformation products. Coexpression of a H(2)O-forming NADPH oxidase (NOX) from Lactobacillus sanfranciscensis led to the implementation of a cofactor regeneration cycle in in vitro oxidation experiments. The presence of NOX in whole cell biotransformations improved reaction velocity but did not result in higher product yields. We could further demonstrate that at least part of the endogenous NAD(P)(+) regeneration capacity in the resting cells results from the respiratory chain. The whole cell catalyst with the high broad range FALDH activity described here is an important biotechnological module for lipid biotransformation processes, especially the shortening of fatty acids. PMID:23180547

  14. Structural Basis of Substrate Recognition by Aldehyde Dehydrogenase 7A1.

    PubMed

    Luo, Min; Tanner, John J

    2015-09-01

    Aldehyde dehydrogenase 7A1 (ALDH7A1) is part of lysine catabolism and catalyzes the NAD(+)-dependent oxidation of α-aminoadipate semialdehyde to α-aminoadipate. Herein, we describe a structural study of human ALDH7A1 focused on substrate recognition. Five crystal structures and small-angle X-ray scattering data are reported, including the first crystal structure of any ALDH7 family member complexed with α-aminoadipate. The product binds with the ε-carboxylate in the oxyanion hole, the aliphatic chain packed into an aromatic box, and the distal end of the product anchored by electrostatic interactions with five conserved residues. This binding mode resembles that of glutamate bound to the proline catabolic enzyme ALDH4A1. Analysis of ALDH7A1 and ALDH4A1 structures suggests key interactions that underlie substrate discrimination. Structures of apo ALDH7A1 reveal dramatic conformational differences from the product complex. Product binding is associated with a 16 Å movement of the C-terminus into the active site, which stabilizes the active conformation of the aldehyde substrate anchor loop. The fact that the C-terminus is part of the active site was hitherto unknown. Interestingly, the C-terminus and aldehyde anchor loop are disordered in a new tetragonal crystal form of the apoenzyme, implying that these parts of the enzyme are highly flexible. Our results suggest that the active site of ALDH7A1 is disassembled when the aldehyde site is vacant, and the C-terminus is a mobile element that forms quaternary structural interactions that aid aldehyde binding. These results are relevant to the c.1512delG genetic deletion associated with pyridoxine-dependent epilepsy, which alters the C-terminus of ALDH7A1. PMID:26260980

  15. Ocular Aldehyde Dehydrogenases: Protection against Ultraviolet Damage and Maintenance of Transparency for Vision

    PubMed Central

    Chen, Ying; Thompson, David C.; Koppaka, Vindhya; Jester, James V.; Vasiliou, Vasilis

    2012-01-01

    Aldehyde dehydrogenase (ALDH) enzymes catalyze the NAD(P)+-dependent oxidation of a wide variety of endogenous and exogenous aldehydes to their corresponding acids. Some members of the ALDH superfamily of enzymes are abundantly expressed in the mammalian cornea and lens in a taxon-specific manner. Considered to be corneal and lens crystallins, they confer protective and transparent properties upon these ocular tissues. ALDH3A1 is highly expressed in the cornea of most mammals, with the exception of rabbit that expresses exclusively ALDH1A1 in the cornea. ALDH1A1 is present in both the cornea and lens of several animal species. As a result of their catalytic and non-catalytic functions, ALDH3A1 and ALDH1A1 proteins protect inner ocular tissues from ultraviolet radiation and reactive oxygen-induced damage. In addition, these corneal crystallins contribute to cellular transparency in corneal stromal keratocytes, supporting a structural role of these ALDH proteins. A putative regulatory function of ALDH3A1 on corneal cell proliferation has also been proposed. Finally, the three retinaldehye dehydrogenases cooperatively mediate retinoic acid signaling during the eye development. PMID:23098688

  16. Betaine Accumulation and Betaine-Aldehyde Dehydrogenase in Spinach Leaves 1

    PubMed Central

    Pan, Shu-Mei; Moreau, Robert A.; Yu, Charles; Huang, Anthony H. C.

    1981-01-01

    Spinach leaf discs accumulated betaine when exposed to a mannitol solution of −20 bars. The accumulation was 12 micromoles per gram original fresh weight in a 24-hour period. Betaine-aldehyde dehydrogenase (EC 1.2.1.8) was assayed in various subcellular fractions prepared from spinach leaves, and it was found only in the soluble fraction. This cytosolic enzyme was purified 175-fold, and its properties were studied. The enzyme was relatively specific for betaine aldehyde as the substrate with an apparent Km value of 2.08 × 10−4 molar. It also exerted activity on other aldehyde analogs tested, but with lower Vmax and higher Km values. The enzyme was relatively specific for nicotinamide adenine dinucleotide as the coenzyme, having an apparent Km value of 9.46 × 10−6 molar; lower activities were observed when nicotinamide adenine dinucleotide phosphate or 3-acetyl pyridine adenine dinucleotide were tested as electron acceptors. The activity was enhanced by dithiothreitol and inhibited by p-chloromercuribenzoate, and the inhibition by p-chloromercuribenzoate was partially reversed by the subsequent addition of dithiothreitol. The activity was inhibited by high concentrations of NaCl and, to a lesser extent, proline. The equilibrium of the enzymic reaction was strongly in favor of betaine formation. The in vitro activity of the enzyme under optimal assay conditions was high enough to account for the amount of betaine accumulated under water stress conditions. The enzyme activity was the same in unstressed leaves and in leaves that had been water stressed for 24 hours. PMID:16661818

  17. Role and structural characterization of plant aldehyde dehydrogenases from family 2 and family 7.

    PubMed

    Končitíková, Radka; Vigouroux, Armelle; Kopečná, Martina; Andree, Tomáš; Bartoš, Jan; Šebela, Marek; Moréra, Solange; Kopečný, David

    2015-05-15

    Aldehyde dehydrogenases (ALDHs) are responsible for oxidation of biogenic aldehyde intermediates as well as for cell detoxification of aldehydes generated during lipid peroxidation. So far, 13 ALDH families have been described in plants. In the present study, we provide a detailed biochemical characterization of plant ALDH2 and ALDH7 families by analysing maize and pea ALDH7 (ZmALDH7 and PsALDH7) and four maize cytosolic ALDH(cALDH)2 isoforms RF2C, RF2D, RF2E and RF2F [the first maize ALDH2 was discovered as a fertility restorer (RF2A)]. We report the crystal structures of ZmALDH7, RF2C and RF2F at high resolution. The ZmALDH7 structure shows that the three conserved residues Glu(120), Arg(300) and Thr(302) in the ALDH7 family are located in the substrate-binding site and are specific to this family. Our kinetic analysis demonstrates that α-aminoadipic semialdehyde, a lysine catabolism intermediate, is the preferred substrate for plant ALDH7. In contrast, aromatic aldehydes including benzaldehyde, anisaldehyde, cinnamaldehyde, coniferaldehyde and sinapaldehyde are the best substrates for cALDH2. In line with these results, the crystal structures of RF2C and RF2F reveal that their substrate-binding sites are similar and are formed by an aromatic cluster mainly composed of phenylalanine residues and several nonpolar residues. Gene expression studies indicate that the RF2C gene, which is strongly expressed in all organs, appears essential, suggesting that the crucial role of the enzyme would certainly be linked to the cell wall formation using aldehydes from phenylpropanoid pathway as substrates. Finally, plant ALDH7 may significantly contribute to osmoprotection because it oxidizes several aminoaldehydes leading to products known as osmolytes. PMID:25734422

  18. An animal model of human aldehyde dehydrogenase deficiency

    SciTech Connect

    Chang, C.; Mann, J.; Yoshida, A.

    1994-09-01

    The genetic deficiency of ALDH2, a major mitochondrial aldehyde dehydrogenase, is intimately related to alcohol sensitivity and the degree of predisposition to alcoholic diseases in humans. The ultimate biological role of ALDH2 can be exposed by knocking out the ALDH2 gene in an animal model. As the first step for this line of studies, we cloned and characterized the ALDH2 gene from mouse C57/6J strain which is associated with a high alcohol preference. The gene spans 26 kbp and is composed of 13 exons. Embryonic stem cells were transfected with a replacement vector which contains a partially deleted exon3, a positive selection cassette (pPgk Neo), exon 4 with an artificial stop codon, exons 5, 6, 7, and a negative selection cassette (pMCI-Tk). Genomic DNAs prepared from drug resistant clones were analyzed by polymerase chain reaction and by Southern blot analysis to distinguish random integration from homologous recombination. Out of 132 clones examined, 8 had undergone homologous recombination at one of the ALDH2 alleles. The cloned transformed embryonic stem cells with a disrupted ALDH2 allele were injected into blastocysts. Transplantation of the blastocysts into surrogate mother mice yielded chimeric mice. The role of ALDH2 in alcohol preference, alcohol sensitivity and other biological and behavioral characteristics can be elucidated by examining the heterozygous and homozygous mutant strains produced by breeding of chimeric mice.

  19. Intracellular localization of aldehyde dehydrogenase in rat liver

    PubMed Central

    Marjanen, Leo

    1972-01-01

    1. Distribution of aldehyde dehydrogenase activity in rat liver was studied by measuring the rate of disappearance of acetaldehyde in the presence of each of the subcellular fractions. These were obtained by rough separation of particulate fractions from the soluble portion of the cell, by differential centrifugation, and by isopycnic gradient centrifugation. 2. The maximal rate of acetaldehyde oxidation was 3.7 μmol/min per g, with an apparent Km value below 10−5m. The highest rate of activity was observed in phosphate buffers of high Pi concentration (above 60mm). 3. The activity measured was completely dependent on NAD+. 4. The microsomal fraction and the nuclei were inactive in the assay. Of the total activity 80% was found in the mitochondrial fraction and the remaining 20% in the cytoplasm. 5. The distribution pattern is important from the point of view of acetaldehyde oxidation during ethanol metabolism. The apparent discrepancy of the results obtained by different workers and the localization of acetaldehyde oxidation in vivo is discussed. PMID:4346744

  20. Structure of betaine aldehyde dehydrogenase at 2.1 A resolution.

    PubMed Central

    Johansson, K.; El-Ahmad, M.; Ramaswamy, S.; Hjelmqvist, L.; Jörnvall, H.; Eklund, H.

    1998-01-01

    The three-dimensional structure of betaine aldehyde dehydrogenase, the most abundant aldehyde dehydrogenase (ALDH) of cod liver, has been determined at 2.1 A resolution by the X-ray crystallographic method of molecular replacement. This enzyme represents a novel structure of the highly multiple ALDH, with at least 12 distinct classes in humans. This betaine ALDH of class 9 is different from the two recently determined ALDH structures (classes 2 and 3). Like these, the betaine ALDH structure has three domains, one coenzyme binding domain, one catalytic domain, and one oligomerization domain. Crystals grown in the presence or absence of NAD+ have very similar structures and no significant conformational change occurs upon coenzyme binding. This is probably due to the tight interactions between domains within the subunit and between subunits in the tetramer. The oligomerization domains link the catalytic domains together into two 20-stranded pleated sheet structures. The overall structure is similar to that of the tetrameric bovine class 2 and dimeric rat class 3 ALDH, but the coenzyme binding with the nicotinamide in anti conformation, resembles that of class 2 rather than of class 3. PMID:9792097

  1. Genomic organization and expression of the human fatty aldehyde dehydrogenase gene (FALDH)

    SciTech Connect

    Rogers, G.R.; Markova, N.G.; Compton, J.G.

    1997-01-15

    Mutations in the fatty aldehyde dehydrogenase (FALDH) gene cause Sjoegren-Larsson syndrome (SLS) - a disease characterized by mental retardation, spasticity, and congenital ichthyosis. To facilitate mutation analysis in SLS and to study the pathogenesis of FALDH deficiency, we have determined the structural organization and characterized expression of the FALDH (proposed designation ALDH10) gene. The gene consists of 10 exons spanning about 30.5 kb. A TATA-less promoter is associated with the major transcription initiation site found to be 258 hp upstream of the ATG codon. The G4C-rich sequences surrounding the transcription initiation site encompassed regulatory elements that interacted with proteins in HeLa nuclear extracts and were able to promote transcription in vitro. FALDH is widely expressed as three transcripts of 2, 3.8, and 4.0 kb, which originate from multiple polyadenylation signals in the 3{prime} UTR. An alternatively spliced mRNA was detected that contains an extra exon and encodes an enzyme that is likely to have altered membrane-binding properties. The FALDH gene lies only 50-85 kb from ALDH3, an aldehyde dehydrogenase gene that has homologous sequence and intron/exon structure. 25 refs., 4 figs., 1 tab.

  2. Alda-1 is an agonist and chemical chaperone for the common human aldehyde dehydrogenase 2 variant

    SciTech Connect

    Perez-Miller, Samantha; Younus, Hina; Vanam, Ram; Chen, Che-Hong; Mochly-Rosen, Daria; Hurley, Thomas D.

    2010-04-19

    In approximately one billion people, a point mutation inactivates a key detoxifying enzyme, aldehyde dehydrogenase (ALDH2). This mitochondrial enzyme metabolizes toxic biogenic and environmental aldehydes, including the endogenously produced 4-hydroxynonenal (4HNE) and the environmental pollutant acrolein, and also bioactivates nitroglycerin. ALDH2 is best known, however, for its role in ethanol metabolism. The accumulation of acetaldehyde following the consumption of even a single alcoholic beverage leads to the Asian alcohol-induced flushing syndrome in ALDH2*2 homozygotes. The ALDH2*2 allele is semidominant, and heterozygotic individuals show a similar but less severe phenotype. We recently identified a small molecule, Alda-1, that activates wild-type ALDH2 and restores near-wild-type activity to ALDH2*2. The structures of Alda-1 bound to ALDH2 and ALDH2*2 reveal how Alda-1 activates the wild-type enzyme and how it restores the activity of ALDH2*2 by acting as a structural chaperone.

  3. Bioactivation of Nitroglycerin by Purified Mitochondrial and Cytosolic Aldehyde Dehydrogenases*

    PubMed Central

    Beretta, Matteo; Gruber, Karl; Kollau, Alexander; Russwurm, Michael; Koesling, Doris; Goessler, Walter; Keung, Wing Ming; Schmidt, Kurt; Mayer, Bernd

    2008-01-01

    Metabolism of nitroglycerin (GTN) to 1,2-glycerol dinitrate (GDN) and nitrite by mitochondrial aldehyde dehydrogenase (ALDH2) is essentially involved in GTN bioactivation resulting in cyclic GMP-mediated vascular relaxation. The link between nitrite formation and activation of soluble guanylate cyclase (sGC) is still unclear. To test the hypothesis that the ALDH2 reaction is sufficient for GTN bioactivation, we measured GTN-induced formation of cGMP by purified sGC in the presence of purified ALDH2 and used a Clark-type electrode to probe for nitric oxide (NO) formation. In addition, we studied whether GTN bioactivation is a specific feature of ALDH2 or is also catalyzed by the cytosolic isoform (ALDH1). Purified ALDH1 and ALDH2 metabolized GTN to 1,2- and 1,3-GDN with predominant formation of the 1,2-isomer that was inhibited by chloral hydrate (ALDH1 and ALDH2) and daidzin (ALDH2). GTN had no effect on sGC activity in the presence of bovine serum albumin but caused pronounced cGMP accumulation in the presence of ALDH1 or ALDH2. The effects of the ALDH isoforms were dependent on the amount of added protein and, like 1,2-GDN formation, were sensitive to ALDH inhibitors. GTN caused biphasic sGC activation with apparent EC50 values of 42 ± 2.9 and 3.1 ± 0.4 μm in the presence of ALDH1 and ALDH2, respectively. Incubation of ALDH1 or ALDH2 with GTN resulted in sustained, chloral hydrate-sensitive formation of NO. These data may explain the coupling of ALDH2-catalyzed GTN metabolism to sGC activation in vascular smooth muscle. PMID:18450747

  4. Biological evaluation and 3D-QSAR studies of curcumin analogues as aldehyde dehydrogenase 1 inhibitors.

    PubMed

    Wang, Hui; Du, Zhiyun; Zhang, Changyuan; Tang, Zhikai; He, Yan; Zhang, Qiuyan; Zhao, Jun; Zheng, Xi

    2014-01-01

    Aldehyde dehydrogenase 1 (ALDH1) is reported as a biomarker for identifying some cancer stem cells, and down-regulation or inhibition of the enzyme can be effective in anti-drug resistance and a potent therapeutic for some tumours. In this paper, the inhibitory activity, mechanism mode, molecular docking and 3D-QSAR (three-dimensional quantitative structure activity relationship) of curcumin analogues (CAs) against ALDH1 were studied. Results demonstrated that curcumin and CAs possessed potent inhibitory activity against ALDH1, and the CAs compound with ortho di-hydroxyl groups showed the most potent inhibitory activity. This study indicates that CAs may represent a new class of ALDH1 inhibitor. PMID:24840575

  5. Aldehyde Dehydrogenase 1a1 Mediates a GABA Synthesis Pathway in Midbrain Dopaminergic Neurons

    PubMed Central

    Kim, Jae-Ick; Ganesan, Subhashree; Luo, Sarah X.; Wu, Yu-Wei; Park, Esther; Huang, Eric J.; Chen, Lu; Ding, Jun B.

    2016-01-01

    Midbrain dopamine neurons are an essential component of the basal ganglia circuitry, playing key roles in the control of fine movement and reward. Recently, it has been demonstrated that γ-aminobutyric acid (GABA), the chief inhibitory neurotransmitter, is co-released by dopamine neurons. Here we show that GABA corelease in dopamine neurons does not utilize the conventional GABA synthesizing enzymes, glutamate decarboxylases GAD65 and GAD67. Our experiments reveal an evolutionarily conserved GABA synthesis pathway mediated by aldehyde dehydrogenase 1a1 (ALDH1a1). Moreover, GABA co-release is modulated by ethanol at binge drinking blood alcohol concentrations and diminished ALDH1a1 leads to enhanced alcohol consumption and preference. These findings provide insights into the functional role of GABA co-release in midbrain dopamine neurons, which may be essential for reward-based behavior and addiction. PMID:26430123

  6. Substrate specificity, substrate channeling, and allostery in BphJ: an acylating aldehyde dehydrogenase associated with the pyruvate aldolase BphI.

    PubMed

    Baker, Perrin; Carere, Jason; Seah, Stephen Y K

    2012-06-01

    BphJ, a nonphosphorylating acylating aldehyde dehydrogenase, catalyzes the conversion of aldehydes to form acyl-coenzyme A in the presence of NAD(+) and coenzyme A (CoA). The enzyme is structurally related to the nonacylating aldehyde dehydrogenases, aspartate-β-semialdehyde dehydrogenase and phosphorylating glyceraldehyde-3-phosphate dehydrogenase. Cys-131 was identified as the catalytic thiol in BphJ, and pH profiles together with site-specific mutagenesis data demonstrated that the catalytic thiol is not activated by an aspartate residue, as previously proposed. In contrast to the wild-type enzyme that had similar specificities for two- or three-carbon aldehydes, an I195A variant was observed to have a 20-fold higher catalytic efficiency for butyraldehyde and pentaldehyde compared to the catalytic efficiency of the wild type toward its natural substrate, acetaldehyde. BphJ forms a heterotetrameric complex with the class II aldolase BphI that channels aldehydes produced in the aldol cleavage reaction to the dehydrogenase via a molecular tunnel. Replacement of Ile-171 and Ile-195 with bulkier amino acid residues resulted in no more than a 35% reduction in acetaldehyde channeling efficiency, showing that these residues are not critical in gating the exit of the channel. Likewise, the replacement of Asn-170 in BphJ with alanine and aspartate did not substantially alter aldehyde channeling efficiencies. Levels of activation of BphI by BphJ N170A, N170D, and I171A were reduced by ≥3-fold in the presence of NADH and ≥4.5-fold when BphJ was undergoing turnover, indicating that allosteric activation of the aldolase has been compromised in these variants. The results demonstrate that the dehydrogenase coordinates the catalytic activity of BphI through allostery rather than through aldehyde channeling. PMID:22574886

  7. Geometric specificity of alcohol dehydrogenases and its potential for separation of trans and cis isomers of unsaturated aldehydes.

    PubMed Central

    Klibanov, A M; Giannousis, P P

    1982-01-01

    The geometric specificity of three different alcohol dehydrogenases (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) (from yeast, from horse liver, and from Leuconostoc mesenteroides) in the reduction of trans- and cis-cinnamaldehydes has been investigated. All three enzymes display a remarkable trans specificity: they react with the trans isomer 7 to 647 times faster than with its cis counterpart. Experiments with the enzymatic reduction of 3-phenylpropionaldehyde, a saturated analog of cinnamaldehyde, have revealed that whereas trans-cinnamaldehyde possesses the "right" configuration for the active centers of the alcohol dehydrogenases, the cis isomer apparently does not fit the active centers well. All three alcohol dehydrogenases studied also exhibit a marked trans specificity in the reaction with alpha-methylcinnamaldehyde. The geometric specificity of alcohol dehydrogenases can be used for the production of otherwise hard to synthesize cis isomers of unsaturated aldehydes from their readily available trans counterparts: trans-cinnamaldehyde was irradiated with ultraviolet light (which converted it to a mixture of trans and cis isomers) then treated with NADH and yeast alcohol dehydrogenase (which selectively reduces only trans aldehyde into the alcohol), and finally the mixture of cis-cinnamaldehyde and trans-cinnamyl alcohol was separated easily by preparative column chromatography. PMID:7048306

  8. Prognostic values of aldehyde dehydrogenase 1 isoenzymes in ovarian cancer

    PubMed Central

    Ma, Yu-mei; Zhao, Shan

    2016-01-01

    Aldehyde dehydrogenase 1 (ALDH1) activity has been used as a functional stem cell marker to isolate cancer stem cells in different cancer types, including ovarian cancer. However, which ALDH1’s isoenzymes are contributing to ALDH1 activity in ovarian cancer remains elusive. In addition, the prognostic value of an individual ALDH1 isoenzyme in ovarian cancer is not clear. Thus, we accessed the prognostic value of ALDH1 isoenzymes in ovarian cancer patients through the “Kaplan–Meier plotter” online database, which can be used to determine the effect of the genes on ovarian cancer prognosis. We found that high mRNA expression of five ALDH1 isoenzymes, such as ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, and ALDH1L1, was not correlated with overall survival (OS) for all 1,306 ovarian cancer patients. In addition, all five of the ALDH1 isoenzymes’ high mRNA expression was found to be uncorrelated with OS in serous cancer or endometrioid cancer patients. However, ALDH1A3’s high mRNA expression is associated with worse OS in grade II ovarian cancer patients, hazard ratio (HR) 1.53 (1.14–2.07), P=0.005. ALDH1A2’s high mRNA expression is significantly associated with worse OS in TP53 wild-type ovarian cancer patients, HR 2.86 (1.56–5.08), P=0.00036. In addition, ALDH1A3’s high mRNA expression is significantly associated with better OS in TP53 wild-type ovarian cancer patients, HR 0.56 (0.32–1.00), P=0.04. Our results indicate that although ALDH1 isoenzyme mRNA might not be a prognostic marker for overall ovarian cancer patients, some isoenzymes, such as ALDH1A2 and ALDH1A3, might be a good prognostic marker for some types of ovarian cancer patients. PMID:27110126

  9. Cloning and heterologous expression of two aryl-aldehyde dehydrogenases from the white-rot basidiomycete Phanerochaete chrysosporium

    SciTech Connect

    Nakamura, Tomofumi; Ichinose, Hirofumi; Wariishi, Hiroyuki

    2010-04-09

    We identified two aryl-aldehyde dehydrogenase proteins (PcALDH1 and PcALDH2) from the white-rot basidiomycete Phanerochaete chrysosporium. Both PcALDHs were translationally up-regulated in response to exogenous addition of vanillin, one of the key aromatic compounds in the pathway of lignin degradation by basidiomycetes. To clarify the catalytic functions of PcALDHs, we isolated full-length cDNAs encoding these proteins and heterologously expressed the recombinant enzymes using a pET/Escherichia coli system. The open reading frames of both PcALDH1 and PcALDH2 consisted of 1503 nucleotides. The deduced amino acid sequences of both proteins showed high homologies with aryl-aldehyde dehydrogenases from other organisms and contained ten conserved domains of ALDHs. Moreover, a novel glycine-rich motif 'GxGxxxG' was located at the NAD{sup +}-binding site. The recombinant PcALDHs catalyzed dehydrogenation reactions of several aryl-aldehyde compounds, including vanillin, to their corresponding aromatic acids. These results strongly suggested that PcALDHs metabolize aryl-aldehyde compounds generated during fungal degradation of lignin and various aromatic xenobiotics.

  10. Androgen regulation of aldehyde dehydrogenase 1A3 (ALDH1A3) in androgen responsive human prostate cancer cell LNCaP.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous gene array data from our laboratory identified the retinoic acid (RA) biosynthesis enzyme aldehyde dehydrogenase 1A3 (ALDH1A3) as a putative androgen-responsive gene in prostate cancer epithelial cells (LNCaP). In the present study we attempted to identify if any of the three ALDH1A/RA synt...

  11. Molecular abnormality of an inactive aldehyde dehydrogenase variant commonly found in Orientals.

    PubMed Central

    Yoshida, A; Huang, I Y; Ikawa, M

    1984-01-01

    Usual human livers contain two major aldehyde dehydrogenase [(ALDH) aldehyde:NAD+ oxidoreductase] isozymes--i.e., a cytosolic ALDH1 component and a mitochondrial ALDH2 component--whereas approximately equal to 50% of Orientals are "atypical" and have only the ALDH1 isozyme and are missing the ALDH2 isozyme. We previously demonstrated that atypical livers contain an enzymatically inactive but immunologically crossreactive material (CRM) corresponding to the ALDH2 component. The enzymatically active ALDH2 obtained from a usual liver and the CRM obtained from an atypical liver were reduced, S-carboxymethylated, and digested by trypsin. Separation of their digests by high-performance reverse-phase chromatography and by two-dimensional paper chromatography and electrophoresis revealed that ALDH2 contained a peptide sequence of -Glu-Leu-Gly-Glu-Ala-Gly-Leu-Gln-Ala-Asn-Val-Gln-Val-Lys- and that the glutamine adjacent to lysine was substituted by lysine in CRM. All other tryptic peptides, including eight peptides containing S-carboxymethylcysteine, were common in ALDH2 and CRM. It is concluded that a point mutation in the human ALDH2 locus produced the glutamine leads to lysine substitution and enzyme inactivation. Images PMID:6582480

  12. IN VITRO ORGANIC NITRATE BIOACTIVATION TO NITRIC OXIDE BY RECOMBINANT ALDEHYDE DEHYDROGENASE 3A1

    PubMed Central

    Lin, Shunxin; Page, Nathaniel A.; Fung, Sun Mi; Fung, Ho-Leung

    2013-01-01

    Organic nitrates (ORNs) are commonly used anti-ischemic and anti-anginal agents, which serve as an exogenous source of the potent vasodilator nitric oxide (NO). Recently, both mitochondrial aldehyde dehydrogenase-2 (ALDH2) and cytosolic aldehyde dehydrogenase-1a1 (ALDH1A1) have been shown to exhibit the ability to selectively bioactivate various ORNs in vitro. The objective of the present research was to examine the potential role of ALDH3A1, another major cytosolic isoform of ALDH, in the in vitro bioactivation of various ORNs, and to estimate the enzyme kinetic parameters toward ORNs through mechanistic modeling. The extent of bioactivation was assayed by exposing recombinant ALDH3A1 to various concentrations of ORNs, and measuring the concentration-time profiles of released NO via a NO-specific electrode. Metabolite formation kinetics was monitored for nitroglycerin (NTG) using LC/MS/MS. Our results showed that ALDH3A1 mRNA and protein were highly expressed in C57BL/6 mouse aortic, cardiac, and hepatic tissues, and it was able to release NO from several ORNs, including NTG, isosorbide dinitrate (ISDN), isosorbide-2-mononitrate (IS-2-MN), and nicorandil with similar Vmax (0.175 – 0.503 nmol/min/mg of ALDH3A1), and Km values of 4.01, 46.5, 818 and 5.75 × 103 μM respectively. However, activation of isosorbide-5-mononitrate (IS-5-MN) by ALDH3A1 was undetectable in vitro. ALDH3A1 was also shown to denitrate NTG, producing primarily glyceryl 1, 2-dinitrate (1, 2-GDN) in preference to glyceryl 1, 3-dinitrate (1, 3-GDN). Therefore, ALDH3A1 may contribute to the bioactivation of ORNs in vivo. PMID:24126018

  13. Alcohol dehydrogenases from Scheffersomyces stipitis involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion.

    PubMed

    Ma, Menggen; Wang, Xu; Zhang, Xiaoping; Zhao, Xianxian

    2013-09-01

    Aldehyde inhibitors such as furfural and 5-hydroxymethylfurfural (HMF) are generated from biomass pretreatment. Scheffersomyces stipitis is able to reduce furfural and HMF to less toxic furanmethanol and furan-2,5-dimethanol; however, the enzymes involved in the reductive reaction still remain unknown. In this study, transcription responses of two known and five putative alcohol dehydrogenase genes from S. stipitis were analyzed under furfural and HMF stress conditions. All the seven alcohol dehydrogenase genes were also cloned and overexpressed for their activity analyses. Our results indicate that transcriptions of SsADH4 and SsADH6 were highly induced under furfural and HMF stress conditions, and the proteins encoded by them exhibited NADH- and/or NADPH-dependent activities for furfural and HMF reduction, respectively. For furfural reduction, NADH-dependent activity was also observed in SsAdh1p and NAD(P)H-dependent activities were also observed in SsAdh5p and SsAdh7p. For HMF reduction, NADPH-dependent activities were also observed in SsAdh5p and SsAdh7p. SsAdh4p displayed the highest NADPH-dependent specific activity and catalytic efficiency for reduction of both furfural and HMF among the seven alcohol dehydrogenases. Enzyme activities of all SsADH proteins were more stable under acidic condition. For most SsADH proteins, the optimum temperature for enzyme activities was 30 °C and more than 50 % enzyme activities remained at 60 °C. Reduction activities of formaldehyde, acetaldehyde, isovaleraldehyde, benzaldehyde, and phenylacetaldehyde were also observed in some SsADH proteins. Our results indicate that multiple alcohol dehydrogenases in S. stipitis are involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion. PMID:23912116

  14. Role of disulfiram in the in vitro inhibition of rat liver mitochondrial aldehyde dehydrogenase.

    PubMed

    Shen, M L; Lipsky, J J; Naylor, S

    2000-10-01

    The alcohol aversion therapy drug disulfiram has been shown to inhibit hepatic aldehyde dehydrogenase (ALDH), one of the key enzymes involved in ethanol metabolism. It is believed by some that disulfiram could be one of the active inhibitors in vivo. However, the actual interaction between disulfiram and ALDH remains ambiguous. We report here that when disulfiram inhibited recombinant rat liver mitochondrial ALDH (rlmALDH) in vitro, no significant molecular mass increase was detected during the first 30 min as determined by on-line HPLC-electrospray ionization mass spectrometry (LC-MS). This indicated that the inhibition in vitro was not caused directly by covalent adduct formation on the enzyme. We subsequently subjected both control and disulfiram-inhibited rlmALDH to Glu-C proteolytic digestion. LC-MS analysis of the Glu-C digestion of disulfiram-inhibited enzyme revealed that one peptide of M(r) = 4821, which contained the putative active site of the enzyme, exhibited a mass decrease of 2 amu as compared with the same peptide found in the Glu-C digestion of the control (M(r) = 4823). We believe that the loss of 2 amu indicated that inhibition of rlmALDH in vitro was due to formation of an intramolecular disulfide bond between two of the three adjacent cysteines in the active site, possibly via a very rapid and unstable mixed disulfide interchange reaction. Further confirmation of the intramolecular disulfide bond formation came from the fact that by adding dithiothreitol (DTT) we were able to recover partial enzyme activity. In addition, the peptide of M(r) = 4821 observed in the Glu-C digestion of the disulfiram-treated ALDH reverted to M(r) = 4823 after treatment with DTT, which indicated that the disulfide bond was reduced. We, thereby, conclude that disulfiram inhibited rlmALDH by forming an intramolecular disulfide, possibly via a fast intermolecular disulfiram interchange reaction. PMID:10974203

  15. High current density PQQ-dependent alcohol and aldehyde dehydrogenase bioanodes.

    PubMed

    Aquino Neto, Sidney; Hickey, David P; Milton, Ross D; De Andrade, Adalgisa R; Minteer, Shelley D

    2015-10-15

    In this paper, we explore the bioelectrooxidation of ethanol using pyrroloquinoline quinone (PQQ)-dependent alcohol and aldehyde dehydrogenase (ADH and AldDH) enzymes for biofuel cell applications. The bioanode architectures were designed with both direct electron transfer (DET) and mediated electron transfer (MET) mechanisms employing high surface area materials such as multi-walled carbon nanotubes (MWCNTs) and MWCNT-decorated gold nanoparticles, along with different immobilization techniques. Three different polymeric matrices were tested (tetrabutyl ammonium bromide (TBAB)-modified Nafion; octyl-modified linear polyethyleneimine (C8-LPEI); and cellulose) in the DET studies. The modified Nafion membrane provided the best electrical communication between enzymes and the electrode surface, with catalytic currents as high as 16.8 ± 2.1 µA cm(-2). Then, a series of ferrocene redox polymers were evaluated for MET. The redox polymer 1,1'-dimethylferrocene-modified linear polyethyleneimine (FcMe2-C3-LPEI) provided the best electrochemical response. Using this polymer, the electrochemical assays conducted in the presence of MWCNTs and MWCNTs-Au indicated a Jmax of 781 ± 59 µA cm(-2) and 925 ± 68 µA cm(-2), respectively. Overall, from the results obtained here, DET using the PQQ-dependent ADH and AldDH still lacks high current density, while the bioanodes that operate via MET employing ferrocene-modified LPEI redox polymers show efficient energy conversion capability in ethanol/air biofuel cells. PMID:25988787

  16. Aldehyde Dehydrogenase Inhibitors: a Comprehensive Review of the Pharmacology, Mechanism of Action, Substrate Specificity, and Clinical Application

    PubMed Central

    Koppaka, Vindhya; Thompson, David C.; Chen, Ying; Ellermann, Manuel; Nicolaou, Kyriacos C.; Juvonen, Risto O.; Petersen, Dennis; Deitrich, Richard A.; Hurley, Thomas D.

    2012-01-01

    Aldehyde dehydrogenases (ALDHs) belong to a superfamily of enzymes that play a key role in the metabolism of aldehydes of both endogenous and exogenous derivation. The human ALDH superfamily comprises 19 isozymes that possess important physiological and toxicological functions. The ALDH1A subfamily plays a pivotal role in embryogenesis and development by mediating retinoic acid signaling. ALDH2, as a key enzyme that oxidizes acetaldehyde, is crucial for alcohol metabolism. ALDH1A1 and ALDH3A1 are lens and corneal crystallins, which are essential elements of the cellular defense mechanism against ultraviolet radiation-induced damage in ocular tissues. Many ALDH isozymes are important in oxidizing reactive aldehydes derived from lipid peroxidation and thereby help maintain cellular homeostasis. Increased expression and activity of ALDH isozymes have been reported in various human cancers and are associated with cancer relapse. As a direct consequence of their significant physiological and toxicological roles, inhibitors of the ALDH enzymes have been developed to treat human diseases. This review summarizes known ALDH inhibitors, their mechanisms of action, isozyme selectivity, potency, and clinical uses. The purpose of this review is to 1) establish the current status of pharmacological inhibition of the ALDHs, 2) provide a rationale for the continued development of ALDH isozyme-selective inhibitors, and 3) identify the challenges and potential therapeutic rewards associated with the creation of such agents. PMID:22544865

  17. Neurodegeneration and motor dysfunction in mice lacking cytosolic and mitochondrial aldehyde dehydrogenases: implications for Parkinson's disease.

    PubMed

    Wey, Margaret Chia-Ying; Fernandez, Elizabeth; Martinez, Paul Anthony; Sullivan, Patricia; Goldstein, David S; Strong, Randy

    2012-01-01

    Previous studies have reported elevated levels of biogenic aldehydes in the brains of patients with Parkinson's disease (PD). In the brain, aldehydes are primarily detoxified by aldehyde dehydrogenases (ALDH). Reduced ALDH1 expression in surviving midbrain dopamine neurons has been reported in brains of patients who died with PD. In addition, impaired complex I activity, which is well documented in PD, reduces the availability of the NAD(+) co-factor required by multiple ALDH isoforms to catalyze the removal of biogenic aldehydes. We hypothesized that chronically decreased function of multiple aldehyde dehydrogenases consequent to exposure to environmental toxins and/or reduced ALDH expression, plays an important role in the pathophysiology of PD. To address this hypothesis, we generated mice null for Aldh1a1 and Aldh2, the two isoforms known to be expressed in substantia nigra dopamine neurons. Aldh1a1(-/-)×Aldh2(-/-) mice exhibited age-dependent deficits in motor performance assessed by gait analysis and by performance on an accelerating rotarod. Intraperitoneal administration of L-DOPA plus benserazide alleviated the deficits in motor performance. We observed a significant loss of neurons immunoreactive for tyrosine hydroxylase (TH) in the substantia nigra and a reduction of dopamine and metabolites in the striatum of Aldh1a1(-/-)×Aldh2(-/-) mice. We also observed significant increases in biogenic aldehydes reported to be neurotoxic, including 4-hydroxynonenal (4-HNE) and the aldehyde intermediate of dopamine metabolism, 3,4-dihydroxyphenylacetaldehyde (DOPAL). These results support the hypothesis that impaired detoxification of biogenic aldehydes may be important in the pathophysiology of PD and suggest that Aldh1a1(-/-)×Aldh2(-/-) mice may be a useful animal model of PD. PMID:22384032

  18. Human Aldehyde Dehydrogenase Genes: Alternatively-Spliced Transcriptional Variants and Their Suggested Nomenclature

    PubMed Central

    Black, William J.; Stagos, Dimitrios; Marchitti, Satori A.; Nebert, Daniel W.; Tipton, Keith F.; Bairoch, Amos; Vasiliou, Vasilis

    2011-01-01

    OBJECTIVE The human aldehyde dehydrogenase (ALDH) gene superfamily consists of 19 genes encoding enzymes critical for NAD(P)+-dependent oxidation of endogenous and exogenous aldehydes, including drugs and environmental toxicants. Mutations in ALDH genes are the molecular basis of several disease states (e.g. Sjögren-Larsson syndrome, pyridoxine-dependent seizures, and type II hyperprolinemia) and may contribute to the etiology of complex diseases such as cancer and Alzheimer’s disease. The aim of this nomenclature update was to identify splice transcriptional variants principally for the human ALDH genes. METHODS Data-mining methods were used to retrieve all human ALDH sequences. Alternatively-spliced transcriptional variants were determined based upon: a) criteria for sequence integrity and genomic alignment; b) evidence of multiple independent cDNA sequences corresponding to a variant sequence; and c) if available, empirical evidence of variants from the literature. RESULTS AND CONCLUSION Alternatively-spliced transcriptional variants and their encoded proteins exist for most of the human ALDH genes; however, their function and significance remain to be established. When compared with the human genome, rat and mouse include an additional gene, Aldh1a7, in the ALDH1A subfamily. In order to avoid confusion when identifying splice variants in various genomes, nomenclature guidelines for the naming of such alternative transcriptional variants and proteins are recommended herein. In addition, a web database (www.aldh.org) has been developed to provide up-to-date information and nomenclature guidelines for the ALDH superfamily. PMID:19823103

  19. Aldehyde Dehydrogenase Gene Superfamily in Populus: Organization and Expression Divergence between Paralogous Gene Pairs

    PubMed Central

    Tian, Feng-Xia; Zang, Jian-Lei; Wang, Tan; Xie, Yu-Li; Zhang, Jin; Hu, Jian-Jun

    2015-01-01

    Aldehyde dehydrogenases (ALDHs) constitute a superfamily of NAD(P)+-dependent enzymes that catalyze the irreversible oxidation of a wide range of reactive aldehydes to their corresponding nontoxic carboxylic acids. ALDHs have been studied in many organisms from bacteria to mammals; however, no systematic analyses incorporating genome organization, gene structure, expression profiles, and cis-acting elements have been conducted in the model tree species Populus trichocarpa thus far. In this study, a comprehensive analysis of the Populus ALDH gene superfamily was performed. A total of 26 Populus ALDH genes were found to be distributed across 12 chromosomes. Genomic organization analysis indicated that purifying selection may have played a pivotal role in the retention and maintenance of PtALDH gene families. The exon-intron organizations of PtALDHs were highly conserved within the same family, suggesting that the members of the same family also may have conserved functionalities. Microarray data and qRT-PCR analysis indicated that most PtALDHs had distinct tissue-specific expression patterns. The specificity of cis-acting elements in the promoter regions of the PtALDHs and the divergence of expression patterns between nine paralogous PtALDH gene pairs suggested that gene duplications may have freed the duplicate genes from the functional constraints. The expression levels of some ALDHs were up- or down-regulated by various abiotic stresses, implying that the products of these genes may be involved in the adaptation of Populus to abiotic stresses. Overall, the data obtained from our investigation contribute to a better understanding of the complexity of the Populus ALDH gene superfamily and provide insights into the function and evolution of ALDH gene families in vascular plants. PMID:25909656

  20. Suppressing glioblastoma stem cell function by aldehyde dehydrogenase inhibition with chloramphenicol or disulfiram as a new treatment adjunct: an hypothesis.

    PubMed

    Kast, Richard E; Belda-Iniesta, Cristobal

    2009-12-01

    Strong expression of aldehyde dehydrogenase is a prominent feature of both normal and cancer stem cells, including the stem cell sub-population of glioblastoma. Aldehyde dehydrogenase function is used by cancer stem cells to repopulate a tumor mass after chemotherapy cytoreduction. Cancer stem cells tend to be chemotherapy compared to the non-stem cell majority cell population in several common human cancers. Such has been demonstrated specifically in glioblastoma. In normal hematopoietic stem cells with unimpaired high levels of aldehyde dehydrogenase, stem cells divide rarely and then asymmetrically to a daughter stem cell and a daughter cell on a path of differentiation or symmetrically with both daughter cells on a differentiated path. If a parallel situation obtains in glioblastoma stem cells, the migrating, far flung paucicellular extensions will be stem cell rich and use aldehyde dehydrogenase to generate the characteristic multiple metastases made up of mostly non-stem cells. With inhibition of aldehyde dehydrogenase, stem cell division to non-stem daughter cells tends to become blocked. We have three old yet potent aldehyde dehydrogenase inhibitors on the market- chloral hydrate, chloramphenicol, and disulfiram- they should be investigated as adjuncts in glioblastoma chemotherapy. If GBM stem cell function can be thwarted by potent aldehyde dehydrogenase inhibition, they will be less able to regenerate a stem cell derived tumor mass after primary resection or chemotherapy. PMID:19500061

  1. Simultaneous Involvement of a Tungsten-Containing Aldehyde:Ferredoxin Oxidoreductase and a Phenylacetaldehyde Dehydrogenase in Anaerobic Phenylalanine Metabolism

    PubMed Central

    Debnar-Daumler, Carlotta; Seubert, Andreas; Schmitt, Georg

    2014-01-01

    Anaerobic phenylalanine metabolism in the denitrifying betaproteobacterium Aromatoleum aromaticum is initiated by conversion of phenylalanine to phenylacetate, which is further metabolized via benzoyl-coenzyme A (CoA). The formation of phenylacetate is catalyzed by phenylalanine transaminase, phenylpyruvate decarboxylase, and a phenylacetaldehyde-oxidizing enzyme. The presence of these enzymes was detected in extracts of cells grown with phenylalanine and nitrate. We found that two distinct enzymes are involved in the oxidation of phenylacetaldehyde to phenylacetate, an aldehyde:ferredoxin oxidoreductase (AOR) and a phenylacetaldehyde dehydrogenase (PDH). Based on sequence comparison, growth studies with various tungstate concentrations, and metal analysis of the enriched enzyme, AOR was shown to be a tungsten-containing enzyme, necessitating specific cofactor biosynthetic pathways for molybdenum- and tungsten-dependent enzymes simultaneously. We predict from the genome sequence that most enzymes of molybdopterin biosynthesis are shared, while the molybdate/tungstate uptake systems are duplicated and specialized paralogs of the sulfur-inserting MoaD and the metal-inserting MoeA proteins seem to be involved in dedicating biosynthesis toward molybdenum or tungsten cofactors. We also characterized PDH biochemically and identified both NAD+ and NADP+ as electron acceptors. We identified the gene coding for the enzyme and purified a recombinant Strep-tagged PDH variant. The homotetrameric enzyme is highly specific for phenylacetaldehyde, has cooperative kinetics toward the substrate, and shows considerable substrate inhibition. Our data suggest that A. aromaticum utilizes PDH as the primary enzyme during anaerobic phenylalanine degradation, whereas AOR is not essential for the metabolic pathway. We hypothesize a function as a detoxifying enzyme if high aldehyde concentrations accumulate in the cytoplasm, which would lead to substrate inhibition of PDH. PMID:24214948

  2. Ectopic overexpression of the aldehyde dehydrogenase ALDH21 from Syntrichia caninervis in tobacco confers salt and drought stress tolerance.

    PubMed

    Yang, Honglan; Zhang, Daoyuan; Li, Haiyan; Dong, Lingfeng; Lan, Haiyan

    2015-10-01

    Aldehyde dehydrogenases are important enzymes that play vital roles in mitigating oxidative/electrophilic stress when plants are exposed to environmental stress. An aldehyde dehydrogenase gene from Syntrichia caninervis, ScALDH21, was introduced into tobacco using Agrobacterium-mediated transformation to generate ScALDH21-overexpressing tobacco plants to investigate its effect on drought and salt resistance. Detached leaves from ScALDH21-overexpressing tobacco plants showed less water loss than those from nontransgenic plants. When subjected to drought and salt stress, transgenic plants displayed higher germination ratios, higher root lengths, greater fresh weight, higher proline accumulation, lower malondialdehyde (MDA) contents and stronger photosynthetic capacities, as well as higher activities of antioxidant enzymes, i.e., superoxide dismutase, catalase and peroxidase, compared with control plants. Therefore, ScALDH21 overexpression in transgenic tobacco plants can enhance drought and salt tolerance and can be used as a candidate gene for the molecular breeding of salt- and drought-tolerant plants. PMID:26202169

  3. Effect of various chemicals on the aldehyde dehydrogenase activity of the rat liver cytosol.

    PubMed

    Marselos, M; Vasiliou, V

    1991-01-01

    The cytosolic activity of aldehyde dehydrogenase (ALDH) was studied in the rat liver, after acute administration of various carcinogenic and chemically related compounds. Male Wistar rats were treated with 27 different chemicals, including polycyclic aromatic hydrocarbons, aromatic amines, nitrosamines, azo dyes, as well as with some known direct-acting carcinogens. The cytosolic ALDH activity of the liver was determined either with propionaldehyde and NAD (P/NAD), or with benzaldehyde and NADP (B/NADP). The activity of ALDH remained unaffected after treatment with 1-naphthylamine, nitrosamines and also with the direct-acting chemical carcinogens tested. On the contrary, polycyclic aromatic hydrocarbons, polychlorinated biphenyls (Arochlor 1254) and 2-naphthylamine produced a remarkable increase of ALDH. In general, the response to the effectors was disproportionate between the two types of enzyme activity, being much in favour for the B/NADP activity. This fact resulted to an inversion of the ratio B/NADP vs. P/NAD, which under constitutive conditions is lower than 1. In this respect, the most potent compounds were found to be polychlorinated biphenyls, 3-methylcholanthrene, benzo(a)pyrene and 1,2,5,6-dibenzoanthracene. Our results suggest that the B/NADP activity of the soluble ALDH is greatly induced after treatment with compounds possessing aromatic ring(s) in their molecule. It is not known, if this response of the hepatocytes is related with the process of chemical carcinogenesis. PMID:2060039

  4. Aldehyde dehydrogenase 2 is associated with cognitive functions in patients with Parkinson’s disease

    PubMed Central

    Yu, Rwei-Ling; Tan, Chun-Hsiang; Lu, Ying-Che; Wu, Ruey-Meei

    2016-01-01

    Neurotransmitter degradation has been proposed to cause the accumulation of neurotoxic metabolites. The metabolism of these metabolites involves aldehyde dehydrogenase 2 (ALDH2). The Asian-specific single nucleotide polymorphism rs671 causes reduced enzyme activity. This study aims to explore whether Parkinson’s disease (PD) patients with reduced ALDH2 activity owing to the rs671 polymorphism are at risk for neuropsychological impairments. A total of 139 PD patients were recruited. Each participant was assessed for medical characteristics and their ALDH2 genotype. The Mini-Mental State Examination (MMSE), the Clinical Dementia Rating Scale and the Frontal Behavioral Inventory were used to measure neuropsychological functions. We found that the MMSE scores were significantly lower in patients with inactive ALDH2 (U = 1873.5, p = 0.02). The presence of cognitive impairments was significantly more frequent in the inactive ALDH2 group (46.0%) than in the active ALDH2 group (26.3%) (χ2 = 5.886, p = 0.01). The inactive group showed significant deterioration in hobbies and exhibited more severe “disorganization” and “hyper-sexuality” behaviours. The additive effects of the allele on the development of cognitive impairments in PD patients may be an important finding that provides further insight into the pathogenic mechanism of cognitive dysfunction in PD. PMID:27453488

  5. Aldehyde dehydrogenase variation enhances effect of pesticides associated with Parkinson disease

    PubMed Central

    Fitzmaurice, Arthur G.; Rhodes, Shannon L.; Cockburn, Myles; Ritz, Beate

    2014-01-01

    Objective: The objective of this study was to determine whether environmental and genetic alterations of neuronal aldehyde dehydrogenase (ALDH) enzymes were associated with increased Parkinson disease (PD) risk in an epidemiologic study. Methods: A novel ex vivo assay was developed to identify pesticides that can inhibit neuronal ALDH activity. These were investigated for PD associations in a population-based case-control study, the Parkinson's Environment & Genes (PEG) Study. Common variants in the mitochondrial ALDH2 gene were genotyped to assess effect measure modification (statistical interaction) of the pesticide effects by genetic variation. Results: All of the metal-coordinating dithiocarbamates tested (e.g., maneb, ziram), 2 imidazoles (benomyl, triflumizole), 2 dicarboxymides (captan, folpet), and 1 organochlorine (dieldrin) inhibited ALDH activity, potentially via metabolic byproducts (e.g., carbon disulfide, thiophosgene). Fifteen screened pesticides did not inhibit ALDH. Exposures to ALDH-inhibiting pesticides were associated with 2- to 6-fold increases in PD risk; genetic variation in ALDH2 exacerbated PD risk in subjects exposed to ALDH-inhibiting pesticides. Conclusion: ALDH inhibition appears to be an important mechanism through which environmental toxicants contribute to PD pathogenesis, especially in genetically vulnerable individuals, suggesting several potential interventions to reduce PD occurrence or slow or reverse its progression. PMID:24491970

  6. Acyloin production from aldehydes in the perfused rat heart: the potential role of pyruvate dehydrogenase.

    PubMed Central

    Montgomery, J A; Jetté, M; Huot, S; Des Rosiers, C

    1993-01-01

    Aldehydes represent an important class of cytotoxic products derived from free radical-induced lipid peroxidation which may contribute to reperfusion injury following myocardial infarct. Metabolism of aldehydes in the heart has not been well characterized aside from conjugation of unsaturated aldehydes with glutathione. However, aliphatic aldehydes like hexanal do not form stable glutathione conjugates. We have recently demonstrated in vitro that pig heart pyruvate dehydrogenase catalyses a reaction between pyruvate and saturated aldehydes to produce acyloins (3-hydroxyalkan-2-ones). In the present study, rat hearts were perfused with various aldehydes and pyruvate. Acyloins were generated from saturated aldehydes (butanal, hexanal or nonanal), but not from 2-hexanal (an unsaturated aldehyde) or malondialdehyde. Hearts perfused with 2 mM pyruvate and 10-100 microM hexanal rapidly took up hexanal in a dose-related manner (140-850 nmol/min), and released 3-hydroxyoctan-2-one (0.7-30 nmol/min), 2,3-octanediol (0-12 nmol/min) and hexanol (10-200 nmol/min). Small quantities of hexanoic acid (about 10 nmol/min) were also released. The rate of release of acyloin metabolites rose with increased concentration of hexanal, whereas hexanol release attained a plateau when hexanal infusion concentrations rose above 50 microM. Up to 50% of hexanal uptake could be accounted for by metabolite release. Less than 0.5% of hexanal uptake was found to be bound to acid-precipitable macromolecules. When hearts perfused with 50 microM hexanal and 2 mM pyruvate were subjected to a 15 min ischaemic period, the rates of release of 2,3-octanediol, 3-hydroxyoctan-2-one, hexanol and hexanoate during the reperfusion period were not significantly different from those in the pre-ischaemic period. Our results indicate that saturated aldehydes can be metabolically converted by the heart into stable diffusible compounds. PMID:8379929

  7. Sjögren-Larsson syndrome. Deficient activity of the fatty aldehyde dehydrogenase component of fatty alcohol:NAD+ oxidoreductase in cultured fibroblasts.

    PubMed Central

    Rizzo, W B; Craft, D A

    1991-01-01

    Sjögren-Larsson syndrome (SLS) is an inherited disorder associated with impaired fatty alcohol oxidation due to deficient activity of fatty alcohol:NAD+ oxidoreductase (FAO). FAO is a complex enzyme which consists of two separate proteins that sequentially catalyze the oxidation of fatty alcohol to fatty aldehyde and fatty acid. To determine which enzymatic component of FAO was deficient in SLS, we assayed fatty aldehyde dehydrogenase (FALDH) and fatty alcohol dehydrogenase in cultured fibroblasts from seven unrelated SLS patients. All SLS cells were selectively deficient in the FALDH component of FAO, and had normal activity of fatty alcohol dehydrogenase. The extent of FALDH deficiency in SLS cells depended on the aliphatic aldehyde used as substrate, ranging from 62% of mean normal activity using propionaldehyde as substrate to 8% of mean normal activity with octadecanal. FALDH activity in obligate SLS heterozygotes was partially decreased to 49 +/- 7% of mean normal activity using octadecanal as substrate. Differential centrifugation studies in fibroblasts indicated that this FALDH enzyme was largely particulate; soluble FALDH activity was normal in SLS cells. Intact SLS fibroblasts oxidized octadecanol to fatty acid at less than 10% of the normal rate, but oxidized free octadecanal normally, suggesting that the FALDH affected in SLS is chiefly involved in the oxidation of fatty alcohol to fatty acid. These results show that the primary enzymatic defect in SLS is the FALDH component of the FAO complex, which leads to deficient oxidation of fatty aldehyde derived from fatty alcohol. PMID:1939650

  8. Aldehyde dehydrogenases inhibition eradicates leukemia stem cells while sparing normal progenitors.

    PubMed

    Venton, G; Pérez-Alea, M; Baier, C; Fournet, G; Quash, G; Labiad, Y; Martin, G; Sanderson, F; Poullin, P; Suchon, P; Farnault, L; Nguyen, C; Brunet, C; Ceylan, I; Costello, R T

    2016-01-01

    The vast majority of patients with acute myeloid leukemia (AML) achieve complete remission (CR) after standard induction chemotherapy. However, the majority subsequently relapse and die of the disease. A leukemia stem cell (LSC) paradigm has been invoked to explain this failure of CR to reliably translate into cure. Indeed, LSCs are highly enriched in CD34+CD38- leukemic cells that exhibit positive aldehyde dehydrogenase activity (ALDH+) on flow cytometry, these LSCs are resistant to currently existing treatments in AML such as cytarabine and anthracycline that, at the cost of great toxicity on normal cells, are highly active against the leukemic bulk, but spare the LSCs responsible for relapse. To try to combat the LSC population selectively, a well-characterized ALDH inhibitor by the trivial name of dimethyl ampal thiolester (DIMATE) was assessed on sorted CD34+CD38- subpopulations from AML patients and healthy patients. ALDH activity and cell viability were monitored by flow cytometry. From enzyme kinetic studies DIMATE is an active enzyme-dependent, competitive, irreversible inhibitor of ALDH1. On cells in culture, DIMATE is a powerful inhibitor of ALDHs 1 and 3, has a major cytotoxic activity on human AML cell lines. Moreover, DIMATE is highly active against leukemic populations enriched in LSCs, but, unlike conventional chemotherapy, DIMATE is not toxic for healthy hematopoietic stem cells which retained, after treatment, their self-renewing and multi-lineage differentiation capacity in immunodeficient mice, xenografted with human leukemic cells. DIMATE eradicates specifically human AML cells and spares healthy mouse hematologic cells. PMID:27611922

  9. Elucidating the contributions of multiple aldehyde/alcohol dehydrogenases to butanol and ethanol production in Clostridium acetobutylicum

    PubMed Central

    Dai, Zongjie; Dong, Hongjun; Zhang, Yanping; Li, Yin

    2016-01-01

    Ethanol and butanol biosynthesis in Clostridium acetobutylicum share common aldehyde/alcohol dehydrogenases. However, little is known about the relative contributions of these multiple dehydrogenases to ethanol and butanol production respectively. The contributions of six aldehyde/alcohol dehydrogenases of C. acetobutylicum on butanol and ethanol production were evaluated through inactivation of the corresponding genes respectively. For butanol production, the relative contributions from these enzymes were: AdhE1 > BdhB > BdhA ≈ YqhD > SMB_P058 > AdhE2. For ethanol production, the contributions were: AdhE1 > BdhB > YqhD > SMB_P058 > AdhE2 > BdhA. AdhE1 and BdhB are two essential enzymes for butanol and ethanol production. AdhE1 was relatively specific for butanol production over ethanol, while BdhB, YqhD, and SMB_P058 favor ethanol production over butanol. Butanol synthesis was increased in the adhE2 mutant, which had a higher butanol/ethanol ratio (8.15:1) compared with wild type strain (6.65:1). Both the SMB_P058 mutant and yqhD mutant produced less ethanol without loss of butanol formation, which led to higher butanol/ethanol ratio, 10.12:1 and 10.17:1, respectively. To engineer a more efficient butanol-producing strain, adhE1 could be overexpressed, furthermore, adhE2, SMB_P058, yqhD are promising gene inactivation targets. This work provides useful information guiding future strain improvement for butanol production. PMID:27321949

  10. Elucidating the contributions of multiple aldehyde/alcohol dehydrogenases to butanol and ethanol production in Clostridium acetobutylicum.

    PubMed

    Dai, Zongjie; Dong, Hongjun; Zhang, Yanping; Li, Yin

    2016-01-01

    Ethanol and butanol biosynthesis in Clostridium acetobutylicum share common aldehyde/alcohol dehydrogenases. However, little is known about the relative contributions of these multiple dehydrogenases to ethanol and butanol production respectively. The contributions of six aldehyde/alcohol dehydrogenases of C. acetobutylicum on butanol and ethanol production were evaluated through inactivation of the corresponding genes respectively. For butanol production, the relative contributions from these enzymes were: AdhE1 > BdhB > BdhA ≈ YqhD > SMB_P058 > AdhE2. For ethanol production, the contributions were: AdhE1 > BdhB > YqhD > SMB_P058 > AdhE2 > BdhA. AdhE1 and BdhB are two essential enzymes for butanol and ethanol production. AdhE1 was relatively specific for butanol production over ethanol, while BdhB, YqhD, and SMB_P058 favor ethanol production over butanol. Butanol synthesis was increased in the adhE2 mutant, which had a higher butanol/ethanol ratio (8.15:1) compared with wild type strain (6.65:1). Both the SMB_P058 mutant and yqhD mutant produced less ethanol without loss of butanol formation, which led to higher butanol/ethanol ratio, 10.12:1 and 10.17:1, respectively. To engineer a more efficient butanol-producing strain, adhE1 could be overexpressed, furthermore, adhE2, SMB_P058, yqhD are promising gene inactivation targets. This work provides useful information guiding future strain improvement for butanol production. PMID:27321949

  11. Comparative genomics of aldehyde dehydrogenase 5a1 (succinate semialdehyde dehydrogenase) and accumulation of gamma-hydroxybutyrate associated with its deficiency

    PubMed Central

    2009-01-01

    Succinic semialdehyde dehydrogenase (SSADH; aldehyde dehydrogenase 5A1 [ALDH5A1]; locus 6p22) occupies a central position in central nervous system (CNS) neurotransmitter metabolism as one of two enzymes necessary for γ-aminobutyric acid (GABA) recycling from the synaptic cleft. Its importance is highlighted by the neurometabolic disease associated with its inherited deficiency in humans, as well as the severe epileptic phenotype observed in Aldh5a1-/- knockout mice. Expanding evidence now suggests, however, that even subtle decreases in human SSADH activity, associated with rare and common single nucleotide polymorphisms, may produce subclinical pathological effects. SSADH, in conjunction with aldo-keto reductase 7A2 (AKR7A2), represent two neural enzymes responsible for further catabolism of succinic semialdehyde, producing either succinate (SSADH) or γ-hydroxybutyrate (GHB; AKR7A2). A GABA analogue, GHB is a short-chain fatty alcohol with unusual properties in the CNS and a long pharmacological history. Moreover, SSADH occupies a further role in the CNS as the enzyme responsible for further metabolism of the lipid peroxidation aldehyde 4-hydroxy-2-nonenal (4-HNE), an intermediate known to induce oxidant stress. Accordingly, subtle decreases in SSADH activity may have the capacity to lead to regional accumulation of neurotoxic intermediates (GHB, 4-HNE). Polymorphisms in SSADH gene structure may also associate with quantitative traits, including intelligence quotient and life expectancy. Further population-based studies of human SSADH activity promise to reveal additional properties of its function and additional roles in CNS tissue. PMID:19164088

  12. Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

    PubMed

    Stiti, Naim; Chandrasekar, Balakumaran; Strubl, Laura; Mohammed, Shabaz; Bartels, Dorothea; van der Hoorn, Renier A L

    2016-06-17

    Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes. PMID:26990764

  13. [Effects of panthenol and carnitine on aldehyde metabolic enzymes in rats with tetrachloromethane-induced liver injury].

    PubMed

    Satanovskaia, V I; Pron'ko, P S; Gaĭshmanova, A V; Miskevich, D A

    2009-01-01

    Tetrachloromethane (2 g/kg, intragastric) produced a decrease in the activity of NAD- and NADH- dependent aldehyde dehydrogenases with high Km for aldehydes in rat liver. Panthenol and L-carnitine administered separately normalized the activity of aldehyde dehydrogenases, while a combination of the drugs did not produce any significant effect. PMID:19441727

  14. Aldehyde Dehydrogenase 1A1: Friend or Foe to Female Metabolism?

    PubMed Central

    Petrosino, Jennifer M.; DiSilvestro, David; Ziouzenkova, Ouliana

    2014-01-01

    In this review, we summarize recent advances in understanding vitamin A-dependent regulation of sex-specific differences in metabolic diseases, inflammation, and certain cancers. We focus on the characterization of the aldehyde dehydrogenase-1 family of enzymes (ALDH1A1, ALDH1A2, ALDH1A3) that catalyze conversion of retinaldehyde to retinoic acid. Additionally, we propose a “horizontal transfer of signaling” from estrogen to retinoids through the action of ALDH1A1. Although estrogen does not directly influence expression of Aldh1a1, it has the ability to suppress Aldh1a2 and Aldh1a3, thereby establishing a female-specific mechanism for retinoic acid generation in target tissues. ALDH1A1 regulates adipogenesis, abdominal fat formation, glucose tolerance, and suppression of thermogenesis in adipocytes; in B cells, ALDH1A1 plays a protective role by inducing oncogene suppressors Rara and Pparg. Considering the conflicting responses of Aldh1a1 in a multitude of physiological processes, only tissue-specific regulation of Aldh1a1 can result in therapeutic effects. We have shown through successful implantation of tissue-specific Aldh1a1−/− preadipocytes that thermogenesis can be induced in wild-type adipose tissues to resolve diet-induced visceral obesity in females. We will briefly discuss the emerging role of ALDH1A1 in multiple myeloma, the regulation of reproduction, and immune responses, and conclude by discussing the role of ALDH1A1 in future therapeutic applications. PMID:24594504

  15. Aldehyde dehydrogenase-independent bioactivation of nitroglycerin in porcine and bovine blood vessels.

    PubMed

    Neubauer, Regina; Wölkart, Gerald; Opelt, Marissa; Schwarzenegger, Christine; Hofinger, Marielies; Neubauer, Andrea; Kollau, Alexander; Schmidt, Kurt; Schrammel, Astrid; Mayer, Bernd

    2015-02-15

    The vascular bioactivation of the antianginal drug nitroglycerin (GTN), yielding 1,2-glycerol dinitrate and nitric oxide or a related activator of soluble guanylate cyclase, is catalyzed by aldehyde dehydrogenase-2 (ALDH2) in rodent and human blood vessels. The essential role of ALDH2 has been confirmed in many studies and is considered as general principle of GTN-induced vasodilation in mammals. However, this view is challenged by an early report showing that diphenyleneiodonium, which we recently characterized as potent ALDH2 inhibitor, has no effect on GTN-induced relaxation of bovine coronary arteries (De La Lande et al., 1996). We investigated this issue and found that inhibition of ALDH2 attenuates GTN-induced coronary vasodilation in isolated perfused rat hearts but has no effect on relaxation to GTN of bovine and porcine coronary arteries. This observation is explained by low levels of ALDH2 protein expression in bovine coronary arteries and several types of porcine blood vessels. ALDH2 mRNA expression and the rates of GTN denitration were similarly low, excluding a significant contribution of ALDH2 to the bioactivation of GTN in these vessels. Attempts to identify the responsible pathway with enzyme inhibitors did not provide conclusive evidence for the involvement of ALDH3A1, cytochrome P450, or GSH-S-transferase. Thus, the present manuscript describes a hitherto unrecognized pathway of GTN bioactivation in bovine and porcine blood vessels. If present in the human vasculature, this pathway might contribute to the therapeutic effects of organic nitrates that are not metabolized by ALDH2. PMID:25576686

  16. Aldehyde dehydrogenase-independent bioactivation of nitroglycerin in porcine and bovine blood vessels

    PubMed Central

    Neubauer, Regina; Wölkart, Gerald; Opelt, Marissa; Schwarzenegger, Christine; Hofinger, Marielies; Neubauer, Andrea; Kollau, Alexander; Schmidt, Kurt; Schrammel, Astrid; Mayer, Bernd

    2015-01-01

    The vascular bioactivation of the antianginal drug nitroglycerin (GTN), yielding 1,2-glycerol dinitrate and nitric oxide or a related activator of soluble guanylate cyclase, is catalyzed by aldehyde dehydrogenase-2 (ALDH2) in rodent and human blood vessels. The essential role of ALDH2 has been confirmed in many studies and is considered as general principle of GTN-induced vasodilation in mammals. However, this view is challenged by an early report showing that diphenyleneiodonium, which we recently characterized as potent ALDH2 inhibitor, has no effect on GTN-induced relaxation of bovine coronary arteries (De La Lande et al., 1996). We investigated this issue and found that inhibition of ALDH2 attenuates GTN-induced coronary vasodilation in isolated perfused rat hearts but has no effect on relaxation to GTN of bovine and porcine coronary arteries. This observation is explained by low levels of ALDH2 protein expression in bovine coronary arteries and several types of porcine blood vessels. ALDH2 mRNA expression and the rates of GTN denitration were similarly low, excluding a significant contribution of ALDH2 to the bioactivation of GTN in these vessels. Attempts to identify the responsible pathway with enzyme inhibitors did not provide conclusive evidence for the involvement of ALDH3A1, cytochrome P450, or GSH-S-transferase. Thus, the present manuscript describes a hitherto unrecognized pathway of GTN bioactivation in bovine and porcine blood vessels. If present in the human vasculature, this pathway might contribute to the therapeutic effects of organic nitrates that are not metabolized by ALDH2. PMID:25576686

  17. Role of aldehyde dehydrogenase in hypoxic vasodilator effects of nitrite in rats and humans

    PubMed Central

    Arif, Sayqa; Borgognone, Alessandra; Lin, Erica Lai-Sze; O'Sullivan, Aine G; Sharma, Vishal; Drury, Nigel E; Menon, Ashvini; Nightingale, Peter; Mascaro, Jorge; Bonser, Robert S; Horowitz, John D; Feelisch, Martin; Frenneaux, Michael P; Madhani, Melanie

    2015-01-01

    Background and Purpose Hypoxic conditions favour the reduction of nitrite to nitric oxide (NO) to elicit vasodilatation, but the mechanism(s) responsible for bioconversion remains ill defined. In the present study, we assess the role of aldehyde dehydrogenase 2 (ALDH2) in nitrite bioactivation under normoxia and hypoxia in the rat and human vasculature. Experimental Approach The role of ALDH2 in vascular responses to nitrite was studied using rat thoracic aorta and gluteal subcutaneous fat resistance vessels from patients with heart failure (HF; 16 patients) in vitro and by measurement of changes in forearm blood flow (FBF) during intra-arterial nitrite infusion (21 patients) in vivo. Specifically, we investigated the effects of (i) ALDH2 inhibition by cyanamide or propionaldehyde and the (ii) tolerance-independent inactivation of ALDH2 by glyceryl trinitrate (GTN) on the vasodilator activity of nitrite. In each setting, nitrite effects were measured via evaluation of the concentration–response relationship under normoxic and hypoxic conditions in the absence or presence of ALDH2 inhibitors. Key Results Both in rat aorta and human resistance vessels, dilatation to nitrite was diminished following ALDH2 inhibition, in particular under hypoxia. In humans there was a non-significant trend towards attenuation of nitrite-mediated increases in FBF. Conclusions and Implications In human and rat vascular tissue in vitro, hypoxic nitrite-mediated vasodilatation involves ALDH2. In patients with HF in vivo, the role of this enzyme in nitrite bioactivation is at the most, modest, suggesting the involvement of other more important mechanisms. PMID:25754766

  18. Immunolocalization of betaine aldehyde dehydrogenase in porcine kidney.

    PubMed

    Figueroa-Soto, C G; Lopez-Cervantes, G; Valenzuela-Soto, E M

    1999-05-19

    Polyclonal anti-BADH serum was raised in rabbits against native BADH purified from porcine kidney. The antiserum cross-reacted strongly with BADH purified from kidney, Amaranthus palmierii, and Pseudomona aeuroginosa (1:1000), and weakly with Amaranthus hypochondriacus L (1:100). Antibodies bound to purified native kidney BADH in immunoblots showed a major band of an apparent molecular mass of 340 kDa and a subunit with an apparent molecular mass of 52 kDa. Data on activity assays showed higher activity in cortex sections (81.3 nmol/min/mg protein) than in medulla sections (21.3 nmol/min/mg protein). Immunolocalization of BADH in kidney tissue sections showed that BADH is found in cortex and medulla. In inner medulla, the enzyme was mainly localized in cells surrounding the tubules. Western blot analysis on extracts from the cortex and medulla sections showed higher concentration of BADH protein in cortex than in medulla. These results were in accordance with immunolocalization and activity analysis. PMID:10329454

  19. Enhanced aldehyde dehydrogenase activity by regenerating NAD+ in Klebsiella pneumoniae and implications for the glycerol dissimilation pathways.

    PubMed

    Li, Ying; Su, Mingyue; Ge, Xizhen; Tian, Pingfang

    2013-10-01

    In Klebsiella pneumoniae, 3-hydroxypropaldehyde is converted to 3-hydroxypropionic acid (3-HP) by aldehyde dehydrogenase (ALDH) with NAD(+) as a cofactor. Although ALDH overexpression stimulates the formation of 3-HP, it ceases to accumulate when NAD(+) is exhausted. Here we show that NAD(+) regeneration, together with ALDH overexpression, facilitates 3-HP production and benefits cell growth. Three distinct NAD(+)-regenerating enzymes: NADH oxidase and NADH dehydrogenase from K. pneumoniae, and glycerol-3-phosphate dehydrogenase (GPD1) from Saccharomyces cerevisiae, were individually expressed in K. pneumoniae. In vitro assay showed their higher activities than that of the control, indicating their capacities to regenerate NAD(+). When they were respectively co-expressed with ALD4, an ALDH from S. cerevisiae, the activities of ALD4 were significantly elevated compared with that expressing ALD4 alone, suggesting that the regenerated NAD(+) enhanced the activity of ALD4. More interestingly, the growth rates of all NAD(+)-regenerating strains were prolonged in comparison with the control, indicating that NAD(+) regeneration stimulated cell proliferation. This study not only reveals the reliance of ALD4 activity on NAD(+) availability but also provides a method for regulating the dha regulon. PMID:23794046

  20. Glu504Lys Single Nucleotide Polymorphism of Aldehyde Dehydrogenase 2 Gene and the Risk of Human Diseases

    PubMed Central

    Zhao, Yan; Wang, Chuancai

    2015-01-01

    Aldehyde dehydrogenase (ALDH) 2 is a mitochondrial enzyme that is known for its important role in oxidation and detoxification of ethanol metabolite acetaldehyde. ALDH2 also metabolizes other reactive aldehydes such as 4-hydroxy-2-nonenal and acrolein. The Glu504Lys single nucleotide polymorphism (SNP) of ALDH2 gene, which is found in approximately 40% of the East Asian populations, causes defect in the enzyme activity of ALDH2, leading to alterations in acetaldehyde metabolism and alcohol-induced “flushing” syndrome. Evidence suggests that ALDH2 Glu504Lys SNP is a potential candidate genetic risk factor for a variety of chronic diseases such as cardiovascular disease, cancer, and late-onset Alzheimer's disease. In addition, the association between ALDH2 Glu504Lys SNP and the development of these chronic diseases appears to be affected by the interaction between the SNP and lifestyle factors such as alcohol consumption as well as by the presence of other genetic variations. PMID:26491656

  1. Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae.

    PubMed

    Moon, Jaewoong; Liu, Z Lewis

    2015-04-01

    The aldehyde reductase gene ARI1 is a recently characterized member of an intermediate subfamily within the short-chain dehydrogenase/reductase (SDR) superfamily that clarified mechanisms of in situ detoxification of 2-furaldehyde and 5-hydroxymethyl-2-furaldehyde by Saccharomyces cerevisiae. Uncharacterized open reading frames (ORFs) are common among tolerant candidate genes identified for lignocellulose-to-advanced biofuels conversion. This study presents partially purified proteins of two ORFs, YDR541C and YGL039W, and direct enzyme assay evidence against aldehyde-inhibitory compounds commonly encountered during lignocellulosic biomass fermentation processes. Each of the partially purified proteins encoded by these ORFs showed a molecular mass of approximately 38 kDa, similar to Ari1p, a protein encoded by aldehyde reductase gene. Both proteins demonstrated strong aldehyde reduction activities toward 14 aldehyde substrates, with high levels of reduction activity for Ydr541cp toward both aromatic and aliphatic aldehydes. While Ydr541cp was observed to have a significantly higher specific enzyme activity at 20 U/mg using co-factor NADPH, Ygl039wp displayed a NADH preference at 25 U/mg in reduction of butylaldehyde. Amino acid sequence analysis identified a characteristic catalytic triad, Ser, Tyr and Lys; a conserved catalytic motif of Tyr-X-X-X-Lys; and a cofactor-binding sequence motif, Gly-X-X-Gly-X-X-Ala, near the N-terminus that are shared by Ydr541cp, Ygl039wp, Yol151wp/GRE2 and Ari1p. Findings of aldehyde reductase genes contribute to the yeast gene annotation and aids development of the next-generation biocatalyst for advanced biofuels production. PMID:25656103

  2. Human liver mitochondrial aldehyde dehydrogenase: three-dimensional structure and the restoration of solubility and activity of chimeric forms.

    PubMed Central

    Ni, L.; Zhou, J.; Hurley, T. D.; Weiner, H.

    1999-01-01

    Human liver cytosolic and mitochondrial isozymes of aldehyde dehydrogenase share 70% sequence identity. However, the first 21 residues are not conserved between the human isozymes (15% identity). The three-dimensional structures of the beef mitochondrial and sheep cytosolic forms have virtually identical three-dimensional structures. Here, we solved the structure of the human mitochondrial enzyme and found it to be identical to the beef enzyme. The first 21 residues are found on the surface of the enzyme and make no contact with other subunits in the tetramer. A pair of chimeric enzymes between the human isozymes was made. Each chimera had the first 21 residues from one isozyme and the remaining 479 from the other. When the first 21 residues were from the mitochondrial isozyme, an enzyme with cytosolic-like properties was produced. The other was expressed but was insoluble. It was possible to restore solubility and activity to the chimera that had the first 21 cytosolic residues fused to the mitochondrial ones by making point mutations to residues at the N-terminal end. When residue 19 was changed from tyrosine to a cysteine, the residue found in the mitochondrial form, an active enzyme could be made though the Km for NAD+ was 35 times higher than the native mitochondrial isozyme and the specific activity was reduced by 75%. This residue interacts with residue 203, a nonconserved, nonactive site residue. A mutation of residue 18, which also interacts with 203, restored solubility, but not activity. Mutation to residue 15, which interacts with 104, also restored solubility but not activity. It appears that to have a soluble or active enzyme a favorable interaction must occur between a residue in a surface loop and a residue elsewhere in the molecule even though neither make contact with the active site region of the enzyme. PMID:10631996

  3. Human aldehyde dehydrogenase 3A1 (ALDH3A1): biochemical characterization and immunohistochemical localization in the cornea.

    PubMed Central

    Pappa, Aglaia; Estey, Tia; Manzer, Rizwan; Brown, Donald; Vasiliou, Vasilis

    2003-01-01

    ALDH3A1 (aldehyde dehydrogenase 3A1) is expressed at high concentrations in the mammalian cornea and it is believed that it protects this vital tissue and the rest of the eye against UV-light-induced damage. The precise biological function(s) and cellular distribution of ALDH3A1 in the corneal tissue remain to be elucidated. Among the hypotheses proposed for ALDH3A1 function in cornea is detoxification of aldehydes formed during UV-induced lipid peroxidation. To investigate in detail the biochemical properties and distribution of this protein in the human cornea, we expressed human ALDH3A1 in Sf9 insect cells using a baculovirus vector and raised monoclonal antibodies against ALDH3A1. Recombinant ALDH3A1 protein was purified to homogeneity with a single-step affinity chromatography method using 5'-AMP-Sepharose 4B. Human ALDH3A1 demonstrated high substrate specificity for medium-chain (6 carbons and more) saturated and unsaturated aldehydes, including 4-hydroxy-2-nonenal, which are generated by the peroxidation of cellular lipids. Short-chain aliphatic aldehydes, such as acetaldehyde, propionaldehyde and malondialdehyde, were found to be very poor substrates for human ALDH3A1. In addition, ALDH3A1 metabolized glyceraldehyde poorly and did not metabolize glucose 6-phosphate, 6-phosphoglucono-delta-lactone and 6-phosphogluconate at all, suggesting that this enzyme is not involved in either glycolysis or the pentose phosphate pathway. Immunohistochemistry in human corneas, using the monoclonal antibodies described herein, revealed ALDH3A1 expression in epithelial cells and stromal keratocytes, but not in endothelial cells. Overall, these cumulative findings support the metabolic function of ALDH3A1 as a part of a corneal cellular defence mechanism against oxidative damage caused by aldehydic products of lipid peroxidation. Both recombinant human ALDH3A1 and the highly specific monoclonal antibodies described in the present paper may prove to be useful in probing

  4. Aldehyde dehydrogenase 2 activation in heart failure restores mitochondrial function and improves ventricular function and remodelling

    PubMed Central

    Gomes, Katia M.S.; Campos, Juliane C.; Bechara, Luiz R.G.; Queliconi, Bruno; Lima, Vanessa M.; Disatnik, Marie-Helene; Magno, Paulo; Chen, Che-Hong; Brum, Patricia C.; Kowaltowski, Alicia J.; Mochly-Rosen, Daria; Ferreira, Julio C.B.

    2014-01-01

    Aims We previously demonstrated that pharmacological activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) protects the heart against acute ischaemia/reperfusion injury. Here, we determined the benefits of chronic activation of ALDH2 on the progression of heart failure (HF) using a post-myocardial infarction model. Methods and results We showed that a 6-week treatment of myocardial infarction-induced HF rats with a selective ALDH2 activator (Alda-1), starting 4 weeks after myocardial infarction at a time when ventricular remodelling and cardiac dysfunction were present, improved cardiomyocyte shortening, cardiac function, left ventricular compliance and diastolic function under basal conditions, and after isoproterenol stimulation. Importantly, sustained Alda-1 treatment showed no toxicity and promoted a cardiac anti-remodelling effect by suppressing myocardial hypertrophy and fibrosis. Moreover, accumulation of 4-hydroxynonenal (4-HNE)-protein adducts and protein carbonyls seen in HF was not observed in Alda-1-treated rats, suggesting that increasing the activity of ALDH2 contributes to the reduction of aldehydic load in failing hearts. ALDH2 activation was associated with improved mitochondrial function, including elevated mitochondrial respiratory control ratios and reduced H2O2 release. Importantly, selective ALDH2 activation decreased mitochondrial Ca2+-induced permeability transition and cytochrome c release in failing hearts. Further supporting a mitochondrial mechanism for ALDH2, Alda-1 treatment preserved mitochondrial function upon in vitro aldehydic load. Conclusions Selective activation of mitochondrial ALDH2 is sufficient to improve the HF outcome by reducing the toxic effects of aldehydic overload on mitochondrial bioenergetics and reactive oxygen species generation, suggesting that ALDH2 activators, such as Alda-1, have a potential therapeutic value for treating HF patients. PMID:24817685

  5. Murine hepatic aldehyde dehydrogenase 1a1 is a major contributor to oxidation of aldehydes formed by lipid peroxidation

    PubMed Central

    Makia, Ngome L.; Bojang, Pasano; Falkner, K. Cameron; Conklin, Daniel J.; Prough, Russell A.

    2015-01-01

    Reactive lipid aldehydes are implicated in the pathogenesis of various oxidative stress-mediated diseases, including non-alcoholic steatohepatitis, atherosclerosis, Alzheimer’s and cataract. In the present study, we sought to define which hepatic Aldh isoform plays a major role in detoxification of lipid-derived aldehydes, such as acrolein and HNE by enzyme kinetic and gene expression studies. The catalytic efficiencies for metabolism of acrolein by Aldh1a1 was comparable to that of Aldh3a1 (Vmax/Km = 23). However, Aldh1a1 exhibits far higher affinity for acrolein (Km = 23.2 μM) compared to Aldh3a1 (Km = 464 μM). Aldh1a1 displays a 3-fold higher catalytic efficiency for HNE than Aldh3a1 (218 vs 69 ml/min/mg). The endogenous Aldh1a1 gene was highly expressed in mouse liver and a liver-derived cell line (Hepa-1c1c7) compared to Aldh2, Aldh1b1 and Aldh3a1. Aldh1a1 mRNA levels was 34-fold and 73-fold higher than Aldh2 in mouse liver and Hepa-1c1c7 cells respectively. Aldh3a1 gene was absent in mouse liver, but moderately expressed in Hepa-1c1c7 cells compared to Aldh1a1. We demonstrated that knockdown of Aldh1a1 expression by siRNA caused Hepa-1c1c7 cells to be more sensitive to acrolein-induced cell death and resulted in increased accumulation of acrolein-protein adducts and caspase 3 activation. These results indicate that Aldh1a1 plays a major role in cellular defense against oxidative damage induced by reactive lipid aldehydes in mouse liver. We also noted that hepatic Aldh1a1 mRNA levels were significantly increased (≈ 3 fold) in acrolein-fed mice compared to control. In addition, hepatic cytosolic ALDH activity was induced by acrolein when 1 mM NAD+ was used as cofactor, suggesting an Aldh1a1-protective mechanism against acrolein toxicity in mice liver. Thus, mechanisms to induce Aldh1a1 gene expression may provide a useful rationale for therapeutic protection against oxidative stress-induced pathologies. PMID:21256123

  6. Structure of a bacterial enzyme regulated by phosphorylation, isocitrate dehydrogenase.

    PubMed

    Hurley, J H; Thorsness, P E; Ramalingam, V; Helmers, N H; Koshland, D E; Stroud, R M

    1989-11-01

    The structure of isocitrate dehydrogenase [threo-DS-isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42] from Escherichia coli has been solved and refined at 2.5 A resolution and is topologically different from that of any other dehydrogenase. This enzyme, a dimer of identical 416-residue subunits, is inactivated by phosphorylation at Ser-113, which lies at the edge of an interdomain pocket that also contains many residues conserved between isocitrate dehydrogenase and isopropylmalate dehydrogenase. Isocitrate dehydrogenase contains an unusual clasp-like domain in which both polypeptide chains in the dimer interlock. Based on the structure of isocitrate dehydrogenase and conservation with isopropylmalate dehydrogenase, we suggest that the active site lies in an interdomain pocket close to the phosphorylation site. PMID:2682654

  7. Aldehyde Dehydrogenase 2 Has Cardioprotective Effects on Myocardial Ischaemia/Reperfusion Injury via Suppressing Mitophagy

    PubMed Central

    Ji, Wenqing; Wei, Shujian; Hao, Panpan; Xing, Junhui; Yuan, Qiuhuan; Wang, Jiali; Xu, Feng; Chen, Yuguo

    2016-01-01

    Mitophagy, a selective form of autophagy, is excessively activated in myocardial ischemia/reperfusion (I/R). The study investigated whether aldehyde dehydrogenase 2 (ALDH2) exerted its cardioprotective effect by regulating mitophagy. Myocardial infarct size and apoptosis after I/R in rats were ameliorated by Alda-1, an ALDH2 activator, and aggravated by ALDH2 inhibition. Both in I/R rats and hypoxia/reoxygenation H9C2 cells, ALDH2 activation suppressed phosphatase and tensin homolog-induced putative kinase 1 (PINK1)/Parkin expression, regulating mitophagy, by preventing 4-hydroxynonenal, reactive oxygen species and mitochondrial superoxide accumulation. Furthermore, the effect was enhanced by ALDH2 inhibition. Thus, ALDH2 may protect hearts against I/R injury by suppressing PINK1/Parkin–dependent mitophagy. PMID:27148058

  8. Molecular mechanism of null expression of aldehyde dehydrogenase-1 in rat liver

    SciTech Connect

    Chen, J.; Yoshida, Akira; Yanagawa, Yuchio

    1996-04-01

    In isozyme systems in general, the pattern of tissue-dependent expression of a given type of isozyme is uniform in various mammalian species. In contrast, a major cytosolic aldehyde dehydrogenase isozyme, termed ALDH1, which is strongly expressed in the livers of humans and other mammals, is hardly detectable in rat liver. Thirteen nucleotides existing in the 5{prime}-promoter region of human, marmoset, and mouse ALDH1 genes are absent in the four rat strains examined. When the 13 nucleotides were deleted from a chloramphenicol acetyltransferase expression construct, which contained the 5{prime} promoter region of the human ALDH1 gene and a low-background promoterless chloramphenicol acetyltransferase expression vector, the expression activity was severely diminished in human hepatic cells. Thus, deletion of the 13 nucleotides in the promoter region of the gene can account for the lack of ALDH1 expression in rat liver. 16 refs., 3 figs.

  9. Azotobacter vinelandii Aldehyde Dehydrogenase Regulated by ς54: Role in Alcohol Catabolism and Encystment

    PubMed Central

    Gama-Castro, Socorro; Núñez, Cinthia; Segura, Daniel; Moreno, Soledad; Guzmán, Josefina; Espín, Guadalupe

    2001-01-01

    Encystment in Azotobacter vinelandii is induced by n-butanol or β-hydroxybutyrate (BHB). We identified a gene, encoding an aldehyde dehydrogenase, that was named aldA. An aldA mutation impaired bacterial growth on n-butanol, ethanol, or hexanol as the sole carbon source. Expression of aldA increased in cells shifted from sucrose to n-butanol and was shown to be dependent on the alternative ς54 factor. A mutation in rpoN encoding the ς54 factor also impaired growth on alcohols. Encystment on n-butanol, but not on BHB, was impaired in aldA or rpoN mutants, indicating that n-butanol is not an inducer of encystment by itself but must be catabolized in order to induce encystment. PMID:11591659

  10. A Personalized Medicine Approach for Asian Americans with the Aldehyde Dehydrogenase 2*2 Variant

    PubMed Central

    Gross, Eric R.; Zambelli, Vanessa O.; Small, Bryce A.; Ferreira, Julio C.B.; Chen, Che-Hong; Mochly-Rosen, Daria

    2015-01-01

    Asian Americans are one of the fastest-growing populations in the United States. A relatively large subset of this population carries a unique loss-of-function point mutation in aldehyde dehydrogenase 2 (ALDH2), ALDH2*2. Found in approximately 560 million people of East Asian descent, ALDH2*2 reduces enzymatic activity by approximately 60% to 80% in heterozygotes. Furthermore, this variant is associated with a higher risk for several diseases affecting many organ systems, including a particularly high incidence relative to the general population of esophageal cancer, myocardial infarction, and osteoporosis. In this review, we discuss the pathophysiology associated with the ALDH2*2 variant, describe why this variant needs to be considered when selecting drug treatments, and suggest a personalized medicine approach for Asian American carriers of this variant. We also discuss future clinical and translational perspectives regarding ALDH2*2 research. PMID:25292432

  11. Pigs fed camelina meal increase hepatic gene expression of cytochrome 8b1, aldehyde dehydrogenase, and thiosulfate transferase

    PubMed Central

    2014-01-01

    Camelina sativa is an oil seed crop which can be grown on marginal lands. Camelina seed oil is rich in omega-3 fatty acids (>35%) and γ-tocopherol but is also high in erucic acid and glucosinolates. Camelina meal, is the by-product after the oil has been extracted. Camelina meal was fed to 28 d old weaned pigs at 3.7% and 7.4% until age 56 d. The camelina meal supplements in the soy based diets, improved feed efficiency but also significantly increased the liver weights. Gene expression analyses of the livers, using intra-species microarrays, identified increased expression of phase 1 and phase 2 drug metabolism enzymes. The porcine versions of the enzymes were confirmed by real time PCR. Cytochrome 8b1 (CYP8B1), aldehyde dehydrogenase 2 (Aldh2), and thiosulfate transferase (TST) were all significantly stimulated. Collectively, these genes implicate the camelina glucosinolate metabolite, methyl-sulfinyldecyl isothiocyanate, as the main xeniobiotic, causing increased hepatic metabolism and increased liver weight. PMID:24383433

  12. Pigs fed camelina meal increase hepatic gene expression of cytochrome 8b1, aldehyde dehydrogenase, and thiosulfate transferase.

    PubMed

    Meadus, William Jon; Duff, Pascale; McDonald, Tanya; Caine, William R

    2014-01-01

    Camelina sativa is an oil seed crop which can be grown on marginal lands. Camelina seed oil is rich in omega-3 fatty acids (>35%) and γ-tocopherol but is also high in erucic acid and glucosinolates. Camelina meal, is the by-product after the oil has been extracted. Camelina meal was fed to 28 d old weaned pigs at 3.7% and 7.4% until age 56 d. The camelina meal supplements in the soy based diets, improved feed efficiency but also significantly increased the liver weights. Gene expression analyses of the livers, using intra-species microarrays, identified increased expression of phase 1 and phase 2 drug metabolism enzymes. The porcine versions of the enzymes were confirmed by real time PCR. Cytochrome 8b1 (CYP8B1), aldehyde dehydrogenase 2 (Aldh2), and thiosulfate transferase (TST) were all significantly stimulated. Collectively, these genes implicate the camelina glucosinolate metabolite, methyl-sulfinyldecyl isothiocyanate, as the main xeniobiotic, causing increased hepatic metabolism and increased liver weight. PMID:24383433

  13. Autocrine function of aldehyde dehydrogenase 1 as a determinant of diet- and sex-specific differences in visceral adiposity.

    PubMed

    Yasmeen, Rumana; Reichert, Barbara; Deiuliis, Jeffrey; Yang, Fangping; Lynch, Alisha; Meyers, Joseph; Sharlach, Molly; Shin, Sangsu; Volz, Katharina S; Green, Kari B; Lee, Kichoon; Alder, Hansjuerg; Duester, Gregg; Zechner, Rudolf; Rajagopalan, Sanjay; Ziouzenkova, Ouliana

    2013-01-01

    Mechanisms for sex- and depot-specific fat formation are unclear. We investigated the role of retinoic acid (RA) production by aldehyde dehydrogenase 1 (Aldh1a1, -a2, and -a3), the major RA-producing enzymes, on sex-specific fat depot formation. Female Aldh1a1(-/-) mice, but not males, were resistant to high-fat (HF) diet-induced visceral adipose formation, whereas subcutaneous fat was reduced similarly in both groups. Sexual dimorphism in visceral fat (VF) was attributable to elevated adipose triglyceride lipase (Atgl) protein expression localized in clusters of multilocular uncoupling protein 1 (Ucp1)-positive cells in female Aldh1a1(-/-) mice compared with males. Estrogen decreased Aldh1a3 expression, limiting conversion of retinaldehyde (Rald) to RA. Rald effectively induced Atgl levels via nongenomic mechanisms, demonstrating indirect regulation by estrogen. Experiments in transgenic mice expressing an RA receptor response element (RARE-lacZ) revealed HF diet-induced RARE activation in VF of females but not males. In humans, stromal cells isolated from VF of obese subjects also expressed higher levels of Aldh1 enzymes compared with lean subjects. Our data suggest that an HF diet mediates VF formation through a sex-specific autocrine Aldh1 switch, in which Rald-mediated lipolysis in Ucp1-positive visceral adipocytes is replaced by RA-mediated lipid accumulation. Our data suggest that Aldh1 is a potential target for sex-specific antiobesity therapy. PMID:22933113

  14. Mitochondrial NAD Dependent Aldehyde Dehydrogenase either from Yeast or Human Replaces Yeast Cytoplasmic NADP Dependent Aldehyde Dehydrogenase for the Aerobic Growth of Yeast on Ethanol

    PubMed Central

    Mukhopadhyay, Abhijit; Wei, Baoxian; Weiner, Henry

    2013-01-01

    Background In a previous study, we deleted three aldehyde dehydrogenase (ALDH) genes, involved in ethanol metabolism, from yeast S. cerevisiae and found that the triple deleted yeast strain did not grow on ethanol as sole carbon source. The ALDHs were NADP dependent cytosolic ALDH1, NAD dependent mitochondrial ALDH2 and NAD/NADP dependent mitochondrial ALDH5. Double deleted strain ΔALDH2+ΔALDH5 or ΔALDH1+ΔALDH5 could grow on ethanol. However, the double deleted strain ΔALDH1+ΔALDH2 did not grow in ethanol. Methods Triple deleted yeast strain was used. Mitochondrial NAD dependent ALDH from yeast or human was placed in yeast cytosol. Results In the present study we found that a mutant form of cytoplasmic ALDH1 with very low activity barely supported the growth of the triple deleted strain (ΔALDH1+ΔALDH2+ΔALDH5) on ethanol. Finding the importance of NADP dependent ALDH1 on the growth of the strain on ethanol we examined if NAD dependent mitochondrial ALDH2 either from yeast or human would be able to support the growth of the triple deleted strain on ethanol if the mitochondrial form was placed in cytosol. We found that the NAD dependent mitochondrial ALDH2 from yeast or human was active in cytosol and supported the growth of the triple deleted strain on ethanol. Conclusion This study showed that coenzyme preference of ALDH is not critical in cytosol of yeast for the growth on ethanol. PMID:23454351

  15. Inhibition of mitochondrial aldehyde dehydrogenase by nitric oxide-mediated S-nitrosylation

    PubMed Central

    Moon, Kwan-Hoon; Kim, Bong-Jo; Song, Byoung J.

    2005-01-01

    Mitochondrial aldehyde dehydrogenase (ALDH2) is responsible for the metabolism of acetaldehyde and other toxic lipid aldehydes. Despite many reports about the inhibition of ALDH2 by toxic chemicals, it is unknown whether nitric oxide (NO) can alter the ALDH2 activity in intact cells or in vivo animals. The aim of this study was to investigate the effects of NO on ALDH2 activity in H4IIE-C3 rat hepatoma cells. NO donors such as S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine, and 3-morpholinosydnonimine significantly increased the nitrite concentration while they inhibited the ALDH2 activity. Addition of GSH-ethylester (GSH-EE) completely blocked the GSNO-mediated ALDH2 inhibition and increased nitrite concentration. To directly demonstrate the NO-mediated S-nitrosylation and inactivation, ALDH2 was immunopurified from control or GSNO-treated cells and subjected to immunoblot analysis. The anti-nitrosocysteine antibody recognized the immunopurified ALDH2 only from the GSNO-treated samples. All these results indicate that S-nitrosylation of ALDH2 in intact cells leads to reversible inhibition of ALDH2 activity. PMID:16242127

  16. The short-chain alcohol dehydrogenase ABA2 catalyzes the conversion of xanthoxin to abscisic aldehyde.

    PubMed

    González-Guzmán, Miguel; Apostolova, Nadezda; Bellés, José M; Barrero, José M; Piqueras, Pedro; Ponce, María R; Micol, José L; Serrano, Ramón; Rodríguez, Pedro L

    2002-08-01

    Mutants able to germinate and perform early growth in medium containing a high NaCl concentration were identified during the course of two independent screenings and named salt resistant (sre) and salobreño (sañ). The sre and sañ mutants also were able to germinate in high-osmoticum medium, indicating that they are osmotolerant in a germination assay. Complementation analyses revealed that sre1-1, sre1-2, sañ3-1, and sañ3-2 were alleles of the abscisic acid (ABA) biosynthesis ABA2 gene. A map-based cloning strategy allowed the identification of the ABA2 gene and molecular characterization of four new aba2 alleles. The ABA2 gene product belongs to the family of short-chain dehydrogenases/reductases, which are known to be NAD- or NADP-dependent oxidoreductases. Recombinant ABA2 protein produced in Escherichia coli exhibits a K(m) value for xanthoxin of 19 micro M and catalyzes in a NAD-dependent manner the conversion of xanthoxin to abscisic aldehyde, as determined by HPLC-mass spectrometry. The ABA2 mRNA is expressed constitutively in all plant organs examined and is not upregulated in response to osmotic stress. The results of this work are discussed in the context of previous genetic and biochemical evidence regarding ABA biosynthesis, confirming the xanthoxin-->abscisic aldehyde-->ABA transition as the last steps of the major ABA biosynthetic pathway. PMID:12172025

  17. Mesenchymal Stem/Stromal Cells Derived From a Reproductive Tissue Niche Under Oxidative Stress Have High Aldehyde Dehydrogenase Activity.

    PubMed

    Kusuma, Gina D; Abumaree, Mohamed H; Pertile, Mark D; Perkins, Anthony V; Brennecke, Shaun P; Kalionis, Bill

    2016-06-01

    The use of mesenchymal stem/stromal cells (MSC) in regenerative medicine often requires MSC to function in environments of high oxidative stress. Human pregnancy is a condition where the mother's tissues, and in particular her circulatory system, are exposed to increased levels of oxidative stress. MSC in the maternal decidua basalis (DMSC) are in a vascular niche, and thus would be exposed to oxidative stress products in the maternal circulation. Aldehyde dehydrogenases (ALDH) are a large family of enzymes which detoxify aldehydes and thereby protect stem cells against oxidative damage. A subpopulation of MSC express high levels of ALDH (ALDH(br)) and these are more potent in repairing and regenerating tissues. DMSC was compared with chorionic villous MSC (CMSC) derived from the human placenta. CMSC reside in vascular niche and are exposed to the fetal circulation, which is in lower oxidative state. We screened an ALDH isozyme cDNA array and determined that relative to CMSC, DMSC expressed high levels of ALDH1 family members, predominantly ALDH1A1. Immunocytochemistry gave qualitative confirmation at the protein level. Immunofluorescence detected ALDH1 immunoreactivity in the DMSC and CMSC vascular niche. The percentage of ALDH(br) cells was calculated by Aldefluor assay and DMSC showed a significantly higher percentage of ALDH(br) cells than CMSC. Finally, flow sorted ALDH(br) cells were functionally potent in colony forming unit assays. DMSC, which are derived from pregnancy tissues that are naturally exposed to high levels of oxidative stress, may be better candidates for regenerative therapies where MSC must function in high oxidative stress environments. PMID:26880140

  18. Mitochondrial Aldehyde Dehydrogenase Activation by Alda‐1 Inhibits Atherosclerosis and Attenuates Hepatic Steatosis in Apolipoprotein E‐Knockout Mice

    PubMed Central

    Stachowicz, Aneta; Olszanecki, Rafał; Suski, Maciej; Wiśniewska, Anna; Totoń‐Żurańska, Justyna; Madej, Józef; Jawień, Jacek; Białas, Magdalena; Okoń, Krzysztof; Gajda, Mariusz; Głombik, Katarzyna; Basta‐Kaim, Agnieszka; Korbut, Ryszard

    2014-01-01

    Background Mitochondrial dysfunction has been shown to play an important role in the development of atherosclerosis and nonalcoholic fatty liver disease (NAFLD). Mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme responsible for the detoxification of reactive aldehydes, is considered to exert protective function in mitochondria. We investigated the influence of Alda‐1, an activator of ALDH2, on atherogenesis and on the liver steatosis in apolipoprotein E knockout (apoE−/−) mice. Methods and Results Alda‐1 caused decrease of atherosclerotic lesions approximately 25% as estimated by “en face” and “cross‐section” methods without influence on plasma lipid profile, atherosclerosis‐related markers of inflammation, and macrophage and smooth muscle content in the plaques. Plaque nitrotyrosine was not changed upon Alda‐1 treatment, and there were no changes in aortic mRNA levels of factors involved in antioxidative defense, regulation of apoptosis, mitogenesis, and autophagy. Hematoxylin/eosin staining showed decrease of steatotic changes in liver of Alda‐1‐treated apoE−/− mice. Alda‐1 attenuated formation of 4‐hydroxy‐2‐nonenal (4‐HNE) protein adducts and decreased triglyceride content in liver tissue. Two‐dimensional electrophoresis coupled with mass spectrometry identified 20 differentially expressed mitochondrial proteins upon Alda‐1 treatment in liver of apoE−/− mice, mostly proteins related to metabolism and oxidative stress. The most up‐regulated were the proteins that participated in beta oxidation of fatty acids. Conclusions Collectively, Alda‐1 inhibited atherosclerosis and attenuated NAFLD in apoE−/− mice. The pattern of changes suggests a beneficial effect of Alda‐1 in NAFLD; however, the exact liver functional consequences of the revealed alterations as well as the mechanism(s) of antiatherosclerotic Alda‐1 action require further investigation. PMID:25392542

  19. Deficient expression of aldehyde dehydrogenase 1A1 is consistent with increased sensitivity of Gorlin syndrome patients to radiation carcinogenesis

    SciTech Connect

    Wright, Aaron T.; Magnaldo, Thierry; Sontag, Ryan L.; Anderson, Lindsey N.; Sadler, Natalie C.; Piehowski, Paul D.; Gache, Yannick; Weber, Thomas J.

    2013-11-27

    Human phenotypes that are highly susceptible to radiation carcinogenesis have been identified. Sensitive phenotypes often display robust regulation of molecular features that modify biological response, which can facilitate identification of relevant pathways/networks. Here we interrogate primary dermal fibroblasts isolated from Gorlin syndrome patients (GDFs), who display a pronounced tumorigenic response to radiation, in comparison to normal human dermal fibroblasts (NHDFs). Our approach exploits newly developed thiol-reactive probes with a flexible click chemistry functional group to define changes in protein thiol profiles in live cell studies, which minimizes artifacts associated with cell lysis. We observe qualitative differences in protein thiol profiles by SDS-PAGE analysis when detection by iodoacetamide vs maleimide probe chemistries are compared, and pretreatment of cells with hydrogen peroxide eliminates detection of the majority of SDS-PAGE bands. Redox probes revealed deficient expression of an apparent 55 kDa protein thiol in GDFs from independent donors, compared with NHDFs. Proteomics tentatively identified this protein as aldehyde dehydrogenase 1A1 (ALDH1A1), a key enzyme regulating retinoic acid synthesis, and this deficiency was confirmed by Western blot. Redox probes revealed additional protein thiol differences between GDFs and NHDFs, including radiation responsive annexin family members. Our results indicate a multifactorial basis for the unusual sensitivity of Gorlin syndrome to radiation carcinogenesis, and the pathways identified have plausible implications for radiation health effects.

  20. Deficient expression of aldehyde dehydrogenase 1A1 is consistent with increased sensitivity of Gorlin syndrome patients to radiation carcinogenesis

    DOE PAGESBeta

    Wright, Aaron T.; Magnaldo, Thierry; Sontag, Ryan L.; Anderson, Lindsey N.; Sadler, Natalie C.; Piehowski, Paul D.; Gache, Yannick; Weber, Thomas J.

    2013-11-27

    Human phenotypes that are highly susceptible to radiation carcinogenesis have been identified. Sensitive phenotypes often display robust regulation of molecular features that modify biological response, which can facilitate identification of relevant pathways/networks. Here we interrogate primary dermal fibroblasts isolated from Gorlin syndrome patients (GDFs), who display a pronounced tumorigenic response to radiation, in comparison to normal human dermal fibroblasts (NHDFs). Our approach exploits newly developed thiol-reactive probes with a flexible click chemistry functional group to define changes in protein thiol profiles in live cell studies, which minimizes artifacts associated with cell lysis. We observe qualitative differences in protein thiol profilesmore » by SDS-PAGE analysis when detection by iodoacetamide vs maleimide probe chemistries are compared, and pretreatment of cells with hydrogen peroxide eliminates detection of the majority of SDS-PAGE bands. Redox probes revealed deficient expression of an apparent 55 kDa protein thiol in GDFs from independent donors, compared with NHDFs. Proteomics tentatively identified this protein as aldehyde dehydrogenase 1A1 (ALDH1A1), a key enzyme regulating retinoic acid synthesis, and this deficiency was confirmed by Western blot. Redox probes revealed additional protein thiol differences between GDFs and NHDFs, including radiation responsive annexin family members. Our results indicate a multifactorial basis for the unusual sensitivity of Gorlin syndrome to radiation carcinogenesis, and the pathways identified have plausible implications for radiation health effects.« less

  1. Deficient Expression of Aldehyde Dehydrogenase 1A1 Is Consistent with Increased Sensitivity of Gorlin Syndrome Patients to Radiation Carcinogenesis

    SciTech Connect

    Wright, Aaron T.; Magnaldo, Thierry; Sontag, Ryan L.; Anderson, Lindsey N.; Sadler, Natalie C.; Piehowski, Paul D.; Gache, Yannick; Weber, Thomas J.

    2015-06-01

    Human phenotypes that are highly susceptible to radiation carcinogenesis have been identified. Sensitive phenotypes often display robust regulation of molecular features that modify biological response, which can facilitate identification of relevant pathways/networks. Here we interrogate primary dermal fibroblasts isolated from Gorlin syndrome patients (GDFs), who display a pronounced tumorigenic response to radiation, in comparison to normal human dermal fibroblasts (NHDFs). Our approach exploits newly developed thiol-reactive probes with a flexible click chemistry functional group to define changes in protein thiol profiles in live cell studies, which minimizes artifacts associated with cell lysis. We observe qualitative differences in protein thiol profiles by SDS-PAGE analysis when detection by iodoacetamide vs maleimide probe chemistries are compared, and pretreatment of cells with hydrogen peroxide eliminates detection of the majority of SDS-PAGE bands. Redox probes revealed deficient expression of an apparent 55 kDa protein thiol in GDFs from independent donors, compared with NHDFs. Proteomics tentatively identified this protein as aldehyde dehydrogenase 1A1 (ALDH1A1), a key enzyme regulating retinoic acid synthesis, and this deficiency was confirmed by Western blot. Redox probes revealed additional protein thiol differences between GDFs and NHDFs, including radiation responsive annexin family members. Our results indicate a multifactorial basis for the unusual sensitivity of Gorlin syndrome to radiation carcinogenesis, and the pathways identified have plausible implications for radiation health effects.

  2. Immunohistochemical analysis of aldehyde dehydrogenase isoforms and their association with estrogen-receptor status and disease progression in breast cancer

    PubMed Central

    Opdenaker, Lynn M; Arnold, Kimberly M; Pohlig, Ryan T; Padmanabhan, Jayasree S; Flynn, Daniel C; Sims-Mourtada, Jennifer

    2014-01-01

    In many types of tumors, especially breast tumors, aldehyde dehydrogenase (ALDH) activity has been used to identify cancer stem-like cells within the tumor. The presence and quantity of these cells are believed to predict the response of tumors to chemotherapy. Therefore, identification and eradication of these cells would be necessary to cure the patient. However, there are 19 different ALDH isoforms that could contribute to the enzyme activity. ALDH1A1 and ALDH1A3 are among the isoforms mostly responsible for the increased ALDH activity observed in these stem-like cells, although the main isoforms vary in different tissues and tumor types. In the study reported here, we attempted to determine if ALDH1A1 or ALDH1A3, specifically, correlate with tumor stage, grade, and hormone-receptor status in breast-cancer patients. While there was no significant correlation between ALDH1A1 and any of the parameters tested, we were able to identify a positive correlation between ALDH1A3 and tumor stage in triple-negative cancers. In addition, ALDH1A3 was negatively correlated with estrogen-receptor status. Our data suggest that ALDH1A3 could be utilized as a marker to identify stem-like cells within triple-negative tumors. PMID:25540596

  3. Low Km aldehyde dehydrogenase (ALDH2) polymorphism, alcohol-drinking behavior, and chromosome alterations in peripheral lymphocytes.

    PubMed Central

    Morimoto, K; Takeshita, T

    1996-01-01

    Excessive drinking of alcohol is now widely known to be one of the major lifestyle choices that ca effect health. Among the various effects of alcohol drinking, cytogenetic and other genotoxic effects are of major concern from the viewpoint of prevention of alcohol-related diseases. Alcohol is first metabolized to acetaldehyde, which directly causes various types of chromosomal DNA lesions and alcohol-related diseases, and is then further detoxified to the much less toxic metabolite acetate. About 50% of Oriental people are deficient in the aldehyde-dehydrogenase 2 isozyme (ALDH2) that can most efficiently detoxify acetaldehyde. We have performed a series of experiments to investigate how the genetic deficiency in ALDH2 affects the behavioral pattern for alcohol drinking and the sensitivity of peripheral lymphocytes to the induction of chromosome alterations by exposure to alcohol and alcohol-related chemicals. We found great effects of the ALDH2 genotypes on alcohol sensitivity and alcohol-drinking behavior. We also show that lymphocytes from habitual drinkers with the deficient ALDH2 enzyme had significantly higher frequencies of sister chromatid exchanges than those from ALDH2-proficient individuals. PMID:8781384

  4. Modeling-Dependent Protein Characterization of the Rice Aldehyde Dehydrogenase (ALDH) Superfamily Reveals Distinct Functional and Structural Features

    PubMed Central

    Kotchoni, Simeon O.; Jimenez-Lopez, Jose C.; Gao, Dongying; Edwards, Vincent; Gachomo, Emma W.; Margam, Venu M.; Seufferheld, Manfredo J.

    2010-01-01

    The completion of the rice genome sequence has made it possible to identify and characterize new genes and to perform comparative genomics studies across taxa. The aldehyde dehydrogenase (ALDH) gene superfamily encoding for NAD(P)+-dependent enzymes is found in all major plant and animal taxa. However, the characterization of plant ALDHs has lagged behind their animal- and prokaryotic-ALDH homologs. In plants, ALDHs are involved in abiotic stress tolerance, male sterility restoration, embryo development and seed viability and maturation. However, there is still no structural property-dependent functional characterization of ALDH protein superfamily in plants. In this paper, we identify members of the rice ALDH gene superfamily and use the evolutionary nesting events of retrotransposons and protein-modeling–based structural reconstitution to report the genetic and molecular and structural features of each member of the rice ALDH superfamily in abiotic/biotic stress responses and developmental processes. Our results indicate that rice-ALDHs are the most expanded plant ALDHs ever characterized. This work represents the first report of specific structural features mediating functionality of the whole families of ALDHs in an organism ever characterized. PMID:20634950

  5. Salt-induction of betaine aldehyde dehydrogenase mRNA, protein, and enzymatic activity in sugar beet. [Beta vulgaris L

    SciTech Connect

    McCue, K.F.; Hanson, A.D. )

    1991-05-01

    In Chenopodiaceae such as sugar beet (Beta vulgaris L.), glycine betaine (betaine) accumulates in response to drought or salinity stress and functions in the cytoplasm as a compatible osmolyte. The last enzyme in the biosynthetic pathway, betaine aldehyde dehydrogenase (BADH), increases as much as 4-fold in response to rising salinity in the external medium. This increase is accompanied by an increase in both protein and mRNA levels. The steady state increases in BADH were examined at a series of NaCl concentrations from 100 to 500 mM NaCl. BADH protein levels were examined by native PAGE, and by western blot analysis using antibodies raised against BADH purified from spinach. mRNA levels were examined by northern plot analysis of total RNA isolated from the leaves and hybridized with a sugar beet BADH cDNA clone. The time course for BADH mRNA induction was determined in a salt shock experiment utilizing 400 mM NaCl added to the external growth medium. Disappearance of BADH was examined in a salt relief experiment using plants step-wise salinized to 500 mM NaCl and then returned to 0 mM NaCl.

  6. Effects of Alda-1, an Aldehyde Dehydrogenase-2 Agonist, on Hypoglycemic Neuronal Death

    PubMed Central

    Ikeda, Tetsuhiko; Takahashi, Tetsuya; Tsujita, Mika; Kanazawa, Masato; Toriyabe, Masafumi; Koyama, Misaki; Itoh, Kosuke; Nakada, Tsutomu; Nishizawa, Masatoyo; Shimohata, Takayoshi

    2015-01-01

    Hypoglycemic encephalopathy (HE) is caused by a lack of glucose availability to neuronal cells, and no neuroprotective drugs have been developed as yet. Studies on the pathogenesis of HE and the development of new neuroprotective drugs have been conducted using animal models such as the hypoglycemic coma model and non-coma hypoglycemia model. However, both models have inherent problems, and establishment of animal models that mimic clinical situations is desirable. In this study, we first developed a short-term hypoglycemic coma model in which rats could be maintained in an isoelectric electroencephalogram (EEG) state for 2 min and subsequent hyperglycemia without requiring anti-seizure drugs and an artificial ventilation. This condition caused the production of 4-hydroxy-2-nonenal (4-HNE), a cytotoxic aldehyde, in neurons of the hippocampus and cerebral cortex, and a marked increase in neuronal death as evaluated by Fluoro-Jade B (FJB) staining. We also investigated whether N-(1,3-benzodioxole-5-ylmethyl)-2,6-dichlorobenzamide (Alda-1), a small-molecule agonist of aldehyde dehydrogenase-2, could attenuate 4-HNE levels and reduce hypoglycemic neuronal death. After confirming that EEG recordings remained isoelectric for 2 min, Alda-1 (8.5 mg/kg) or vehicle (dimethyl sulfoxide; DMSO) was administered intravenously with glucose to maintain a blood glucose level of 250 to 270 mg/dL. Fewer 4-HNE and FJB-positive cells were observed in the cerebral cortex of Alda-1-treated rats than in DMSO-treated rats 24 h after glucose administration (P = 0.002 and P = 0.020). Thus, activation of the ALDH2 pathway could be a molecular target for HE treatment, and Alda-1 is a potentially neuroprotective agent that exerts a beneficial effect on neurons when intravenously administered simultaneously with glucose. PMID:26083658

  7. Eukaryotic aldehyde dehydrogenase (ALDH) genes: human polymorphisms, and recommended nomenclature based on divergent evolution and chromosomal mapping.

    PubMed

    Vasiliou, V; Bairoch, A; Tipton, K F; Nebert, D W

    1999-08-01

    As currently being performed with an increasing number of superfamilies, a standardized gene nomenclature system is proposed here, based on divergent evolution, using multiple alignment analysis of all 86 eukaryotic aldehyde dehydrogenase (ALDH) amino-acid sequences known at this time. The ALDHs represent a superfamily of NAD(P)(+)-dependent enzymes having similar primary structures that oxidize a wide spectrum of endogenous and exogenous aliphatic and aromatic aldehydes. To date, a total of 54 animal, 15 plant, 14 yeast, and three fungal ALDH genes or cDNAs have been sequenced. These ALDHs can be divided into a total of 18 families (comprising 37 subfamilies), and all nonhuman ALDH genes are named here after the established human ALDH genes, when possible. An ALDH protein from one gene family is defined as having approximately < or = 40% amino-acid identity to that from another family. Two members of the same subfamily exhibit approximately > or = 60% amino-acid identity and are expected to be located at the same subchromosomal site. For naming each gene, it is proposed that the root symbol 'ALDH' denoting 'aldehyde dehydrogenase' be followed by an Arabic number representing the family and, when needed, a letter designating the subfamily and an Arabic number denoting the individual gene within the subfamily; all letters are capitalized in all mammals except mouse and fruit fly, e.g. 'human ALDH3A1 (mouse, Drosophila Aldh3a1).' It is suggested that the Human Gene Nomenclature Guidelines (http://++www.gene.ucl.ac.uk/nomenclature/guidelines.h tml) be used for all species other than mouse and Drosophila. Following these guidelines, the gene is italicized, whereas the corresponding cDNA, mRNA, protein or enzyme activity is written with upper-case letters and without italics, e.g. 'human, mouse or Drosophila ALDH3A1 cDNA, mRNA, or activity'. If an orthologous gene between species cannot be identified with certainty, sequential naming of these genes will be carried out

  8. Metabolic basis of ethylene glycol monobutyl ether (2-butoxyethanol) toxicity: role of alcohol and aldehyde dehydrogenases

    SciTech Connect

    Ghanayem, B.I.; Burka, L.T.; Matthews, H.B.

    1987-07-01

    2-Butoxyethanol (BE) is a massively produced glycol ether of which more than 230 million pounds was produced in the United States in 1983. It is extensively used in aerosols and cleaning agents intended for household use. This creates a high potential for human exposure during its manufacturing and use. A single exposure of rats to BE causes severe hemolytic anemia accompanied by secondary hemoglobinuria as well as liver and kidney damage. Butoxyacetic acid (BAA) was earlier identified as a urinary metabolite of BE. In addition, we have recently identified two additional urinary metabolites of BE, namely, BE-glucuronide and BE-sulfate conjugates. The current studies were undertaken to investigate the metabolic basis of BE-induced hematotoxicity in male F344 rats. Treatment of rats with pyrazole (alcohol dehydrogenase inhibitor) protected rats against BE-induced hematotoxicity and inhibited BE metabolism to BAA. Pyrazole inhibition of BE metabolism to BAA was accompanied by increased BE metabolism to BE-glucuronide and BE-sulfate as determined by quantitative high-performance liquid chromatography analysis of BE metabolites in urine. There was approximately a 10-fold decrease in the ratio of BAA to BE-glucuronide + BE-sulfate in the urine of rats treated with pyrazole + BE compared to rats treated with BE alone. Pretreatment of rats with cyanamide (aldehyde dehydrogenase inhibitor) also significantly protected rats against BE-induced hematotoxicity and modified BE metabolism in a manner similar to that caused by pyrazole. Administration of equimolar doses of BE, the metabolic intermediate butoxyacetaldehyde, or the ultimate metabolite BAA caused similar hematotoxic effects. Cyanamide also protected rats against butoxyacetaldehyde-induced hematotoxicity.

  9. Revascularization of ischemic limbs after transplantation of human bone marrow cells with high aldehyde dehydrogenase activity

    PubMed Central

    Capoccia, Benjamin J.; Robson, Debra L.; Levac, Krysta D.; Maxwell, Dustin J.; Hohm, Sarah A.; Neelamkavil, Marian J.; Bell, Gillian I.; Xenocostas, Anargyros; Link, Daniel C.; Piwnica-Worms, David; Nolta, Jan A.

    2009-01-01

    The development of cell therapies to treat peripheral vascular disease has proven difficult because of the contribution of multiple cell types that coordinate revascularization. We characterized the vascular regenerative potential of transplanted human bone marrow (BM) cells purified by high aldehyde dehydrogenase (ALDHhi) activity, a progenitor cell function conserved between several lineages. BM ALDHhi cells were enriched for myelo-erythroid progenitors that produced multipotent hematopoietic reconstitution after transplantation and contained nonhematopoietic precursors that established colonies in mesenchymal-stromal and endothelial culture conditions. The regenerative capacity of human ALDHhi cells was assessed by intravenous transplantation into immune-deficient mice with limb ischemia induced by femoral artery ligation/transection. Compared with recipients injected with unpurified nucleated cells containing the equivalent of 2- to 4-fold more ALDHhi cells, mice transplanted with purified ALDHhi cells showed augmented recovery of perfusion and increased blood vessel density in ischemic limbs. ALDHhi cells transiently recruited to ischemic regions but did not significantly integrate into ischemic tissue, suggesting that transient ALDHhi cell engraftment stimulated endogenous revascularization. Thus, human BM ALDHhi cells represent a progenitor-enriched population of several cell lineages that improves perfusion in ischemic limbs after transplantation. These clinically relevant cells may prove useful in the treatment of critical ischemia in humans. PMID:19324906

  10. Aldehyde Dehydrogenase Expression Drives Human Regulatory T Cell Resistance to Posttransplantation Cyclophosphamide

    PubMed Central

    Kanakry, Christopher G.; Ganguly, Sudipto; Zahurak, Marianna; Bolaños-Meade, Javier; Thoburn, Christopher; Perkins, Brandy; Fuchs, Ephraim J.; Jones, Richard J.; Hess, Allan D.; Luznik, Leo

    2014-01-01

    High-dose, posttransplantation cyclophosphamide (PTCy) is an effective strategy for preventing graft-versus-host disease (GVHD) after allogeneic blood or marrow transplantation (alloBMT). However, the mechanisms by which PTCy modulates alloimmune responses are not well understood. We studied early T cell reconstitution in patients undergoing alloBMT with PTCy and the effects of mafosfamide, a cyclophosphamide (Cy) analog, on CD4+ T cells in allogeneic mixed lymphocyte reactions (MLRs) in vitro. Patients exhibited reductions in naïve, potentially alloreactive conventional CD4+ T cells with relative preservation of memory CD4+Foxp3+ T cells. In particular, CD4+CD45RA−Foxp3+hi effector regulatory T cells (Tregs) recovered rapidly after alloBMT and, unexpectedly, were present at higher levels in patients with GVHD. CD4+Foxp3+ T cells from patients and from allogeneic MLRs expressed relatively high levels of aldehyde dehydrogenase (ALDH), the major in vivo mechanism of Cy resistance. Treatment of MLR cultures with the ALDH inhibitor diethylaminobenzaldehyde reduced the activation and proliferation of CD4+ T cells and sensitized Tregs to mafosfamide. Finally, removing Tregs from peripheral blood lymphocyte grafts obviated PTCy's GVHD-protective effect in a xenogeneic transplant model. Together, these findings suggest that Treg resistance to Cy through expression of ALDH may contribute to the clinical activity of PTCy in preventing GVHD. PMID:24225944

  11. Rotenone Decreases Intracellular Aldehyde Dehydrogenase Activity: Implications for the Pathogenesis of Parkinson Disease

    PubMed Central

    Goldstein, David S.; Sullivan, Patti; Cooney, Adele; Jinsmaa, Yunden; Kopin, Irwin J.; Sharabi, Yehonatan

    2015-01-01

    Repeated systemic administration of the mitochondrial complex I inhibitor rotenone produces a rodent model of Parkinson disease (PD). Mechanisms of relatively selective rotenone-induced damage to nigrostriatal dopaminergic neurons remain incompletely understood. According to the “catecholaldehyde hypothesis,” buildup of the autotoxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) contributes to PD pathogenesis. Vesicular uptake blockade increases DOPAL levels, and DOPAL is detoxified mainly by aldehyde dehydrogenase (ALDH). We tested whether rotenone interferes with vesicular uptake and intracellular ALDH activity. Endogenous and F-labeled catechols were measured in PC12 cells incubated with rotenone (0-1000 nM, 180 minutes), without or with F-dopamine (2 μM) to track vesicular uptake and catecholamine metabolism. Rotenone dose-dependently increased DOPAL, F-DOPAL, and 3,4-dihydroxyphenylethanol (DOPET) levels while decreasing dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels and the ratio of dopamine to the sum of its deaminated metabolites. In test tubes, rotenone did not affect conversion of DOPAL to DOPAC by ALDH when NAD+ was supplied, whereas the direct-acting ALDH inhibitor benomyl markedly increased DOPAL and decreased DOPAC concentrations in the reaction mixtures. We propose that rotenone builds up intracellular DOPAL by decreasing ALDH activity and attenuating vesicular sequestration of cytoplasmic catecholamines. The results provide a novel mechanism for selective rotenone-induced toxicity in dopaminergic neurons. PMID:25645689

  12. Aldehyde Dehydrogenase Activity Identifies a Population of Human Skeletal Muscle Cells With High Myogenic Capacities

    PubMed Central

    Vauchez, Karine; Marolleau, Jean-Pierre; Schmid, Michel; Khattar, Patricia; Chapel, Alain; Catelain, Cyril; Lecourt, Séverine; Larghéro, Jérôme; Fiszman, Marc; Vilquin, Jean-Thomas

    2009-01-01

    Aldehyde dehydrogenase 1A1 (ALDH) activity is one hallmark of human bone marrow (BM), umbilical cord blood (UCB), and peripheral blood (PB) primitive progenitors presenting high reconstitution capacities in vivo. In this study, we have identified ALDH+ cells within human skeletal muscles, and have analyzed their phenotypical and functional characteristics. Immunohistofluorescence analysis of human muscle tissue sections revealed rare endomysial cells. Flow cytometry analysis using the fluorescent substrate of ALDH, Aldefluor, identified brightly stained (ALDHbr) cells with low side scatter (SSClo), in enzymatically dissociated muscle biopsies, thereafter abbreviated as SMALD+ (for skeletal muscle ALDH+) cells. Phenotypical analysis discriminated two sub-populations according to CD34 expression: SMALD+/CD34− and SMALD+/CD34+ cells. These sub-populations did not initially express endothelial (CD31), hematopoietic (CD45), and myogenic (CD56) markers. Upon sorting, however, whereas SMALD+/CD34+ cells developed in vitro as a heterogeneous population of CD56− cells able to differentiate in adipoblasts, the SMALD+/CD34− fraction developed in vitro as a highly enriched population of CD56+ myoblasts able to form myotubes. Moreover, only the SMALD+/CD34− population maintained a strong myogenic potential in vivo upon intramuscular transplantation. Our results suggest that ALDH activity is a novel marker for a population of new human skeletal muscle progenitors presenting a potential for cell biology and cell therapy. PMID:19738599

  13. Aldehyde dehydrogenase 1a3 defines a subset of failing pancreatic β cells in diabetic mice.

    PubMed

    Kim-Muller, Ja Young; Fan, Jason; Kim, Young Jung R; Lee, Seung-Ah; Ishida, Emi; Blaner, William S; Accili, Domenico

    2016-01-01

    Insulin-producing β cells become dedifferentiated during diabetes progression. An impaired ability to select substrates for oxidative phosphorylation, or metabolic inflexibility, initiates progression from β-cell dysfunction to β-cell dedifferentiation. The identification of pathways involved in dedifferentiation may provide clues to its reversal. Here we isolate and functionally characterize failing β cells from various experimental models of diabetes and report a striking enrichment in the expression of aldehyde dehydrogenase 1 isoform A3 (ALDH(+)) as β cells become dedifferentiated. Flow-sorted ALDH(+) islet cells demonstrate impaired glucose-induced insulin secretion, are depleted of Foxo1 and MafA, and include a Neurogenin3-positive subset. RNA sequencing analysis demonstrates that ALDH(+) cells are characterized by: (i) impaired oxidative phosphorylation and mitochondrial complex I, IV and V; (ii) activated RICTOR; and (iii) progenitor cell markers. We propose that impaired mitochondrial function marks the progression from metabolic inflexibility to dedifferentiation in the natural history of β-cell failure. PMID:27572106

  14. Heterologous Expression of Aldehyde Dehydrogenase from Saccharomyces cerevisiae in Klebsiella pneumoniae for 3-Hydroxypropionic Acid Production from Glycerol.

    PubMed

    Wang, Kang; Wang, Xi; Ge, Xizhen; Tian, Pingfang

    2012-09-01

    3-Hydroxypropionic acid (3-HP) is a commercially valuable platform compound. Klebsiella pneumoniae has been concerned as an appropriate host for 3-HP production because of its robust capacity to metabolize glycerol. Glycerol conversion to 3-HP in K. pneumoniae comprises two successive reactions: glycerol dehydratase catalyzes glycerol to 3-hydroxypropionaldehyde (3-HPA); aldehyde dehydrogenase catalyzes 3-HPA to 3-HP. Previous studies focusing on inducible expression of aldehyde dehydrogenase have shown defects of high cost of inducer and low catalytic activity due to inclusion body. Here we show a different strategy that a native promoter in the host K. pneumoniae was used to drive the heterologous expression of aldehyde dehydrogenase gene ald4 from Saccharomyces cerevisiae. The 3-HP yield of the recombinant reached a peak of 4.23 g/L at log phase, but it decreased during later period of fermentation. Except the validation of high activity of ald4, particularly, the 3-HP formation was uncovered to be closely coupled with cell division, and the lacking of NAD and ATP at latter fermentation phase became the bottleneck for cell growth and 3-HP accumulation. Furthermore, 3-HP is postulated to be converted to 3-HPA via feedback inhibition or other metabolite via unknown mechanism. Since glycerol dissimilation is a common mechanism in a variety of bacteria, the expression strategy using native promoter and implications may provide significant insight into the metabolic engineering for 3-HP production. PMID:23997342

  15. Molecular and Catalytic Properties of the Aldehyde Dehydrogenase of Gluconacetobacter diazotrophicus, a Quinoheme Protein Containing Pyrroloquinoline Quinone, Cytochrome b, and Cytochrome c▿

    PubMed Central

    Gómez-Manzo, S.; Chavez-Pacheco, J. L.; Contreras-Zentella, M.; Sosa-Torres, M. E.; Arreguín-Espinosa, R.; Pérez de la Mora, M.; Membrillo-Hernández, J.; Escamilla, J. E.

    2010-01-01

    Several aldehyde dehydrogenase (ALDH) complexes have been purified from the membranes of acetic acid bacteria. The enzyme structures and the chemical nature of the prosthetic groups associated with these enzymes remain a matter of debate. We report here on the molecular and catalytic properties of the membrane-bound ALDH complex of the diazotrophic bacterium Gluconacetobacter diazotrophicus. The purified ALDH complex is a heterodimer comprising two subunits of 79.7 and 50 kDa, respectively. Reversed-phase high-pressure liquid chromatography (HPLC) and electron paramagnetic resonance spectroscopy led us to demonstrate, for the first time, the unequivocal presence of a pyrroloquinoline quinone prosthetic group associated with an ALDH complex from acetic acid bacteria. In addition, heme b was detected by UV-visible light (UV-Vis) spectroscopy and confirmed by reversed-phase HPLC. The smaller subunit bears three cytochromes c. Aliphatic aldehydes, but not formaldehyde, were suitable substrates. Using ferricyanide as an electron acceptor, the enzyme showed an optimum pH of 3.5 that shifted to pH 7.0 when phenazine methosulfate plus 2,6-dichlorophenolindophenol were the electron acceptors. Acetaldehyde did not reduce measurable levels of the cytochrome b and c centers; however, the dithionite-reduced hemes were conveniently oxidized by ubiquinone-1; this finding suggests that cytochrome b and the cytochromes c constitute an intramolecular redox sequence that delivers electrons to the membrane ubiquinone. PMID:20802042

  16. New aldehyde tag sequences identified by screening formylglycine generating enzymes in vitro and in vivo.

    PubMed

    Rush, Jason S; Bertozzi, Carolyn R

    2008-09-17

    Formylglycine generating enzyme (FGE) performs a critical posttranslational modification of type I sulfatases, converting cysteine within the motif CxPxR to the aldehyde-bearing residue formylglycine (FGly). This concise motif can be installed within heterologous proteins as a genetically encoded "aldehyde tag" for site-specific labeling with aminooxy- or hydrazide-functionalized probes. In this report, we screened FGEs from M. tuberculosis and S. coelicolor against synthetic peptide libraries and identified new substrate sequences that diverge from the canonical motif. We found that E. coli's FGE-like activity is similarly promiscuous, enabling the use of novel aldehyde tag sequences for in vivo modification of recombinant proteins. PMID:18722427

  17. Characterization of the Saccharomyces cerevisiae YMR318C (ADH6) gene product as a broad specificity NADPH-dependent alcohol dehydrogenase: relevance in aldehyde reduction.

    PubMed Central

    Larroy, Carol; Fernández, M Rosario; González, Eva; Parés, Xavier; Biosca, Josep A

    2002-01-01

    YMR318C represents an open reading frame from Saccharomyces cerevisiae with unknown function. It possesses a conserved sequence motif, the zinc-containing alcohol dehydrogenase (ADH) signature, specific to the medium-chain zinc-containing ADHs. In the present study, the YMR318C gene product has been purified to homogeneity from overexpressing yeast cells, and found to be a homodimeric ADH, composed of 40 kDa subunits and with a pI of 5.0-5.4. The enzyme was strictly specific for NADPH and was active with a wide variety of substrates, including aliphatic (linear and branched-chain) and aromatic primary alcohols and aldehydes. Aldehydes were processed with a 50-fold higher catalytic efficiency than that for the corresponding alcohols. The highest k(cat)/K(m) values were found with pentanal>veratraldehyde > hexanal > 3-methylbutanal >cinnamaldehyde. Taking into consideration the substrate specificity and sequence characteristics of the YMR318C gene product, we have proposed this gene to be called ADH6. The disruption of ADH6 was not lethal for the yeast under laboratory conditions. Although S. cerevisiae is considered a non lignin-degrading organism, the catalytic activity of ADHVI can direct veratraldehyde and anisaldehyde, arising from the oxidation of lignocellulose by fungal lignin peroxidases, to the lignin biodegradation pathway. ADHVI is the only S. cerevisiae enzyme able to significantly reduce veratraldehyde in vivo, and its overexpression allowed yeast to grow under toxic concentrations of this aldehyde. The enzyme may also be involved in the synthesis of fusel alcohols. To our knowledge this is the first NADPH-dependent medium-chain ADH to be characterized in S. cerevisiae. PMID:11742541

  18. Aldehyde dehydrogenase 1A1 circumscribes high invasive glioma cells and predicts poor prognosis.

    PubMed

    Xu, Sen-Lin; Liu, Sha; Cui, Wei; Shi, Yu; Liu, Qin; Duan, Jiang-Jie; Yu, Shi-Cang; Zhang, Xia; Cui, You-Hong; Kung, Hsiang-Fu; Bian, Xiu-Wu

    2015-01-01

    Glioma is the most aggressive brain tumor with high invasiveness and poor prognosis. More reliable, sensitive and practical biomarkers to reveal glioma high invasiveness remain to be explored for the guidance of therapy. We herein evaluated the diagnostic and prognostic value of aldehyde dehydrogenase 1A1 (ALDH1A1) in the glioma specimens from 237 patients, and found that ADLH1A1 was frequently overexpressed in the high-grade glioma (WHO grade III-IV) as compared to the low-grade glioma (WHO grade I-II) patients. The tumor cells with ALDH1A1 expression were more abundant in the region between tumor and the borderline of adjacent tissue as compared to the central part of the tumor. ALDH1A1 overexpression was associated with poor differentiation and dismal prognosis. Notably, the overall and disease-free survivals of the patients who had ALDH1A1(+) tumor cells sparsely located in the adjacent tissue were much worse. Furthermore, ALDH1A1 expression was correlated with the "classical-like" (CL) subtype as we examined GBM specimens from 72 patients. Multivariate Cox regression analysis revealed that ALDH1A1 was an independent marker for glioma patients' outcome. Mechanistically, both in vitro and in vivo studies revealed that ALDH1A1(+) cells isolated from either a glioblastoma cell line U251 or primary glioblastoma cells displayed significant invasiveness, clonogenicity, and proliferation as compared to ALDH1A1(-) cells, due to increased levels of mRNA and protein for matrix metalloproteinase 2, 7 and 9 (MMP2, MMP7 and MMP9). These results indicate that ALDH1A1(+) cells contribute to the progression of glioma including invasion, proliferation and poor prognosis, and suggest that targeting ALDH1A1 may have important implications for the treatment of highly invasive glioma. PMID:26101711

  19. Effects on sister chromatid exchange frequency of aldehyde dehydrogenase 2 genotype and smoking in vinyl chloride workers.

    PubMed

    Wong, R H; Wang, J D; Hsieh, L L; Du, C L; Cheng, T J

    1998-12-01

    Vinyl chloride monomer (VCM) is a human carcinogen. However, the exact mechanism of carcinogenesis remains unclear. VCM may be metabolized by cytochrome P450 2E1 (CYP2E1), aldehyde dehydrogenase 2 (ALDH2) and glutathione S-transferases (GSTs). Thus workers with inherited variant metabolic enzyme activities may have an altered risk of genotoxicity. This study was designed to investigate which risk factors might affect sister chromatid exchange (SCE) frequency in polyvinyl chloride (PVC) workers. Study subjects were 44 male workers from three PVC factories. Questionnaires were administered to obtain detailed histories of cigarette smoking, alcohol consumption, occupations, and medications. SCE frequency in peripheral lymphocytes was determined using a standardized method, and CYP2E1, GSTM1, GSTT1 and ALDH2 genotypes were identified by the polymerase chain reaction (PCR). Analysis revealed that smoking status and exposure to VCM were significantly associated with increased SCE frequency. The presence of ALDH2 1-2/2-2 genotypes was also significantly associated with an elevation of SCE frequency (9. 5 vs. 8.1, p<0.01). However, CYP2E1, GSTM1 or GSTT1 genotypes were not significantly associated with SCE frequency. When various genotypes were considered together, combination of CYP2E1 c1c2/c2c2 with ALDH2 1-2/2-2 showed an additive effect on SCE frequency. Similar results were also found for the combination of smoking with CYP2E1, or smoking with ALDH2. These results suggest that VCM workers with ALDH2 1-2/2-2 genotypes, who also smoke, may have increased risk of DNA damage. PMID:9838066

  20. Murine and Human Myogenic Cells Identified by Elevated Aldehyde Dehydrogenase Activity: Implications for Muscle Regeneration and Repair

    PubMed Central

    Vella, Joseph B.; Thompson, Seth D.; Bucsek, Mark J.; Song, Minjung; Huard, Johnny

    2011-01-01

    Background Despite the initial promise of myoblast transfer therapy to restore dystrophin in Duchenne muscular dystrophy patients, clinical efficacy has been limited, primarily by poor cell survival post-transplantation. Murine muscle derived stem cells (MDSCs) isolated from slowly adhering cells (SACs) via the preplate technique, induce greater muscle regeneration than murine myoblasts, primarily due to improved post-transplantation survival, which is conferred by their increased stress resistance capacity. Aldehyde dehydrogenase (ALDH) represents a family of enzymes with important morphogenic as well as oxidative damage mitigating roles and has been found to be a marker of stem cells in both normal and malignant tissue. In this study, we hypothesized that elevated ALDH levels could identify murine and human muscle derived cell (hMDC) progenitors, endowed with enhanced stress resistance and muscle regeneration capacity. Methodology/Principal Findings Skeletal muscle progenitors were isolated from murine and human skeletal muscle by a modified preplate technique and unfractionated enzymatic digestion, respectively. ALDHhi subpopulations isolated by fluorescence activate cell sorting demonstrated increased proliferation and myogenic differentiation capacities compared to their ALDHlo counterparts when cultivated in oxidative and inflammatory stress media conditions. This behavior correlated with increased intracellular levels of reduced glutathione and superoxide dismutase. ALDHhi murine myoblasts were observed to exhibit an increased muscle regenerative potential compared to ALDHlo myoblasts, undergo multipotent differentiation (osteogenic and chondrogenic), and were found predominately in the SAC fraction, characteristics that are also observed in murine MDSCs. Likewise, human ALDHhi hMDCs demonstrated superior muscle regenerative capacity compared to ALDHlo hMDCs. Conclusions The methodology of isolating myogenic cells on the basis of elevated ALDH activity

  1. Improved Tolerance to Various Abiotic Stresses in Transgenic Sweet Potato (Ipomoea batatas) Expressing Spinach Betaine Aldehyde Dehydrogenase

    PubMed Central

    Fan, Weijuan; Zhang, Min; Zhang, Hongxia; Zhang, Peng

    2012-01-01

    Abiotic stresses are critical delimiters for the increased productivity and cultivation expansion of sweet potato (Ipomoea batatas), a root crop with worldwide importance. The increased production of glycine betaine (GB) improves plant tolerance to various abiotic stresses without strong phenotypic changes, providing a feasible approach to improve stable yield production under unfavorable conditions. The gene encoding betaine aldehyde dehydrogenase (BADH) is involved in the biosynthesis of GB in plants, and the accumulation of GB by the heterologous overexpression of BADH improves abiotic stress tolerance in plants. This study is to improve sweet potato, a GB accumulator, resistant to multiple abiotic stresses by promoted GB biosynthesis. A chloroplastic BADH gene from Spinacia oleracea (SoBADH) was introduced into the sweet potato cultivar Sushu-2 via Agrobacterium-mediated transformation. The overexpression of SoBADH in the transgenic sweet potato improved tolerance to various abiotic stresses, including salt, oxidative stress, and low temperature. The increased BADH activity and GB accumulation in the transgenic plant lines under normal and multiple environmental stresses resulted in increased protection against cell damage through the maintenance of cell membrane integrity, stronger photosynthetic activity, reduced reactive oxygen species (ROS) production, and induction or activation of ROS scavenging by the increased activity of free radical-scavenging enzymes. The increased proline accumulation and systemic upregulation of many ROS-scavenging genes in stress-treated transgenic plants also indicated that GB accumulation might stimulate the ROS-scavenging system and proline biosynthesis via an integrative mechanism. This study demonstrates that the enhancement of GB biosynthesis in sweet potato is an effective and feasible approach to improve its tolerance to multiple abiotic stresses without causing phenotypic defects. This strategy for trait improvement in

  2. Novel NAD+-Farnesal Dehydrogenase from Polygonum minus Leaves. Purification and Characterization of Enzyme in Juvenile Hormone III Biosynthetic Pathway in Plant

    PubMed Central

    Mohamed-Hussein, Zeti-Azura; Ng, Chyan Leong

    2016-01-01

    Juvenile Hormone III is of great concern due to negative effects on major developmental and reproductive maturation in insect pests. Thus, the elucidation of enzymes involved JH III biosynthetic pathway has become increasing important in recent years. One of the enzymes in the JH III biosynthetic pathway that remains to be isolated and characterized is farnesal dehydrogenase, an enzyme responsible to catalyze the oxidation of farnesal into farnesoic acid. A novel NAD+-farnesal dehydrogenase of Polygonum minus was purified (315-fold) to apparent homogeneity in five chromatographic steps. The purification procedures included Gigacap S-Toyopearl 650M, Gigacap Q-Toyopearl 650M, and AF-Blue Toyopearl 650ML, followed by TSK Gel G3000SW chromatographies. The enzyme, with isoelectric point of 6.6 is a monomeric enzyme with a molecular mass of 70 kDa. The enzyme was relatively active at 40°C, but was rapidly inactivated above 45°C. The optimal temperature and pH of the enzyme were found to be 35°C and 9.5, respectively. The enzyme activity was inhibited by sulfhydryl agent, chelating agent, and metal ion. The enzyme was highly specific for farnesal and NAD+. Other terpene aldehydes such as trans- cinnamaldehyde, citral and α- methyl cinnamaldehyde were also oxidized but in lower activity. The Km values for farnesal, citral, trans- cinnamaldehyde, α- methyl cinnamaldehyde and NAD+ were 0.13, 0.69, 0.86, 1.28 and 0.31 mM, respectively. The putative P. minus farnesal dehydrogenase that’s highly specific towards farnesal but not to aliphatic aldehydes substrates suggested that the enzyme is significantly different from other aldehyde dehydrogenases that have been reported. The MALDI-TOF/TOF-MS/MS spectrometry further identified two peptides that share similarity to those of previously reported aldehyde dehydrogenases. In conclusion, the P. minus farnesal dehydrogenase may represent a novel plant farnesal dehydrogenase that exhibits distinctive substrate specificity

  3. Novel NAD+-Farnesal Dehydrogenase from Polygonum minus Leaves. Purification and Characterization of Enzyme in Juvenile Hormone III Biosynthetic Pathway in Plant.

    PubMed

    Seman-Kamarulzaman, Ahmad-Faris; Mohamed-Hussein, Zeti-Azura; Ng, Chyan Leong; Hassan, Maizom

    2016-01-01

    Juvenile Hormone III is of great concern due to negative effects on major developmental and reproductive maturation in insect pests. Thus, the elucidation of enzymes involved JH III biosynthetic pathway has become increasing important in recent years. One of the enzymes in the JH III biosynthetic pathway that remains to be isolated and characterized is farnesal dehydrogenase, an enzyme responsible to catalyze the oxidation of farnesal into farnesoic acid. A novel NAD+-farnesal dehydrogenase of Polygonum minus was purified (315-fold) to apparent homogeneity in five chromatographic steps. The purification procedures included Gigacap S-Toyopearl 650M, Gigacap Q-Toyopearl 650M, and AF-Blue Toyopearl 650ML, followed by TSK Gel G3000SW chromatographies. The enzyme, with isoelectric point of 6.6 is a monomeric enzyme with a molecular mass of 70 kDa. The enzyme was relatively active at 40°C, but was rapidly inactivated above 45°C. The optimal temperature and pH of the enzyme were found to be 35°C and 9.5, respectively. The enzyme activity was inhibited by sulfhydryl agent, chelating agent, and metal ion. The enzyme was highly specific for farnesal and NAD+. Other terpene aldehydes such as trans- cinnamaldehyde, citral and α- methyl cinnamaldehyde were also oxidized but in lower activity. The Km values for farnesal, citral, trans- cinnamaldehyde, α- methyl cinnamaldehyde and NAD+ were 0.13, 0.69, 0.86, 1.28 and 0.31 mM, respectively. The putative P. minus farnesal dehydrogenase that's highly specific towards farnesal but not to aliphatic aldehydes substrates suggested that the enzyme is significantly different from other aldehyde dehydrogenases that have been reported. The MALDI-TOF/TOF-MS/MS spectrometry further identified two peptides that share similarity to those of previously reported aldehyde dehydrogenases. In conclusion, the P. minus farnesal dehydrogenase may represent a novel plant farnesal dehydrogenase that exhibits distinctive substrate specificity towards

  4. Sca-1+ cells from fetal heart with high aldehyde dehydrogenase activity exhibit enhanced gene expression for self-renewal, proliferation, and survival.

    PubMed

    Dey, Devaveena; Pan, Guodong; Varma, Nadimpalli Ravi S; Palaniyandi, Suresh Selvaraj

    2015-01-01

    Stem/progenitor cells from multiple tissues have been isolated based on enhanced activity of cytosolic aldehyde dehydrogenase (ALDH) enzyme. ALDH activity has emerged as a reliable marker for stem/progenitor cells, such that ALDH(bright/high) cells from multiple tissues have been shown to possess enhanced stemness properties (self-renewal and multipotency). So far though, not much is known about ALDH activity in specific fetal organs. In this study, we sought to analyze the presence and activity of the ALDH enzyme in the stem cell antigen-1-positive (Sca-1+) cells of fetal human heart. Biochemical assays showed that a subpopulation of Sca-1+ cells (15%) possess significantly high ALDH1 activity. This subpopulation showed increased expression of self-renewal markers compared to the ALDH(low) fraction. The ALDH(high) fraction also exhibited significant increase in proliferation and pro-survival gene expression. In addition, only the ALDH(high) and not the ALDH(low) fraction could give rise to all the cell types of the original population, demonstrating multipotency. ALDH(high) cells showed increased resistance against aldehyde challenge compared to ALDH(low) cells. These results indicate that ALDH(high) subpopulation of the cultured human fetal cells has enhanced self-renewal, multipotency, high proliferation, and survival, indicating that this might represent a primitive stem cell population within the fetal human heart. PMID:25861413

  5. Mitigation of Radiation-Induced Dermatitis by Activation of Aldehyde Dehydrogenase 2 Using Topical Alda-1 in Mice1

    PubMed Central

    Ning, Shoucheng; Budas, Grant R.; Churchill, Eric N.; Chen, Che-Hong; Knox, Susan J.; Mochly-Rosen, Daria

    2012-01-01

    Ning, S., Budas, G. R., Churchill, E. N., Chen, C., Knox, S. J. and Mochly-Rosen, D. Mitigation of Radiation-Induced Dermatitis by Activation of Aldehyde Dehydrogenase 2 Using Topical Alda-1 in Mice. Radiation-induced dermatitis is a debilitating clinical problem in cancer patients undergoing cancer radiation therapy. It is also a possible outcome of exposure to high levels of radiation due to accident or hostile activity. We report that activation of aldehyde dehydrogenase 2 (ALDH2) enzymatic activity using the allosteric agonist, Alda-1, significantly reduced 4-hydroxynonenal adducts accumulation, delayed the onset of radiation dermatitis and substantially reduced symptoms in a clinically-relevant model of radiation-induced dermatitis. Importantly, Alda-1 did not radioprotect tumors in mice. Rather, it increased the sensitivity of the tumors to radiation therapy. This is the first report of reactive aldehydes playing a role in the intrinsic radiosensitivity of normal and tumor tissues. Our findings suggest that ALDH2 represents a novel target for the treatment of radiation dermatitis without reducing the benefit of radiotherapy. PMID:22404739

  6. Aldehyde dehydrogenase 3A1 promotes multi-modality resistance and alters gene expression profile in human breast adenocarcinoma MCF-7 cells.

    PubMed

    Voulgaridou, Georgia-Persephoni; Kiziridou, Magdalini; Mantso, Theodora; Chlichlia, Katerina; Galanis, Alex; Koukourakis, Michael I; Franco, Rodrigo; Panayiotidis, Mihalis I; Pappa, Aglaia

    2016-08-01

    Aldehyde dehydrogenases participate in a variety of cellular homeostatic mechanisms like metabolism, proliferation, differentiation, apoptosis, whereas recently, they have been implicated in normal and cancer cell stemness. We explored roles for ALDH3A1 in conferring resistance to chemotherapeutics/radiation/oxidative stress and whether ectopic overexpression of ALDH3A1 could lead to alterations of gene expression profile associated with cancer stem cell-like phenotype. MCF-7 cells were stably transfected either with an empty vector (mock) or human aldehyde dehydrogenase 3A1 cDNA. The expression of aldehyde dehydrogenase 3A1 in MCF-7 cells was associated with altered cell proliferation rate and enhanced cell resistance against various chemotherapeutic drugs (4-hydroxyperoxycyclophosphamide, doxorubicin, etoposide, and 5-fluorouracil). Aldehyde dehydrogenase 3A1 expression also led to increased tolerance of MCF-7 cells to gamma radiation and hydrogen peroxide-induced stress. Furthermore, aldehyde dehydrogenase 3A1-expressing MCF-7 cells exhibited gene up-regulation of cyclins A, B1, B2, and down-regulation of cyclin D1 as well as transcription factors p21, CXR4, Notch1, SOX2, SOX4, OCT4, and JAG1. When compared to mock cells, no changes were observed in mRNA levels of ABCA2 and ABCB1 protein pumps with only a minor decrease of the ABCG2 pump in the aldehyde dehydrogenase 3A1-expressing cells. Also, the adhesion molecules EpCAM and CD49F were also found to be up-regulated in aldehyde dehydrogenase 3A1expressing cells. Taken together, ALDH3A1 confers a multi-modality resistance phenotype in MCF-7 cells associated with slower growth rate, increased clonogenic capacity, and altered gene expression profile, underlining its significance in cell homeostasis. PMID:27276244

  7. Metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, in mouse liver by alcohol dehydrogenase Adh1 and aldehyde reductase AKR1A4

    SciTech Connect

    Short, Duncan M.; Lyon, Robert; Watson, David G.; Barski, Oleg A.; McGarvie, Gail; Ellis, Elizabeth M. . E-mail: Elizabeth.ellis@strath.ac.uk

    2006-01-15

    The reductive metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, was studied in mouse liver. Using an HPLC-based stopped assay, the primary reduced metabolite was identified as 6-hydroxy-trans, trans-2,4-hexadienal (OH/CHO) and the secondary metabolite as 1,6-dihydroxy-trans, trans-2,4-hexadiene (OH/OH). The main enzymes responsible for the highest levels of reductase activity towards trans, trans-muconaldehyde were purified from mouse liver soluble fraction first by Q-sepharose chromatography followed by either blue or red dye affinity chromatography. In mouse liver, trans, trans-muconaldehyde is predominantly reduced by an NADH-dependent enzyme, which was identified as alcohol dehydrogenase (Adh1). Kinetic constants obtained for trans, trans-muconaldehyde with the native Adh1 enzyme showed a V {sub max} of 2141 {+-} 500 nmol/min/mg and a K {sub m} of 11 {+-} 4 {mu}M. This enzyme was inhibited by pyrazole with a K {sub I} of 3.1 {+-} 0.57 {mu}M. Other fractions were found to contain muconaldehyde reductase activity independent of Adh1, and one enzyme was identified as the NADPH-dependent aldehyde reductase AKR1A4. This showed a V {sub max} of 115 nmol/min/mg and a K {sub m} of 15 {+-} 2 {mu}M and was not inhibited by pyrazole.

  8. Alcohol oxidase is a novel pathogenicity factor for Cladosporium fulvum, but aldehyde dehydrogenase is dispensable.

    PubMed

    Segers, G; Bradshaw, N; Archer, D; Blissett, K; Oliver, R P

    2001-03-01

    Cladosporiumfulvum is a mitosporic ascomycete pathogen of tomato. A study of fungal genes expressed during carbon starvation in vitro identified several genes that were up regulated during growth in planta. These included genes predicted to encode acetaldehyde dehydrogenase (Aldh1) and alcohol oxidase (Aox1). An Aldh1 deletion mutant was constructed. This mutant lacked all detectable ALDH activity, had lost the ability to grow with ethanol as a carbon source, but was unaffected in pathogenicity. Aox1 expression was induced by carbon starvation and during the later stages of infection. The alcohol oxidase enzyme activity has broadly similar properties (Km values, substrate specificity, pH, and heat stability) to yeast enzymes. Antibodies raised to Hansenula polymorpha alcohol oxidase (AOX) detected antigens in Western blots of starved C. fulvum mycelium and infected plant material. Antigen reacting with the antibodies was localized to organelles resembling peroxisomes in starved mycelium and infected plants. Disruption mutants of Aox1 lacked detectable AOX activity and had markedly reduced pathogenicity as assayed by two different measures of fungal growth. These results identify alcohol oxidase as a novel pathogenicity factor and are discussed in relation to peroxisomal metabolism of fungal pathogens during growth in planta. PMID:11277434

  9. Aldehyde dehydrogenase activity selects for the holoclone phenotype in prostate cancer cells

    SciTech Connect

    Doherty, R.E.; Haywood-Small, S.L.; Sisley, K.; Cross, N.A.

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Isolated ALDH{sup Hi} PC3 cells preferentially form primitive holoclone-type colonies. Black-Right-Pointing-Pointer Primitive holoclone colonies are predominantly ALDH{sup Lo} but contain rare ALDH{sup Hi} cells. Black-Right-Pointing-Pointer Holoclone-forming cells are not restricted to the ALDH{sup Hi} population. Black-Right-Pointing-Pointer ALDH phenotypic plasticity occurs in PC3 cells (ALDH{sup Lo} to ALDH{sup Hi} and vice versa). Black-Right-Pointing-Pointer ALDH{sup Hi} cells are observed but very rare in PC3 spheroids grown in stem cell medium. -- Abstract: Aldehyde dehydrogenase 1 (ALDH) activity is considered to be a marker of cancer stem cells (CSCs) in many tumour models, since these cells are more proliferative and tumourigenic than ALDH{sup Lo} cells in experimental models. However it is unclear whether all CSC-like cells are within the ALDH{sup Hi} population, or whether all ALDH{sup Hi} cells are highly proliferative and tumourigenic. The ability to establish a stem cell hierarchy in vitro, whereby sub-populations of cells have differing proliferative and differentiation capacities, is an alternate indication of the presence of stem cell-like populations within cell lines. In this study, we have examined the interaction between ALDH status and the ability to establish a stem cell hierarchy in PC3 prostate cancer cells. We demonstrate that PC3 cells contain a stem cell hierarchy, and isolation of ALDH{sup Hi} cells enriches for the most primitive holoclone population, however holoclone formation is not restricted to ALDH{sup Hi} cells. In addition, we show that ALDH activity undergoes phenotypic plasticity, since the ALDH{sup Lo} population can develop ALDH{sup Hi} populations comparable to parental cells within 2 weeks in culture. Furthermore, we show that the majority of ALDH{sup Hi} cells are found within the least primitive paraclone population, which is circumvented by culturing PC3 cells as spheroids in

  10. Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones

    PubMed Central

    Ying, Xiangxian; Wang, Yifang; Xiong, Bin; Wu, Tingting; Xie, Liping; Yu, Meilan

    2014-01-01

    A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the C=O bond of allylic aldehydes/ketones to the corresponding α,β-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (S)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg−1 for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg−1 using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower Km values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP+, suggesting the nature of being an aldehyde reductase. PMID:24509923

  11. Characterization of an allylic/benzyl alcohol dehydrogenase from Yokenella sp. strain WZY002, an organism potentially useful for the synthesis of α,β-unsaturated alcohols from allylic aldehydes and ketones.

    PubMed

    Ying, Xiangxian; Wang, Yifang; Xiong, Bin; Wu, Tingting; Xie, Liping; Yu, Meilan; Wang, Zhao

    2014-04-01

    A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the C=O bond of allylic aldehydes/ketones to the corresponding α,β-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (S)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg(-1) for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg(-1) using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower Km values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP(+), suggesting the nature of being an aldehyde reductase. PMID:24509923

  12. Enzymic and structural studies on Drosophila alcohol dehydrogenase and other short-chain dehydrogenases/reductases.

    PubMed

    Smilda, T; Kamminga, A H; Reinders, P; Baron, W; van Hylckama Vlieg, J E; Beintema, J J

    2001-05-01

    Enzymic and structural studies on Drosophila alcohol dehydrogenases and other short-chain dehydrogenases/reductases (SDRs) are presented. Like alcohol dehydrogenases from other Drosophila species, the enzyme from D. simulans is more active on secondary than on primary alcohols, although ethanol is its only known physiological substrate. Several secondary alcohols were used to determine the kinetic parameters kcat and Km. The results of these experiments indicate that the substrate-binding region of the enzyme allows optimal binding of a short ethyl side-chain in a small binding pocket, and of a propyl or butyl side-chain in large binding pocket, with stereospecificity for R(-) alcohols. At a high concentration of R(-) alcohols substrate activation occurs. The kcat and Km values determined under these conditions are about two-fold, and two orders of magnitude, respectively, higher than those at low substrate concentrations. Sequence alignment of several SDRs of known, and unknown three-dimensional structures, indicate the presence of several conserved residues in addition to those involved in the catalyzed reactions. Structural roles of these conserved residues could be derived from observations made on superpositioned structures of several SDRs with known structures. Several residues are conserved in tetrameric SDRs, but not in dimeric ones. Two halohydrin-halide-lyases show significant homology with SDRs in the catalytic domains of these enzymes, but they do not have the structural features required for binding NAD+. Probably these lyases descend from an SDR, which has lost the capability to bind NAD+, but the enzyme reaction mechanisms may still be similar. PMID:11443349

  13. Evidence that the C-terminal domain of a type B PutA protein contributes to aldehyde dehydrogenase activity and substrate channeling.

    PubMed

    Luo, Min; Christgen, Shelbi; Sanyal, Nikhilesh; Arentson, Benjamin W; Becker, Donald F; Tanner, John J

    2014-09-01

    Proline utilization A (PutA) is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate. Structures of type A PutAs have revealed the catalytic core consisting of proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) modules connected by a substrate-channeling tunnel. Type B PutAs also have a C-terminal domain of unknown function (CTDUF) that is absent in type A PutAs. Small-angle X-ray scattering (SAXS), mutagenesis, and kinetics are used to determine the contributions of this domain to PutA structure and function. The 1127-residue Rhodobacter capsulatus PutA (RcPutA) is used as a representative CTDUF-containing type B PutA. The reaction progress curve for the coupled PRODH-P5CDH activity of RcPutA does not exhibit a time lag, implying a substrate channeling mechanism. RcPutA is monomeric in solution, which is unprecedented for PutAs. SAXS rigid body modeling with target-decoy validation is used to build a model of RcPutA. On the basis of homology to aldehyde dehydrogenases (ALDHs), the CTDUF is predicted to consist of a β-hairpin fused to a noncatalytic Rossmann fold domain. The predicted tertiary structural interactions of the CTDUF resemble the quaternary structural interactions in the type A PutA dimer interface. The model is tested by mutagenesis of the dimerization hairpin of a type A PutA and the CTDUF hairpin of RcPutA. Similar functional phenotypes are observed in the two sets of variants, supporting the hypothesis that the CTDUF mimics the type A PutA dimer interface. These results suggest annotation of the CTDUF as an ALDH superfamily domain that facilitates P5CDH activity and substrate channeling by stabilizing the aldehyde-binding site and sealing the substrate-channeling tunnel from the bulk medium. PMID:25137435

  14. Evidence That the C-Terminal Domain of a Type B PutA Protein Contributes to Aldehyde Dehydrogenase Activity and Substrate Channeling

    PubMed Central

    2015-01-01

    Proline utilization A (PutA) is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate. Structures of type A PutAs have revealed the catalytic core consisting of proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) modules connected by a substrate-channeling tunnel. Type B PutAs also have a C-terminal domain of unknown function (CTDUF) that is absent in type A PutAs. Small-angle X-ray scattering (SAXS), mutagenesis, and kinetics are used to determine the contributions of this domain to PutA structure and function. The 1127-residue Rhodobacter capsulatus PutA (RcPutA) is used as a representative CTDUF-containing type B PutA. The reaction progress curve for the coupled PRODH–P5CDH activity of RcPutA does not exhibit a time lag, implying a substrate channeling mechanism. RcPutA is monomeric in solution, which is unprecedented for PutAs. SAXS rigid body modeling with target–decoy validation is used to build a model of RcPutA. On the basis of homology to aldehyde dehydrogenases (ALDHs), the CTDUF is predicted to consist of a β-hairpin fused to a noncatalytic Rossmann fold domain. The predicted tertiary structural interactions of the CTDUF resemble the quaternary structural interactions in the type A PutA dimer interface. The model is tested by mutagenesis of the dimerization hairpin of a type A PutA and the CTDUF hairpin of RcPutA. Similar functional phenotypes are observed in the two sets of variants, supporting the hypothesis that the CTDUF mimics the type A PutA dimer interface. These results suggest annotation of the CTDUF as an ALDH superfamily domain that facilitates P5CDH activity and substrate channeling by stabilizing the aldehyde-binding site and sealing the substrate-channeling tunnel from the bulk medium. PMID:25137435

  15. Structural and Functional Consequences of Coenzyme Binding to the Inactive Asian Variant of Mitochondrial Aldehyde Dehydrogenase: Roles of Residues 475 and 487

    SciTech Connect

    Larson,H.; Zhou, J.; Chen, Z.; Stamler, J.; Weiner, H.; Hurley, T.

    2007-01-01

    The common mitochondrial aldehyde dehydrogenase (ALDH2) ALDH2*2 polymorphism is associated with impaired ethanol metabolism and decreased efficacy of nitroglycerin treatment. These physiological effects are due to the substitution of Lys for Glu-487 that reduces the k{sub cat} for these processes and increases the K{sub m} for NAD{sup +}, as compared with ALDH2. In this study, we sought to understand the nature of the interactions that give rise to the loss of structural integrity and low activity in ALDH2*2 even when complexed with coenzyme. Consequently, we have solved the crystal structure of ALDH2*2 complexed with coenzyme to 2.5 {angstrom}. We have also solved the structures of a mutated form of ALDH2 where Arg-475 is replaced by Gln (R475Q). The structural and functional properties of the R475Q enzyme are intermediate between those of wild-type and the ALDH2*2 enzymes. In both cases, the binding of coenzyme restores most of the structural deficits observed in the apoenzyme structures. The binding of coenzyme to the R475Q enzyme restores its structure and catalytic properties to near wild-type levels. In contrast, the disordered helix within the coenzyme binding pocket of ALDH2*2 is reordered, but the active site is only partially reordered. Consistent with the structural data, ALDH2*2 showed a concentration-dependent increase in esterase activity and nitroglycerin reductase activity upon addition of coenzyme, but the levels of activity do not approach those of the wild-type enzyme or that of the R475Q enzyme. The data presented shows that Glu-487 maintains a critical function in linking the structure of the coenzyme binding site to that of the active site through its interactions with Arg-264 and Arg-475, and in doing so, creates the stable structural scaffold conducive to catalysis.

  16. Inhibition of human alcohol and aldehyde dehydrogenases by aspirin and salicylate: assessment of the effects on first-pass metabolism of ethanol.

    PubMed

    Lee, Shou-Lun; Lee, Yung-Pin; Wu, Min-Li; Chi, Yu-Chou; Liu, Chiu-Ming; Lai, Ching-Long; Yin, Shih-Jiun

    2015-05-01

    Previous studies have reported that aspirin significantly reduced the first-pass metabolism (FPM) of ethanol in humans thereby increasing adverse effects of alcohol. The underlying causes, however, remain poorly understood. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), principal enzymes responsible for metabolism of ethanol, are complex enzyme families that exhibit functional polymorphisms among ethnic groups and distinct tissue distributions. We investigated the inhibition profiles by aspirin and its major metabolite salicylate of ethanol oxidation by recombinant human ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2, ADH2, and ADH4, and acetaldehyde oxidation by ALDH1A1 and ALDH2, at pH 7.5 and 0.5 mM NAD(+). Competitive inhibition pattern was found to be a predominant type among the ADHs and ALDHs studied, although noncompetitive and uncompetitive inhibitions were also detected in a few cases. The inhibition constants of salicylate for the ADHs and ALDHs were considerably lower than that of aspirin with the exception of ADH1A that can be ascribed to a substitution of Ala-93 at the bottom of substrate pocket as revealed by molecular docking experiments. Kinetic inhibition equation-based simulations show at higher therapeutic levels of blood plasma salicylate (1.5 mM) that the decrease of activities at 2-10 mM ethanol for ADH1A/ADH2 and ADH1B2/ADH1B3 are predicted to be 75-86% and 31-52%, respectively, and that the activity decline for ALDH1A1 and ALDH2 at 10-50 μM acetaldehyde to be 62-73%. Our findings suggest that salicylate may substantially inhibit hepatic FPM of alcohol at both the ADH and ALDH steps when concurrent intaking aspirin. PMID:25772736

  17. Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aldehyde reductase gene ARI1 is a recently characterized member of intermediate subfamily under SDR (short-chain dehydrogenase/reductase) superfamily that revealed mechanisms of in situ detoxification of furfural and HMF for tolerance of Saccharomyces cerevisiae. Uncharacterized open reading frames ...

  18. The longitudinal effect of the aldehyde dehydrogenase 2*2 allele on the risk for nonalcoholic fatty liver disease.

    PubMed

    Oniki, K; Morita, K; Watanabe, T; Kajiwara, A; Otake, K; Nakagawa, K; Sasaki, Y; Ogata, Y; Saruwatari, J

    2016-01-01

    Aldehyde dehydrogenase 2 (ALDH2) detoxifies toxic aldehydes and has a key role in protecting the liver. An elevated gamma-glutamyl transferase (GGT) level is related to oxidative stress and nonalcoholic fatty liver disease (NAFLD). We herein investigated the association between inactive ALDH2*2 allele (rs671) and the risk of NAFLD, including the relationship to the GGT level. A retrospective follow-up study (mean 5.4±1.1 years) was conducted among 341 Japanese health screening program participants. The receiver operating characteristic curve indicated that the GGT level predicted the development of NAFLD (area under the curve: 0.65, P<0.05) with a cutoff value of 25.5 IUl(-1). The longitudinal risk of NAFLD was higher in the ALDH2*2 allele carriers than in the noncarriers (odds ratio (OR): 2.30, 95% confidence interval (CI): 1.21-4.40), and the risk was further increased among the *2 allele carriers with GGT values ⩾25.5 IUl(-1) (OR: 4.28, 95% CI: 1.80-10.19). On the other hand, there were no significant changes in the subjects' body weight and body mass index during observation period. The ALDH2*2 allele, in relation to the GGT level, may potentially be a novel risk factor for NAFLD. PMID:27214654

  19. The longitudinal effect of the aldehyde dehydrogenase 2*2 allele on the risk for nonalcoholic fatty liver disease

    PubMed Central

    Oniki, K; Morita, K; Watanabe, T; Kajiwara, A; Otake, K; Nakagawa, K; Sasaki, Y; Ogata, Y; Saruwatari, J

    2016-01-01

    Aldehyde dehydrogenase 2 (ALDH2) detoxifies toxic aldehydes and has a key role in protecting the liver. An elevated gamma-glutamyl transferase (GGT) level is related to oxidative stress and nonalcoholic fatty liver disease (NAFLD). We herein investigated the association between inactive ALDH2*2 allele (rs671) and the risk of NAFLD, including the relationship to the GGT level. A retrospective follow-up study (mean 5.4±1.1 years) was conducted among 341 Japanese health screening program participants. The receiver operating characteristic curve indicated that the GGT level predicted the development of NAFLD (area under the curve: 0.65, P<0.05) with a cutoff value of 25.5 IUl−1. The longitudinal risk of NAFLD was higher in the ALDH2*2 allele carriers than in the noncarriers (odds ratio (OR): 2.30, 95% confidence interval (CI): 1.21–4.40), and the risk was further increased among the *2 allele carriers with GGT values ⩾25.5 IUl−1 (OR: 4.28, 95% CI: 1.80–10.19). On the other hand, there were no significant changes in the subjects' body weight and body mass index during observation period. The ALDH2*2 allele, in relation to the GGT level, may potentially be a novel risk factor for NAFLD. PMID:27214654

  20. Isolated tumoral pyruvate dehydrogenase can synthesize acetoin which inhibits pyruvate oxidation as well as other aldehydes.

    PubMed

    Baggetto, L G; Lehninger, A L

    1987-05-29

    Oxidation of 1 mM pyruvate by Ehrlich and AS30-D tumor mitochondria is inhibited by acetoin, an unusual and important metabolite of pyruvate utilization by cancer cells, by acetaldehyde, methylglyoxal and excess pyruvate. The respiratory inhibition is reversed by other substrates added to pyruvate and also by 0.5 mM ATP. Kinetic properties of pyruvate dehydrogenase complex isolated from these tumor mitochondria have been studied. This complex appears to be able to synthesize acetoin from acetaldehyde plus pyruvate and is competitively inhibited by acetoin. The role of a new regulatory pattern for tumoral pyruvate dehydrogenase is presented. PMID:3593337

  1. The impact of mitochondrial aldehyde dehydrogenase (ALDH2) activation by Alda-1 on the behavioral and biochemical disturbances in animal model of depression.

    PubMed

    Stachowicz, Aneta; Głombik, Katarzyna; Olszanecki, Rafał; Basta-Kaim, Agnieszka; Suski, Maciej; Lasoń, Władysław; Korbut, Ryszard

    2016-01-01

    The etiology of depression remains still unclear. Recently, it has been proposed, that mitochondrial dysfunction may be associated with development of mood disorders, such as depression, bipolar disorder and anxiety disorders. Mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme responsible for the detoxification of reactive aldehydes, is considered to exert protective function in mitochondria. We investigated the influence of Alda-1, a small-molecule activator of ALDH2, on depressive- and anxiety-like behaviors in an animal model of depression - the prenatally stressed rats - using behavioral, molecular and proteomic methods. Prolonged Alda-1 administration significantly increased the climbing time, tended to reduce the immobility time and increased the swimming time of the prenatally stressed rats in the forced swim test. Moreover, treatment of prenatally stressed rats with Alda-1 significantly increased number of entries into the open arms of the maze and the time spent therein, as assessed by elevated plus-maze test. Such actions were associated with reduction of plasma 4-HNE-protein content, decrease of TNF-α mRNA and increase of PGC-1α (regulator of mitochondrial biogenesis) mRNA level in the frontal cortex and hippocampus of the prenatally stressed rats as well as with normalization of peripheral immune parameters and significant changes in expression of 6 and 4 proteins related to mitochondrial functions in the frontal cortex and hippocampus, respectively. Collectively, ALDH2 activation by Alda-1 led to a significant attenuation of depressive- and anxiety-like behaviors in the prenatally stressed rats. The pattern of changes suggested mitoprotective effect of Alda-1, however the exact functional consequences of the revealed alterations require further investigation. PMID:26254233

  2. Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

    PubMed Central

    Zheng, Tianyong; Olson, Daniel G.; Tian, Liang; Bomble, Yannick J.; Himmel, Michael E.; Lo, Jonathan; Hon, Shuen; Shaw, A. Joe; van Dijken, Johannes P.

    2015-01-01

    ABSTRACT Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are thermophilic bacteria that have been engineered to produce ethanol from the cellulose and hemicellulose fractions of biomass, respectively. Although engineered strains of T. saccharolyticum produce ethanol with a yield of 90% of the theoretical maximum, engineered strains of C. thermocellum produce ethanol at lower yields (∼50% of the theoretical maximum). In the course of engineering these strains, a number of mutations have been discovered in their adhE genes, which encode both alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. To understand the effects of these mutations, the adhE genes from six strains of C. thermocellum and T. saccharolyticum were cloned and expressed in Escherichia coli, the enzymes produced were purified by affinity chromatography, and enzyme activity was measured. In wild-type strains of both organisms, NADH was the preferred cofactor for both ALDH and ADH activities. In high-ethanol-producing (ethanologen) strains of T. saccharolyticum, both ALDH and ADH activities showed increased NADPH-linked activity. Interestingly, the AdhE protein of the ethanologenic strain of C. thermocellum has acquired high NADPH-linked ADH activity while maintaining NADH-linked ALDH and ADH activities at wild-type levels. When single amino acid mutations in AdhE that caused increased NADPH-linked ADH activity were introduced into C. thermocellum and T. saccharolyticum, ethanol production increased in both organisms. Structural analysis of the wild-type and mutant AdhE proteins was performed to provide explanations for the cofactor specificity change on a molecular level. IMPORTANCE This work describes the characterization of the AdhE enzyme from different strains of C. thermocellum and T. saccharolyticum. C. thermocellum and T. saccharolyticum are thermophilic anaerobes that have been engineered to make high yields of ethanol and can solubilize components of

  3. Reconstitution of Formylglycine-generating Enzyme with Copper(II) for Aldehyde Tag Conversion.

    PubMed

    Holder, Patrick G; Jones, Lesley C; Drake, Penelope M; Barfield, Robyn M; Bañas, Stefanie; de Hart, Gregory W; Baker, Jeanne; Rabuka, David

    2015-06-19

    To further our aim of synthesizing aldehyde-tagged proteins for research and biotechnology applications, we developed methods for recombinant production of aerobic formylglycine-generating enzyme (FGE) in good yield. We then optimized the FGE biocatalytic reaction conditions for conversion of cysteine to formylglycine in aldehyde tags on intact monoclonal antibodies. During the development of these conditions, we discovered that pretreating FGE with copper(II) is required for high turnover rates and yields. After further investigation, we confirmed that both aerobic prokaryotic (Streptomyces coelicolor) and eukaryotic (Homo sapiens) FGEs contain a copper cofactor. The complete kinetic parameters for both forms of FGE are described, along with a proposed mechanism for FGE catalysis that accounts for the copper-dependent activity. PMID:25931126

  4. Reconstitution of Formylglycine-generating Enzyme with Copper(II) for Aldehyde Tag Conversion

    PubMed Central

    Holder, Patrick G.; Jones, Lesley C.; Drake, Penelope M.; Barfield, Robyn M.; Bañas, Stefanie; de Hart, Gregory W.; Baker, Jeanne; Rabuka, David

    2015-01-01

    To further our aim of synthesizing aldehyde-tagged proteins for research and biotechnology applications, we developed methods for recombinant production of aerobic formylglycine-generating enzyme (FGE) in good yield. We then optimized the FGE biocatalytic reaction conditions for conversion of cysteine to formylglycine in aldehyde tags on intact monoclonal antibodies. During the development of these conditions, we discovered that pretreating FGE with copper(II) is required for high turnover rates and yields. After further investigation, we confirmed that both aerobic prokaryotic (Streptomyces coelicolor) and eukaryotic (Homo sapiens) FGEs contain a copper cofactor. The complete kinetic parameters for both forms of FGE are described, along with a proposed mechanism for FGE catalysis that accounts for the copper-dependent activity. PMID:25931126

  5. Dihydrolipoamide dehydrogenase from halophilic archaebacteria: purification and properties of the enzyme from halobacterium halobium

    SciTech Connect

    Danson, J.J.; McQuattie, A.; Stevenson, K.J.

    1986-07-01

    Halophilic archaebacteria possess dihydrolipoamide dehydrogenase activity but apparently lack the 2-oxoacid dehydrogenase multienzyme complexes of which it is usually an integral component. In this paper, the purification of dihydrolipoamide dehydrogenase from Halobacterium halobium is reported. The enzyme is a dimer with a polypeptide chain M/sub r/ of 58,000 (+/-3000). The amino acid composition of the enzyme is compared with those of the eubacterial and eukaryotic dihydrolipoamide dehydrogenases, and evidence is presented to suggest that the N-terminal amino acid of the H. halobium enzyme is blocked. Chemical modification with the trivalent arsenical reagent (p-aminophenyl)dichloroarsine indicates the involvement of a reversibly reducible disulfide bond in the enzyme's catalytic mechanism. The possible metabolic role of this dihydrolipoamide dehydrogenase in the absence of 2-oxoacid dehydrogenase complexes is discussed.

  6. Aldehyde dehydrogenase 2 (ALDH2) and alcohol dehydrogenase 1B (ADH1B) polymorphisms exacerbate bladder cancer risk associated with alcohol drinking: gene-environment interaction.

    PubMed

    Masaoka, Hiroyuki; Ito, Hidemi; Soga, Norihito; Hosono, Satoyo; Oze, Isao; Watanabe, Miki; Tanaka, Hideo; Yokomizo, Akira; Hayashi, Norio; Eto, Masatoshi; Matsuo, Keitaro

    2016-06-01

    Although a range of chemical exposures (cigarette smoking and occupational exposure) are recognized risk factors for the development of bladder cancer (BCa), many epidemiological studies have demonstrated that alcohol drinking is not associated with BCa risk. Aldehyde dehydrogenase 2 (ALDH2; rs671, Glu504Lys) and alcohol dehydrogenase 1B (ADH1B; rs1229984, His47Arg) polymorphisms impact the accumulation of acetaldehyde, resulting in an increased risk of various cancers. To date, however, no studies evaluating the association between BCa risk and alcohol drinking have considered these polymorphisms. Here, we conducted a matched case-control study to investigate whether ALDH2 and ADH1B polymorphisms influence BCa risk associated with alcohol drinking. Cases were 74 BCa patients and controls were 740 first-visit outpatients without cancer at Aichi Cancer Center Hospital between January 2001 and December 2005. Odds ratio (OR), 95% confidence interval (CI) and gene-environment interaction were assessed by conditional logistic regression analysis with adjustment for potential confounders. Results showed that ALDH2 Glu/Lys was associated with a significantly increased risk of BCa compared with Glu/Glu (OR 2.03, 95% CI 1.14-3.62, P = 0.017). In contrast, ALDH2 Glu/Lys showed no increase in risk among the stratum of never drinkers compared with Glu/Glu, indicating a gene-environment interaction. ADH1B His/Arg had an OR of 1.98 (1.20-3.24, P = 0.007) compared with His/His. ADH1B Arg+ showed a similar OR and 95% CI. Individuals with ALDH2 Glu/Lys and ADH1B Arg+ had the highest risk of BCa compared with ALDH2 Glu/Glu and ADH1B His/His [OR 4.00 (1.81-8.87), P = 0.001]. PMID:26992901

  7. Single-dose ethanol administration downregulates expression of cytochrome p450 2E1 mRNA in aldehyde dehydrogenase 2 knockout mice.

    PubMed

    Matsumoto, Akiko; Kawamoto, Toshihiro; Horita, Mikako; Takahashi, Tatsuya; Isse, Toyohi; Oyama, Tsunehiro; Ichiba, Masayoshi

    2007-12-01

    The polymorphism of aldehyde dehydrogenase 2 (ALDH2), denoted ALDH2*2, is very common in East Asian origin. Acetaldehyde, an intermediate metabolite of ethanol, is metabolized very slowly in people with ALDH2*2 because the mutant ALDH2 protein lacks the activity of acetaldehyde metabolism. On the other hand, it is well established that one of the cytochrome P450 enzymes, CYP2E1, is an activator of carcinogens (e.g., nitorosamines) and a generator of oxidative stress, and it is shown that CYP2E1 was induced by ethanol via gene transcriptional regulation. In the present study, to examine the consequences of ALDH2 polymorphism on transcriptional regulation of CYP2E1 in liver tissue, Aldh2+/+ and Aldh2-/- mice were orally administered 5 g/kg body weight of ethanol and the levels of CYP2E1 mRNA in liver tissue then analyzed. The level of CYP2E1 mRNA 12h after the ethanol administration tended to be higher than the 0-h group in Aldh2+/+ mice, however, it was significantly lower than the 0-h group in Aldh2-/- mice. These findings suggest that single-dose ethanol administration downregulates the expression of cytochrome p450 2E1 mRNA in the presence of inactive ALDH2. PMID:17980998

  8. Pharmacological recruitment of aldehyde dehydrogenase 3A1 (ALDH3A1) to assist ALDH2 in acetaldehyde and ethanol metabolism in vivo

    PubMed Central

    Chen, Che-Hong; Cruz, Leslie A.; Mochly-Rosen, Daria

    2015-01-01

    Correcting a genetic mutation that leads to a loss of function has been a challenge. One such mutation is in aldehyde dehydrogenase 2 (ALDH2), denoted ALDH2*2. This mutation is present in ∼0.6 billion East Asians and results in accumulation of toxic acetaldehyde after consumption of ethanol. To temporarily increase metabolism of acetaldehyde in vivo, we describe an approach in which a pharmacologic agent recruited another ALDH to metabolize acetaldehyde. We focused on ALDH3A1, which is enriched in the upper aerodigestive track, and identified Alda-89 as a small molecule that enables ALDH3A1 to metabolize acetaldehyde. When given together with the ALDH2-specific activator, Alda-1, Alda-89 reduced acetaldehyde-induced behavioral impairment by causing a rapid reduction in blood ethanol and acetaldehyde levels after acute ethanol intoxication in both wild-type and ALDH2-deficient, ALDH2*1/*2, heterozygotic knock-in mice. The use of a pharmacologic agent to recruit an enzyme to metabolize a substrate that it usually does not metabolize may represent a novel means to temporarily increase elimination of toxic agents in vivo. PMID:25713355

  9. Association of Genetically Determined Aldehyde Dehydrogenase 2 Activity with Diabetic Complications in Relation to Alcohol Consumption in Japanese Patients with Type 2 Diabetes Mellitus: The Fukuoka Diabetes Registry.

    PubMed

    Idewaki, Yasuhiro; Iwase, Masanori; Fujii, Hiroki; Ohkuma, Toshiaki; Ide, Hitoshi; Kaizu, Shinako; Jodai, Tamaki; Kikuchi, Yohei; Hirano, Atsushi; Nakamura, Udai; Kubo, Michiaki; Kitazono, Takanari

    2015-01-01

    Aldehyde dehydrogenase 2 (ALDH2) detoxifies aldehyde produced during ethanol metabolism and oxidative stress. A genetic defect in this enzyme is common in East Asians and determines alcohol consumption behaviors. We investigated the impact of genetically determined ALDH2 activity on diabetic microvascular and macrovascular complications in relation to drinking habits in Japanese patients with type 2 diabetes mellitus. An ALDH2 single-nucleotide polymorphism (rs671) was genotyped in 4,400 patients. Additionally, the relationship of clinical characteristics with ALDH2 activity (ALDH2 *1/*1 active enzyme activity vs. *1/*2 or *2/*2 inactive enzyme activity) and drinking habits (lifetime abstainers vs. former or current drinkers) was investigated cross-sectionally (n = 691 in *1/*1 abstainers, n = 1,315 in abstainers with *2, n = 1,711 in *1/*1 drinkers, n = 683 in drinkers with *2). The multiple logistic regression analysis for diabetic complications was adjusted for age, sex, current smoking habits, leisure-time physical activity, depressive symptoms, diabetes duration, body mass index, hemoglobin A1c, insulin use, high-density lipoprotein cholesterol, systolic blood pressure and renin-angiotensin system inhibitors use. Albuminuria prevalence was significantly lower in the drinkers with *2 than that of other groups (odds ratio [95% confidence interval (CI)]: *1/*1 abstainers as the referent, 0.94 [0.76-1.16] in abstainers with *2, 1.00 [0.80-1.26] in *1/*1 drinkers, 0.71 [0.54-0.93] in drinkers with *2). Retinal photocoagulation prevalence was also lower in drinkers with ALDH2 *2 than that of other groups. In contrast, myocardial infarction was significantly increased in ALDH2 *2 carriers compared with that in ALDH2 *1/*1 abstainers (odds ratio [95% CI]: *1/*1 abstainers as the referent, 2.63 [1.28-6.13] in abstainers with *2, 1.89 [0.89-4.51] in *1/*1 drinkers, 2.35 [1.06-5.79] in drinkers with *2). In summary, patients with type 2 diabetes and ALDH2 *2 displayed a

  10. Association of Genetically Determined Aldehyde Dehydrogenase 2 Activity with Diabetic Complications in Relation to Alcohol Consumption in Japanese Patients with Type 2 Diabetes Mellitus: The Fukuoka Diabetes Registry

    PubMed Central

    Idewaki, Yasuhiro; Iwase, Masanori; Fujii, Hiroki; Ohkuma, Toshiaki; Ide, Hitoshi; Kaizu, Shinako; Jodai, Tamaki; Kikuchi, Yohei; Hirano, Atsushi; Nakamura, Udai; Kubo, Michiaki; Kitazono, Takanari

    2015-01-01

    Aldehyde dehydrogenase 2 (ALDH2) detoxifies aldehyde produced during ethanol metabolism and oxidative stress. A genetic defect in this enzyme is common in East Asians and determines alcohol consumption behaviors. We investigated the impact of genetically determined ALDH2 activity on diabetic microvascular and macrovascular complications in relation to drinking habits in Japanese patients with type 2 diabetes mellitus. An ALDH2 single-nucleotide polymorphism (rs671) was genotyped in 4,400 patients. Additionally, the relationship of clinical characteristics with ALDH2 activity (ALDH2 *1/*1 active enzyme activity vs. *1/*2 or *2/*2 inactive enzyme activity) and drinking habits (lifetime abstainers vs. former or current drinkers) was investigated cross-sectionally (n = 691 in *1/*1 abstainers, n = 1,315 in abstainers with *2, n = 1,711 in *1/*1 drinkers, n = 683 in drinkers with *2). The multiple logistic regression analysis for diabetic complications was adjusted for age, sex, current smoking habits, leisure-time physical activity, depressive symptoms, diabetes duration, body mass index, hemoglobin A1c, insulin use, high-density lipoprotein cholesterol, systolic blood pressure and renin-angiotensin system inhibitors use. Albuminuria prevalence was significantly lower in the drinkers with *2 than that of other groups (odds ratio [95% confidence interval (CI)]: *1/*1 abstainers as the referent, 0.94 [0.76–1.16] in abstainers with *2, 1.00 [0.80–1.26] in *1/*1 drinkers, 0.71 [0.54–0.93] in drinkers with *2). Retinal photocoagulation prevalence was also lower in drinkers with ALDH2 *2 than that of other groups. In contrast, myocardial infarction was significantly increased in ALDH2 *2 carriers compared with that in ALDH2 *1/*1 abstainers (odds ratio [95% CI]: *1/*1 abstainers as the referent, 2.63 [1.28–6.13] in abstainers with *2, 1.89 [0.89–4.51] in *1/*1 drinkers, 2.35 [1.06–5.79] in drinkers with *2). In summary, patients with type 2 diabetes and ALDH2 *2

  11. Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes

    DOEpatents

    Smith, Robert E. [557 Escondido Cir., Livermore, CA 94550; Dolbeare, Frank A. [5178 Diane La., Livermore, CA 94550

    1980-10-21

    Analysis of proteinases is accomplished using conventional amino acid containing aromatic amine substrates. Aromatic amines such as 4-methoxy-2-naphthylamine (4M2NA), 2-naphthylamine, aminoisophthalic acid dimethyl ester, p-nitroaniline, 4-methoxy-1-aminofluorene and coumarin derivatives resulting from enzymatic hydrolysis of the substrate couples with aromatic aldehydes such as 5-nitrosalicylaldehyde (5-NSA), benzaldehyde and p-nitrobenzaldehyde to produce Schiff-base complexes which are water insoluble. Certain Schiff-base complexes produce a shift from blue to orange-red (visible) fluorescence. Such complexes are useful in the assay of enzymes.

  12. Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes

    DOEpatents

    Smith, Robert E.; Dolbeare, Frank A.

    1979-01-01

    Analysis of proteinases is accomplished using conventional amino acid containing aromatic amine substrates. Aromatic amines such as 4-methoxy-2-naphthylamine (4M2NA), 2-naphthylamine, aminoisophthalic acid dimethyl ester, p-nitroaniline, 5-methoxy-1-aminofluorene and coumarin derivatives resulting from enzymatic hydrolysis of the substrate couples with aromatic aldehydes such as 5-nitrosalicylaldehyde (5-NSA), benzaldehyde and p-nitrobenzaldehyde to produce Schiff-base complexes which are water insoluble. Certain Schiff-base complexes produce a shift from blue to orange-red (visible) fluorescence. Such complexes are useful in the assay of enzymes.

  13. Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes

    DOEpatents

    Smith, R.E.; Dolbeare, F.A.

    1980-10-21

    Analysis of proteinases is accomplished using conventional amino acid containing aromatic amine substrates. Aromatic amines such as 4-methoxy-2-naphthylamine (4M2NA), 2-naphthylamine, aminoisophthalic acid dimethyl ester, p-nitroaniline, 4-methoxy-1-aminofluorene and coumarin derivatives resulting from enzymatic hydrolysis of the substrate couples with aromatic aldehydes such as 5-nitrosalicylaldehyde (5-NSA), benzaldehyde and p-nitrobenzaldehyde to produce Schiff-base complexes which are water insoluble. Certain Schiff-base complexes produce a shift from blue to orange-red (visible) fluorescence. Such complexes are useful in the assay of enzymes. No Drawings

  14. Discovery of NCT-501, a Potent and Selective Theophylline-Based Inhibitor of Aldehyde Dehydrogenase 1A1 (ALDH1A1).

    PubMed

    Yang, Shyh-Ming; Yasgar, Adam; Miller, Bettina; Lal-Nag, Madhu; Brimacombe, Kyle; Hu, Xin; Sun, Hongmao; Wang, Amy; Xu, Xin; Nguyen, Kimloan; Oppermann, Udo; Ferrer, Marc; Vasiliou, Vasilis; Simeonov, Anton; Jadhav, Ajit; Maloney, David J

    2015-08-13

    Aldehyde dehydrogenases (ALDHs) metabolize reactive aldehydes and possess important physiological and toxicological functions in areas such as CNS, metabolic disorders, and cancers. Increased ALDH (e.g., ALDH1A1) gene expression and catalytic activity are vital biomarkers in a number of malignancies and cancer stem cells, highlighting the need for the identification and development of small molecule ALDH inhibitors. A new series of theophylline-based analogs as potent ALDH1A1 inhibitors is described. The optimization of hits identified from a quantitative high throughput screening (qHTS) campaign led to analogs with improved potency and early ADME properties. This chemotype exhibits highly selective inhibition against ALDH1A1 over ALDH3A1, ALDH1B1, and ALDH2 isozymes as well as other dehydrogenases such as HPGD and HSD17β4. Moreover, the pharmacokinetic evaluation of selected analog 64 (NCT-501) is also highlighted. PMID:26207746

  15. Folate, alcohol, and aldehyde dehydrogenase 2 polymorphism and the risk of oral and pharyngeal cancer in Japanese.

    PubMed

    Matsuo, Keitaro; Rossi, Marta; Negri, Eva; Oze, Isao; Hosono, Satoyo; Ito, Hidemi; Watanabe, Miki; Yatabe, Yasushi; Hasegawa, Yasuhisa; Tanaka, Hideo; Tajima, Kazuo; La Vecchia, Carlo

    2012-03-01

    Folate consumption is inversely associated with the risk of oral and pharyngeal cancer (OPC) and potentially interacts with alcohol drinking in the risk of OPC. Aldehyde dehydrogenase 2 (ALDH2) gene polymorphism is known to interact with alcohol consumption. The aim of this study was to investigate potential interaction between folate, alcohol drinking, and ALDH2 polymorphism in the risk of OPC in a Japanese population. The study group comprised 409 head and neck cancer cases and 1227 age-matched and sex-matched noncancer controls; of these, 251 cases and 759 controls were evaluated for ALDH rs671 polymorphism. Associations were assessed by odds ratios and 95% confidence intervals in multiple logistic regression models. We observed an inverse association between folate consumption and OPC risk. The odds ratio for high folate intake was 0.53 (95% confidence interval: 0.36-0.77) relative to low intake (P trend=0.003). This association was consistent across strata of sex, age, smoking, and ALDH2 genotypes. Interaction between folate consumption, drinking, and ALDH2 genotype was remarkable (three-way interaction, P<0.001). We observed significant interaction among folate, drinking, and ALDH2 genotype in the Japanese population. PMID:21946912

  16. E. coli metabolic protein aldehyde-alcohol dehydrogenase-E binds to the ribosome: a unique moonlighting action revealed.

    PubMed

    Shasmal, Manidip; Dey, Sandip; Shaikh, Tanvir R; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosome. PMID:26822933

  17. E. coli metabolic protein aldehyde-alcohol dehydrogenase-E binds to the ribosome: a unique moonlighting action revealed

    PubMed Central

    Shasmal, Manidip; Dey, Sandip; Shaikh, Tanvir R.; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosome. PMID:26822933

  18. Implication of an Aldehyde Dehydrogenase Gene and a Phosphinothricin N-Acetyltransferase Gene in the Diversity of Pseudomonas cichorii Virulence

    PubMed Central

    Tanaka, Masayuki; Wali, Ullah Md; Nakayashiki, Hitoshi; Fukuda, Tatsuya; Mizumoto, Hiroyuki; Ohnishi, Kouhei; Kiba, Akinori; Hikichi, Yasufumi

    2011-01-01

    Pseudomonas cichorii harbors the hrp genes. hrp-mutants lose their virulence on eggplant but not on lettuce. A phosphinothricin N-acetyltransferase gene (pat) is located between hrpL and an aldehyde dehydrogenase gene (aldH) in the genome of P. cichorii. Comparison of nucleotide sequences and composition of the genes among pseudomonads suggests a common ancestor of hrp and pat between P. cichorii strains and P. viridiflava strains harboring the single hrp pathogenicity island. In contrast, phylogenetic diversification of aldH corresponded to species diversification amongst pseudomonads. In this study, the involvement of aldH and pat in P. cichorii virulence was analyzed. An aldH-deleted mutant (ΔaldH) and a pat-deleted mutant (Δpat) lost their virulence on eggplant but not on lettuce. P. cichorii expressed both genes in eggplant leaves, independent of HrpL, the transcriptional activator for the hrp. Inoculation into Asteraceae species susceptible to P. cichorii showed that the involvement of hrp, pat and aldH in P. cichorii virulence is independent of each other and has no relationship with the phylogeny of Asteraceae species based on the nucleotide sequences of ndhF and rbcL. It is thus thought that not only the hrp genes but also pat and aldH are implicated in the diversity of P. cichorii virulence on susceptible host plant species. PMID:24704843

  19. Activity and electrophoretic profiles of liver aldehyde dehydrogenases from mice of inbred strains with different alcohol preference.

    PubMed

    Yamazaki, H; Nishiguchi, K; Miyamoto, R; Ogita, Z I; Nakanishi, S

    1983-01-01

    1. The activity of low Km-aldehyde dehydrogenase (ALDH) in the liver mitochondrial fraction (MT-fraction) from male C57BL/6J strain mice (alcohol preferring) was significantly higher than that from DBA/2 mice (alcohol avoiding). The F1 hybrids (C57BL/6J X DBA/2) did not exhibit the intermediate activity to these two strains. 2. Strain differences in liver mitochondrial ALDH isozymes were observed by isoelectric focusing. C57BL/6J strain had two isozymes at pH 7.1 while DBA/2 had no band at this pH. F1 hybrid mice had similar two bands with lower density to those of C57BL/6J at pH 7.1. There was no difference in zymograms of the soluble fraction between C57BL/6J and DBA/2 strains. 3. The present results suggest that the difference in alcohol preference of mice may depend on some restricted ALDH isozymes with different pl or electric mobility rather than the enzymatic activity in the liver MT-fraction. PMID:6822317

  20. Aldehyde Dehydrogenase 2 (ALDH2) Polymorphism and the Risk of Alcoholic Liver Cirrhosis among East Asians: A Meta-Analysis

    PubMed Central

    He, Lei; Luo, Hesheng

    2016-01-01

    Purpose The aldehyde dehydrogenase 2 (ALDH2) gene has been implicated in the development of alcoholic liver cirrhosis (ALC) in East Asians. However, the results are inconsistent. In this study, a meta-analysis was performed to assess the associations between the ALDH2 polymorphism and the risk of ALC. Materials and Methods Relevant studies were retrieved by searching PubMed, Web of Science, CNKI, Wanfang and Veipu databases up to January 10, 2015. Pooled odds ratio (OR) and 95% confidence interval (CI) were calculated using either the fixed- or random effects model. Results A total of twelve case-control studies included 1003 cases and 2011 controls were included. Overall, the ALDH2 polymorphism was associated with a decreased risk of ALC (*1/*2 vs. *1/*1: OR=0.78, 95% CI: 0.61–0.99). However, in stratification analysis by country, we failed to detect any association among Chinese, Korean or Japanese populations. Conclusion The pooled evidence suggests that ALDH2 polymorphism may be an important protective factor for ALC in East Asians. PMID:27189280

  1. Solanum Incanum Extract Downregulates Aldehyde Dehydrogenase 1-Mediated Stemness and Inhibits Tumor Formation in Ovarian Cancer Cells

    PubMed Central

    Wu, Yi-Hui; Chiu, Wen-Tai; Young, Ming-Jer; Chang, Tzu-Hao; Huang, Yu-Fang; Chou, Cheng-Yang

    2015-01-01

    Solanum incanum extract (SR-T100), containing the active ingredient solamargine, can induce apoptosis via upregulation of tumor necrosis factor receptor expression and activation of the mitochondrial apoptosis pathway, and has therapeutic effects in patients with actinic keratosis. Here, we evaluate the novel molecular mechanisms underlying SR-T100-regulated stemness and chemoresistance. The concentration of SR-T100 that inhibited 50% cell viability (IC50) was lower in ovarian cancer cells than in nonmalignant cells. Furthermore, the SR-T100 IC50 in chemoresistant cells was similar to the IC50 in chemosensitive cells. Additionally, SR-T100 increased cisplatin and paclitaxel sensitivity in chemoresistant cells. SR-T100 downregulated the expression of stem cell markers, including aldehyde dehydrogenase 1 (ALDH1), Notch1, and FoxM1, and reduced sphere formation in ovarian cancer cells. Using microarray analyses, immunoblotting, luciferase activity, and chromatin immunoprecipitation (ChIP) assays, we showed that SR-T100 suppressed the expression of c/EBPβ and COL11A1, and its promoter activity, in resistant cells, but not sensitive cells. SR-T100, paclitaxel, and cisplatin inhibited the growth of A2780CP70 cells in mouse xenografts, as compared to the vehicle control, and the combination of cisplatin and SR-T100 was more effective than either treatment alone. SR-T100 may represent a potential therapeutic adjunct to chemotherapy for ovarian cancer treatment. PMID:26366215

  2. Mitochondrial aldehyde dehydrogenase mediates vasodilator responses of glyceryl trinitrate and sodium nitrite in the pulmonary vascular bed of the rat.

    PubMed

    Badejo, Adeleke M; Hodnette, Chris; Dhaliwal, Jasdeep S; Casey, David B; Pankey, Edward; Murthy, Subramanyam N; Nossaman, Bobby D; Hyman, Albert L; Kadowitz, Philip J

    2010-09-01

    It has been reported that mitochondrial aldehyde dehydrogenase (ALDH2) catalyzes the formation of glyceryl dinitrate and inorganic nitrite from glyceryl trinitrate (GTN), leading to an increase in cGMP and vasodilation in the coronary and systemic vascular beds. However, the role of nitric oxide (NO) formed from nitrite in mediating the response to GTN in the pulmonary vascular bed is uncertain. The purpose of the present study was to determine if nitrite plays a role in mediating vasodilator responses to GTN. In this study, intravenous injections of GTN and sodium nitrite decreased pulmonary and systemic arterial pressures and increased cardiac output. The decreases in pulmonary arterial pressure under baseline and elevated tone conditions and decreases in systemic arterial pressure in response to GTN and sodium nitrite were attenuated by cyanamide, an ALDH2 inhibitor, whereas responses to the NO donor, sodium nitroprusside (SNP), were not altered. The decreases in pulmonary and systemic arterial pressure in response to GTN and SNP were not altered by allopurinol, an inhibitor of xanthine oxidoreductase, whereas responses to sodium nitrite were attenuated. GTN was approximately 1,000-fold more potent than sodium nitrite in decreasing pulmonary and systemic arterial pressures. These results suggest that ALDH2 plays an important role in the bioactivation of GTN and nitrite in the pulmonary and systemic vascular beds and that the reduction of nitrite to vasoactive NO does not play an important role in mediating vasodilator responses to GTN in the intact chest rat. PMID:20543077

  3. Mitochondrial aldehyde dehydrogenase mediates vasodilator responses of glyceryl trinitrate and sodium nitrite in the pulmonary vascular bed of the rat

    PubMed Central

    Badejo, Adeleke M.; Hodnette, Chris; Dhaliwal, Jasdeep S.; Casey, David B.; Pankey, Edward; Murthy, Subramanyam N.; Nossaman, Bobby D.; Hyman, Albert L.

    2010-01-01

    It has been reported that mitochondrial aldehyde dehydrogenase (ALDH2) catalyzes the formation of glyceryl dinitrate and inorganic nitrite from glyceryl trinitrate (GTN), leading to an increase in cGMP and vasodilation in the coronary and systemic vascular beds. However, the role of nitric oxide (NO) formed from nitrite in mediating the response to GTN in the pulmonary vascular bed is uncertain. The purpose of the present study was to determine if nitrite plays a role in mediating vasodilator responses to GTN. In this study, intravenous injections of GTN and sodium nitrite decreased pulmonary and systemic arterial pressures and increased cardiac output. The decreases in pulmonary arterial pressure under baseline and elevated tone conditions and decreases in systemic arterial pressure in response to GTN and sodium nitrite were attenuated by cyanamide, an ALDH2 inhibitor, whereas responses to the NO donor, sodium nitroprusside (SNP), were not altered. The decreases in pulmonary and systemic arterial pressure in response to GTN and SNP were not altered by allopurinol, an inhibitor of xanthine oxidoreductase, whereas responses to sodium nitrite were attenuated. GTN was ∼1,000-fold more potent than sodium nitrite in decreasing pulmonary and systemic arterial pressures. These results suggest that ALDH2 plays an important role in the bioactivation of GTN and nitrite in the pulmonary and systemic vascular beds and that the reduction of nitrite to vasoactive NO does not play an important role in mediating vasodilator responses to GTN in the intact chest rat. PMID:20543077

  4. Aberrant expression of aldehyde dehydrogenase 1A (ALDH1A) subfamily genes in acute lymphoblastic leukaemia is a common feature of T-lineage tumours.

    PubMed

    Longville, Brooke A C; Anderson, Denise; Welch, Mathew D; Kees, Ursula R; Greene, Wayne K

    2015-01-01

    The class 1A aldehyde dehydrogenase (ALDH1A) subfamily of genes encode enzymes that function at the apex of the retinoic acid (RA) signalling pathway. We detected aberrant expression of ALDH1A genes, particularly ALDH1A2, in a majority (72%) of primary paediatric T cell acute lymphoblastic leukaemia (T-ALL) specimens. ALDH1A expression was almost exclusive to T-lineage, but not B-lineage, ALL. To determine whether ALDH1A expression may have relevance to T-ALL cell growth and survival, the effect of inhibiting ALDH1A function was measured on a panel of human ALL cell lines. This revealed that T-ALL proliferation had a higher sensitivity to modulation of ALDH1A activity and RA signalling as compared to ALL cell lines of B-lineage. Consistent with these findings, the genes most highly correlated with ALDH1A2 expression were involved in cell proliferation and apoptosis. Evidence that such genes may be targets of regulation via RA signalling initiated by ALDH1A activity was provided by the TNFRSF10B gene, encoding the apoptotic death receptor TNFRSF10B (also termed TRAIL-R2), which negatively correlated with ALDH1A2 and showed elevated transcription following treatment of T-ALL cell lines with the ALDH1A inhibitor citral (3,7-dimethyl-2,6-octadienal). These data indicate that ALDH1A expression is a common event in T-ALL and supports a role for these enzymes in the pathobiology of this disease. PMID:25208926

  5. Alcohol and Aldehyde Dehydrogenases Contribute to Sex-Related Differences in Clearance of Zolpidem in Rats

    PubMed Central

    Peer, Cody J.; Strope, Jonathan D.; Beedie, Shaunna; Ley, Ariel M.; Holly, Alesia; Calis, Karim; Farkas, Ronald; Parepally, Jagan; Men, Angela; Fadiran, Emmanuel O.; Scott, Pamela; Jenkins, Marjorie; Theodore, William H.; Sissung, Tristan M.

    2016-01-01

    Objectives: The recommended zolpidem starting dose was lowered in females (5 mg vs. 10 mg) since side effects were more frequent and severe than those of males; the mechanism underlying sex differences in pharmacokinetics (PK) is unknown. We hypothesized that such differences were caused by known sex-related variability in alcohol dehydrogenase (ADH) expression. Methods: Male, female, and castrated male rats were administered 2.6 mg/kg zolpidem, ± disulfiram (ADH/ALDH pathway inhibitor) to compare PK changes induced by sex and gonadal hormones. PK analyses were conducted in rat plasma and rat brain. Key findings: Sex differences in PK were evident: females had a higher CMAX (112.4 vs. 68.1 ug/L) and AUC (537.8 vs. 231.8 h∗ug/L) than uncastrated males. Castration induced an earlier TMAX (0.25 vs. 1 h), greater CMAX (109.1 vs. 68.1 ug/L), and a corresponding AUC increase (339.7 vs. 231.8 h∗ug/L). Administration of disulfiram caused more drastic CMAX and TMAX changes in male vs. female rats that mirrored the effects of castration on first-pass metabolism, suggesting that the observed PK differences may be caused by ADH/ALDH expression. Brain concentrations paralleled plasma concentrations. Conclusion: These findings indicate that sex differences in zolpidem PK are influenced by variation in the expression of ADH/ALDH due to gonadal androgens. PMID:27574509

  6. Alcohol Dehydrogenase-1B (rs1229984) and Aldehyde Dehydrogenase-2 (rs671) Genotypes Are Strong Determinants of the Serum Triglyceride and Cholesterol Levels of Japanese Alcoholic Men

    PubMed Central

    Yokoyama, Akira; Yokoyama, Tetsuji; Matsui, Toshifumi; Mizukami, Takeshi; Kimura, Mitsuru; Matsushita, Sachio; Higuchi, Susumu; Maruyama, Katsuya

    2015-01-01

    Background Elevated serum triglyceride (TG) and high-density-lipoprotein cholesterol (HDL-C) levels are common in drinkers. The fast-metabolizing alcohol dehydrogenase-1B encoded by the ADH1B*2 allele (vs. ADH1B*1/*1 genotype) and inactive aldehyde dehydrogenase-2 encoded by the ALDH2*2 allele (vs. ALDH2*1/*1 genotype) modify ethanol metabolism and are prevalent (≈90% and ≈40%, respectively) in East Asians. We attempted to evaluate the associations between the ADH1B and ALDH2 genotypes and lipid levels in alcoholics. Methods The population consisted of 1806 Japanese alcoholic men (≥40 years) who had undergone ADH1B and ALDH2 genotyping and whose serum TG, total cholesterol, and HDL-C levels in the fasting state had been measured within 3 days after admission. Results High serum levels of TG (≥150 mg/dl), HDL-C (>80 mg/dl), and low-density-lipoprotein cholesterol (LDL-C calculated by the Friedewald formula ≥140 mg/dl) were observed in 24.3%, 16.8%, and 15.6%, respectively, of the subjects. Diabetes, cirrhosis, smoking, and body mass index (BMI) affected the serum lipid levels. Multivariate analysis revealed that the presence of the ADH1B*2 allele and the active ALDH2*1/*1 genotype increased the odds ratio (OR; 95% confidence interval) for a high TG level (2.22 [1.67–2.94] and 1.39 [0.99–1.96], respectively), and decreased the OR for a high HDL-C level (0.37 [0.28–0.49] and 0.51 [0.37–0.69], respectively). The presence of the ADH1B*2 allele decreased the OR for a high LDL-C level (0.60 [0.45–0.80]). The ADH1B*2 plus ALDH2*1/*1 combination yielded the highest ORs for high TG levels and lowest OR for a high HDL-C level. The genotype effects were more prominent in relation to the higher levels of TG (≥220 mg/dl) and HDL-C (≥100 mg/dl). Conclusions The fast-metabolizing ADH1B and active ALDH2, and especially a combination of the two were strongly associated with higher serum TG levels and lower serum HDL-C levels of alcoholics. The fast

  7. Hydroperoxidic inhibitor of horse liver alcohol dehydrogenase activity, tightly bound to the enzyme-NAD+ complex, characteristically degrades the coenzyme.

    PubMed

    Skurský, L; Rezác, M; Khan, A N; Zídek, L; Rocek, J

    1992-01-01

    The strong inhibition of horse liver alcohol dehydrogenase (HLAD) by p-methylbenzyl hydroperoxide (XyHP) is only transient, XyHP behaves also as a pseudo-substrate of the enzyme and in the presence of NAD+, is degraded by HLAD to (as yet unidentified) non-inhibiting products while the NAD+ is converted to a derivative similar to the "NADX", originally observed in an analogous reaction of HLAD with hydrogen peroxide. The apparent KM for XyHP is approximately 10(4) times smaller than that for H2O2. The catalytic constant kcat for HLAD degradation of XyHP is two orders of magnitude less than that for ethanol dehydrogenation. XyHP inhibits both directions of the alcohol-aldehyde interconversion with equal potency. The first step of the inhibition mechanism is a tight binding of XyHP to the binary HLAD-NAD+ complex. PMID:1284958

  8. Inhibition of telomerase activity preferentially targets aldehyde dehydrogenase-positive cancer stem-like cells in lung cancer

    PubMed Central

    2011-01-01

    Background Mortality rates for advanced lung cancer have not declined for decades, even with the implementation of novel chemotherapeutic regimens or the use of tyrosine kinase inhibitors. Cancer Stem Cells (CSCs) are thought to be responsible for resistance to chemo/radiotherapy. Therefore, targeting CSCs with novel compounds may be an effective approach to reduce lung tumor growth and metastasis. We have isolated and characterized CSCs from non-small cell lung cancer (NSCLC) cell lines and measured their telomerase activity, telomere length, and sensitivity to the novel telomerase inhibitor MST312. Results The aldehyde dehydrogenase (ALDH) positive lung cancer cell fraction is enriched in markers of stemness and endowed with stem cell properties. ALDH+ CSCs display longer telomeres than the non-CSC population. Interestingly, MST312 has a strong antiproliferative effect on lung CSCs and induces p21, p27 and apoptosis in the whole tumor population. MST312 acts through activation of the ATM/pH2AX DNA damage pathway (short-term effect) and through decrease in telomere length (long-term effect). Administration of this telomerase inhibitor (40 mg/kg) in the H460 xenograft model results in significant tumor shrinkage (70% reduction, compared to controls). Combination therapy consisting of irradiation (10Gy) plus administration of MST312 did not improve the therapeutic efficacy of the telomerase inhibitor alone. Treatment with MST312 reduces significantly the number of ALDH+ CSCs and their telomeric length in vivo. Conclusions We conclude that antitelomeric therapy using MST312 mainly targets lung CSCs and may represent a novel approach for effective treatment of lung cancer. PMID:21827695

  9. Aldehyde Dehydrogenase-2 (ALDH2) Ameliorates Chronic Alcohol Ingestion-Induced Hepatic Steatosis and Inflammation: Role of Autophagy

    PubMed Central

    Guo, Rui; Xu, Xihui; Babcock, Sara A.; Zhang, Yingmei; Ren, Jun

    2014-01-01

    Background & Aims Mitochondrial aldehyde dehydrogenase (ALDH2) plays a critical role in the detoxification of the ethanol metabolite acetaldehyde. This study was designed to examine the impact of global ALDH2 overexpression on alcohol-induced hepatic steatosis. Methods Wild-type friendly virus B (FVB) and ALDH2 transgenic mice were placed on a 4% alcohol or control diet for 12 weeks. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin and cholesterol, hepatic triglyceride, steatosis, fat metabolism-related proteins, pro-inflammatory cytokines, glutathione (GSH), oxidized glutathione (GSSG), autophagy and autophagy signaling were examined. The role of autophagy was evaluated in ADH1-transfected human hepatocellular liver carcinoma cells (VA-13) treated with or without autophagy inducer rapamycin and lysosomal inhibitors. Results Chronic alcohol intake led to elevated AST, ALT, bilirubin, AST/ALT ratio, cholesterol, hepatic triglycerides, hepatic fat deposition as evidenced by H&E and oil Red O staining, associated with disturbed fat metabolism-related proteins (fatty acid synthase, SCD1), upregulated interleukin-6, TNF-α, cyclooxygenase, oxidative stress, and loss of autophagy, the effects of which were attenuated or ablated by ALDH2 transgene. Moreover, ethanol (100 mM) and acetaldehyde (100, 500 μM) increased levels of IL-6 and IFN-γ, and suppressed autophagy in VA-13 cells, the effects of which were markedly alleviated by rapamycin. In addition, lysosomal inhibitors mimicked ethanol-induced p62 accumulation with little additive effect with ethanol. Ethanol significantly suppressed LC3 conversion in the presence of lysosomal inhibitors. Conclusions In summary, our results revealed that ALDH2 plays a beneficial role in ameliorating chronic alcohol intake-induced hepatic steatosis and inflammation through regulation of autophagy. PMID:25457208

  10. Aldehyde dehydrogenase 3A1 is robustly upregulated in gastric cancer stem-like cells and associated with tumorigenesis.

    PubMed

    Wu, Di; Mou, Yi-Ping; Chen, Ke; Cai, Jia-Qin; Zhou, Yu-Cheng; Pan, Yu; Xu, Xiao-Wu; Zhou, Wei; Gao, Jia-Qi; Chen, Ding-Wei; Zhang, Ren-Chao

    2016-08-01

    Enhanced aldehyde dehydrogenase (ALDH) activity has been shown to serve as a hallmark for cancer stem cells (CSCs). Recent evidence suggests that its role as a stem cell-related marker has come down to the specific isoform. However, little is known about the specific ALDH isoform contributing to aldefluor activity in gastric cancer. In this study, we isolated ALDHbright cells from 2 human gastric cancer cell lines MKN-45 and SGC‑7901 by using an Aldefluor assay and found elevated self-renewal, differentiation and tumorigenicity, as demonstration of stemness characteristics. We also found that ALDHbright cells expressed decreased levels of E-cadherin but increased levels of Snail and Vimentin, indication of an epithelial-mesenchymal transition (EMT) phenotype which may be responsible for the enhanced metastatic potential. Since further research and prognostic application based on ALDH prevalence require the quantification of the specific ALDH isoform, we characterized the expression of all 19 ALDH isoforms in the sorted gastric cancer cell lines by quantitative real-time polymerase chain reaction (qRT-PCR). Compared with the non-stem counterparts, robust upregulation of ALDH-3A1 was observed in these gastric cancer stem-like cells. Furthermore, we performed immunohistological analysis on 93 fixed patient gastric tumor samples and found that ALDH-3A1 expression correlated well with gastric cancer dysplasia and grades, differentiation, lymph node metastasis and cancer stage. Our data, therefore, provide strong evidence that ALDH-3A1 is a novel gastric cancer stem cell related marker with potential prognostic values and demonstrate a clear association between ALDH-3A1 prevalence and gastric cancer progression. PMID:27279633

  11. Aldehyde dehydrogenase 2 and head and neck cancer: a meta-analysis implementing a Mendelian randomization approach.

    PubMed

    Boccia, Stefania; Hashibe, Mia; Gallì, Paola; De Feo, Emma; Asakage, Takahiro; Hashimoto, Tomoko; Hiraki, Akio; Katoh, Takahiko; Nomura, Takeshi; Yokoyama, Akira; van Duijn, Cornelia M; Ricciardi, Gualtiero; Boffetta, Paolo

    2009-01-01

    Alcohol drinking at high doses is a risk factor for head and neck cancer, and exposure to acetaldehyde, the principle metabolite of alcohol, is supposed to account for the increased risk. Individuals homozygous for the 2 variant allele of aldehyde dehydrogenase 2 (ALDH2) are unable to metabolize acetaldehyde, which prevents them from alcohol drinking, whereas 1 2 have 6-fold higher blood acetaldehyde concentration postalcohol consumption with respect to 1 1. According to the concept of Mendelian randomization, because this polymorphism is distributed randomly during gamete formation, its association with head and neck cancer should be not confounded by smoking. We carried out a meta-analysis of ALDH2 and head and neck cancer searching for relevant studies on Medline and Embase up to January 31, 2008, and investigated the consistency between the expected odds ratio (OR) among drinkers from the largest pooled analysis among never smokers and the observed OR from this meta-analysis by an interaction test. Six studies were selected (945 cases, 2,917 controls). The OR of head and neck cancer among 2 2 was 0.53 [95% confidence interval (95% CI), 0.28-1.00] relative to 1 1 and 1.83 (95% CI, 1.21-2.77) among 1 2. The expected OR for head and neck cancer due to alcohol intake among 1 1 was 1.38 (95% CI, 0.88-2.17) and the observed OR among 1 1 compared with 2*2 from this meta-analysis was 1.88 (95% CI, 1.00-3.57; P for interaction = 0.43). Besides showing the effectiveness of the Mendelian randomization approach, these findings support the theory that alcohol increases head and neck cancer risk through the carcinogenic action of acetaldehyde. PMID:19124505

  12. Mitochondrial aldehyde dehydrogenase protects against doxorubicin cardiotoxicity through a transient receptor potential channel vanilloid 1-mediated mechanism.

    PubMed

    Ge, Wei; Yuan, Ming; Ceylan, Asli F; Wang, Xiaoming; Ren, Jun

    2016-04-01

    Cardiotoxicity is one of the major life-threatening effects encountered in cancer chemotherapy with doxorubicin and other anthracyclines. Mitochondrial aldehyde dehydrogenase (ALDH2) may alleviate doxorubicin toxicity although the mechanism remains elusive. This study was designed to evaluate the impact of ALDH2 overexpression on doxorubicin-induced myocardial damage with a focus on mitochondrial injury. Wild-type (WT) and transgenic mice overexpressing ALDH2 driven by chicken β-actin promoter were challenged with doxorubicin (15mg/kg, single i.p. injection, for 6days) and cardiac mechanical function was assessed using the echocardiographic and IonOptix systems. Western blot analysis was used to evaluate intracellular Ca(2+) regulatory and mitochondrial proteins, PKA and its downstream signal eNOS. Doxorubicin challenge altered cardiac geometry and function evidenced by enlarged left ventricular end systolic and diastolic diameters, decreased factional shortening, cell shortening and intracellular Ca(2+) rise, prolonged relengthening and intracellular Ca(2+) decay, the effects of which were attenuated by ALDH2. Doxorubicin challenge compromised mitochondrial integrity and upregulated 4-HNE and UCP-2 levels while downregulating levels of TRPV1, SERCA2a and PGC-1α, the effects of which were alleviated by ALDH2. Doxorubicin-induced cardiac functional defect and apoptosis were reversed by the TRPV1 agonist SA13353 and the ALDH-2 agonist Alda-1 whereas the TRPV1 antagonist capsazepine nullified ALDH2/Alda-1-induced protection. Doxorubicin suppressed phosphorylation of PKA and eNOS, the effect of which was reversed by ALDH2. Moreover, 4-HNE mimicked doxorubicin-induced cardiomyocyte anomalies, the effect of which was ablated by SA13353. Taken together, our results suggested that ALDH2 may rescue against doxorubicin cardiac toxicity possibly through a TRPV1-mediated protection of mitochondrial integrity. PMID:26692169

  13. Aldehyde dehydrogenase 2 activation in aged heart improves the autophagy by reducing the carbonyl modification on SIRT1.

    PubMed

    Wu, Bing; Yu, Lu; Wang, Yishi; Wang, Hongtao; Li, Chen; Yin, Yue; Yang, Jingrun; Wang, Zhifa; Zheng, Qiangsun; Ma, Heng

    2016-01-19

    Cardiac aging is characterized by accumulation of damaged proteins and decline of autophagic efficiency. Here, by forestalling SIRT1 carbonylated inactivation in aged heart, we determined the benefits of activation of aldehyde dehydrogenase 2 (ALDH2) on the autophagy. In this study, the ALDH2 KO mice progressively developed age-related heart dysfunction and showed reduction in the life span, which strongly suggests that ALDH2 ablation leads to cardiac aging. What's more, aged hearts displayed a significant decrease ALDH2 activity, resulting in accumulation of 4-HNE-protein adducts and protein carbonyls, impairment in the autophagy flux, and, consequently, deteriorated cardiac function after starvation. Sustained Alda-1 (selective ALDH2 activator) treatment increased cardiac ALDH2 activity and abrogated these effects. Using SIRT1 deficient heterozygous (Sirt1+/-) mice, we found that SIRT1 was necessary for ALDH2 activation-induced autophagy. We further demonstrated that ALDH2 activation attenuated SIRT1 carbonylation and improved SIRT1 activity, thereby increasing the deacetylation of nuclear LC3 and FoxO1. Sequentially, ALDH2 enhanced SIRT1 regulates LC3-Atg7 interaction and FoxO1 increased Rab7 expression, which were both necessary and sufficient for restoring autophagy flux. These results highlight that both accumulation of proteotoxic carbonyl stress linkage with autophagy decline contribute to heart senescence. ALDH2 activation is adequate to improve the autophagy flux by reducing the carbonyl modification on SIRT1, which in turn plays an important role in maintaining cardiac health during aging. PMID:26741505

  14. In vitro oxidative metabolism of 6-mercaptopurine in human liver: insights into the role of the molybdoflavoenzymes aldehyde oxidase, xanthine oxidase, and xanthine dehydrogenase.

    PubMed

    Choughule, Kanika V; Barnaba, Carlo; Joswig-Jones, Carolyn A; Jones, Jeffrey P

    2014-08-01

    Anticancer agent 6-mercaptopurine (6MP) has been in use since 1953 for the treatment of childhood acute lymphoblastic leukemia (ALL) and inflammatory bowel disease. Despite being available for 60 years, several aspects of 6MP drug metabolism and pharmacokinetics in humans are unknown. Molybdoflavoenzymes such as aldehyde oxidase (AO) and xanthine oxidase (XO) have previously been implicated in the metabolism of this drug. In this study, we investigated the in vitro metabolism of 6MP to 6-thiouric acid (6TUA) in pooled human liver cytosol. We discovered that 6MP is metabolized to 6TUA through sequential metabolism via the 6-thioxanthine (6TX) intermediate. The role of human AO and XO in the metabolism of 6MP was established using the specific inhibitors raloxifene and febuxostat. Both AO and XO were involved in the metabolism of the 6TX intermediate, whereas only XO was responsible for the conversion of 6TX to 6TUA. These findings were further confirmed using purified human AO and Escherichia coli lysate containing expressed recombinant human XO. Xanthine dehydrogenase (XDH), which belongs to the family of xanthine oxidoreductases and preferentially reduces nicotinamide adenine dinucleotide (NAD(+)), was shown to contribute to the overall production of the 6TX intermediate as well as the final product 6TUA in the presence of NAD(+) in human liver cytosol. In conclusion, we present evidence that three enzymes, AO, XO, and XDH, contribute to the production of 6TX intermediate, whereas only XO and XDH are involved in the conversion of 6TX to 6TUA in pooled HLC. PMID:24824603

  15. Aldehyde-alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations.

    PubMed

    Sillers, Ryan; Al-Hinai, Mohab Ali; Papoutsakis, Eleftherios T

    2009-01-01

    Metabolic engineering (ME) of Clostridium acetobutylicum has led to increased solvent (butanol, acetone, and ethanol) production and solvent tolerance, thus demonstrating that further efforts have the potential to create strains of industrial importance. With recently developed ME tools, it is now possible to combine genetic modifications and thus implement more advanced ME strategies. We have previously shown that antisense RNA (asRNA)-based downregulation of CoA transferase (CoAT, the first enzyme in the acetone-formation pathway) results in increased butanol to acetone selectivity, but overall reduced butanol yields and titers. In this study the alcohol/aldehyde dehydrogenase (aad) gene (encoding the bifunctional protein AAD responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA, respectively) was expressed from the phosphotransbutyrylase (ptb) promoter to enhance butanol formation and selectivity, while CoAT downregulation was used to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers, overall solvent titers of 30 g/L, and a higher alcohol/acetone ratio. Metabolic flux analysis revealed the likely depletion of butyryl-CoA. In order to increase then the flux towards butyryl-CoA, we examined the impact of thiolase (THL, thl) overexpression. THL converts acetyl-CoA to acetoacetyl-CoA, the first step of the pathway from acetyl-CoA to butyryl-CoA, and thus, combining thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. thl overexpression in strains with asRNA CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to enhance the intracellular butyryl-CoA pool and reduce the acetyl-CoA pool in order to achieve improved butanol titers and selectivity. PMID:18726959

  16. Impact of smoking on lung cancer risk is stronger in those with the homozygous aldehyde dehydrogenase 2 null allele in a Japanese population.

    PubMed

    Park, Ji Young; Matsuo, Keitaro; Suzuki, Takeshi; Ito, Hidemi; Hosono, Satoyo; Kawase, Takakazu; Watanabe, Miki; Oze, Isao; Hida, Toyoaki; Yatabe, Yasushi; Mitsudomi, Tetsuya; Takezaki, Toshiro; Tajima, Kazuo; Tanaka, Hideo

    2010-04-01

    The main lifestyle contributor to acetaldehyde exposure is the drinking of alcoholic beverages, but tobacco smoke also makes some contribution. Although acetaldehyde is associated with upper aerodigestive tract cancer risk, in accordance with genetically determined acetaldehyde metabolism, it is unclear whether lung cancer, a representative smoking-related cancer, is associated with acetaldehyde or genes impacting its metabolism. We conducted a case-control study to examine possible interaction between smoking and aldehyde dehydrogenase 2 (ALDH2) Glu504Lys polymorphism (rs671) on the risk of lung cancer in Japanese. Subjects were 718 lung cancer cases and 1416 non-cancer controls enrolled in the Hospital-based Epidemiologic Research Program at Aichi Cancer Center. Lifestyle factors, including smoking, were determined by self-administered questionnaire. We applied pack-years (PY; categorized into five levels: never, <15, <30, <45 and > or =45) as a marker of cumulative exposure to smoking. The impact of smoking, ALDH2 genotype, and their interaction on lung cancer risk were assessed by odds ratio (OR) and 95% confidence interval adjusted for potential confounders. Adjusted ORs for PY <15, <30, <45 and > or =45 relative to never smokers among those with Glu/Glu or Glu/Lys were 1.39, 1.80, 3.44 and 6.25, respectively (P-trend = 1.4 x 10(-30)). In contrast, ORs among Lys/Lys were 1.01, 10.2, 11.4 and 23.2, respectively (P-trend = 2.6 x 10(-7)). Interaction between ALDH2 genotype (Glu/Glu + Glu/Lys versus Lys/Lys) and cumulative smoking dose was statistically significant (P = 0.036) and was consistently observed in the analysis among never-drinkers (interaction P = 0.041). These results suggest that ALDH2 Lys/Lys, a null enzyme activity genotype, modifies the impact of smoking on the risk of lung cancer. PMID:20093384

  17. Genetic analysis of the phenobarbital regulation of the cytochrome P-450 2b-9 and aldehyde dehydrogenase type 2 mRNAs in mouse liver.

    PubMed Central

    Damon, M; Fautrel, A; Guillouzo, A; Corcos, L

    1996-01-01

    The aim of this study was to investigate the effect of the genetic background on the phenobarbital inducibility of cytochrome P-450 2b-9, cytochrome P-450 2b-10 and aldehyde dehydrogenase type 2 mRNAs in mice. We analysed the basal expression and the phenobarbital inducibility of both cytochrome P-450 mRNAs by semi-quantitative specific reverse transcription-PCR analyses in five inbred mouse strains (A/J,BALB/cByJ,C57BL/6J, DBA/2J and SWR/J). Male mice constitutively expressed cytochrome P-450 2b-9 and cytochrome P-450 2b-10 mRNAs, but a number of differences in their response to phenobarbital were observed. In all these mouse strains, phenobarbital induced cytochrome P-450 2b-10 mRNA whereas it could have either a positive or a negative effect on cytochrome P-450 2b-9 expression, depending on the strain and the sex of the mice. Specifically, phenobarbital increased cytochrome P-450 2b-9 expression in C57BL/6J males while it decreased it in DBA/2J mice. Interestingly, dexamethasone was able to mimic the phenobarbital effect on both cytochromes P-450 in these two strains. Aldehyde dehydrogenase type 2 mRNA was always induced by phenobarbital, except in the C57BL/6J strain. Genetic analysis revealed that the phenobarbital-inducible phenotype was either a semi-dominant or a recessive trait in F1 animals from a C57BL/6J x DBA/2J cross for the cytochrome P-450 2b-9 and the aldehyde dehydrogenase type 2 genes, respectively. This study suggests that the genetic basis for phenobarbital induction in mice depends on the target gene, and that more than one regulatory step would by involved in this response pathway. PMID:8713075

  18. Prognostic Value of Drinking Status and Aldehyde Dehydrogenase 2 Polymorphism in Patients With Head and Neck Squamous Cell Carcinoma

    PubMed Central

    Kawakita, Daisuke; Oze, Isao; Hosono, Satoyo; Ito, Hidemi; Watanabe, Miki; Yatabe, Yasushi; Hasegawa, Yasuhisa; Murakami, Shingo; Tanaka, Hideo; Matsuo, Keitaro

    2016-01-01

    Background The association between alcohol drinking, aldehyde dehydrogenase 2 (ALDH2) polymorphism, and survival in patients with head and neck squamous cell carcinoma (HNSCC) remains unclear. Methods We performed a retrospective cohort study of 267 HNSCC patients at Aichi Cancer Center. Of these, 65 patients (24%) were non-drinkers, 104 (39%) were light drinkers (ethanol <46 g or <5 days/week), 46 (17%) were moderate drinkers (ethanol intake 46–68 g/day and ≥5 days/week), and 52 (20%) were heavy drinkers (ethanol intake ≥69 g and ≥5 days/week). The prognostic value of pre-treatment drinking status and ALDH2 polymorphism was investigated using multivariate proportional hazard models. Results Drinking status was associated with disease-free survival (DFS) in HNSCC patients, with marginal statistical significance (5-year DFS: 67.9% [95% confidence interval {CI}, 53.8–78.4%] for non-drinkers, 57.6% [95% CI, 47.4–66.6%] for light drinkers, 46.1% [95% CI, 30.8–60.1%] for moderate drinkers, and 43.5% [95% CI, 29.3–56.9%] for heavy drinkers; P = 0.088). However, this association lost significance when multivariate analyses were adjusted for established prognostic factors. ALDH2 genotype was not significantly associated with DFS in HNSCC patients (5-year DFS: 85.7% [95% CI, 53.9–96.2%] for Lys/Lys, 56.2% [95% CI, 47.4–64.1%] for Glu/Lys, and 50.5% [95% CI, 40.3–59.7%] for Glu/Glu; P = 0.154). After stratification by ALDH2 genotype, we observed a significant positive dose-response relationship between drinking status and DFS in HNSCC patients with ALDH2 Glu/Glu (P trend = 0.029). Conclusions In this study, we identified a significant positive dose-response relationship between pre-treatment drinking status and DFS in HNSCC patients with ALDH2 Glu/Glu. To confirm this association, further study is warranted. PMID:26804037

  19. Increased Expression of Aldehyde Dehydrogenase 2 Reduces Renal Cell Apoptosis During Ischemia/Reperfusion Injury After Hypothermic Machine Perfusion.

    PubMed

    Zhong, Zibiao; Hu, Qianchao; Fu, Zhen; Wang, Ren; Xiong, Yan; Zhang, Yang; Liu, Zhongzhong; Wang, Yanfeng; Ye, Qifa

    2016-06-01

    Hypothermic machine perfusion (MP) can reduce graft's injury after kidney transplantation; however, the mechanism has not been elucidated. In the past decade, many studies showed that aldehyde dehydrogenase 2 (ALDH2) is a protease which can inhibit cell apoptosis. Therefore, this study aims to explore whether ALDH2 takes part in reducing organ damage after MP. Eighteen healthy male New Zealand rabbits (12 weeks old, weight 3.0 ± 0.3 kg) were randomly divided into three groups: normal group, MP group, and cold storage (CS) group (n = 6). The left kidney of rabbits underwent warm ischemia for 35 min through clamping the left renal pedicle and then reperfusion for 1 h. Left kidneys were preserved by MP or CS (4°C for 4 h) in vivo followed by the right nephrectomy and 24-h reperfusion, and then the specimens and blood were collected. Finally, concentration of urine creatinine (Cr), blood urea nitrogen (BUN), and 4-HNE were tested. Renal apoptosis was detected by TUNEL staining, and the expression of ALDH2, cleaved-caspase 3, bcl-2/ bax, MAPK in renal tissue was detected by immunohistochemistry or Western blot; 24 h after surgery, the concentration of Cr in MP group was 355 ± 71μmol/L, in CS group was 511 ± 44 μmol/L (P < 0.05), while the BUN was 15.02 ± 2.34 mmol/L in MP group, 22.64 ± 3.58 mmol/L in CS group (P < 0.05). The rate of apoptosis and expression of cleaved caspase-3, p-P38, p-ERK, and p-JNK in MP group was significantly lower than that in CS group (P < 0.05), while expression of ALDH2 and bcl-2/bax in MP group was significantly higher than that in CS group (P < 0.05); expression of cleaved caspase-3 in both MP and CS group significantly increased as compared with that in normal group (P < 0.05). In conclusion, increased expression of ALDH2 can reduce the renal cell apoptosis through inhibiting MAPK pathway during ischemia/reperfusion injury (IRI) after hypothermic MP. PMID:26582147

  20. Malignant phyllodes tumors display mesenchymal stem cell features and aldehyde dehydrogenase/disialoganglioside identify their tumor stem cells

    PubMed Central

    2014-01-01

    Introduction Although breast phyllodes tumors are rare, there is no effective therapy other than surgery. Little is known about their tumor biology. A malignant phyllodes tumor contains heterologous stromal elements, and can transform into rhabdomyosarcoma, liposarcoma and osteosarcoma. These versatile properties prompted us to explore their possible relationship to mesenchymal stem cells (MSCs) and to search for the presence of cancer stem cells (CSCs) in phyllodes tumors. Methods Paraffin sections of malignant phyllodes tumors were examined for various markers by immunohistochemical staining. Xenografts of human primary phyllodes tumors were established by injecting freshly isolated tumor cells into the mammary fat pad of non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. To search for CSCs, xenografted tumor cells were sorted into various subpopulations by flow cytometry and examined for their in vitro mammosphere forming capacity, in vivo tumorigenicity in NOD-SCID mice and their ability to undergo differentiation. Results Immunohistochemical analysis revealed the expression of the following 10 markers: CD44, CD29, CD106, CD166, CD105, CD90, disialoganglioside (GD2), CD117, Aldehyde dehydrogenase 1 (ALDH), and Oct-4, and 7 clinically relevant markers (CD10, CD34, p53, p63, Ki-67, Bcl-2, vimentin, and Globo H) in all 51 malignant phyllodes tumors examined, albeit to different extents. Four xenografts were successfully established from human primary phyllodes tumors. In vitro, ALDH+ cells sorted from xenografts displayed approximately 10-fold greater mammosphere-forming capacity than ALDH- cells. GD2+ cells showed a 3.9-fold greater capacity than GD2- cells. ALDH+/GD2+cells displayed 12.8-fold greater mammosphere forming ability than ALDH-/GD2- cells. In vivo, the tumor-initiating frequency of ALDH+/GD2+ cells were up to 33-fold higher than that of ALDH+ cells, with as few as 50 ALDH+/GD2+ cells being sufficient for engraftment. Moreover, we

  1. In vivo ethanol elimination in man, monkey and rat: A lack of relationship between the ethanol metabolism and the hepatic activities of alcohol and aldehyde dehydrogenases

    SciTech Connect

    Zorzano, A. ); Herrera, E. )

    1990-01-01

    The in vivo ethanol elimination in human subjects, monkeys and rats was investigated after an oral ethanol dosage. After 0.4 g. ethanol/kg of body weight, ethanol elimination was much slower in human subjects than in monkeys. In order to detect a rise in monkey plasma ethanol concentrations as early as observed in human subjects, ethanol had to be administered at a dose of 3 g/kg body weight. Ethanol metabolism in rats was also much faster than in human subjects. However, human liver showed higher alcohol dehydrogenase activity and higher low Km aldehyde dehydrogenase activity than rat liver. Thus, our data suggest a lack of relationship between hepatic ethanol-metabolizing activities and the in vivo ethanol elimination rate.

  2. Ethylbenzene Dehydrogenase and Related Molybdenum Enzymes Involved in Oxygen-Independent Alkyl Chain Hydroxylation.

    PubMed

    Heider, Johann; Szaleniec, Maciej; Sünwoldt, Katharina; Boll, Matthias

    2016-01-01

    Ethylbenzene dehydrogenase initiates the anaerobic bacterial degradation of ethylbenzene and propylbenzene. Although the enzyme is currently only known from a few closely related denitrifying bacterial strains affiliated to the Rhodocyclaceae, it clearly marks a universally occurring mechanism used for attacking recalcitrant substrates in the absence of oxygen. Ethylbenzene dehydrogenase belongs to subfamily 2 of the DMSO reductase-type molybdenum enzymes together with paralogous enzymes involved in the oxygen-independent hydroxylation of p-cymene, the isoprenoid side chains of sterols and even possibly n-alkanes; the subfamily also extends to dimethylsulfide dehydrogenases, selenite, chlorate and perchlorate reductases and, most significantly, dissimilatory nitrate reductases. The biochemical, spectroscopic and structural properties of the oxygen-independent hydroxylases among these enzymes are summarized and compared. All of them consist of three subunits, contain a molybdenum-bis-molybdopterin guanine dinucleotide cofactor, five Fe-S clusters and a heme b cofactor of unusual ligation, and are localized in the periplasmic space as soluble enzymes. In the case of ethylbenzene dehydrogenase, it has been determined that the heme b cofactor has a rather high redox potential, which may also be inferred for the paralogous hydroxylases. The known structure of ethylbenzene dehydrogenase allowed the calculation of detailed models of the reaction mechanism based on the density function theory as well as QM-MM (quantum mechanics - molecular mechanics) methods, which yield predictions of mechanistic properties such as kinetic isotope effects that appeared consistent with experimental data. PMID:26960184

  3. Expression of Drosophila melanogaster xanthine dehydrogenase in Aspergillus nidulans and some properties of the recombinant enzyme.

    PubMed Central

    Adams, Benjamin; Lowe, David J; Smith, Andrew T; Scazzocchio, Claudio; Demais, Stephane; Bray, Robert C

    2002-01-01

    Recent crystal structures of xanthine dehydrogenase, xanthine oxidase and related enzymes have paved the way for a detailed structural and functional analysis of these enzymes. One problem encountered when working with these proteins, especially with recombinant protein, is that the preparations tend to be heterogeneous, with only a fraction of the enzyme molecules being active. This is due to the incompleteness of post-translational modification, which for this protein is a complex, and incompletely understood, process involving incorporation of the Mo and Fe/S centres. The enzyme has been expressed previously in both Drosophila and insect cells using baculovirus. The insect cell system has been exploited by Iwasaki et al. [Iwasaki, Okamoto, Nishino, Mizushima and Hori (2000) J. Biochem (Tokyo) 127, 771-778], but, for the rat enzyme, yields a complex mixture of enzyme forms, containing around 10% of functional enzyme. The expression of Drosophila melanogaster xanthine dehydrogenase in Aspergillus nidulans is described. The purified protein has been analysed both functionally and spectroscopically. Its specific activity is indistinguishable from that of the enzyme purified from fruit flies [Doyle, Burke, Chovnick, Dutton, Whittle and Bray (1996) Eur. J. Biochem. 239, 782-795], and it appears to be more active than recombinant xanthine dehydrogenase produced with the baculovirus system. EPR spectra of the recombinant Drosophila enzyme are reported, including parameters for the Fe/S centres. Only a very weak "Fe/SIII" signal (g(1,2,3), 2.057, 1.930, 1.858) was observed, in contrast to the strong analogous signal reported for the enzyme from baculovirus. Since this signal appears to be associated with incomplete post-translational modification, this is consistent with relatively more complete cofactor incorporation in the Aspergillus-produced enzyme. Thus we have developed a recombinant expression system for D. melanogaster xanthine dehydrogenase, which can be used

  4. Pepper aldehyde dehydrogenase CaALDH1 interacts with Xanthomonas effector AvrBsT and promotes effector-triggered cell death and defence responses.

    PubMed

    Kim, Nak Hyun; Hwang, Byung Kook

    2015-06-01

    Xanthomonas type III effector AvrBsT induces hypersensitive cell death and defence responses in pepper (Capsicum annuum) and Nicotiana benthamiana. Little is known about the host factors that interact with AvrBsT. Here, we identified pepper aldehyde dehydrogenase 1 (CaALDH1) as an AvrBsT-interacting protein. Bimolecular fluorescence complementation and co-immunoprecipitation assays confirmed the interaction between CaALDH1 and AvrBsT in planta. CaALDH1:smGFP fluorescence was detected in the cytoplasm. CaALDH1 expression in pepper was rapidly and strongly induced by avirulent Xanthomonas campestris pv. vesicatoria (Xcv) Ds1 (avrBsT) infection. Transient co-expression of CaALDH1 with avrBsT significantly enhanced avrBsT-triggered cell death in N. benthamiana leaves. Aldehyde dehydrogenase activity was higher in leaves transiently expressing CaALDH1, suggesting that CaALDH1 acts as a cell death enhancer, independently of AvrBsT. CaALDH1 silencing disrupted phenolic compound accumulation, H2O2 production, defence response gene expression, and cell death during avirulent Xcv Ds1 (avrBsT) infection. Transgenic Arabidopsis thaliana overexpressing CaALDH1 exhibited enhanced defence response to Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis infection. These results indicate that cytoplasmic CaALDH1 interacts with AvrBsT and promotes plant cell death and defence responses. PMID:25873668

  5. Extended flow cytometry characterization of normal bone marrow progenitor cells by simultaneous detection of aldehyde dehydrogenase and early hematopoietic antigens: implication for erythroid differentiation studies

    PubMed Central

    Mirabelli, Peppino; Di Noto, Rosa; Lo Pardo, Catia; Morabito, Paolo; Abate, Giovanna; Gorrese, Marisa; Raia, Maddalena; Pascariello, Caterina; Scalia, Giulia; Gemei, Marica; Mariotti, Elisabetta; Del Vecchio, Luigi

    2008-01-01

    Background Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients. Results In normal bone marrow, we identified three populations of cells, namely ALDH+CD34+, ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH-CD34+ cells, ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH+CD34- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH+CD34- cells showed a CD71bright, CD105+, CD45- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA). Conclusion Our study, comparing surface antigen expression of ALDH+/CD34+, ALDH

  6. Amphibian alcohol dehydrogenase, the major frog liver enzyme. Relationships to other forms and assessment of an early gene duplication separating vertebrate class I and class III alcohol dehydrogenases

    SciTech Connect

    Cederlund, E.; Joernvall, H. ); Peralba, J.M.; Pares, X. )

    1991-03-19

    Submammalian alcohol dehydrogenase structures can be used to evaluate the origins and functions of different types of the mammalian enzyme. Two avian forms were recently reported, and the authors now define the major amphibian alcohol dehydrogenase. The enzyme from the liver of the Green frog Rana perezi was purified, carboxymethylated, and submitted to amino acid sequence determination by peptide analysis of six different digest. The protein has a 375-residue subunit and is a class I alcohol dehydrogenase, bridging the gap toward the original separation of the classes that are observable in the human alcohol dehydrogenase system. In relation to the human class I enzyme, the amphibian protein has residue identities exactly halfway (68%) between those for the corresponding avian enzyme (74%) and the human class III enzyme (62%), suggesting an origin of the alcohol dehnydrogenase classes very early in or close to the evolution of the vertebrate line. This conclusion suggests that these enzyme classes are more universal among animals than previously realized and constitutes the first real assessment of the origin of the duplications leading to the alcohol dehydrogenase classes. In conclusion, the amphibian enzyme allows a rough positioning of the divergence of the alcohol dehydrogenase classes, shows that the class I type is widesprread in vertebrates, and functionally conforms with greater variations at the substrate-binding than the coenzyme-binding site.

  7. Aldehyde dehydrogenase type 2 activation by adenosine and histamine inhibits ischemic norepinephrine release in cardiac sympathetic neurons: mediation by protein kinase Cε.

    PubMed

    Robador, Pablo A; Seyedi, Nahid; Chan, Noel Yan-Ki; Koda, Kenichiro; Levi, Roberto

    2012-10-01

    During myocardial ischemia/reperfusion, lipid peroxidation leads to the formation of toxic aldehydes that contribute to ischemic dysfunction. Mitochondrial aldehyde dehydrogenase type 2 (ALDH2) alleviates ischemic heart damage and reperfusion arrhythmias via aldehyde detoxification. Because excessive norepinephrine release in the heart is a pivotal arrhythmogenic mechanism, we hypothesized that neuronal ALDH2 activation might diminish ischemic norepinephrine release. Incubation of cardiac sympathetic nerve endings with acetaldehyde, at concentrations achieved in myocardial ischemia, caused a concentration-dependent increase in norepinephrine release. A major increase in norepinephrine release also occurred when sympathetic nerve endings were incubated in hypoxic conditions. ALDH2 activation substantially reduced acetaldehyde- and hypoxia-induced norepinephrine release, an action prevented by inhibition of ALDH2 or protein kinase Cε (PKCε). Selective activation of G(i/o)-coupled adenosine A(1), A(3), or histamine H(3) receptors markedly inhibited both acetaldehyde- and hypoxia-induced norepinephrine release. These effects were also abolished by PKCε and/or ALDH2 inhibition. Moreover, A(1)-, A(3)-, or H(3)-receptor activation increased ALDH2 activity in a sympathetic neuron model (differentiated PC12 cells stably transfected with H(3) receptors). This action was prevented by the inhibition of PKCε and ALDH2. Our findings suggest the existence in sympathetic neurons of a protective pathway initiated by A(1)-, A(3)-, and H(3)-receptor activation by adenosine and histamine released in close proximity of these terminals. This pathway comprises the sequential activation of PKCε and ALDH2, culminating in aldehyde detoxification and inhibition of hypoxic norepinephrine release. Thus, pharmacological activation of PKCε and ALDH2 in cardiac sympathetic nerves may have significant protective effects by alleviating norepinephrine-induced life-threatening arrhythmias that

  8. Histamine H4-receptors inhibit mast cell renin release in ischemia/reperfusion via protein kinase C ε-dependent aldehyde dehydrogenase type-2 activation.

    PubMed

    Aldi, Silvia; Takano, Ken-ichi; Tomita, Kengo; Koda, Kenichiro; Chan, Noel Y-K; Marino, Alice; Salazar-Rodriguez, Mariselis; Thurmond, Robin L; Levi, Roberto

    2014-06-01

    Renin released by ischemia/reperfusion (I/R) from cardiac mast cells (MCs) activates a local renin-angiotensin system (RAS) causing arrhythmic dysfunction. Ischemic preconditioning (IPC) inhibits MC renin release and consequent activation of this local RAS. We postulated that MC histamine H4-receptors (H4Rs), being Gαi/o-coupled, might activate a protein kinase C isotype-ε (PKCε)-aldehyde dehydrogenase type-2 (ALDH2) cascade, ultimately eliminating MC-degranulating and renin-releasing effects of aldehydes formed in I/R and associated arrhythmias. We tested this hypothesis in ex vivo hearts, human mastocytoma cells, and bone marrow-derived MCs from wild-type and H4R knockout mice. We found that activation of MC H4Rs mimics the cardioprotective anti-RAS effects of IPC and that protection depends on the sequential activation of PKCε and ALDH2 in MCs, reducing aldehyde-induced MC degranulation and renin release and alleviating reperfusion arrhythmias. These cardioprotective effects are mimicked by selective H4R agonists and disappear when H4Rs are pharmacologically blocked or genetically deleted. Our results uncover a novel cardioprotective pathway in I/R, whereby activation of H4Rs on the MC membrane, possibly by MC-derived histamine, leads sequentially to PKCε and ALDH2 activation, reduction of toxic aldehyde-induced MC renin release, prevention of RAS activation, reduction of norepinephrine release, and ultimately to alleviation of reperfusion arrhythmias. This newly discovered protective pathway suggests that MC H4Rs may represent a new pharmacologic and therapeutic target for the direct alleviation of RAS-induced cardiac dysfunctions, including ischemic heart disease and congestive heart failure. PMID:24696042

  9. Isolation and Characterization of Anaerobic Ethylbenzene Dehydrogenase, a Novel Mo-Fe-S Enzyme

    PubMed Central

    Johnson, Hope A.; Pelletier, Dale A.; Spormann, Alfred M.

    2001-01-01

    The first step in anaerobic ethylbenzene mineralization in denitrifying Azoarcus sp. strain EB1 is the oxidation of ethylbenzene to (S)-(−)-1-phenylethanol. Ethylbenzene dehydrogenase, which catalyzes this reaction, is a unique enzyme in that it mediates the stereoselective hydroxylation of an aromatic hydrocarbon in the absence of molecular oxygen. We purified ethylbenzene dehydrogenase to apparent homogeneity and showed that the enzyme is a heterotrimer (αβγ) with subunit masses of 100 kDa (α), 35 kDa (β), and 25 kDa (γ). Purified ethylbenzene dehydrogenase contains approximately 0.5 mol of molybdenum, 16 mol of iron, and 15 mol of acid-labile sulfur per mol of holoenzyme, as well as a molydopterin cofactor. In addition to ethylbenzene, purified ethylbenzene dehydrogenase was found to oxidize 4-fluoro-ethylbenzene and the nonaromatic hydrocarbons 3-methyl-2-pentene and ethylidenecyclohexane. Sequencing of the encoding genes revealed that ebdA encodes the α subunit, a 974-amino-acid polypeptide containing a molybdopterin-binding domain. The ebdB gene encodes the β subunit, a 352-amino-acid polypeptide with several 4Fe-4S binding domains. The ebdC gene encodes the γ subunit, a 214-amino-acid polypeptide that is a potential membrane anchor subunit. Sequence analysis and biochemical data suggest that ethylbenzene dehydrogenase is a novel member of the dimethyl sulfoxide reductase family of molybdopterin-containing enzymes. PMID:11443088

  10. Ergot alkaloid biosynthesis in Aspergillus fumigatus: conversion of chanoclavine-I to chanoclavine-I aldehyde catalyzed by a short-chain alcohol dehydrogenase FgaDH.

    PubMed

    Wallwey, Christiane; Matuschek, Marco; Li, Shu-Ming

    2010-02-01

    Ergot alkaloids are toxins and important pharmaceuticals which are produced biotechnologically on an industrial scale. A putative gene fgaDH has been identified in the biosynthetic gene cluster of fumigaclavine C, an ergot alkaloid of the clavine-type. The deduced gene product FgaDH comprises 261 amino acids with a molecular mass of about 27.8 kDa and contains the conserved motifs of classical short-chain dehydrogenases/reductases (SDRs), but shares no worth mentioning sequence similarity with SDRs and other known proteins. The coding region of fgaDH consisting of two exons was amplified by PCR from a cDNA library of Aspergillus fumigatus, cloned into pQE60 and overexpressed in E. coli. The soluble tetrameric His(6)-FgaDH was purified to apparent homogeneity and characterized biochemically. It has been shown that FgaDH catalyzes the oxidation of chanoclavine-I in the presence of NAD(+) resulting in the formation of chanoclavine-I aldehyde, which was unequivocally identified by NMR and MS analyzes. Therefore, FgaDH functions as a chanoclavine-I dehydrogenase and represents a new group of short-chain dehydrogenases. K (M) values for chanoclavine-I and NAD(+) were determined at 0.27 and 1.1 mM, respectively. The turnover number was 0.38 s(-1). PMID:20039019

  11. Studies of a Halophilic NADH Dehydrogenase. 1: Purification and Properties of the Enzyme

    NASA Technical Reports Server (NTRS)

    Hochstein, Lawrence I.; Dalton, Bonnie P.

    1973-01-01

    An NADH dehydrogenase obtained from an extremely halophilic bacterium was purified 570-fold by a combination of gel filtration, chromatography on hydroxyapatite, and ion-exchange chromatography on QAE-Sephadex. The purified enzyme appeared to be FAD-linked and bad an apparent molecular weight of 64000. Even though enzyme activity was stimulated by NaCl, considerable activity (430 % of the maximum activity observed in the presence of 2.5 M NaCl) was observed in the absence of added NaCl. The enzyme was unstable when incubated in solutions of low ionic strength. The presence of NADH enhanced the stability of the enzyme.

  12. Subcellular Localization and Biochemical Comparison of Cytosolic and Secreted Cytokinin Dehydrogenase Enzymes from Maize

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cytokinin dehydrogenase (CKX, EC 1.5.99.12) degrades cytokinin hormones in plants. There are several differently targeted isoforms of CKX in cells of each plant. While most CKX enzymes appear to be localized in the apoplast or vacuoles, there is generally only one CKX per plant genome that lacks a t...

  13. Action of shear on enzymes: studies with alcohol dehydrogenase.

    PubMed

    Thomas, C R; Nienow, A W; Dunnill, P

    1979-12-01

    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. PMID:42450

  14. Purification and Characterization of the Bifunctional Enzyme Lysine-Ketoglutarate Reductase-Saccharopine Dehydrogenase from Maize.

    PubMed Central

    Goncalves-Butruille, M.; Szajner, P.; Torigoi, E.; Leite, A.; Arruda, P.

    1996-01-01

    The first enzyme of the lysine degradation pathway in maize (Zea mays L.), lysine-ketoglutarate reductase, condenses lysine and [alpha]-ketoglutarate into saccharopine using NADPH as a cofactor, whereas the second, saccharopine dehydrogenase, converts saccharopine to [alpha]-aminoadipic-[delta]-semialdehyde and glutamic acid using NAD+ or NADP+ as a cofactor. The reductase and dehydrogenase activities are optimal at pH 7.0 and 9.0, respectively. Both enzyme activities, co-purified on diethylaminoethyl-cellulose and gel filtration columns, were detected on nondenaturing polyacrylamide gels as single bands with identical electrophoretic mobilities and share tissue specificity for the endosperm. The highly purified preparation containing the reductase and dehydrogenase activities showed a single polypeptide band of 125 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native form of the enzyme is a dimer of 260 kD. Limited proteolysis with elastase indicated that lysine-ketoglutarate reductase and saccharopine dehydrogenase from maize endosperm are located in two functionally independent domains of a bifunctional polypeptide. PMID:12226216

  15. Molecular characterization and organ-specific expression of the gene that encodes betaine aldehyde dehydrogenase from the white shrimp Litopenaeus vannamei in response to osmotic stress.

    PubMed

    Delgado-Gaytán, María F; Hernández-Palomares, Magally L E; Soñanez-Organis, José G; Muhlia-Almazán, Adriana; Sánchez-Paz, Arturo; Stephens-Camacho, Norma A; Valenzuela-Soto, Elisa M; Rosas-Rodríguez, Jesús A

    2015-11-01

    Crustaceans overcome osmotic disturbances by regulating their intracellular concentration of ions and osmolytes. Glycine betaine (GB), an osmolyte accumulated in response to hyperosmotic stress, is synthesized by betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) through the oxidation of betaine aldehyde. A partial BADH cDNA sequence from the white shrimp Litopenaeus vannamei was obtained and its organ-specific expression during osmotic stress (low and high salinity) was evaluated. The partial BADH cDNA sequence (LvBADH) is 1103bp long and encodes an open reading frame for 217 protein residues. The amino acid sequence of LvBADH is related to that of other BADHs, TMABA-DH and ALDH9 from invertebrate and vertebrate homologues, and includes the essential domains of their function and regulation. LvBADH activity and mRNA expression were detected in the gills, hepatopancreas and muscle with the highest levels in the hepatopancreas. LvBADH mRNA expression increased 2-3-fold in the hepatopancreas and gills after 7days of osmotic variation (25 and 40ppt). In contrast, LvBADH mRNA expression in muscle decreased 4-fold and 15-fold after 7days at low and high salinity, respectively. The results indicate that LvBADH is ubiquitously expressed, but its levels are organ-specific and regulated by osmotic stress, and that LvBADH is involved in the cellular response of crustaceans to variations in environmental salinity. PMID:26219579

  16. Escherichia coli d-Malate Dehydrogenase, a Generalist Enzyme Active in the Leucine Biosynthesis Pathway*

    PubMed Central

    Vorobieva, Anastassia A.; Khan, Mohammad Shahneawz; Soumillion, Patrice

    2014-01-01

    The enzymes of the β-decarboxylating dehydrogenase superfamily catalyze the oxidative decarboxylation of d-malate-based substrates with various specificities. Here, we show that, in addition to its natural function affording bacterial growth on d-malate as a carbon source, the d-malate dehydrogenase of Escherichia coli (EcDmlA) naturally expressed from its chromosomal gene is capable of complementing leucine auxotrophy in a leuB− strain lacking the paralogous isopropylmalate dehydrogenase enzyme. To our knowledge, this is the first example of an enzyme that contributes with a physiologically relevant level of activity to two distinct pathways of the core metabolism while expressed from its chromosomal locus. EcDmlA features relatively high catalytic activity on at least three different substrates (l(+)-tartrate, d-malate, and 3-isopropylmalate). Because of these properties both in vivo and in vitro, EcDmlA may be defined as a generalist enzyme. Phylogenetic analysis highlights an ancient origin of DmlA, indicating that the enzyme has maintained its generalist character throughout evolution. We discuss the implication of these findings for protein evolution. PMID:25160617

  17. Biochemical and molecular characterization of the Clostridium magnum acetoin dehydrogenase enzyme system.

    PubMed Central

    Krüger, N; Oppermann, F B; Lorenzl, H; Steinbüchel, A

    1994-01-01

    E2 (dihydrolipoamide acetyltransferase) and E3 (dihydrolipoamide dehydrogenase) of the Clostridium magnum acetoin dehydrogenase enzyme system were copurified in a three-step procedure from acetoin-grown cells. The denatured E2-E3 preparation comprised two polypeptides with M(r)s of 49,000 and 67,000, respectively. Microsequencing of both proteins revealed identical amino acid sequences. By use of oligonucleotide probes based on the N-terminal sequences of the alpha and beta subunits of E1 (acetoin dehydrogenase, thymine PPi dependent), which were purified recently (H. Lorenzl, F.B. Oppermann, B. Schmidt, and A. Steinbüchel, Antonie van Leeuwenhoek 63:219-225, 1993), and of E2-E3, structural genes acoA (encoding E1 alpha), acoB (encoding E1 beta), acoC (encoding E2), and acoL (encoding E3) were identified on a single ClaI restriction fragment and expressed in Escherichia coli. The nucleotide sequences of acoA (978 bp), acoB (999 bp), acoC (1,332 bp), and acoL (1,734 bp), as well as those of acoX (996 bp) and acoR (1,956 bp), were determined. The amino acid sequences deduced from acoA, acoB, acoC, and acoL for E1 alpha (M(r), 35,532), E1 beta (M(r), 35,541), E2 (M(r), 48,149), and E3 (M(r), 61,255) exhibited striking similarities to the amino acid sequences of the corresponding components of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system and the Alcaligenes eutrophus acetoin-cleaving system, respectively. Significant homologies to the enzyme components of various 2-oxo acid dehydrogenase complexes were also found, indicating a close relationship between the two enzyme systems. As a result of the partial repetition of the 5' coding region of acoC into the corresponding part of acoL, the E3 component of the C. magnum acetoin dehydrogenase enzyme system contains an N-terminal lipoyl domain, which is unique among dihydrolipoamide dehydrogenases. We found strong similarities between the AcoR and AcoX sequences and the A. eutrophus acoR gene product

  18. Engineering a domain-locking disulfide into a bacterial malate dehydrogenase produces a redox-sensitive enzyme.

    SciTech Connect

    Muslin, E. H.; Li, D.; Stevens, F. J.; Donnelly, M.; Schiffer, M.; Anderson, L. E.; Univ. of Illinois at Chicago

    1995-06-01

    Light-dependent reduction of cystine disulfide bonds results in activation of several of the enzymes of photosynthetic carbon metabolism within the chloroplast. We have modeled the tertiary structure of four of these light-activated enzymes, namely NADP-linked malate dehydrogenase, glyceraldehyde-3-P dehydrogenase, fructosebisphosphatase, and sedoheptulosebisphosphatase, and identified cysteines in each enzyme that be expected to form inactivating disulfide bonds (Li, D., F. J. Stevens, M. Schiffer, and L. E. Anderson, 1994. Biophys. J. 67:29-35). We have now converted two residues in the Escherichia coli NAD-linked malate dehydrogenase to cysteines and produced a redox-sensitive enzyme. Oxidation of domain-locking cysteine residues in the mutant enzyme clearly mimics dark inactivation of the redox-sensitive chloroplast dehydrogenase. This result is completely consistent with our proposed mechanism.

  19. Novel biohybrids of layered double hydroxide and lactate dehydrogenase enzyme: Synthesis, characterization and catalytic activity studies

    NASA Astrophysics Data System (ADS)

    Djebbi, Mohamed Amine; Braiek, Mohamed; Hidouri, Slah; Namour, Philippe; Jaffrezic-Renault, Nicole; Ben Haj Amara, Abdesslem

    2016-02-01

    The present work introduces new biohybrid materials involving layered double hydroxides (LDH) and biomolecule such as enzyme to produce bioinorganic system. Lactate dehydrogenase (Lac Deh) has been chosen as a model enzyme, being immobilized onto MgAl and ZnAl LDH materials via direct ion-exchange (adsorption) and co-precipitation methods. The immobilization efficiency was largely dependent upon the immobilization methods. A comparative study shows that the co-precipitation method favors the immobilization of great and tunable amount of enzyme. The structural behavior, chemical bonding composition and morphology of the resulting biohybrids were determined by X-ray diffraction (XRD) study, Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM), respectively. The free and immobilized enzyme activity and kinetic parameters were also reported using UV-Visible spectroscopy. However, the modified LDH materials showed a decrease in crystallinity as compared to the unmodified LDH. The change in activity of the immobilized lactate dehydrogenase was considered to be due, to the reduced accessibility of substrate molecules to the active sites of the enzyme and the partial conformational change of the Lac Deh molecules as a result of the immobilization way. Finally, it was proven that there is a correlation between structure/microstructure and enzyme activity dependent on the immobilization process.

  20. Catabolism of circulating enzymes: plasma clearance, endocytosis, and breakdown of lactate dehydrogenase-1 in rabbits.

    PubMed

    Smit, M J; Beekhuis, H; Duursma, A M; Bouma, J M; Gruber, M

    1988-12-01

    Lactate dehydrogenase-1 (EC 1.1.1.27), intravenously injected into rabbits, was cleared with first-order kinetics (half-life 27 min), until at least 80% of the injected activity had disappeared from plasma. Radioactivity from injected 125I-labeled enzyme disappeared at this same rate. Trichloroacetic-acid-soluble breakdown products started to appear in the circulation shortly after injection of the labeled enzyme. Body scans of the rabbits for 80 min after injection of 131I-labeled enzyme revealed rapid accumulation of label in the liver, peaking 10-20 min after injection. Subsequently, activity in the liver declined and radioactivity (probably labeled breakdown products of low molecular mass) steadily accumulated in the bladder. Tissue fractionation of liver, 19 min after injection of labeled enzyme, indicated that the radioactivity was present both in endosomes and in lysosomes, suggesting uptake by endocytosis, followed by breakdown in the lysosomes. Measurements of radioactivity in liver and plasma suggest that the liver is responsible for the breakdown of at least 75% of the injected enzyme. Radioautography of tissue sections of liver and spleen showed accumulated radioactivity in sinusoidal liver cells and red pulpa, respectively. These results are very similar to those for lactate dehydrogenase-5, creatine kinase MM, and several other enzymes that we have previously studied in rats. PMID:3197286

  1. Expression of a heat-stable NADPH-dependent alcohol dehydrogenase in Caldicellulosiruptor bescii results in furan aldehyde detoxification

    SciTech Connect

    Chung, Daehwan; Verbeke, Tobin J.; Cross, Karissa L.; Westpheling, Janet; Elkins, James G.

    2015-07-22

    Compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) are generated through the dehydration of xylose and glucose, respectively, during dilute-acid pretreatment of lignocellulosic biomass and are also potent microbial growth and fermentation inhibitors. The enzymatic reduction of these furan aldehydes to their corresponding, and less toxic, alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenicEscherichia coli, but has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP). Developing CBP-relevant biocatalysts that are either naturally resistant to such inhibitors, or are amenable to engineered resistance, is therefore, an important component in making biofuels production from lignocellulosic biomass feasible.

  2. Expression of a heat-stable NADPH-dependent alcohol dehydrogenase in Caldicellulosiruptor bescii results in furan aldehyde detoxification

    DOE PAGESBeta

    Chung, Daehwan; Verbeke, Tobin J.; Cross, Karissa L.; Westpheling, Janet; Elkins, James G.

    2015-07-22

    Compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) are generated through the dehydration of xylose and glucose, respectively, during dilute-acid pretreatment of lignocellulosic biomass and are also potent microbial growth and fermentation inhibitors. The enzymatic reduction of these furan aldehydes to their corresponding, and less toxic, alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenicEscherichia coli, but has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP). Developing CBP-relevant biocatalysts that are either naturally resistant to such inhibitors, or are amenable to engineered resistance, is therefore, an important componentmore » in making biofuels production from lignocellulosic biomass feasible.« less

  3. Inhibitory effects of ofloxacin and cefepime on enzyme activity of 6-phosphogluconate dehydrogenase from chicken liver.

    PubMed

    Erat, Mustafa; Sakiroğlu, Halis

    2007-01-01

    In this study, effects of some antibiotics, namely, ofloxacin, cefepime, cefazolin, and ampicillin on the in vitro enzyme activity of 6-phosphogluconate dehydrogenase have been investigated. For this purpose, 6-phosphogluconate dehydrogenase was purified from chicken liver 535-fold with a yield of 18% by using ammonium sulphate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. In order to check the purity of the enzyme, SDS polyacylamide gel electrophoresis (SDS-PAGE) was performed. This analysis revealed a highly pure enzyme band on the gel. Among the antibiotics, ofloxacin and cefepime exhibited inhibitory effects, but cefazolin and ampicillin showed neither important inhibitory nor activatory effects on the enzyme activity. The measured I(50) values by plotting activity percent vs. inhibitor concentration, [I(50)] were 0.1713 mM for ofloxacin and 6.0028 mM for cefepime. Inhibition constants, K(i), for ofloxacin and cefepime were also calculated as 0.2740 +/- 0.1080 mM and 12.869 +/- 16.6540 mM by means of Lineweaver-Burk graphs, and inhibition types of the antibiotics were found out to be non-competitive and competitive, respectively. It has been understood from the calculated inhibitory parameters that the purified chicken enzyme has been quite inhibited by these two antimicrobials. PMID:17305608

  4. Conversion of Suspected Food Carcinogen 5-Hydroxymethylfurfural by Sulfotransferases and Aldehyde Dehydrogenases in Postmitochondrial Tissue Preparations of Humans, Mice, and Rats.

    PubMed

    Sachse, Benjamin; Meinl, Walter; Glatt, Hansruedi; Monien, Bernhard H

    2016-01-01

    The food contaminant 5-hydroxymethylfurfural (HMF) is formed by heat- and acid-catalyzed reactions from carbohydrates. More than 80% of HMF is metabolized by oxidation of the aldehyde group in mice and rats. Sulfo conjugation yields mutagenic 5-sulfoxymethylfurfural, the probable cause for the neoplastic effects observed in HMF-treated rodents. Considerable metabolic differences between species hinder assessing the tumorigenic risk associated with human dietary HMF uptake. Here, we assayed HMF turnover catalyzed by sulfotransferases or by aldehyde dehydrogenases (ALDHs) in postmitochondrial preparations from liver, kidney, colon, and lung of humans, mice, and rats. The tissues-specific clearance capacities of HMF sulfo conjugation (CL(SC)) and ALDH-catalyzed oxidation (CL(OX)) were concentrated to the liver. The hepatic clearance CL(SC) in mice (males: 487 µl/min/kg bw, females: 2520 µl/min/kg bw) and rats (males: 430 µl/min/kg bw, females: 198 µl/min/kg bw) were considerably higher than those in humans (males: 21.2 µl/min/kg bw, females: 32.2 µl/min/kg bw). The ALDH-related clearance rates CLOX in mice (males: 3400 ml/min/kg bw, females: 1410 ml/min/kg bw) were higher than those of humans (males: 436 ml/min/kg bw, females: 646 ml/min/kg bw) and rats (males: 627 ml/min/kg bw, females: 679 ml/min/kg bw). The ratio of CL(OX) to CL(SC) was lowest in female mice. This finding indicated that HMF sulfo conjugation was most substantial in the liver of female mice, a target tissue for HMF-induced neoplastic effects, and that humans may be less sensitive regarding HMF sulfo conjugation compared with the rodent models. PMID:26454887

  5. Development of Selective Inhibitors for Human Aldehyde Dehydrogenase 3A1 (ALDH3A1) for the Enhancement of Cyclophosphamide Cytotoxicity

    PubMed Central

    Parajuli, Bibek; Georgiadis, Taxiarchis M.; Fishel, Melissa L.; Hurley, Thomas D.

    2014-01-01

    Aldehyde dehydrogenase 3A1 (ALDH3A1) plays an important role in many cellular oxidative processes, including cancer chemo-resistance by metabolizing activated forms of oxazaphosphorine drugs such as cyclophosphamide (CP) and its analogues such as mafosfamide (MF), ifosfamide (IFM), 4-hydroperoxycyclophosphamide (4-HPCP). Compounds that can selectively target ALDH3A1 may permit delineation of its roles in these processes and could restore chemosensitivity in cancer cells that express this isoenzyme. Here we report the detailed kinetic and structural characterization of an ALDH3A1 selective inhibitor, CB29, previously identified in a high throughput screen. Kinetic and crystallographic studies demonstrate that CB29 binds within the aldehyde substrate-binding site of ALDH3A1. Cellular proliferation of ALDH3A1-expressing lung adenocarcinoma (A549) and glioblastoma (SF767) cell lines, as well as the ALDH3A1 non-expressing lung fibroblast cells, CCD-13Lu, is unaffected by treatment with CB29 and its analogues alone. However, the sensitivity toward the anti-proliferative effects of mafosfamide is enhanced by treatment with CB29 and its analogue in the tumour cells. In contrast, the sensitivity of CCD-13Lu cells toward mafosfamide was unaffected by the addition of these same compounds. CB29 is chemically distinct from the previously reported small molecule inhibitors of ALDH isoenzymes and does not inhibit ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1 or ALDH2 isoenzymes at concentrations up to 250 μM. Thus, CB29 is a novel small molecule inhibitor of ALDH3A1, which may be useful as a chemical tool to delineate the role of ALDH3A1 in numerous metabolic pathways, including sensitizing ALDH3A1-positive cancer cells to oxazaphosphorines. PMID:24677340

  6. A potent specific inhibitor of 6-phosphogluconate dehydrogenase of Cryptococcus neoformans and of certain other fungal enzymes.

    PubMed

    Niehaus, W G; Flynn, T

    1993-09-01

    A particular lot of the zwitterionic buffer, 2(N-morpholino) ethane sulfonic acid (MES), contained a contaminant that inhibited a number of fungal NADP-dependent dehydrogenases. Enzymes that were particularly sensitive include 6-phosphogluconate dehydrogenases from Cryptococcus neoformans and Schizophyllum commune and glucose-6-phosphate dehydrogenase from Schizophyllum commune. A number of NADP-dependent dehydrogenases of animal origin were tested and all were completely insensitive to inhibition except for rat liver 6-phosphogluconate dehydrogenase, which was 10-fold less sensitive than the Cryptococcal enzyme. The pattern of inhibition in all cases was linear competitive versus NADP. The inhibitor has been purified and identified as an ethylenesulfonic acid oligomer. This inhibitor holds promise as a model compound for the development of a specific antifungal agent. PMID:8302365

  7. Computational optimization of AG18051 inhibitor for amyloid-beta binding alcohol dehydrogenase enzyme

    NASA Astrophysics Data System (ADS)

    Marques, Alexandra T.; Antunes, Agostinho; Fernandes, Pedro A.; Ramos, Maria J.

    Amyloid-beta (Abeta) binding alcohol dehydrogenase (ABAD) is a multifunctional enzyme involved in maintaining the homeostasis. The enzyme can also mediate some diseases, including genetic diseases, Alzheimer's disease, and possibly some prostate cancers. Potent inhibitors of ABAD might facilitate a better clarification of the functions of the enzyme under normal and pathogenic conditions and might also be used for therapeutic intervention in disease conditions mediated by the enzyme. The AG18051 is the only presently available inhibitor of ABAD. It binds in the active-site cavity of the enzyme and reacts with the NAD+ cofactor to form a covalent adduct. In this work, we use computational methods to perform a rational optimization of the AG18051 inhibitor, through the introduction of chemical substitutions directed to improve the affinity of the inhibitor to the enzyme. The molecular mechanics-Poisson-Boltzmann surface area methodology was used to predict the relative free binding energy of the different modified inhibitor-NAD-enzyme complexes. We show that it is possible to increase significantly the affinity of the inhibitor to the enzyme with small modifications, without changing the overall structure and ADME (absorption, distribution, metabolism, and excretion) properties of the original inhibitor.

  8. A bifunctional enzyme from Rhodococcus erythropolis exhibiting secondary alcohol dehydrogenase-catalase activities.

    PubMed

    Martinez-Rojas, Enriqueta; Kurt, Tutku; Schmidt, Udo; Meyer, Vera; Garbe, Leif-Alexander

    2014-11-01

    Alcohol dehydrogenases have long been recognized as potential biocatalyst for production of chiral fine and bulk chemicals. They are relevant for industry in enantiospecific production of chiral compounds. In this study, we identified and purified a nicotinamide adenine dinucleotide (NAD)-dependent secondary alcohol dehydrogenase (SdcA) from Rhodococcus erythropolis oxidizing γ-lactols into γ-lactones. SdcA showed broad substrate specificity on γ-lactols; secondary aliphatic alcohols with 8 and 10 carbon atoms were also substrates and oxidized with (2S)-stereospecificity. The enzyme exhibited moderate stability with a half-life of 5 h at 40 °C and 20 days at 4 °C. Mass spectrometric identification revealed high sequence coverage of SdcA amino acid sequence to a highly conserved catalase from R. erythropolis. The corresponding encoding gene was isolated from genomic DNA and subsequently overexpressed in Escherichia coli BL21 DE3 cells. In addition, the recombinant SdcA was purified and characterized in order to confirm that the secondary alcohol dehydrogenase and catalase activity correspond to the same enzyme. PMID:24846734

  9. A new role for α-ketoglutarate dehydrogenase complex: regulating metabolism through post-translational modification of other enzymes.

    PubMed

    McKenna, Mary C; Rae, Caroline D

    2015-07-01

    This Editorial highlights a study by Gibson et al. published in this issue of JNeurochem, in which the authors reveal a novel role for the α-ketoglutarate dehydrogenase complex (KGDHC) in post-translational modification of proteins. KGDHC may catalyze post-translational modification of itself as well as several other proteins by succinylation of lysine residues. The authors' report of an enzyme responsible for succinylation of key mitochondrial enzymes represents a major step toward our understanding of the complex functional metabolome. TCA, tricarboxylic acid; KG, α-ketoglutarate; KGDHC, α-ketoglutarate dehydrogenase complex; FUM, fumarase; MDH, malate dehydrogenase; ME, malic enzyme; GDH, glutamate dehydrogenase; AAT, aspartate aminotransferase; GS, glutamine synthetase; PAG, phosphate-activated glutaminase; SIRT3, silent information regulator 3; SIRT5, silent information regulator 5. PMID:26052752

  10. Structural Basis for Flip-Flop Action of Thiamin Pyrophosphate-dependent Enzymes Revealed by Human Pyruvate Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Ciszak, Ewa M.; Korotchkina, Lioubov G.; Dominiak, Paulina M.; Sidhu, Sukdeep; Patel, Mulchand S.

    2003-01-01

    The derivative of vitamin B1, thiamin pyrophosphate, is a cofactor of enzymes performing catalysis in pathways of energy production. In alpha (sub 2) beta (sub 2)-heterotetrameric human pyruvate dehydrogenase, this cofactor is used to cleave the C(sup alpha) -C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites has not yet been understood. To understand the mechanism of action of this enzyme, we determined the crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95-Angstrom resolution. We propose a model for the flip-flop action of this enzyme through a concerted approximately 2-Angstrom shuttle-like motion of its heterodimers. Similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase with functionally related enzymes suggests that this newly defined shuttle-like motion of domains is common to the family of thiamin pyrophosphate-dependent enzymes.

  11. The absence of aldehyde dehydrogenase 1 A1-positive cells in benign mammary stroma is associated with risk factors for breast cancer

    PubMed Central

    Isfoss, Björn Logi; Holmqvist, Bo; Jernström, Helena; Alm, Per; Olsson, Håkan

    2016-01-01

    In this study, aldehyde dehydrogenase 1 (ALDH1)-expressing cells in stroma of histologically normal breast tissue from premenopausal women were investigated in situ regarding cellular morphology, cell distribution, and relation to the additional stem cell markers, CD44 (+) and CD24 (−). These results were correlated with hormonal and genetic risk factors for breast cancer. Triple immunofluorescence labeling was performed on tissues from premenopausal women with a family history of breast cancer, and breast reduction specimens from premenopausal women with no family history of breast cancer were used as a control group. The majority of ALDH1-immunoreactive cells in stroma were spindle-shaped or polygonal, and such cells that were CD44− and CD24− were absent in the breast stroma of a significantly larger number of nulliparous than parous women. A less common morphological type of ALDH1-positive cells in stroma was round or oval in shape, and such cells that were CD44+ and CD24− were absent in a significant number of women with a family history of breast cancer. The CD44+/CD24− immunophenotype is consistent with stem cells, and the round/oval morphology suggests mesenchymal cells. This study demonstrates that there are two morphologically distinct types of ALDH1-positive cells in histologically benign mammary stroma, and the absence of these cells is correlated with clinical risk factors for breast cancer in premenopausal women. PMID:27313475

  12. Two clinical drugs deubiquitinase inhibitor auranofin and aldehyde dehydrogenase inhibitor disulfiram trigger synergistic anti-tumor effects in vitro and in vivo.

    PubMed

    Huang, Hongbiao; Liao, Yuning; Liu, Ningning; Hua, Xianliang; Cai, Jianyu; Yang, Changshan; Long, Huidan; Zhao, Chong; Chen, Xin; Lan, Xiaoying; Zang, Dan; Wu, Jinjie; Li, Xiaofen; Shi, Xianping; Wang, Xuejun; Liu, Jinbao

    2016-01-19

    Inhibition of proteasome-associated deubiquitinases (DUBs) is emerging as a novel strategy for cancer therapy. It was recently reported that auranofin (Aur), a gold (I)-containing compound used clinically to treat rheumatoid arthritis, is a proteasome-associated DUB inhibitor. Disulfiram (DSF), an inhibitor of aldehyde dehydrogenase, is currently in clinical use for treating alcoholism. Recent studies have indicated that DSF can also act as an antitumor agent. We investigated the effect of combining DSF and Aur on apoptosis induction and tumor growth in hepatoma cancer cells. Here we report that (i) the combined treatment of Aur and DSF results in synergistic cytotoxicity to hepatoma cells in vitro and in vivo; (ii) Aur and DSF in combination induces caspase activation, endoplasmic reticulum (ER) stress, and reactive oxygen species (ROS) production; (iii) pan-caspase inhibitor z-VAD-FMK could efficiently block apoptosis but not proteasome inhibition induced by Aur and DSF combined treatment, and ROS is not required for Aur+DSF to induce apoptosis. Collectively, we demonstrate a model of synergism between DSF and proteasome-associated DUB inhibitor Aur in the induction of apoptosis in hepatoma cancer cells, identifying a potential novel anticancer strategy for clinical use in the future. PMID:26625200

  13. The absence of aldehyde dehydrogenase 1 A1-positive cells in benign mammary stroma is associated with risk factors for breast cancer.

    PubMed

    Isfoss, Björn Logi; Holmqvist, Bo; Jernström, Helena; Alm, Per; Olsson, Håkan

    2016-01-01

    In this study, aldehyde dehydrogenase 1 (ALDH1)-expressing cells in stroma of histologically normal breast tissue from premenopausal women were investigated in situ regarding cellular morphology, cell distribution, and relation to the additional stem cell markers, CD44 (+) and CD24 (-). These results were correlated with hormonal and genetic risk factors for breast cancer. Triple immunofluorescence labeling was performed on tissues from premenopausal women with a family history of breast cancer, and breast reduction specimens from premenopausal women with no family history of breast cancer were used as a control group. The majority of ALDH1-immunoreactive cells in stroma were spindle-shaped or polygonal, and such cells that were CD44(-) and CD24(-) were absent in the breast stroma of a significantly larger number of nulliparous than parous women. A less common morphological type of ALDH1-positive cells in stroma was round or oval in shape, and such cells that were CD44(+) and CD24(-) were absent in a significant number of women with a family history of breast cancer. The CD44(+)/CD24(-) immunophenotype is consistent with stem cells, and the round/oval morphology suggests mesenchymal cells. This study demonstrates that there are two morphologically distinct types of ALDH1-positive cells in histologically benign mammary stroma, and the absence of these cells is correlated with clinical risk factors for breast cancer in premenopausal women. PMID:27313475

  14. Reliability of a flushing questionnaire and the ethanol patch test in screening for inactive aldehyde dehydrogenase-2 and alcohol-related cancer risk.

    PubMed

    Yokoyama, A; Muramatsu, T; Ohmori, T; Kumagai, Y; Higuchi, S; Ishii, H

    1997-12-01

    Molecular epidemiology of esophageal and upper aerodigestive tract cancers revealed that alcohol is more carcinogenic in persons with inactive aldehyde dehydrogenase-2 (ALDH2) than in those with active ALDH2. A simple questionnaire has been developed to screen for the facial flushing that occurs in persons with inactive ALDH2 when they drink even a single glass of beer. In this study, 266 of 284 consecutive male Japanese clinic patients (age > or = 50 years) completed the flushing questionnaire, and 239 underwent the ethanol patch test (a cutaneous model for the flushing response). Blinded genotyping showed inactive ALDH2 for 94.4% (102 of 108) of subjects who reported always flushing (early in their drinking history or currently) and for 47.7% (21 of 44) of those who reported sometimes flushing, whereas 95.6% (109 of 114) of subjects reporting that they never exhibited facial flushing had active ALDH2. When all three categories of flushing (current always, former always, and sometimes) were collapsed into one, the questionnaire's sensitivity and specificity for identifying inactive ALDH2 were 96.1 and 79.0%, respectively, compared with 72.4 and 71.4% for the ethanol patch test. The results suggest the utility of this simple flushing questionnaire in daily practice, as well as large-scale studies to assess cancer risks associated with drinking and ALDH2 and for activities aimed at preventing alcohol-related cancer. PMID:9419411

  15. Two clinical drugs deubiquitinase inhibitor auranofin and aldehyde dehydrogenase inhibitor disulfiram trigger synergistic anti-tumor effects in vitro and in vivo

    PubMed Central

    Liu, Ningning; Hua, Xianliang; Cai, Jianyu; Yang, Changshan; Long, Huidan; Zhao, Chong; Chen, Xin; Lan, Xiaoying; Zang, Dan; Wu, Jinjie; Li, Xiaofen; Shi, Xianping; Wang, Xuejun; Liu, Jinbao

    2016-01-01

    Inhibition of proteasome-associated deubiquitinases (DUBs) is emerging as a novel strategy for cancer therapy. It was recently reported that auranofin (Aur), a gold (I)-containing compound used clinically to treat rheumatoid arthritis, is a proteasome-associated DUB inhibitor. Disulfiram (DSF), an inhibitor of aldehyde dehydrogenase, is currently in clinical use for treating alcoholism. Recent studies have indicated that DSF can also act as an antitumor agent. We investigated the effect of combining DSF and Aur on apoptosis induction and tumor growth in hepatoma cancer cells. Here we report that (i) the combined treatment of Aur and DSF results in synergistic cytotoxicity to hepatoma cells in vitro and in vivo; (ii) Aur and DSF in combination induces caspase activation, endoplasmic reticulum (ER) stress, and reactive oxygen species (ROS) production; (iii) pan-caspase inhibitor z-VAD-FMK could efficiently block apoptosis but not proteasome inhibition induced by Aur and DSF combined treatment, and ROS is not required for Aur+DSF to induce apoptosis. Collectively, we demonstrate a model of synergism between DSF and proteasome-associated DUB inhibitor Aur in the induction of apoptosis in hepatoma cancer cells, identifying a potential novel anticancer strategy for clinical use in the future. PMID:26625200

  16. A proposal for nomenclature of aldehyde dehydrogenases in Saccharomyces cerevisiae and characterization of the stress-inducible ALD2 and ALD3 genes.

    PubMed

    Navarro-Aviño, J P; Prasad, R; Miralles, V J; Benito, R M; Serrano, R

    1999-07-01

    The complete sequencing of the genome of Saccharomyces cerevisiae indicated that this organism contains five genes encoding aldehyde dehydrogenases. YOR374w and YER073w correspond to the mitochondrial isoforms and we propose as gene names ALD4 and ALD5, respectively. YPL061w has been described as the cytoplasmic constitutive isoform and named ALD6. We characterize here the tandem-repeated ORFs YMR170c and YMR169c as the cytoplasmic stress-inducible isoforms, with gene names ALD2 and ALD3, respectively. The expression of ALD2 and ALD3 is dependent on the general-stress transcription factors Msn2,4 but independent of the HOG MAP kinase pathway. ALD3 is induced by a variety of stresses, including osmotic shock, heat shock, glucose exhaustion, oxidative stress and drugs. ALD2 is only induced by osmotic stress and glucose exhaustion. A double null mutant, ald2 ald3, exhibited unchanged sensitivity to any of the above stresses. The only phenotype detected in this mutant was a reduced growth rate in ethanol medium as compared to the wild type. PMID:10407263

  17. 9-O-acetylated sialic acids differentiating normal haematopoietic precursors from leukemic stem cells with high aldehyde dehydrogenase activity in children with acute lymphoblastic leukaemia.

    PubMed

    Chowdhury, Suchandra; Chandra, Sarmila; Mandal, Chitra

    2014-10-01

    Childhood acute lymphoblastic leukaemia (ALL) originates from mutations in haematopoietic progenitor cells (HPCs). For high-risk patients, treated with intensified post-remission chemotherapy, haematopoietic stem cell (HSC) transplantation is considered. Autologous HSC transplantation needs improvisation till date. Previous studies established enhanced disease-associated expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs) on lymphoblasts of these patients at diagnosis, followed by its decrease with clinical remission and reappearance with relapse. Based on this differential expression of Neu5,9Ac2-GPs, identification of a normal HPC population was targeted from patients at diagnosis. This study identifies two distinct haematopoietic progenitor populations from bone marrow of diagnostic ALL patients, exploring the differential expression of Neu5,9Ac2-GPs with stem cell (CD34, CD90, CD117, CD133), haematopoietic (CD45), lineage-commitment (CD38) antigens and cytosolic aldehyde dehydrogenase (ALDH). Normal haematopoietic progenitor cells (ALDH(+)SSC(lo)CD45(hi)Neu5,9Ac2 -GPs(lo)CD34(+)CD38(-)CD90(+)CD117(+)CD133(+)) differentiated into morphologically different, lineage-specific colonies, being crucial for autologous HSC transplantation while leukemic stem cells (ALDH(+)SSC(lo)CD45(lo)Neu5,9Ac2 -GPs(hi)CD34(+)CD38(+)CD90(-)CD117(-)CD133(-)) lacking this ability can be potential targets for minimal residual disease detection and drug-targeted immunotherapy. PMID:25283637

  18. MUC1-C Oncoprotein Activates ERK→C/EBPβ Signaling and Induction of Aldehyde Dehydrogenase 1A1 in Breast Cancer Cells*

    PubMed Central

    Alam, Maroof; Ahmad, Rehan; Rajabi, Hasan; Kharbanda, Akriti; Kufe, Donald

    2013-01-01

    Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is used as a marker of breast cancer stem cells; however, little is known about the regulation of ALDH1A1 expression. Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly overexpressed in most human breast cancers. In studies of breast cancer cells stably silenced for MUC1 or overexpressing the oncogenic MUC1-C subunit, we demonstrate that MUC1-C is sufficient for induction of MEK→ERK signaling and that treatment with a MUC1-C inhibitor suppresses ERK activation. In turn, MUC1-C induces ERK-mediated phosphorylation and activation of the CCAAT/enhancer-binding protein β (C/EBPβ) transcription factor. The results further show that MUC1-C and C/EBPβ form a complex on the ALDH1A1 gene promoter and activate ALDH1A1 gene transcription. MUC1-C-induced up-regulation of ALDH1A1 expression is associated with increases in ALDH activity and is detectable in stem-like cells when expanded as mammospheres. These findings demonstrate that MUC1-C (i) activates a previously unrecognized ERK→C/EBPβ→ALDH1A1 pathway, and (ii) promotes the induction of ALDH activity in breast cancer cells. PMID:24043631

  19. Biochemical Analysis of Recombinant AlkJ from Pseudomonas putida Reveals a Membrane-Associated, Flavin Adenine Dinucleotide-Dependent Dehydrogenase Suitable for the Biosynthetic Production of Aliphatic Aldehydes

    PubMed Central

    Kirmair, Ludwig

    2014-01-01

    The noncanonical alcohol dehydrogenase AlkJ is encoded on the alkane-metabolizing alk operon of the mesophilic bacterium Pseudomonas putida GPo1. To gain insight into the enzymology of AlkJ, we have produced the recombinant protein in Escherichia coli and purified it to homogeneity using His6 tag affinity and size exclusion chromatography (SEC). Despite synthesis in the cytoplasm, AlkJ was associated with the bacterial cell membrane, and solubilization with n-dodecyl-β-d-maltoside was necessary to liberate the enzyme. SEC and spectrophotometric analysis revealed a dimeric quaternary structure with stoichiometrically bound reduced flavin adenine dinucleotide (FADH2). The holoenzyme showed thermal denaturation at moderate temperatures around 35°C, according to both activity assay and temperature-dependent circular dichroism spectroscopy. The tightly bound coenzyme was released only upon denaturation with SDS or treatment with urea-KBr and, after air oxidation, exhibited the characteristic absorption spectrum of FAD. The enzymatic activity of purified AlkJ for 1-butanol, 1-hexanol, and 1-octanol as well as the n-alkanol derivative ω-hydroxy lauric acid methyl ester (HLAMe) was quantified in the presence of the artificial electron acceptors phenazine methosulfate (PMS) and 2,6-dichlorophenolindophenol (DCPIP), indicating broad substrate specificity with the lowest activity on the shortest alcohol, 1-butanol. Furthermore, AlkJ was able to accept as cosubstrates/oxidants the ubiquinone derivatives Q0 and Q1, also in conjunction with cytochrome c, which suggests coupling to the bacterial respiratory chain of this membrane-associated enzyme in its physiological environment. Using gas chromatographic analysis, we demonstrated specific biocatalytic conversion by AlkJ of the substrate HLAMe to the industrially relevant aldehyde, thus enabling the biotechnological production of 12-amino lauric acid methyl ester via subsequent enzymatic transamination. PMID:24509930

  20. Identification and analysis of the genes coding for the putative pyruvate dehydrogenase enzyme complex in Acholeplasma laidlawii.

    PubMed Central

    Wallbrandt, P; Tegman, V; Jonsson, B H; Wieslander, A

    1992-01-01

    A monospecific antibody recognizing two membrane proteins in Acholeplasma laidlawii identified a plasmid clone from a genomic library. The nucleotide sequence of the 4.6-kbp insert contained four sequential genes coding for proteins of 39 kDa (E1 alpha, N terminus not cloned), 36 kDa (E1 beta), 57 kDa (E2), and 36 kDa (E3; C terminus not cloned). The N termini of the cloned E2, E1 beta, and native A. laidlawii E2 proteins were verified by amino acid sequencing. Computer-aided searches showed that the translated DNA sequences were homologous to the four subenzymes of the pyruvate dehydrogenase complexes from gram-positive bacteria and humans. The plasmid-encoded 57-kDa (E2) protein was recognized by antibodies against the E2 subenzymes of the pyruvate and oxoglutarate dehydrogenase complexes from Bacillus subtilis. A substantial fraction of the E2 protein as well as part of the pyruvate dehydrogenase enzymatic activity was associated with the cytoplasmic membrane in A. laidlawii. In vivo complementation with three different Escherichia coli pyruvate dehydrogenase-defective mutants showed that the four plasmid-encoded proteins were able to restore pyruvate dehydrogenase enzyme activity in E. coli. Since A. laidlawii lacks oxoglutarate dehydrogenase and most likely branched-chain dehydrogenase enzyme complex activities, these results strongly suggest that the sequenced genes code for the pyruvate dehydrogenase complex. Images PMID:1735725

  1. GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE-S, A SPERM-SPECIFIC GLYCOLYTIC ENZYME, IS REQUIRED FOR SPERM MOTILITY AND MALE FERTILITY

    EPA Science Inventory

    While glycolysis is highly conserved, it is remarkable that several novel isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like it...

  2. Lactate dehydrogenase is the key enzyme for pneumococcal pyruvate metabolism and pneumococcal survival in blood.

    PubMed

    Gaspar, Paula; Al-Bayati, Firas A Y; Andrew, Peter W; Neves, Ana Rute; Yesilkaya, Hasan

    2014-12-01

    Streptococcus pneumoniae is a fermentative microorganism and causes serious diseases in humans, including otitis media, bacteremia, meningitis, and pneumonia. However, the mechanisms enabling pneumococcal survival in the host and causing disease in different tissues are incompletely understood. The available evidence indicates a strong link between the central metabolism and pneumococcal virulence. To further our knowledge on pneumococcal virulence, we investigated the role of lactate dehydrogenase (LDH), which converts pyruvate to lactate and is an essential enzyme for redox balance, in the pneumococcal central metabolism and virulence using an isogenic ldh mutant. Loss of LDH led to a dramatic reduction of the growth rate, pinpointing the key role of this enzyme in fermentative metabolism. The pattern of end products was altered, and lactate production was totally blocked. The fermentation profile was confirmed by in vivo nuclear magnetic resonance (NMR) measurements of glucose metabolism in nongrowing cell suspensions of the ldh mutant. In this strain, a bottleneck in the fermentative steps is evident from the accumulation of pyruvate, revealing LDH as the most efficient enzyme in pyruvate conversion. An increase in ethanol production was also observed, indicating that in the absence of LDH the redox balance is maintained through alcohol dehydrogenase activity. We also found that the absence of LDH renders the pneumococci avirulent after intravenous infection and leads to a significant reduction in virulence in a model of pneumonia that develops after intranasal infection, likely due to a decrease in energy generation and virulence gene expression. PMID:25245810

  3. Lactate Dehydrogenase Is the Key Enzyme for Pneumococcal Pyruvate Metabolism and Pneumococcal Survival in Blood

    PubMed Central

    Gaspar, Paula; Al-Bayati, Firas A. Y.; Andrew, Peter W.; Neves, Ana Rute

    2014-01-01

    Streptococcus pneumoniae is a fermentative microorganism and causes serious diseases in humans, including otitis media, bacteremia, meningitis, and pneumonia. However, the mechanisms enabling pneumococcal survival in the host and causing disease in different tissues are incompletely understood. The available evidence indicates a strong link between the central metabolism and pneumococcal virulence. To further our knowledge on pneumococcal virulence, we investigated the role of lactate dehydrogenase (LDH), which converts pyruvate to lactate and is an essential enzyme for redox balance, in the pneumococcal central metabolism and virulence using an isogenic ldh mutant. Loss of LDH led to a dramatic reduction of the growth rate, pinpointing the key role of this enzyme in fermentative metabolism. The pattern of end products was altered, and lactate production was totally blocked. The fermentation profile was confirmed by in vivo nuclear magnetic resonance (NMR) measurements of glucose metabolism in nongrowing cell suspensions of the ldh mutant. In this strain, a bottleneck in the fermentative steps is evident from the accumulation of pyruvate, revealing LDH as the most efficient enzyme in pyruvate conversion. An increase in ethanol production was also observed, indicating that in the absence of LDH the redox balance is maintained through alcohol dehydrogenase activity. We also found that the absence of LDH renders the pneumococci avirulent after intravenous infection and leads to a significant reduction in virulence in a model of pneumonia that develops after intranasal infection, likely due to a decrease in energy generation and virulence gene expression. PMID:25245810

  4. Betaine aldehyde, betaine, and choline levels in rat livers during ethanol metabolism.

    PubMed

    Chern, M K; Gage, D A; Pietruszko, R

    2000-12-01

    Betaine aldehyde levels were determined in rat livers following 4 weeks of ethanol feeding, employing the Lieber-De Carli liquid diet. The results showed that the levels of betaine aldehyde are unaffected by alcohol feeding to rats. These levels in both experimental and control animals were found to be quite low, 5.5 nmol/g liver. Betaine aldehyde levels have not been determined previously in mammalian liver because of methodological difficulties. This investigation employed fast atom bombardment-mass spectroscopy to determine the levels of betaine aldehyde, betaine, and choline. The decrease in betaine levels following ethanol administration confirmed the results of other investigators. Choline levels determined during this investigation were lower than previously reported. The reason for starting this investigation was the fact that the enzyme that catalyzes betaine aldehyde dehydrogenation to betaine, which is distributed in both mitochondria and the cytoplasm, was found to also metabolize acetaldehyde with K(m) and V(max) values lower than those for betaine aldehyde. Thus, it appeared likely that the metabolism of acetaldehyde during ethanol metabolism might inhibit betaine aldehyde conversion to betaine and thereby result in decreased betaine levels (Barak et al., Alcohol 13: 395-398, 1996). The fact that betaine aldehyde levels in alcohol-fed animals were similar to those in controls demonstrates that competition between acetaldehyde and betaine aldehyde for the same enzyme does not occur. This complete lack of competition suggests that betaine aldehyde dehydrogenase in the mitochondrial matrix may totally metabolize betaine aldehyde to betaine without any involvement of cytoplasmic betaine aldehyde dehydrogenase. PMID:11077045

  5. Phospholipid protection against proteolysis of D-beta-hydroxybutyrate dehydrogenase, a lecithin-requiring enzyme.

    PubMed

    Maurer, A; McIntyre, J O; Churchill, S; Fleischer, S

    1985-02-10

    D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme which is localized on the inner face of the mitochondrial inner membrane. The apodehydrogenase, i.e. the purified enzyme devoid of lipid, has been purified from beef heart mitochondria and as such is inactive. It can be reactivated by insertion into phospholipid vesicles containing lecithin. Proteolytic digestion with different proteases has been carried out to obtain insight into the orientation of the enzyme in the membrane and to assess the extent of immersion of the protein into the phospholipid bilayer. Digestion of the apodehydrogenase with either trypsin, chymotrypsin, Staphylococcus aureus protease, thermolysin, carboxypeptidases A and Y, or Pronase (from Streptomyces griseus) leads to loss of activity, as assayed with phospholipid. Limited digestion with carboxypeptidase results in complete inactivation. Of the proteases tested, only Pronase and chymotrypsin cleave and inactivate the enzyme inserted into phospholipid vesicles (enzyme-phospholipid complex). For the enzyme-phospholipid complex, the loss of activity with Pronase digestion follows a single exponential decay to less than 10% of the initial activity. With chymotrypsin digestion, the staining intensity of the original approximately 31,500-dalton polypeptide decreases more rapidly than the loss of enzymic activity. The enzyme-phospholipid complex, after limited cleavage with chymotrypsin, retains enzymic activity and resonance energy transfer from protein to bound NADH and an approximately 26,000-dalton polypeptide is observed. Phospholipid alters the cleavage pattern with both chymotrypsin and Pronase, and the rate of inactivation of the enzyme-phospholipid complex is slowed in the presence of NAD(H). Moreover, the rate of inactivation of the apodehydrogenase with chymotrypsin is diminished approximately 3-fold in the presence of NAD+. Digestion of submitochondrial vesicles with either trypsin, chymotrypsin, or Pronase rapidly

  6. Lactate dehydrogenase is not a mitochondrial enzyme in human and mouse vastus lateralis muscle

    PubMed Central

    Rasmussen, Hans N; van Hall, Gerrit; Rasmussen, Ulla F

    2002-01-01

    The presence of lactate dehydrogenase in skeletal muscle mitochondria was investigated to clarify whether lactate is a possible substrate for mitochondrial respiration. Mitochondria were prepared from 100 mg samples of human and mouse vastus lateralis muscle. All fractions from the preparation procedure were assayed for marker enzymes and lactate dehydrogenase (LDH). The mitochondrial fraction contained no LDH activity (detection limit ∼0.05 % of the tissue activity) and the distribution of LDH activity among the fractions paralleled that of pyruvate kinase, i.e. LDH was fractionated as a cytoplasmic enzyme. Respiratory experiments with the mitochondrial fraction also indicated the absence of LDH. Lactate did not cause respiration, nor did it affect the respiration of pyruvate + malate. The major part of the native cytochrome c was retained in the isolated mitochondria, which, furthermore, showed high specific rates of state 3 respiration. This excluded artificial loss from the mitochondria of all activity of a possible LDH. It was concluded that skeletal muscle mitochondria are devoid of LDH and unable to metabolize lactate. PMID:12042361

  7. Assessment of freshness and freeze-thawing of sea bream fillets (Sparus aurata) by a cytosolic enzyme: Lactate dehydrogenase.

    PubMed

    Diop, Mamadou; Watier, Denis; Masson, Pierre-Yves; Diouf, Amadou; Amara, Rachid; Grard, Thierry; Lencel, Philippe

    2016-11-01

    The evaluation of freshness and freeze-thawing of fish fillets was carried out by assessment of autolysis of cells using a cytosolic enzyme lactate dehydrogenase. Autolysis plays an important role in spoilage of fish and postmortem changes in fish tissue are due to the breakdown of the cellular structures and release of cytoplasmic contents. The outflow of a cytosolic enzyme, lactate dehydrogenase, was studied in sea bream fillets and the Sparus aurata fibroblasts (SAF-1) cell-line during an 8day storage period at +4°C. A significant increase of lactate dehydrogenase release was observed, especially after 5days of storage. The ratio between the free and the total lactate dehydrogenase activity is a promising predictive marker to measure the quality of fresh fish fillets. The effect of freeze-thawing on cytosolic lactate dehydrogenase and lysosomal α-d-glucosidase activities was also tested. Despite the protecting effect of the tissue compared to the cell-line, a loss of lactate dehydrogenase activity, but not of α-d-glucosidase, was observed. In conclusion, lactate dehydrogenase may be used as a marker to both assess freshness of fish and distinguish between fresh and frozen-thawed fish fillets. PMID:27211667

  8. Interaction between glutamate dehydrogenase (GDH) and L-leucine catabolic enzymes: intersecting metabolic pathways.

    PubMed

    Hutson, Susan M; Islam, Mohammad Mainul; Zaganas, Ioannis

    2011-09-01

    Branched-chain amino acids (BCAAs) catabolism follows sequential reactions and their metabolites intersect with other metabolic pathways. The initial enzymes in BCAA metabolism, the mitochondrial branched-chain aminotransferase (BCATm), which deaminates the BCAAs to branched-chain α-keto acids (BCKAs); and the branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC), which oxidatively decarboxylates the BCKAs, are organized in a supramolecular complex termed metabolon. Glutamate dehydrogenase (GDH1) is found in the metabolon in rat tissues. Bovine GDH1 binds to the pyridoxamine 5'-phosphate (PMP)-form of human BCATm (PMP-BCATm) but not to pyridoxal 5'-phosphate (PLP)-BCATm in vitro. This protein interaction facilitates reamination of the α-ketoglutarate (αKG) product of the GDH1 oxidative deamination reaction. Human GDH1 appears to act like bovine GDH1 but human GDH2 does not show the same enhancement of BCKDC enzyme activities. Another metabolic enzyme is also found in the metabolon is pyruvate carboxylase (PC). Kinetic results suggest that PC binds to the E1 decarboxylase of BCKDC but does not effect BCAA catabolism. The protein interaction of BCATm and GDH1 promotes regeneration of PLP-BCATm which then binds to BCKDC resulting in channeling of the BCKA products from BCATm first half reaction to E1 and promoting BCAA oxidation and net nitrogen transfer from BCAAs. The cycling of nitrogen through glutamate via the actions of BCATm and GDH1 releases free ammonia. Formation of ammonia may be important for astrocyte glutamine synthesis in the central nervous system. In peripheral tissue association of BCATm and GDH1 would promote BCAA oxidation at physiologically relevant BCAA concentrations. PMID:21621574

  9. Structural Basis for Flip-Flop Action of Thiamin-Dependent Enzymes Revealed by Crystal Structure of Human Pyruvate Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Ciszak, Ewa; Korotchkina, Lioubov G.; Dominiak, Paulina M.; Sidhu, Sukdeep; Patel, Mulchand S.

    2003-01-01

    The biologically active derivative of vitamin B1; thiamin pyrophosphate; is used as cofactor by many enzymes that perform a wide range of catalytic functions in the pathways of energy production. In alpha2beta2-heterotetrameric human pyruvate dehydrogenase, the first catalytic component enzyme of human pyruvate dehydrogenase complex, this cofactor is used to cleave the C(sup alpha)-C(=0) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase, the second catalytic component of the complex. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites have puzzled researchers from earlier functional studies of this enzyme. In order to gain insight into the mechanism of action of this enzyme, we determined the crystal structure of the holoform of human pyruvate dehydrogenase at 1.958, resolution. We propose a kinetic model for the flip-flop action of this enzyme through the concerted approx. 2A, shuttle-like motion of the heterodimers. The similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase and other functionally related enzymes suggests this newly defined mechanism of shuttle-like motion of domains to be common for the family of thiamin pyrophosphate-dependent enzymes.

  10. Optimization of enzyme assisted extraction of Fructus Mori polysaccharides and its activities on antioxidant and alcohol dehydrogenase.

    PubMed

    Deng, Qingfang; Zhou, Xin; Chen, Huaguo

    2014-10-13

    In the present study, enzyme assisted extraction of Fructus Mori polysaccharides (FMPS) from F. mori using four kinds of enzymes and three compound enzymes were examined. Research found that glucose oxidase offered a better performance in enhancement of the extraction yields of FMPS, antioxidant and activate alcohol dehydrogenase activities. The glucose oxidase assisted extraction process was further optimized by using response surface method (RSM) to obtain maximum yield of crude FMPS. The results showed that optimized extraction conditions were ratio of enzyme amount 0.40%, enzyme treated time 38 min, treated temperature 58 °C and liquid-solid radio 11.0. Under these conditions, the mean experimental value of extraction yield (16.16 ± 0.14%) corresponded well with the predicted values and increased 160% than none enzyme treated ones. Pharmacological verification tests showed that F. mori crude polysaccharides had good antioxidant and activate alcohol dehydrogenase activities in vitro. PMID:25037415

  11. Strong Protective Effect of The Aldehyde Dehydrogenase Gene (ALDH2) 504lys (*2) Allele Against Alcoholism And Alcohol-Induced Medical Diseases in Asians

    PubMed Central

    Li, Dawei; Zhao, Hongyu; Gelernter, Joel

    2013-01-01

    Alcohol is oxidized to acetaldehyde, which in turn is oxidized to acetate. The aldehyde dehydrogenase 2 gene (ALDH2) is the most important gene responsible for acetaldehyde metabolism. Individuals heterozygous or homozygous for the lys (A or *2) allele at the single nucleotide polymorphism (SNP) glu504lys (rs671) of ALDH2 have greatly reduced ability to metabolize acetaldehyde, which greatly decreases their risk for alcohol dependence (AD). Case-control studies have shown association between this SNP and alcohol dependence as well as alcohol-induced liver disease. However, some studies have produced insignificant results. Using cumulative data from the past 20 years predominately from Asian populations (from both English and Chinese publications), this meta-analysis sought to examine and update whether the aggregate data provide new evidence of statistical significance for the proposed association. Our results (9,678 cases and 7,331 controls from 53 studies) support a strong association of alcohol abuse and dependence, with allelic P value of 3×10−56 and OR of 0.23 (0.2, 0.28) under the random effects model. The dominant model (lys-lys + lys-glu vs. glu-glu) also showed strong association with P value of 1×10−44 and OR of 0.22 (0.18, 0.27). When stricter criteria and various sub-group analyses were applied, the association remained strong (for example, OR = 0.23 (0.18, 0.3) and P = 2×10−28 for the alcoholic patients with alcoholic liver disease, cirrhosis, or pancreatitis). These findings provide confirmation of the involvement of the human ALDH2 gene in the pathogenesis of AD as well as alcohol-induced medical illnesses in East-Asians. PMID:22102315

  12. Crosstalk-eliminated quantitative determination of aflatoxin B1-induced hepatocellular cancer stem cells based on concurrent monitoring of CD133, CD44, and aldehyde dehydrogenase1.

    PubMed

    Ju, Hee; Shim, Yumi; Arumugam, Parthasarathy; Song, Joon Myong

    2016-01-22

    Cancer stem cells (CSCs), known as tumor initiating cells, have become a critically important issue for cancer therapy. Although much research has demonstrated the induction of hepato cellular carcinoma by aflatoxin B1, the formation of hepatocellular CSCs and their quantitative determination is hardly reported. In this work, it was found that hepatocellular CSCs were produced from HepG2 cells by aflatoxin B1-induced mutation, and their amount was quantitatively determined using crosstalk-eliminated multicolor cellular imaging based on quantum dot (Qdot) nanoprobes and an acousto-optical tunable filter (AOTF). Hepatocellular CSCs were acquired via magnetic bead-based sorting and observed using concurrent detection of three different markers: CD133, CD44, and aldehyde dehydrogenase1 (ALDH1). The DNA mutation of HepG2 cells caused by aflatoxin B1 was quantitatively observed via absorbance spectra of aflatoxin B1-8, 9-epoxide-DNA adducts. The percentages of hepatocellular CSCs formed in the entire HepG2 cells were determined to be 9.77±0.65%, 10.9±1.39%, 11.4±1.32%, and 12.8±0.7%, respectively, at 0 μM, 5 μM, 10 μM, and 20 μM of aflatoxin B1. The results matched well with those obtained utilizing flow cytometry. This study demonstrates that aflatoxin mediated mutation induced the conversion of hepatic cancer cell to hepatic CSCs by using a Qdot based constructed multicolor cellular imaging system. PMID:26739636

  13. Association between Carotid Intima-media Thickness and Aldehyde Dehydrogenase 2 Glu504Lys Polymorphism in Chinese Han with Essential Hypertension

    PubMed Central

    Ma, Xiao-Xiang; Zheng, Shu-Zhan; Shu, Yan; Wang, Yong; Chen, Xiao-Ping

    2016-01-01

    Background: Aldehyde dehydrogenase 2 (ALDH2) is involved in the pathophysiological processes of cardiovascular diseases. Recent studies showed that mutant ALDH2 could increase oxidative stress and is a susceptible factor for hypertension. In addition, wild-type ALDH2 could improve the endothelial functions, therefore reducing the risk of developing atherosclerosis. The aim of the present study was to explore the frequency of the Glu504Lys polymorphism of the ALDH2 gene and its relation to carotid intima-media thickness (CIMT) in a group of patients with essential hypertension (EH) and to investigate the association between the Glu504Lys polymorphism and CIMT in Chinese Han patients with EH. Methods: In this study, 410 Chinese Han patients with EH who received physical examinations at the People's Hospital of Sichuan Province (China) were selected. DNA microarray chip was used for the genotyping of the Glu504Lys polymorphism of the ALDH2 gene. The differences in CIMT among patients with different Glu504Lys ALDH2 genotypes were analyzed. Results: The mean CIMT of the patients carrying AA/AG and GG genotypes was 1.02 ± 0.31 mm and 0.78 ± 0.28 mm, respectively. One-way ANOVA showed that the CIMT of the patients carrying the AA/AG genotype was significantly higher than in the ones carrying the GG genotype (P < 0.001). Multivariate logistic regression showed that the Glu504Lys AA/AG genotype of the ALDH2 gene was one of the major factors influencing the CIMT in patients with EH (odds ratio = 3.731, 95% confidence interval = 1.589–8.124, P = 0.001). Conclusions: The Glu504Lys polymorphism of the ALDH2 gene is associated with the CIMT of Chinese Han patients with EH in Sichuan, China. PMID:27270535

  14. Aldehyde dehydrogenase inhibitors: alpha,beta-acetylenic N-substituted aminothiolesters are reversible growth inhibitors of normal epithelial but irreversible apoptogens for cancer epithelial cells from human prostate in culture.

    PubMed

    Quash, Gerard; Fournet, Guy; Courvoisier, Charlotte; Martinez, Rosa M; Chantepie, Jacqueline; Paret, Marie Julie; Pharaboz, Julie; Joly-Pharaboz, Marie Odile; Goré, Jacques; André, Jean; Reichert, Uwe

    2008-05-01

    The pharmacomodulation of the N atom of alpha,beta-acetylenic aminothiolesters or the replacement of the thiolester moiety by more electrophilic groups did not permit any clear rationale to be established for improving the selective growth-inhibitory activity of this family of compounds over that of the previously synthesized alpha,beta-acetylenic aminothiolesters DIMATE and MATE [G. Quash, G. Fournet, J. Chantepie, J. Goré, C. Ardiet, D. Ardail, Y. Michal, U. Reichert, Biochem Pharmacol 64 (2002) 1279-92]. Hence DIMATE and MATE were investigated more thoroughly for selectivity and growth-inhibitory activity using human prostate epithelial normal cells (HPENC) on the one hand and human prostate epithelial cancer cells (DU145) on the other. Unequivocal evidence was obtained showing that both compounds were reversible growth inhibitors of HPENC but irreversible growth inhibitors of DU145. Growth-inhibition of DU145 was due to the induction of early apoptosis as revealed by the flow cytometric analytical profile of inhibitor-treated cells, of the decrease in the redox potential and increase in superoxide anion content of their mitochondria. Of the two intracellular enzymes: aldehyde dehydrogenases 1 and 3 (ALDH1 and ALDH3) targeted by DIMATE and MATE, ALDH3 was inhibited to the same extent by both compounds whereas ALDH1 was less susceptible to inhibition by MATE. As the induction of ALDH3 by xenobiotics is hormone-dependent, MATE, the more selective of the two inhibitors, is a useful tool not only for examining the role of the ALDH3 isoform in hormone-sensitive and resistant prostate cancer cells in culture but also for investigating if it can inhibit the growth of xenografts of prostate cancer in immunodeficient mice. PMID:17692435

  15. An enzyme-amplified microtiter plate assay for ethanol: Its application to the detection of peanut ethanol and alcohol dehydrogenase

    SciTech Connect

    Chung, S.Y.; Vercellotti, J.R.; Sanders, T.H.

    1995-12-01

    A calorimetric microliter plate assay for ethanol amplified by aldehyde dehydrogenase (ALDH) was developed. In the assay ethanol from a sample took part in a chain-reaction catalyzed by alcohol dehydrogenase (ADH) and amplified by ALDH in the presence of NAD{sup +}, diaphorase, and p-ibdonitrotetrazolium-violet (INT-violet)(a precursor of red product). The resultant reaction gave a red color, the intensity of which was proportional to the amount of ethanol present. Using the technique, the content of activity from peanuts of differing maturity and curing stages were determined respectively. Data showed that immature peanuts had a higher level of ethanol and a lower ADH activity than mature peanuts, and that the level of ethanol and ADH activity decreased with the curing time. This indicates that peanut maturity and curing have an effect on ethanol. Also, this implies that other peanut volatiles could be affected in the same way as ethanol, a major volatile in peanuts.

  16. Structure of glycerol-3-phosphate dehydrogenase, an essential monotopic membrane enzyme involved in respiration and metabolism

    SciTech Connect

    Yeh, Joanne I.; Chinte, Unmesh; Du, Shoucheng

    2008-04-02

    Sn-glycerol-3-phosphate dehydrogenase (GlpD) is an essential membrane enzyme, functioning at the central junction of respiration, glycolysis, and phospholipid biosynthesis. Its critical role is indicated by the multitiered regulatory mechanisms that stringently controls its expression and function. Once expressed, GlpD activity is regulated through lipid-enzyme interactions in Escherichia coli. Here, we report seven previously undescribed structures of the fully active E. coli GlpD, up to 1.75 {angstrom} resolution. In addition to elucidating the structure of the native enzyme, we have determined the structures of GlpD complexed with substrate analogues phosphoenolpyruvate, glyceric acid 2-phosphate, glyceraldehyde-3-phosphate, and product, dihydroxyacetone phosphate. These structural results reveal conformational states of the enzyme, delineating the residues involved in substrate binding and catalysis at the glycerol-3-phosphate site. Two probable mechanisms for catalyzing the dehydrogenation of glycerol-3-phosphate are envisioned, based on the conformational states of the complexes. To further correlate catalytic dehydrogenation to respiration, we have additionally determined the structures of GlpD bound with ubiquinone analogues menadione and 2-n-heptyl-4-hydroxyquinoline N-oxide, identifying a hydrophobic plateau that is likely the ubiquinone-binding site. These structures illuminate probable mechanisms of catalysis and suggest how GlpD shuttles electrons into the respiratory pathway. Glycerol metabolism has been implicated in insulin signaling and perturbations in glycerol uptake and catabolism are linked to obesity in humans. Homologs of GlpD are found in practically all organisms, from prokaryotes to humans, with >45% consensus protein sequences, signifying that these structural results on the prokaryotic enzyme may be readily applied to the eukaryotic GlpD enzymes.

  17. Structure of glycerol-3-phosphate dehydrogenase, an essential monotopic membrane enzyme involved in respiration and metabolism

    PubMed Central

    Yeh, Joanne I.; Chinte, Unmesh; Du, Shoucheng

    2008-01-01

    Sn-glycerol-3-phosphate dehydrogenase (GlpD) is an essential membrane enzyme, functioning at the central junction of respiration, glycolysis, and phospholipid biosynthesis. Its critical role is indicated by the multitiered regulatory mechanisms that stringently controls its expression and function. Once expressed, GlpD activity is regulated through lipid-enzyme interactions in Escherichia coli. Here, we report seven previously undescribed structures of the fully active E. coli GlpD, up to 1.75 Å resolution. In addition to elucidating the structure of the native enzyme, we have determined the structures of GlpD complexed with substrate analogues phosphoenolpyruvate, glyceric acid 2-phosphate, glyceraldehyde-3-phosphate, and product, dihydroxyacetone phosphate. These structural results reveal conformational states of the enzyme, delineating the residues involved in substrate binding and catalysis at the glycerol-3-phosphate site. Two probable mechanisms for catalyzing the dehydrogenation of glycerol-3-phosphate are envisioned, based on the conformational states of the complexes. To further correlate catalytic dehydrogenation to respiration, we have additionally determined the structures of GlpD bound with ubiquinone analogues menadione and 2-n-heptyl-4-hydroxyquinoline N-oxide, identifying a hydrophobic plateau that is likely the ubiquinone-binding site. These structures illuminate probable mechanisms of catalysis and suggest how GlpD shuttles electrons into the respiratory pathway. Glycerol metabolism has been implicated in insulin signaling and perturbations in glycerol uptake and catabolism are linked to obesity in humans. Homologs of GlpD are found in practically all organisms, from prokaryotes to humans, with >45% consensus protein sequences, signifying that these structural results on the prokaryotic enzyme may be readily applied to the eukaryotic GlpD enzymes. PMID:18296637

  18. Radiochemical detection of dihydrodiol dehydrogenase: distribution of the indomethacin sensitive enzyme in rat tissues

    SciTech Connect

    Ivins, J.; Penning, T.

    1986-05-01

    Dihydrodiol dehydrogenase catalyzes the NADP/sup +/ dependent oxidation of trans-dihydrodiols of polycyclic aromatic hydrocarbons (PAH) which are potent proximate carcinogens. The authors have developed a highly sensitive radiochemical assay for this enzyme in which the oxidation of trans-1,2-dihydroxy-3,5-cyclohexadiene, a model substrate for trans-dihydrodiol proximate carcinogens, is coupled to O-methylation catalyzed by catechol O-methyl transferase. Using S-adenosyl-(/sup 3/H-methyl)-methionine as methyl donor at a specific activity of 0.1 nCi/pmol and extracting the product, /sup 3/H-o-methoxyphenol, the assay provides a 5000 fold increase in sensitivity over the existing spectrophotometric method. The radiochemical assay was validated by comparing the K/sub m/ and V/sub max/ values for rat liver cytosol with those derived spectrophotometrically. In both instances there was close agreement between values (K/sub m/ = 0.77 +/- 0.11 mM and V/sub max/ = 2.14 +/- 0.13 nmoles/min/mg protein determined radiochemically; K/sub m/ = 0.96 +/- 0.10 mM and V/sub max/ = 6.31 +/- 0.50 nmoles/min/mg protein determined spectrophotometrically). Using the radiochemical method, dihydrodiol dehydrogenase activity was detected in the following rat tissues: liver > lung > heart > small intestine > testis > seminal vesicle > bladder > prostate > spleen. Specific activities ranged between 0.944 and 0.016 nmoles/min/mg protein. In liver, lung, and testis, which are sites of PAH metabolism, the dehydrogenase is sensitive to inhibition by low ..mu..M concentrations of indomethacin, suggesting that this drug can prevent the detoxification of proximate carcinogens by this route.

  19. NADP+-dependent farnesol dehydrogenase, a corpora allata enzyme involved in juvenile hormone synthesis

    PubMed Central

    Mayoral, Jaime G.; Nouzova, Marcela; Navare, Arti; Noriega, Fernando G.

    2009-01-01

    The synthesis of juvenile hormone (JH) is an attractive target for control of insect pests and vectors of disease, but the minute size of the corpora allata (CA), the glands that synthesize JH, has made it difficult to identify important biosynthetic enzymes by classical biochemical approaches. Here, we report identification and characterization of an insect farnesol dehydrogenase (AaSDR-1) that oxidizes farnesol into farnesal, a precursor of JH, in the CA. AaSDR-1 was isolated as an EST in a library of the corpora allata-corpora cardiaca of the mosquito Aedes aegypti. The 245-amino acid protein presents the typical short-chain dehydrogenase (SDR) Rossmann-fold motif for nucleotide binding. This feature, together with other conserved sequence motifs, place AaSDR-1 into the “classical” NADP+-dependent cP2 SDR subfamily. The gene is part of a group of highly conserved paralogs that cluster together in the mosquito genome; similar clusters of orthologs were found in other insect species. AaSDR-1 acts as a homodimer and efficiently oxidizes C10 to C15 isoprenoid and aliphatic alcohols, showing the highest affinity for the conversion of farnesol into farnesal. Farnesol dehydrogenase activity was not detected in the CA of newly emerged mosquitoes but significant activity was detected 24 h later. Real time PCR experiments revealed that AaSDR-1 mRNA levels were very low in the inactive CA of the newly emerged female, but increased >30-fold 24 h later during the peak of JH synthesis. These results suggest that oxidation of farnesol might be a rate-limiting step in JH III synthesis in adult mosquitoes. PMID:19940247

  20. Methanoarchaeal sulfolactate dehydrogenase: prototype of a new family of NADH-dependent enzymes

    PubMed Central

    Irimia, Adriana; Madern, Dominique; Zaccaï, Giuseppe; Vellieux, Frédéric MD

    2004-01-01

    The crystal structure of the sulfolactate dehydrogenase from the hyperthermophilic and methanogenic archaeon Methanocaldococcus jannaschii was solved at 2.5 Å resolution (PDB id. 1RFM). The asymmetric unit contains a tetramer of tight dimers. This structure, complexed with NADH, does not contain a cofactor-binding domain with ‘Rossmann-fold' topology. Instead, the tertiary and quaternary structures indicate a novel fold. The NADH is bound in an extended conformation in each active site, in a manner that explains the pro-S specificity. Cofactor binding involves residues belonging to both subunits within the tight dimers, which are therefore the smallest enzymatically active units. The protein was found to be a homodimer in solution by size-exclusion chromatography, analytical ultracentrifugation and small-angle neutron scattering. Various compounds were tested as putative substrates. The results indicate the existence of a substrate discrimination mechanism, which involves electrostatic interactions. Based on sequence homology and phylogenetic analyses, several other enzymes were classified as belonging to this novel family of homologous (S)-2-hydroxyacid dehydrogenases. PMID:15014443

  1. Polyol specificity of recombinant Arabidopsis thaliana sorbitol dehydrogenase studied by enzyme kinetics and in silico modeling

    PubMed Central

    Aguayo, M. Francisca; Cáceres, Juan Carlos; Fuentealba, Matías; Muñoz, Rodrigo; Stange, Claudia; Cabrera, Ricardo; Handford, Michael

    2015-01-01

    Polyols are enzymatically-produced plant compounds which can act as compatible solutes during periods of abiotic stress. Nicotinamide adenine dinucleotide+-dependent SORBITOL DEHYDROGENASE (SDH, E. C. 1.1.1.14) from Arabidopsis thaliana L. sorbitol dehydrogenase (AtSDH) is capable of oxidizing several polyols including sorbitol, ribitol, and xylitol. In the present study, enzymatic assays using recombinant AtSDH demonstrated a higher specificity constant for xylitol compared to sorbitol and ribitol, all of which are C2 (S) and C4 (R) polyols. Enzyme activity was reduced by preincubation with ethylenediaminetetraacetic acid, indicating a requirement for zinc ions. In humans, it has been proposed that sorbitol becomes part of a pentahedric coordination sphere of the catalytic zinc during the reaction mechanism. In order to determine the validity of this pentahedric coordination model in a plant SDH, homology modeling, and Molecular Dynamics simulations of AtSDH ternary complexes with the three polyols were performed using crystal structures of human and Bemisia argentifolii (Genn.) (Hemiptera: Aleyrodidae) SDHs as scaffolds. The results indicate that the differences in interaction with structural water molecules correlate very well with the observed enzymatic parameters, validate the proposed pentahedric coordination of the catalytic zinc ion in a plant SDH, and provide an explanation for why AtSDH shows a preference for polyols with a chirality of C2 (S) and C4 (R). PMID:25755662

  2. Multiple Independent Fusions of Glucose-6-Phosphate Dehydrogenase with Enzymes in the Pentose Phosphate Pathway

    PubMed Central

    Stover, Nicholas A.; Dixon, Thomas A.; Cavalcanti, Andre R. O.

    2011-01-01

    Fusions of the first two enzymes in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconolactonase (6PGL), have been previously described in two distant clades, chordates and species of the malarial parasite Plasmodium. We have analyzed genome and expressed sequence data from a variety of organisms to identify the origins of these gene fusion events. Based on the orientation of the domains and range of species in which homologs can be found, the fusions appear to have occurred independently, near the base of the metazoan and apicomplexan lineages. Only one of the two metazoan paralogs of G6PD is fused, showing that the fusion occurred after a duplication event, which we have traced back to an ancestor of choanoflagellates and metazoans. The Plasmodium genes are known to contain a functionally important insertion that is not seen in the other apicomplexan fusions, highlighting this as a unique characteristic of this group. Surprisingly, our search revealed two additional fusion events, one that combined 6PGL and G6PD in an ancestor of the protozoan parasites Trichomonas and Giardia, and another fusing G6PD with phosphogluconate dehydrogenase (6PGD) in a species of diatoms. This study extends the range of species known to contain fusions in the pentose phosphate pathway to many new medically and economically important organisms. PMID:21829610

  3. Biochemical characterization of isocitrate dehydrogenase from Methylococcus capsulatus reveals a unique NAD+-dependent homotetrameric enzyme.

    PubMed

    Stokke, Runar; Madern, Dominique; Fedøy, Anita-Elin; Karlsen, Solveig; Birkeland, Nils-Kåre; Steen, Ida Helene

    2007-05-01

    The gene encoding isocitrate dehydrogenase (IDH) of Methylococcus capsulatus (McIDH) was cloned and overexpressed in Escherichia coli. The purified enzyme was NAD+-dependent with a thermal optimum for activity at 55-60 degrees C and an apparent midpoint melting temperature (Tm) of 70 degrees C. Analytical ultracentrifugation (AUC) revealed a homotetrameric state, and McIDH thus represents the first homotetrameric NAD+-dependent IDH that has been characterized. Based on a structural alignment of McIDH and homotetrameric homoisocitrate dehydrogenase (HDH) from Thermus thermophilus (TtHDH), we identified the clasp-like domain of McIDH as a likely site for tetramerization. McIDH showed moreover, higher sequence identity (48%) to TtHDH than to previously characterized IDHs. Putative NAD+-IDHs with high sequence identity (48-57%) to McIDH were however identified in a variety of bacteria showing that NAD+-dependent IDHs are indeed widespread within the domain, Bacteria. Phylogenetic analysis including these new sequences revealed a close relationship with eukaryal allosterically regulated NAD+-IDH and the subfamily III of IDH was redefined to include bacterial NAD+- and NADP+-dependent IDHs. This apparent relationship suggests that the mitochondrial genes encoding NAD+-IDH are derived from the McIDH-like IDHs. PMID:17160675

  4. Effect of selective thiol-group derivatization on enzyme kinetics of (R)-3-hydroxybutyrate dehydrogenase.

    PubMed Central

    Dalton, L A; McIntyre, J O; Fleischer, S

    1993-01-01

    (R)-3-Hydroxybutyrate dehydrogenase (BDH) is a phosphatidylcholine-requiring tetrameric enzyme with two thiol groups (SH-1 and SH-2) per protomer. By first protecting the more rapidly reacting thiol group (SH-1) with diamide [1,1'-azobis-(NN'-dimethylformamide), DM] to form DM(SH-1)BDH, SH-2 can be selectively derivatized by reaction with maleimide reagents such as 4-maleimido-2,2,6,6-tetramethyl-piperidine-N-oxyl (MSL), which gives DM(SH-1)MSL(SH-2)BDH. Reduction with dithiothreitol (DTT) regenerates SH-1, yielding MAL(SH-2)BDH (where MAL is the diamagnetic reduction product of MSL-BDH and DTT). The enzymic activity of DM(SH-1)BDH is decreased to approx. 4% relative to the native purified enzyme, and the apparent Km for substrate, KmBOH, is increased approx. 100-fold. Reduction of DM(SH-1)BDH with DTT regenerates SH-1 and restores normal enzymic function. Modification of SH-2 with piperidinylmaleimide [MAL(SH-2)BDH] diminishes enzymic activity to approx. 35% of its original value, but has no significant effect on apparent KmBOH. The doubly derivatized enzyme, DM(SH-1)MSL(SH-2)BDH, has lower enzymic activity [about half that for DM(SH-2)BDH] and a yet higher apparent KmBOH than DM(SH-1)BDH. Derivatization of SH-2 with different maleimide reagents results in diminished activity approximately proportional to the size of the maleimide substituent, suggesting that this inhibition is steric. Whereas modification of SH-1 results in marked changes in kinetic parameters (increased apparent Km and reduced apparent Vmax), derivatization of SH-2 has a lesser effect on enzymic function. Thus SH-1 is postulated to be closer to the active centre than is SH-2, although neither is involved in catalysis, since: (1) the activity of the derivatized enzyme is not abolished; and (2) activity can be enhanced by increasing substrate (and cofactor) concentrations. PMID:8280053

  5. Suitability of the hydrocarbon-hydroxylating molybdenum-enzyme ethylbenzene dehydrogenase for industrial chiral alcohol production.

    PubMed

    Tataruch, M; Heider, J; Bryjak, J; Nowak, P; Knack, D; Czerniak, A; Liesiene, J; Szaleniec, M

    2014-12-20

    The molybdenum/iron-sulfur/heme protein ethylbenzene dehydrogenase (EbDH) was successfully applied to catalyze enantiospecific hydroxylation of alkylaromatic and alkylheterocyclic compounds. The optimization of the synthetic procedure involves use of the enzyme in a crude purification state that saves significant preparation effort and is more stable than purified EbDH without exhibiting unwanted side reactions. Moreover, immobilization of the enzyme on a crystalline cellulose support and changes in reaction conditions were introduced in order to increase the amounts of product formed (anaerobic atmosphere, electrochemical electron acceptor recycling or utilization of ferricyanide as alternative electron acceptor in high concentrations). We report here on an extension of effective enzyme activity from 4h to more than 10 days and final product yields of up to 0.4-0.5g/l, which represent a decent starting point for further optimization. Therefore, we expect that the hydrocarbon-hydroxylation capabilities of EbDH may be developed into a new process of industrial production of chiral alcohols. PMID:24998764

  6. A new cofactor in prokaryotic enzyme: Tryptophan tryptophylquinone as the redox prosthetic group in methylamine dehydrogenase

    SciTech Connect

    McIntire, W.S. Univ. of California, San Francisco ); Wemmer, D.E. ); Chistoserdov, A.; Lidstrom, M.E. )

    1991-05-10

    Methylamine dehydrogenase (MADH), an {alpha}{sub 2}{beta}{sub 2} enzyme from numerous methylotrophic soil bacteria, contains a novel quinonoid redox prosthetic group that is covalently bound to its small {beta} subunit through two amino acyl residues. A comparison of the amino acid sequence deduced from the gene sequence of the small subunit for the enzyme from Methylobacterium extorquens AM1 with the published amino acid sequence obtained by Edman degradation method, allowed the identification of the amino acyl constituents of the cofactor as two tryptophyl residues. This information was crucial for interpreting {sup 1}H and {sup 13}C nuclear magnetic resonance, and mass spectral data collected for the semicarbazide- and carboxymethyl-derivatized bis(tripeptidyl)-cofactor of MADH from bacterium W3A1. The cofactor is composed of two cross-linked tryptophyl residues. Although there are many possible isomers, only one is consistent with all the data: The first tryptophyl residue in the peptide sequence exists as an indole-6,7-dione, and is attached at its 4 position to the 2 position of the second, otherwise unmodified, indole side group. Contrary to earlier reports, the cofactor of MADH is not 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), a derivative thereof, of pro-PQQ. This appears to be the only example of two cross-linked, modified amino acyl residues having a functional role in the active site of an enzyme, in the absence of other cofactors or metal ions.

  7. Structural and Kinetic Evidence That Catalytic Reaction of Human UDP-glucose 6-Dehydrogenase Involves Covalent Thiohemiacetal and Thioester Enzyme Intermediates*

    PubMed Central

    Egger, Sigrid; Chaikuad, Apirat; Klimacek, Mario; Kavanagh, Kathryn L.; Oppermann, Udo; Nidetzky, Bernd

    2012-01-01

    Biosynthesis of UDP-glucuronic acid by UDP-glucose 6-dehydrogenase (UGDH) occurs through the four-electron oxidation of the UDP-glucose C6 primary alcohol in two NAD+-dependent steps. The catalytic reaction of UGDH is thought to involve a Cys nucleophile that promotes formation of a thiohemiacetal enzyme intermediate in the course of the first oxidation step. The thiohemiacetal undergoes further oxidation into a thioester, and hydrolysis of the thioester completes the catalytic cycle. Herein we present crystallographic and kinetic evidence for the human form of UGDH that clarifies participation of covalent catalysis in the enzymatic mechanism. Substitution of the putative catalytic base for water attack on the thioester (Glu161) by an incompetent analog (Gln161) gave a UGDH variant (E161Q) in which the hydrolysis step had become completely rate-limiting so that a thioester enzyme intermediate accumulated at steady state. By crystallizing E161Q in the presence of 5 mm UDP-glucose and 2 mm NAD+, we succeeded in trapping a thiohemiacetal enzyme intermediate and determined its structure at 2.3 Å resolution. Cys276 was covalently modified in the structure, establishing its role as catalytic nucleophile of the reaction. The thiohemiacetal reactive C6 was in a position suitable to become further oxidized by hydride transfer to NAD+. The proposed catalytic mechanism of human UGDH involves Lys220 as general base for UDP-glucose alcohol oxidation and for oxyanion stabilization during formation and breakdown of the thiohemiacetal and thioester enzyme intermediates. Water coordinated to Asp280 deprotonates Cys276 to function as an aldehyde trap and also provides oxyanion stabilization. Glu161 is the Brønsted base catalytically promoting the thioester hydrolysis. PMID:22123821

  8. Inhibition of Human Aldehyde Oxidase Activity by Diet-Derived Constituents: Structural Influence, Enzyme-Ligand Interactions, and Clinical Relevance

    PubMed Central

    Barr, John T.; Jones, Jeffrey P.; Oberlies, Nicholas H.

    2015-01-01

    The mechanistic understanding of interactions between diet-derived substances and conventional medications in humans is nascent. Most investigations have examined cytochrome P450–mediated interactions. Interactions mediated by other phase I enzymes are understudied. Aldehyde oxidase (AO) is a phase I hydroxylase that is gaining recognition in drug design and development programs. Taken together, a panel of structurally diverse phytoconstituents (n = 24) was screened for inhibitors of the AO-mediated oxidation of the probe substrate O6-benzylguanine. Based on the estimated IC50 (<100 μM), 17 constituents were advanced for Ki determination. Three constituents were described best by a competitive inhibition model, whereas 14 constituents were described best by a mixed-mode model. The latter model consists of two Ki terms, Kis and Kii, which ranged from 0.26–73 and 0.80–120 μM, respectively. Molecular modeling was used to glean mechanistic insight into AO inhibition. Docking studies indicated that the tested constituents bound within the AO active site and elucidated key enzyme-inhibitor interactions. Quantitative structure-activity relationship modeling identified three structural descriptors that correlated with inhibition potency (r2 = 0.85), providing a framework for developing in silico models to predict the AO inhibitory activity of a xenobiotic based solely on chemical structure. Finally, a simple static model was used to assess potential clinically relevant AO-mediated dietary substance–drug interactions. Epicatechin gallate and epigallocatechin gallate, prominent constituents in green tea, were predicted to have moderate to high risk. Further characterization of this uncharted type of interaction is warranted, including dynamic modeling and, potentially, clinical evaluation. PMID:25326286

  9. Inhibition of human aldehyde oxidase activity by diet-derived constituents: structural influence, enzyme-ligand interactions, and clinical relevance.

    PubMed

    Barr, John T; Jones, Jeffrey P; Oberlies, Nicholas H; Paine, Mary F

    2015-01-01

    The mechanistic understanding of interactions between diet-derived substances and conventional medications in humans is nascent. Most investigations have examined cytochrome P450-mediated interactions. Interactions mediated by other phase I enzymes are understudied. Aldehyde oxidase (AO) is a phase I hydroxylase that is gaining recognition in drug design and development programs. Taken together, a panel of structurally diverse phytoconstituents (n = 24) was screened for inhibitors of the AO-mediated oxidation of the probe substrate O(6)-benzylguanine. Based on the estimated IC50 (<100 μM), 17 constituents were advanced for Ki determination. Three constituents were described best by a competitive inhibition model, whereas 14 constituents were described best by a mixed-mode model. The latter model consists of two Ki terms, Kis and Kii, which ranged from 0.26-73 and 0.80-120 μM, respectively. Molecular modeling was used to glean mechanistic insight into AO inhibition. Docking studies indicated that the tested constituents bound within the AO active site and elucidated key enzyme-inhibitor interactions. Quantitative structure-activity relationship modeling identified three structural descriptors that correlated with inhibition potency (r(2) = 0.85), providing a framework for developing in silico models to predict the AO inhibitory activity of a xenobiotic based solely on chemical structure. Finally, a simple static model was used to assess potential clinically relevant AO-mediated dietary substance-drug interactions. Epicatechin gallate and epigallocatechin gallate, prominent constituents in green tea, were predicted to have moderate to high risk. Further characterization of this uncharted type of interaction is warranted, including dynamic modeling and, potentially, clinical evaluation. PMID:25326286

  10. Microbial Engineering for Aldehyde Synthesis

    PubMed Central

    Kunjapur, Aditya M.

    2015-01-01

    Aldehydes are a class of chemicals with many industrial uses. Several aldehydes are responsible for flavors and fragrances present in plants, but aldehydes are not known to accumulate in most natural microorganisms. In many cases, microbial production of aldehydes presents an attractive alternative to extraction from plants or chemical synthesis. During the past 2 decades, a variety of aldehyde biosynthetic enzymes have undergone detailed characterization. Although metabolic pathways that result in alcohol synthesis via aldehyde intermediates were long known, only recent investigations in model microbes such as Escherichia coli have succeeded in minimizing the rapid endogenous conversion of aldehydes into their corresponding alcohols. Such efforts have provided a foundation for microbial aldehyde synthesis and broader utilization of aldehydes as intermediates for other synthetically challenging biochemical classes. However, aldehyde toxicity imposes a practical limit on achievable aldehyde titers and remains an issue of academic and commercial interest. In this minireview, we summarize published efforts of microbial engineering for aldehyde synthesis, with an emphasis on de novo synthesis, engineered aldehyde accumulation in E. coli, and the challenge of aldehyde toxicity. PMID:25576610

  11. Biosynthesis of LL-Z1272β: Discovery of a New Member of NRPS-like Enzymes for Aryl-Aldehyde Formation.

    PubMed

    Li, Chang; Matsuda, Yudai; Gao, Hao; Hu, Dan; Yao, Xin Sheng; Abe, Ikuro

    2016-05-17

    LL-Z1272β (1) is a prenylated aryl-aldehyde produced by several fungi; it also serves as a key pathway intermediate for many fungal meroterpenoids. Despite its importance in the biosynthesis of natural products, the molecular basis for the biosynthesis of 1 has yet to be elucidated. Here we identified the biosynthetic gene cluster for 1 from Stachybotrys bisbyi PYH05-7, and elucidated the biosynthetic route to 1. The biosynthesis involves a polyketide synthase, a prenyltransferase, and a nonribosomal peptide synthetase (NRPS)-like enzyme, which is responsible for the generation of the aldehyde functionality. Interestingly, the NRPS-like enzyme only accepts the farnesylated substrate to catalyze the carboxylate reduction; this represents a new example of a substrate for adenylation domains. PMID:26972702

  12. Promiscuous catalysis of asymmetric Michael-type additions of linear aldehydes to β-nitrostyrene by the proline-based enzyme 4-oxalocrotonate tautomerase.

    PubMed

    Miao, Yufeng; Geertsema, Edzard M; Tepper, Pieter G; Zandvoort, Ellen; Poelarends, Gerrit J

    2013-01-21

    Exploiting catalytic promiscuity: The proline-based enzyme 4-oxalocrotonate tautomerase (4-OT) promiscuously catalyzes asymmetric Michael-type additions of linear aldehydes--ranging from acetaldehyde to octanal--to trans-β-nitrostyrene in aqueous solvent. The presence of 1.4 mol% of 4-OT effected formation of the anticipated γ-nitroaldehydes in fair to good yields with dr values of up to 93:7 and ee values of up to 81 %. PMID:23303727

  13. Glutamate dehydrogenase in brain mitochondria: do lipid modifications and transient metabolon formation influence enzyme activity?

    PubMed Central

    McKenna, Mary C.

    2011-01-01

    Metabolism of glutamate, the primary excitatory neurotransmitter in brain, is complex and of paramount importance to overall brain function. Thus, understanding the regulation of enzymes involved in formation and disposal of glutamate and related metabolites is crucial to understanding glutamate metabolism. Glutamate dehydrogenase (GDH) is a pivotal enzyme that links amino acid metabolism and TCA cycle activity in brain and other tissues. The allosteric regulation of GDH has been extensively studied and characterized. Less is known about the influence of lipid modifications on GDH activity, and the participation of GDH in transient heteroenzyme complexes (metabolons) that can greatly influence metabolism by altering kinetic parameters and lead to channeling of metabolites. This review summarizes evidence for palmitoylation and acylation of GDH, information on protein binding, and information regarding the participation of GDH in transient heteroenzyme complexes. Recent studies suggest that a number of other proteins can bind to GDH altering activity and overall metabolism. It is likely that these modifications and interactions contribute additional levels of regulation of GDH activity and glutamate metabolism. PMID:21771624

  14. Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase

    PubMed Central

    Borgnia, Mario J.; Banerjee, Soojay; Merk, Alan; Matthies, Doreen; Bartesaghi, Alberto; Rao, Prashant; Pierson, Jason; Earl, Lesley A.; Falconieri, Veronica

    2016-01-01

    Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an “open” or “closed” state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes. PMID:27036132

  15. Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase.

    PubMed

    Borgnia, Mario J; Banerjee, Soojay; Merk, Alan; Matthies, Doreen; Bartesaghi, Alberto; Rao, Prashant; Pierson, Jason; Earl, Lesley A; Falconieri, Veronica; Subramaniam, Sriram; Milne, Jacqueline L S

    2016-06-01

    Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an "open" or "closed" state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes. PMID:27036132

  16. Structural characterization of tartrate dehydrogenase: a versatile enzyme catalyzing multiple reactions

    PubMed Central

    Malik, Radhika; Viola, Ronald E.

    2010-01-01

    The first structure of an NAD-dependent tartrate dehydrogenase (TDH) has been solved to 2 Å resolution by single anomalous diffraction (SAD) phasing as a complex with the intermediate analog oxalate, Mg2+ and NADH. This TDH structure from Pseudomonas putida has a similar overall fold and domain organization to other structurally characterized members of the hydroxy-acid dehydrogenase family. How­ever, there are considerable differences between TDH and these functionally related enzymes in the regions connecting the core secondary structure and in the relative positioning of important loops and helices. The active site in these complexes is highly ordered, allowing the identification of the substrate-binding and cofactor-binding groups and the ligands to the metal ions. Residues from the adjacent subunit are involved in both the substrate and divalent metal ion binding sites, establishing a dimer as the functional unit and providing structural support for an alternating-site reaction mechanism. The divalent metal ion plays a prominent role in substrate binding and orientation, together with several active-site arginines. Functional groups from both subunits form the cofactor-binding site and the ammonium ion aids in the orientation of the nicotinamide ring of the cofactor. A lysyl amino group (Lys192) is the base responsible for the water-mediated proton abstraction from the C2 hydroxyl group of the substrate that begins the catalytic reaction, followed by hydride transfer to NAD. A tyrosyl hydroxyl group (Tyr141) functions as a general acid to protonate the enolate inter­mediate. Each substrate undergoes the initial hydride transfer, but differences in substrate orientation are proposed to account for the different reactions catalyzed by TDH. PMID:20516620

  17. Structural characterization of tartrate dehydrogenase: a versatile enzyme catalyzing multiple reactions

    SciTech Connect

    Malik, Radhika; Viola, Ronald E.

    2010-10-28

    The first structure of an NAD-dependent tartrate dehydrogenase (TDH) has been solved to 2 {angstrom} resolution by single anomalous diffraction (SAD) phasing as a complex with the intermediate analog oxalate, Mg{sup 2+} and NADH. This TDH structure from Pseudomonas putida has a similar overall fold and domain organization to other structurally characterized members of the hydroxy-acid dehydrogenase family. However, there are considerable differences between TDH and these functionally related enzymes in the regions connecting the core secondary structure and in the relative positioning of important loops and helices. The active site in these complexes is highly ordered, allowing the identification of the substrate-binding and cofactor-binding groups and the ligands to the metal ions. Residues from the adjacent subunit are involved in both the substrate and divalent metal ion binding sites, establishing a dimer as the functional unit and providing structural support for an alternating-site reaction mechanism. The divalent metal ion plays a prominent role in substrate binding and orientation, together with several active-site arginines. Functional groups from both subunits form the cofactor-binding site and the ammonium ion aids in the orientation of the nicotinamide ring of the cofactor. A lysyl amino group (Lys192) is the base responsible for the water-mediated proton abstraction from the C2 hydroxyl group of the substrate that begins the catalytic reaction, followed by hydride transfer to NAD. A tyrosyl hydroxyl group (Tyr141) functions as a general acid to protonate the enolate intermediate. Each substrate undergoes the initial hydride transfer, but differences in substrate orientation are proposed to account for the different reactions catalyzed by TDH.

  18. Response surface methodology to optimize partition and purification of two recombinant oxidoreductase enzymes, glucose dehydrogenase and d-galactose dehydrogenase in aqueous two-phase systems.

    PubMed

    Shahbaz Mohammadi, Hamid; Mostafavi, Seyede Samaneh; Soleimani, Saeideh; Bozorgian, Sajad; Pooraskari, Maryam; Kianmehr, Anvarsadat

    2015-04-01

    Oxidoreductases are an important family of enzymes that are used in many biotechnological processes. An experimental design was applied to optimize partition and purification of two recombinant oxidoreductases, glucose dehydrogenase (GDH) from Bacillus subtilis and d-galactose dehydrogenase (GalDH) from Pseudomonas fluorescens AK92 in aqueous two-phase systems (ATPS). Response surface methodology (RSM) with a central composite rotatable design (CCRD) was performed to optimize critical factors like polyethylene glycol (PEG) concentration, concentration of salt and pH value. The best partitioning conditions was achieved in an ATPS composed of 12% PEG-6000, 15% K2HPO4 with pH 7.5 at 25°C, which ensured partition coefficient (KE) of 66.6 and 45.7 for GDH and GalDH, respectively. Under these experimental conditions, the activity of GDH and GalDH was 569.5U/ml and 673.7U/ml, respectively. It was found that these enzymes preferentially partitioned into the top PEG-rich phase and appeared as single bands on SDS-PAGE gel. Meanwhile the validity of the response model was confirmed by a good agreement between predicted and experimental results. Collectively, according to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of any enzyme from oxidoreductase family. PMID:25591389

  19. Cloning of the Arabidopsis and Rice Formaldehyde Dehydrogenase Genes: Implications for the Origin of Plant Adh Enzymes

    PubMed Central

    Dolferus, R.; Osterman, J. C.; Peacock, W. J.; Dennis, E. S.

    1997-01-01

    This article reports the cloning of the genes encoding the Arabidopsis and rice class III ADH enzymes, members of the alcohol dehydrogenase or medium chain reductase/dehydrogenase superfamily of proteins with glutathione-dependent formaldehyde dehydrogenase activity (GSH-FDH). Both genes contain eight introns in exactly the same positions, and these positions are conserved in plant ethanol-active Adh genes (class P). These data provide further evidence that plant class P genes have evolved from class III genes by gene duplication and acquisition of new substrate specificities. The position of introns and similarities in the nucleic acid and amino acid sequences of the different classes of ADH enzymes in plants and humans suggest that plant and animal class III enzymes diverged before they duplicated to give rise to plant and animal ethanol-active ADH enzymes. Plant class P ADH enzymes have gained substrate specificities and evolved promoters with different expression properties, in keeping with their metabolic function as part of the alcohol fermentation pathway. PMID:9215914

  20. The Crystal Structure of a Ternary Complex of Betaine Aldehyde Dehydrogenase from Pseudomonas aeruginosa Provides New Insight Into the Reaction Mechansim and Shows A Novel Binding Mode of the 2'-Phosphate of NADP+ and A Novel Cation Binding Site

    SciTech Connect

    Gonzalez-Segura, L.; Rudino-Pinera, E; Munoz-Clares, R; Horjales, E

    2009-01-01

    In the human pathogen Pseudomonas aeruginosa, the NAD(P)+-dependent betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating carbon and nitrogen from choline or choline precursors-abundant at infection sites-and producing glycine betaine and NADPH, potentially protective against the high-osmolarity and oxidative stresses prevalent in the infected tissues. Disruption of the PaBADH gene negatively affects the growth of bacteria, suggesting that this enzyme could be a target for antibiotic design. PaBADH is one of the few ALDHs that efficiently use NADP+ and one of the even fewer that require K+ ions for stability. Crystals of PaBADH were obtained under aerobic conditions in the presence of 2-mercaptoethanol, glycerol, NADP+ and K+ ions. The three-dimensional structure was determined at 2.1-A resolution. The catalytic cysteine (C286, corresponding to C302 of ALDH2) is oxidized to sulfenic acid or forms a mixed disulfide with 2-mercaptoethanol. The glutamyl residue involved in the deacylation step (E252, corresponding to E268 of ALDH2) is in two conformations, suggesting a proton relay system formed by two well-conserved residues (E464 and K162, corresponding to E476 and K178, respectively, of ALDH2) that connects E252 with the bulk water. In some active sites, a bound glycerol molecule mimics the thiohemiacetal intermediate; its hydroxyl oxygen is hydrogen bonded to the nitrogen of the amide groups of the side chain of the conserved N153 (N169 of ALDH2) and those of the main chain of C286, which form the 'oxyanion hole.' The nicotinamide moiety of the nucleotide is not observed in the crystal, and the adenine moiety binds in the usual way. A salt bridge between E179 (E195 of ALDH2) and R40 (E53 of ALDH2) moves the carboxylate group of the former away from the 2?-phosphate of the NADP+, thus avoiding steric clashes and/or electrostatic repulsion between the two groups. Finally, the crystal shows two K+ binding sites per subunit. One is in an

  1. Oxidoreductive cellulose depolymerization by the enzymes cellobiose dehydrogenase and glycoside hydrolase 61.

    PubMed

    Langston, James A; Shaghasi, Tarana; Abbate, Eric; Xu, Feng; Vlasenko, Elena; Sweeney, Matt D

    2011-10-01

    Several members of the glycoside hydrolase 61 (GH61) family of proteins have recently been shown to dramatically increase the breakdown of lignocellulosic biomass by microbial hydrolytic cellulases. However, purified GH61 proteins have neither demonstrable direct hydrolase activity on various polysaccharide or lignacious components of biomass nor an apparent hydrolase active site. Cellobiose dehydrogenase (CDH) is a secreted flavocytochrome produced by many cellulose-degrading fungi with no well-understood biological function. Here we demonstrate that the binary combination of Thermoascus aurantiacus GH61A (TaGH61A) and Humicola insolens CDH (HiCDH) cleaves cellulose into soluble, oxidized oligosaccharides. TaGH61A-HiCDH activity on cellulose is shown to be nonredundant with the activities of canonical endocellulase and exocellulase enzymes in microcrystalline cellulose cleavage, and while the combination of TaGH61A and HiCDH cleaves highly crystalline bacterial cellulose, it does not cleave soluble cellodextrins. GH61 and CDH proteins are coexpressed and secreted by the thermophilic ascomycete Thielavia terrestris in response to environmental cellulose, and the combined activities of T. terrestris GH61 and T. terrestris CDH are shown to synergize with T. terrestris cellulose hydrolases in the breakdown of cellulose. The action of GH61 and CDH on cellulose may constitute an important, but overlooked, biological oxidoreductive system that functions in microbial lignocellulose degradation and has applications in industrial biomass utilization. PMID:21821740

  2. A hemolysis trigger in glucose-6-phosphate dehydrogenase enzyme deficiency. Vicia sativa (Vetch).

    PubMed

    Bicakci, Zafer

    2009-02-01

    Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme, playing an important role in the redox metabolism of all aerobic cells. It was reported that certain medications, fava beans, and infections can trigger acute hemolytic anemia in patients with G6PD deficiency. An 8-year-old male patient was admitted to the hospital with blood in the urine, headache, dizziness, fatigue, loss of appetite, and jaundice in the eyes, 24 hours after eating large amounts of fresh, vetch grains. Laboratory investigation revealed hemolytic anemia, hyperbilirubinemia, and G6PD deficiency. Approximately 0.5% of fava bean seeds have 2 pyrimidine beta-glycosides called, vicine and convicine. Vetch has 0.731% vicine, 0.081% convicine, and 0.530% beta cyanoalanine glycosides. The aim of this case report is to emphasize the importance of vetch seeds as a cause for hemolytic crisis in our country, where approximately one million tons of vetch is produced per year, especially in the agricultural regions. PMID:19198723

  3. Mitochondrial Probe Methyltriphenylphosphonium (TPMP) Inhibits the Krebs Cycle Enzyme 2-Oxoglutarate Dehydrogenase

    PubMed Central

    Elkalaf, Moustafa; Tůma, Petr; Weiszenstein, Martin; Polák, Jan

    2016-01-01

    Methyltriphenylphosphonium (TPMP) salts have been widely used to measure the mitochondrial membrane potential and the triphenylphosphonium (TPP+) moiety has been attached to many bioactive compounds including antioxidants to target them into mitochondria thanks to their high affinity to accumulate in the mitochondrial matrix. The adverse effects of these compounds on cellular metabolism have been insufficiently studied and are still poorly understood. Micromolar concentrations of TPMP cause a progressive inhibition of cellular respiration in adherent cells without a marked effect on mitochondrial coupling. In permeabilized cells the inhibition was limited to NADH-linked respiration. We found a mixed inhibition of the Krebs cycle enzyme 2-oxoglutarate dehydrogenase complex (OGDHC) with an estimated IC50 3.93 [3.70–4.17] mM, which is pharmacologically plausible since it corresponds to micromolar extracellular concentrations. Increasing the lipophilic character of the used TPP+ compound further potentiates the inhibition of OGDHC activity. This effect of TPMP on the Krebs cycle ought to be taken into account when interpreting observations on cells and mitochondria in the presence of TPP+ derivatives. Compounds based on or similar to TPP+ derivatives may also be used to alter OGDHC activity for experimental or therapeutic purposes. PMID:27537184

  4. Clostridium difficile glutamate dehydrogenase is a secreted enzyme that confers resistance to H2O2

    PubMed Central

    Girinathan, Brintha Prasummanna; Braun, Sterling E.

    2014-01-01

    Clostridium difficile produces an NAD-specific glutamate dehydrogenase (GDH), which converts l-glutamate into α-ketoglutarate through an irreversible reaction. The enzyme GDH is detected in the stool samples of patients with C. difficile-associated disease and serves as one of the diagnostic tools to detect C. difficile infection (CDI). We demonstrate here that supernatant fluids of C. difficile cultures contain GDH. To understand the role of GDH in the physiology of C. difficile, an isogenic insertional mutant of gluD was created in strain JIR8094. The mutant failed to produce and secrete GDH as shown by Western blot analysis. Various phenotypic assays were performed to understand the importance of GDH in C. difficile physiology. In TY (tryptose yeast extract) medium, the gluD mutant grew slower than the parent strain. Complementation of the gluD mutant with the functional gluD gene reversed the growth defect in TY medium. The presence of extracellular GDH may have a functional role in the pathogenesis of CDI. In support of this assumption we found higher sensitivity to H2O2 in the gluD mutant as compared to the parent strain. Complementation of the gluD mutant with the functional gluD gene reversed the H2O2 sensitivity. PMID:24145018

  5. Mitochondrial Probe Methyltriphenylphosphonium (TPMP) Inhibits the Krebs Cycle Enzyme 2-Oxoglutarate Dehydrogenase.

    PubMed

    Elkalaf, Moustafa; Tůma, Petr; Weiszenstein, Martin; Polák, Jan; Trnka, Jan

    2016-01-01

    Methyltriphenylphosphonium (TPMP) salts have been widely used to measure the mitochondrial membrane potential and the triphenylphosphonium (TPP+) moiety has been attached to many bioactive compounds including antioxidants to target them into mitochondria thanks to their high affinity to accumulate in the mitochondrial matrix. The adverse effects of these compounds on cellular metabolism have been insufficiently studied and are still poorly understood. Micromolar concentrations of TPMP cause a progressive inhibition of cellular respiration in adherent cells without a marked effect on mitochondrial coupling. In permeabilized cells the inhibition was limited to NADH-linked respiration. We found a mixed inhibition of the Krebs cycle enzyme 2-oxoglutarate dehydrogenase complex (OGDHC) with an estimated IC50 3.93 [3.70-4.17] mM, which is pharmacologically plausible since it corresponds to micromolar extracellular concentrations. Increasing the lipophilic character of the used TPP+ compound further potentiates the inhibition of OGDHC activity. This effect of TPMP on the Krebs cycle ought to be taken into account when interpreting observations on cells and mitochondria in the presence of TPP+ derivatives. Compounds based on or similar to TPP+ derivatives may also be used to alter OGDHC activity for experimental or therapeutic purposes. PMID:27537184

  6. Structure and Evolution of the Archaeal Lipid Synthesis Enzyme sn-Glycerol-1-phosphate Dehydrogenase*

    PubMed Central

    Carbone, Vincenzo; Schofield, Linley R.; Zhang, Yanli; Sang, Carrie; Dey, Debjit; Hannus, Ingegerd M.; Martin, William F.; Sutherland-Smith, Andrew J.; Ronimus, Ron S.

    2015-01-01

    One of the most critical events in the origins of cellular life was the development of lipid membranes. Archaea use isoprenoid chains linked via ether bonds to sn-glycerol 1-phosphate (G1P), whereas bacteria and eukaryotes use fatty acids attached via ester bonds to enantiomeric sn-glycerol 3-phosphate. NAD(P)H-dependent G1P dehydrogenase (G1PDH) forms G1P and has been proposed to have played a crucial role in the speciation of the Archaea. We present here, to our knowledge, the first structures of archaeal G1PDH from the hyperthermophilic methanogen Methanocaldococcus jannaschii with bound substrate dihydroxyacetone phosphate, product G1P, NADPH, and Zn2+ cofactor. We also biochemically characterized the enzyme with respect to pH optimum, cation specificity, and kinetic parameters for dihydroxyacetone phosphate and NAD(P)H. The structures provide key evidence for the reaction mechanism in the stereospecific addition for the NAD(P)H-based pro-R hydrogen transfer and the coordination of the Zn2+ cofactor during catalysis. Structure-based phylogenetic analyses also provide insight into the origins of G1PDH. PMID:26175150

  7. Characterization of lactate dehydrogenase enzyme in seminal plasma of Japanese quail (Coturnix coturnix japonica).

    PubMed

    Singh, R P; Sastry, K V H; Pandey, N K; Shit, N; Agrawal, R; Singh, K B; Mohan, Jag; Saxena, V K; Moudgal, R P

    2011-02-01

    Lactate dehydrogenase enzyme present in quail seminal plasma has been characterized. Polyacrylamide gel electrophoresis and subsequently with LDH specific staining of seminal plasma revealed a single isozyme in quail semen. Studies on substrate inhibition, pH for optimum activity and inhibitor (urea) indicated the isozyme present in the quail semen has catalytic properties like LDH-1 viz. H-type. Furthermore, unlike other mammalian species, electrophoretic and kinetic investigations did not support the existence of semen specific LDH-X isozyme in quail semen. The effect of exogenous lactate and pyruvate on sperm metabolic activity was also studied. The addition of 1 mM lactate or pyruvate to quail semen increased sperm metabolic activity. Our results suggested that both pyruvate and lactate could be used by quail spermatozoa to maintain their basic functions. Since the H-type isozyme is important for conversion of lactate to pyruvate under anaerobic conditions it was postulated that exogenous lactate being converted into pyruvate via LDH present in semen may be used by sperm mitochondria to generate ATP. During conversion of lactate to pyruvate NADH is being generated that may be useful for maintaining sperm mitochondrial membrane potential. PMID:21074838

  8. Wired pyrroloquinoline quinone soluble glucose dehydrogenase enzyme electrodes operating at unprecedented low redox potential.

    PubMed

    Flexer, Victoria; Mano, Nicolas

    2014-03-01

    We report unprecedented high current densities for the enzymatic oxidation of glucose already at 0 V versus Ag/AgCl. The modified electrodes were made by assembling pyrroloquinoline quinone (PQQ)-soluble glucose dehydrogenase (PQQ-sGDH) from Acinetobacter calcoaceticus with osmium-based redox polymers and a cross-linker. Both redox mediators are made of a poly(4-vinylpyridine) (PVP) polymer with Os complexes tethered to the polymer backbone via long C chains, giving the Os complexes flexibility and mobility inside the redox hydrogels. Current densities larger than 1 mA cm(-2) were measured already below 0 V with a plateau value of 4.4 mA cm(-2). Similar hydrogel electrodes comprising the same redox polymers and glucose oxidase (GOx) showed less than half the current densities of the PQQ-sGDH electrodes. The current versus potential curve dependence showed a sigmoidal shape characteristic of mediated enzyme catalysis but with a current increase versus potential less sharp than expected. Surprisingly, the midwave redox potential was positively shifted with respect to the potential of the redox mediator. PMID:24475934

  9. Characterization of Anammox Hydrazine Dehydrogenase, a Key N2-producing Enzyme in the Global Nitrogen Cycle.

    PubMed

    Maalcke, Wouter J; Reimann, Joachim; de Vries, Simon; Butt, Julea N; Dietl, Andreas; Kip, Nardy; Mersdorf, Ulrike; Barends, Thomas R M; Jetten, Mike S M; Keltjens, Jan T; Kartal, Boran

    2016-08-12

    Anaerobic ammonium-oxidizing (anammox) bacteria derive their energy for growth from the oxidation of ammonium with nitrite as the electron acceptor. N2, the end product of this metabolism, is produced from the oxidation of the intermediate, hydrazine (N2H4). Previously, we identified N2-producing hydrazine dehydrogenase (KsHDH) from the anammox organism Kuenenia stuttgartiensis as the gene product of kustc0694 and determined some of its catalytic properties. In the genome of K. stuttgartiensis, kustc0694 is one of 10 paralogs related to octaheme hydroxylamine (NH2OH) oxidoreductase (HAO). Here, we characterized KsHDH as a covalently cross-linked homotrimeric octaheme protein as found for HAO and HAO-related hydroxylamine-oxidizing enzyme kustc1061 from K. stuttgartiensis Interestingly, the HDH trimers formed octamers in solution, each octamer harboring an amazing 192 c-type heme moieties. Whereas HAO and kustc1061 are capable of hydrazine oxidation as well, KsHDH was highly specific for this activity. To understand this specificity, we performed detailed amino acid sequence analyses and investigated the catalytic and spectroscopic (electronic absorbance, EPR) properties of KsHDH in comparison with the well defined HAO and kustc1061. We conclude that HDH specificity is most likely derived from structural changes around the catalytic heme 4 (P460) and of the electron-wiring circuit comprising seven His/His-ligated c-type hemes in each subunit. These nuances make HDH a globally prominent N2-producing enzyme, next to nitrous oxide (N2O) reductase from denitrifying microorganisms. PMID:27317665

  10. Catecholamines enhance dihydrolipoamide dehydrogenase inactivation by the copper Fenton system. Enzyme protection by copper chelators.

    PubMed

    Correa, J G; Stoppani, A O

    1996-04-01

    Catecholamines (CAs: epinephrine, norepinephrine, dopamine, L-DOPA, 6-hydroxydopamine) and o-diphenols (DOPAC and catechol) enhanced dihydrolipoamide dehydrogenase (LADH) inactivation by Cu(II)/H2O2 (Cu-Fenton system). The inhibition of LADH activity correlated with Cu(II), H2O2 and CA concentrations. Similar inhibitions were obtained with the assayed CAs and o-diphenols. CAs enhanced HO. radical production by Cu(II)/H2O2, as demonstrated by benzoate hydroxylation and deoxyribose oxidation; LADH counteracted the pro-oxidant effect of CAs by scavenging hydroxyl radicals. Captopril, dihydrolipoamide, dihydrolipoic acid, DL-dithiothreitol, GSSG, trypanothione and histidine effectively preserved LADH from oxidative damage, whereas N-acetylcysteine, N-(2-mercaptopropionylglycine) and lipoamide were less effective protectors. Catalase (though neither bovine serum albumin nor superoxide dismutase) protected LADH against the Cu(II)/H2O2/CAs systems. Denatured catalase protected less than the native enzyme, its action possibly depending on Cu-binding. LADH increased and Captopril inhibited epinephrine oxidation by Cu(II)/H2O2 and Cu(II). The summarized evidence supports the following steps for LADH inactivation: (1) reduction of LADH linked-Cu(II) to Cu(I) by CAs; (2) production of HO. from H2O2 by LADH-linked Cu(I) (Haber-Weiss reaction) and (3) oxidation of aminoacid residues at the enzyme active site by site-specifically generated HO. radicals. Hydrogen peroxide formation from CAs autoxidation may contribute to LADH inactivation. PMID:8731015

  11. Isolation of carbon monoxide dehydrogenase from Acetobacterium woodii and comparison of its properties with those of the Clostridium thermoaceticum enzyme.

    PubMed

    Ragsdale, S W; Ljungdahl, L G; DerVartanian, D V

    1983-09-01

    An oxygen-labile carbon monoxide dehydrogenase was purified to at least 98% homogeneity from fructose-grown cells of Acetobacterium woodii. Gel filtration and electrophoresis experiments gave molecular weights of 480,000 and 153,000, respectively, of the active enzyme. The molecular weights for the subunits are 80,000 and 68,000; the subunits occur in equal proportion. The small subunit of the A. woodii enzyme differs in size from that of the Clostridium thermoaceticum enzyme; however, the large subunits are similar. The specific activity of the A. woodii enzyme, measured at 30 degrees C and pH 7.6, is 500 mumol of CO oxidized min-1 mg-1 with 20 mM methyl viologen as the electron acceptor. Analysis revealed (number per dimer) iron (9), acid-labile sulfide (12), nickel (1.4), and magnesium or zinc (1). This metal content is quite similar to that of the C. thermoaceticum enzyme (Ragsdale et al., J. Biol. Chem. 258:2364-2369, 1983). The nickel as well as the iron-sulfur clusters are redox-active, as was found for the C. thermoaceticum enzyme (Ragsdale et al., Biochem. Biophys. Res. Commun. 108:658-663, 1982). CO can reduce and CO2 can oxidize the iron-sulfur clusters. The enzyme is inhibited by cyanide, but CO2 in the presence of reduced methyl viologen or CO alone can reverse or prevent this inhibition. Several ferredoxins, flavodoxin, and rubredoxin and some artificial electron carriers were tested for their relative rates of reaction with the CO dehydrogenases from A. woodii, C. thermoaceticum, and Clostridium formicoaceticum. Rubredoxin was by far the most reactive acceptor and is proposed to be the primary natural electron carrier for the acetogenic CO dehydrogenases. PMID:6309745

  12. Role of ammonia in the activiation of methanol dehydrogenase/cytochrome C(L) enzyme

    NASA Astrophysics Data System (ADS)

    Kunjumon, Ancy

    Recent advancement in enzyme catalysis has opened ways to design efficient biocatalysts, bio-sensors and bio-fuel cells. An in-depth knowledge about the mechanism of the reaction taking place within the enzymes is of great importance to achieve these goals. In this dissertation, various computation methods are applied to investigate the mechanism behind enzyme catalysis in the presence of compounds called activators. Methanol dehydrogenase (MDH) is a well-known bio-catalyst that can oxidize excess of methanol from the environment to formaldehyde. The enzyme works well within the bacterial environment, but under in vitro, it loses activity. Ammonia is used as an activator to restore the activity of MDH. The Monte Carlo search using simulated annealing metaheuristic method is conducted to explore the binding of MDH with its natural electron acceptor Cytochrome cL in varying concentration of ammonia. The main aim behind this is to explore the interaction energy between the enzymes under the influence of its activator. The concentration of ammonia is varied from 0 to 5 ammonia molecules. Moving deeper into the active site of MDH, molecular mechanics and dynamics calculations were performed to investigate the position and effect of ammonia in the active site amino acids of MDH. The concentration of ammonia was varied from 0 to 55.39 mM. It was proposed that ammonia may form a complex conjugate with the cofactor of MDH (Pyrroloquinoline quinone) to assist in the oxidation of methanol. Two of the most debated methanol oxidation mechanisms, Addition-Elimination reaction and Hydride-Transfer mechanism, were used to investigate the role of ammonia in the oxidation of methanol. Density functional theory (DFT) was applied to explore the methanol oxidation mechanism in the presence of ammonia. Models of varying size that best represent the active site of MDH were tested for this purpose. The interaction energy obtained after the docking of MDH and Cytochrome cL (CL) indicate

  13. Cooperativity in highly aggregated enzyme systems. A slow transition model for the pyruvate dehydrogenase complex from Escherichia coli.

    PubMed

    Bisswanger, H

    1984-02-25

    Three models are compared describing cooperative phenomena in enzymatic reactions in order to explain sigmoidal saturation curves found with the pyruvate dehydrogenase complex from Escherichia coli: the concerted model, the sequential model, and the slow transition model. Both the concerted and the sequential model were considered especially with regard to the increasing number of identical interaction subunits (protomers) in order to get close to the situation found with the pyruvate dehydrogenase complex which consists of 24 protomers. Applying the sequential model to a great number of protomers results in a weak increase of the Hill coefficient, while, in addition to this effect, the concerted model drastically shifts the sigmoidal range of the saturation function to very low ligand concentrations. Such shift is seen with saturation curves of pyruvate and thiamine disphosphate with the pyruvate dehydrogenase complex and a good fit with theoretical curves derived from the concerted model is obtained. However, subcomplexes with a reduced number of protomers exhibited no change in saturation behavior, thus providing evidence against concerted conformational changes of all subunits of the enzyme complex. A scheme for the initial reaction of the pyruvate dehydrogenase complex based on slow transitions is presented and a rate equation has been derived. Ordered binding of thiamine diphosphate and pyruvate and a ligand-induced slow transition between a less active and a fully active enzyme form has been assumed. The curves simulated with this model are in agreement with all essential kinetic data, which are observed with the pyruvate dehydrogenase complex: the atypical shape of the saturation curves of pyruvate and thiamine diphosphate, the respective Hill coefficients and Michaelis constants, the hyperbolic binding behavior of thiamine diphosphate, and the inhibition pattern found for acetyl coenzyme A. PMID:6365912

  14. On-plate enzyme and inhibition assay of glucose-6-phosphate dehydrogenase using thin-layer chromatography.

    PubMed

    Tian, Miaomiao; Mohamed, Amara Camara; Wang, Shengtian; Yang, Li

    2015-08-01

    We performed on-plate enzyme and inhibition assays of glucose 6-phosphate dehydrogenase using thin-layer chromatography. The assays were accomplished based on different retardation factors of the substrates, enzyme, and products. All the necessary steps were integrated on-plate in one developing process, including substrate/enzyme mixing, reaction starting, and quenching as well as product separation. In order to quantitatively measure the enzyme reaction, the developed plate was then densitometrically evaluated to determine the peak area of the product. Rapid and high-throughput assays were achieved by loading different substrate spots and/or enzyme (and inhibition) spots in different tracks on the plate. The on-plate enzyme assay could be finished in a developing time of only 4 min, with good track-to-track and plate-to-plate repeatability. Moreover, we determined the Km values of the enzyme reaction and Ki values of the inhibition (Pb(2+) Cd(2+) and Cu(2+) as inhibitors), as well as the corresponding kinetics using the on-plate assay. Taken together, our method expanded the application of thin-layer chromatography in enzyme assays, and it could be potentially used in research fields for rapid and quantitative measurement of enzyme activity and inhibition. PMID:26017233

  15. Determination of the inhibitory effect of green tea extract on glucose-6-phosphate dehydrogenase based on multilayer capillary enzyme microreactor.

    PubMed

    Camara, Mohamed Amara; Tian, Miaomiao; Liu, Xiaoxia; Liu, Xin; Wang, Yujia; Yang, Jiqing; Yang, Li

    2016-08-01

    Natural herbal medicines are an important source of enzyme inhibitors for the discovery of new drugs. A number of natural extracts such as green tea have been used in prevention and treatment of diseases due to their low-cost, low toxicity and good performance. The present study reports an online assay of the activity and inhibition of the green tea extract of the Glucose 6-phosphate dehydrogenase (G6PDH) enzyme using multilayer capillary electrophoresis based immobilized enzyme microreactors (CE-IMERs). The multilayer CE-IMERs were produced with layer-by-layer electrostatic assembly, which can easily enhance the enzyme loading capacity of the microreactor. The activity of the G6PDH enzyme was determined and the enzyme inhibition by the inhibitors from green tea extract was investigated using online assay of the multilayer CE-IMERs. The Michaelis constant (Km ) of the enzyme, the IC50 and Ki values of the inhibitors were achieved and found to agree with those obtained using offline assays. The results show a competitive inhibition of green tea extract on the G6PDH enzyme. The present study provides an efficient and easy-to-operate approach for determining G6PDH enzyme reaction and the inhibition of green tea extract, which may be beneficial in research and the development of natural herbal medicines. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26659432

  16. Subcellular localization and biochemical comparison of cytosolic and secreted cytokinin dehydrogenase enzymes from maize.

    PubMed

    Smehilová, Mária; Galuszka, Petr; Bilyeu, Kristin D; Jaworek, Pavel; Kowalska, Marta; Sebela, Marek; Sedlárová, Michaela; English, James T; Frébort, Ivo

    2009-01-01

    Cytokinin dehydrogenase (CKX; EC 1.5.99.12) degrades cytokinin hormones in plants. There are several differently targeted isoforms of CKX in plant cells. While most CKX enzymes appear to be localized in the apoplast or vacuoles, there is generally only one CKX per plant genome that lacks a translocation signal and presumably functions in the cytosol. The only extensively characterized maize CKX is the apoplastic ZmCKX1; a maize gene encoding a non-secreted CKX has not previously been cloned or characterized. Thus, the aim of this work was to characterize the maize non-secreted CKX gene (ZmCKX10), elucidate the subcellular localization of ZmCKX10, and compare its biochemical properties with those of ZmCKX1. Expression profiling of ZmCKX1 and ZmCKX10 was performed in maize tissues to determine their transcript abundance and organ-specific expression. For determination of the subcellular localization, the CKX genes were fused with green fluorescent protein (GFP) and overexpressed in tomato hairy roots. Using confocal microscopy, the ZmCKX1-GFP signal was confirmed to be present in the apoplast, whereas ZmCKX10-GFP was detected in the cytosol. No interactions of ZmCKX1 with the plasma membrane were observed. While roots overexpressing ZmCKX1-GFP formed significantly more mass in comparison with the control, non-secreted CKX overexpression resulted in a small reduction in root mass accumulation. Biochemical characterization of ZmCKX10 was performed using recombinant protein produced in Pichia pastoris. In contrast to the preference for 2,6-dichlorophenolindophenol (DCPIP) as an electron acceptor and trans-zeatin, N(6)-(Delta(2)-isopentenyl)adenine (iP) and N(6)-(Delta(2)-isopentenyl)adenosine (iPR) as substrates for ZmCKX1, the non-secreted ZmCKX10 had a range of suitable electron acceptors, and the enzyme had a higher preference for cis-zeatin and cytokinin N-glucosides as substrates. PMID:19436049

  17. Biochemical and molecular characterization of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase from Toxoplasma gondii.

    PubMed

    Hortua Triana, Miryam Andrea; Huynh, My-Hang; Garavito, Manuel F; Fox, Barbara A; Bzik, David J; Carruthers, Vern B; Löffler, Monika; Zimmermann, Barbara H

    2012-08-01

    The pyrimidine biosynthesis pathway in the protozoan pathogen Toxoplasma gondii is essential for parasite growth during infection. To investigate the properties of dihydroorotate dehydrogenase (TgDHOD), the fourth enzyme in the T. gondii pyrimidine pathway, we expressed and purified recombinant TgDHOD. TgDHOD exhibited a specific activity of 84U/mg, a k(cat) of 89s(-1), a K(m)=60μM for l-dihydroorotate, and a K(m)=29μM for decylubiquinone (Q(D)). Quinones lacking or having short isoprenoid side chains yielded lower k(cat)s than Q(D). As expected, fumarate was a poor electron acceptor for this family 2 DHOD. The IC(50)s determined for A77-1726, the active derivative of the human DHOD inhibitor leflunomide, and related compounds MD249 and MD209 were, 91μM, 96μM, and 60μM, respectively. The enzyme was not significantly affected by brequinar or TTFA, known inhibitors of human DHOD, or by atovaquone. DSM190, a known inhibitor of Plasmodium falciparum DHOD, was a poor inhibitor of TgDHOD. TgDHOD exhibits a lengthy 157-residue N-terminal extension, consistent with a potential organellar targeting signal. We constructed C-terminally c-myc tagged TgDHODs to examine subcellular localization of TgDHOD in transgenic parasites expressing the tagged protein. Using both exogenous and endogenous expression strategies, anti-myc fluorescence signal colocalized with antibodies against the mitochondrial marker ATPase. These findings demonstrate that TgDHOD is associated with the parasite's mitochondrion, revealing this organelle as the site of orotate production in T. gondii. The TgDHOD gene appears to be essential because while gene tagging was successful at the TgDHOD gene locus, attempts to delete the TgDHOD gene were not successful in the KU80 background. Collectively, our study suggests that TgDHOD is an excellent target for the development of anti-Toxoplasma drugs. PMID:22580100

  18. Biochemical and molecular characterization of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase from Toxoplasma gondii

    PubMed Central

    Triana, Miryam Andrea Hortua; Huynh, My-Hang; Garavito, Manuel F.; Fox, Barbara A.; Bzik, David J.; Carruthers, Vern B.; Löffler, Monika; Zimmermann, Barbara H.

    2013-01-01

    Summary The pyrimidine biosynthesis pathway in the protozoan pathogen Toxoplasma gondii is essential for parasite growth during infection. To investigate the properties of dihydroorotate dehydrogenase (TgDHOD), the fourth enzyme in the T. gondii pyrimidine pathway, we expressed and purified recombinant TgDHOD. TgDHOD exhibited a specific activity of 84 U/mg, a kcat of 89 sec−1, a Km = 60 μM for L-dihydroorotate, and a Km = 29 μM for decylubiquinone (QD). Quinones lacking or having short isoprenoid side chains yielded lower kcats than QD. As expected, fumarate was a poor electron acceptor for this family 2 DHOD. The The IC50s determined for A77-1726, the active derivative of the human DHOD inhibitor leflunomide, and related compounds MD249 and MD209 were, 91 μM, 96 μM, and 60 μM, respectively. The enzyme was not significantly affected by brequinar or TTFA, known inhibitors of human DHOD, or by atovaquone. DSM190, a known inhibitor of Plasmodium falciparum DHOD, was a poor inhibitor of TgDHOD. TgDHOD exhibits a lengthy 157-residue N-terminal extension, consistent with a potential organellar targeting signal. We constructed C-terminally c-myc tagged TgDHODs to examine subcellular localization of TgDHOD in transgenic parasites expressing the tagged protein. Using both exogenous and endogenous expression strategies, anti-myc fluorescence signal colocalized with antibodies against the mitochondrial marker ATPase. These findings demonstrate that TgDHOD is associated with the parasite’s mitochondrion, revealing this organelle as the site of orotate production in T gondii. The TgDHOD gene appears to be essential because while gene tagging was successful at the TgDHOD gene locus, attempts to delete the TgDHOD gene were not successful in the KU80 background. Collectively, our study suggests that TgDHOD is an excellent target for the development of anti-Toxoplasma drugs. PMID:22580100

  19. Structural Biology of Proteins of the Multi-enzyme Assembly Human Pyruvate Dehydrogenase Complex

    NASA Technical Reports Server (NTRS)

    2003-01-01

    Objectives and research challenges of this effort include: 1. Need to establish Human Pyruvate Dehydrogenase Complex protein crystals; 2. Need to test value of microgravity for improving crystal quality of Human Pyruvate Dehydrogenase Complex protein crystals; 3. Need to improve flight hardware in order to control and understand the effects of microgravity on crystallization of Human Pyruvate Dehydrogenase Complex proteins; 4. Need to integrate sets of national collaborations with the restricted and specific requirements of flight experiments; 5. Need to establish a highly controlled experiment in microgravity with a rigor not yet obtained; 6. Need to communicate both the rigor of microgravity experiments and the scientific value of results obtained from microgravity experiments to the national community; and 7. Need to advance the understanding of Human Pyruvate Dehydrogenase Complex structures so that scientific and commercial advance is identified for these proteins.

  20. Determining Structure and Function of Steroid Dehydrogenase Enzymes by Sequence Analysis, Homology Modeling, and Rational Mutational Analysis

    PubMed Central

    DUAX, WILLIAM L.; THOMAS, JAMES; PLETNEV, VLADIMIR; ADDLAGATTA, ANTHONY; HUETHER, ROBERT; HABEGGER, LUKAS; WEEKS, CHARLES M.

    2006-01-01

    The short-chain oxidoreductase (SCOR) family of enzymes includes over 6,000 members identified in sequenced genomes. Of these enzymes, ~300 have been characterized functionally, and the three-dimensional crystal structures of ~40 have been reported. Since some SCOR enzymes are steroid dehydrogenases involved in hypertension, diabetes, breast cancer, and polycystic kidney disease, it is important to characterize the other members of the family for which the biological functions are currently unknown and to determine their three-dimensional structure and mechanism of action. Although the SCOR family appears to have only a single fully conserved residue, it was possible, using bioinformatics methods, to determine characteristic fingerprints composed of 30–40 residues that are conserved at the 70% or greater level in SCOR subgroups. These fingerprints permit reliable prediction of several important structure-function features including cofactor preference, catalytic residues, and substrate specificity. Human type 1 3β-hydroxysteroid dehydrogenase isomerase (3β-HSDI) has 30% sequence identity with a human UDP galactose 4-epimerase (UDPGE), a SCOR family enzyme for which an X-ray structure has been reported. Both UDPGE and 3-HSDI appear to trace their origins back to bacterial 3α,20β-HSD. Combining three-dimensional structural information and sequence data on the 3α,20β-HSD, UDPGE, and 3β-HSDI subfamilies with mutational analysis, we were able to identify the residues critical to the dehydrogenase function of 3-HSDI. We also identified the residues most probably responsible for the isomerase activity of 3β-HSDI. We test our predictions by specific mutations based on sequence analysis and our structure-based model. PMID:16467263

  1. Purification and properties of the inducible coenzyme A-linked butyraldehyde dehydrogenase from Clostridium acetobutylicum.

    PubMed Central

    Palosaari, N R; Rogers, P

    1988-01-01

    The coenzyme A (CoA)-linked butyraldehyde dehydrogenase (BAD) from Clostridium acetobutylicum was characterized and purified to homogeneity. The enzyme was induced over 200-fold, coincident with a shift from an acidogenic to a solventogenic fermentation, during batch culture growth. The increase in enzyme activity was found to require new protein synthesis since induction was blocked by the addition of rifampin and antibody against the purified enzyme showed the appearance of enzyme antigen beginning at the shift of the fermentation and increasing coordinately with the increase in enzyme specific activity. The CoA-linked acetaldehyde dehydrogenase was copurified with BAD during an 89-fold purification, indicating that one enzyme accounts for the synthesis of the two aldehyde intermediates for both butanol and ethanol production. Butanol dehydrogenase activity was clearly separate from the BAD enzyme activity on TEAE cellulose. A molecular weight of 115,000 was determined for the native enzyme, and the enzyme subunit had a molecular weight of 56,000 indicating that the active form is a homodimer. Kinetic constants were determined in both the forward and reverse directions. In the reverse direction both the Vmax and the apparent affinity for butyraldehyde and caproaldehyde were significantly greater than they were for acetaldehyde, while in the forward direction, the Vmax for butyryl-CoA was fivefold that for acetyl-CoA. These and other properties of BAD indicate that this enzyme is distinctly different from other reported CoA-dependent aldehyde dehydrogenases. Images PMID:3384801

  2. Structural Basis for Flip-Flop Action of Thiamin Pyrophosphate-Dependent Enzymes Revealed by Human Pyruvate Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Dominiak, Paulina; Ciszak, Ewa M.; Korotchkina, Lioubov; Sidhu, Sukhdeep; Patel, Mulchand

    2003-01-01

    Thiamin pyrophosphate (TPP), the biologically active form of vitamin BI, is a cofactor of enzymes catalyzing reactions involving the cleavage of a carbon-carbon bond adjacent to an oxo group. TPP-dependent enzymes show a common mechanism of TPP activation by: (1) forming the ionic N-H...O(sup -) hydrogen bonding between the N1' atom of the aminopirymidine ring of the coenzyme and intrinsic gamma-carboxylate group of glutamate and (2) imposing an "active" V-conformation that brings the N4' atom of the aminopirymidine to the distance required for the intramolecular C-H.. .N hydrogen bonding with the thiazolium C2 atom. Within these two hydrogen bonds that rapidly exchange protons, protonation of the N1' atom is strictly coordinated with the deprotonation of the 4' -amino group and eventually abstraction of the proton from C2. The human pyruvate dehydrogenase Elp, component of human pyruvate dehydrogenase complex, catalyzes the irreversible decarboxylation of the pyruvate followed by the reductive acetylation of the lipoyl group of dihydrolipoyl acyltransferase. Elp is alpha(sub 2)beta(sub2)-heterotetrameric with a molecular mass of I54 kDa, which has two catalytic sites, each providing TPP and magnesium ion as cofactors and each formed on the interface between the PP and PYR domains. The dynamic nonequivalence of two otherwise chemically equivalent catalytic sites has been observed and the flip-flop mechanism was suggested, according to which two active sites affect each other and in which different steps of the catalytic reaction are performed in each of the sites at any given moment. Based on specific futures of human pyruvate dehydrogenase including rigid and flexible connections between domains that bind the cofactor we propose a mechanistic model for the flip-flop action of this enzyme. We postulate that the dynamic protein environment drives the exchange of tautomers in the 4' -aminopyrimidine ring of the cofactor through a concerted shuttl-like motion of

  3. Transcription analysis of pyranose dehydrogenase from the basidiomycete Agaricus bisporus and characterization of the recombinantly expressed enzyme.

    PubMed

    Gonaus, Christoph; Kittl, Roman; Sygmund, Christoph; Haltrich, Dietmar; Peterbauer, Clemens

    2016-03-01

    Agaricus bisporus is a litter degrading basidiomycete commonly found in humic-rich environments. It is used as model organism and cultivated in large scale for food industry. Due to its ecological niche it produces a variety of enzymes for detoxification and degradation of humified plant litter. One of these, pyranose dehydrogenase, is thought to play a role in detoxification and lignocellulose degradation. It is a member of the glucose-methanol-choline family of flavin-dependent enzymes and oxidizes a wide range of sugars with concomitant reduction of electron acceptors like quinones. In this work, transcription of pdh in A. bisporus was investigated with real-time PCR revealing influence of the carbon source on pdh expression levels. The gene was isolated and heterologously expressed in Pichia pastoris. Characterization of the recombinant enzyme showed a higher affinity towards disaccharides compared to other tested pyranose dehydrogenases from related Agariceae. Homology modeling and sequence alignments indicated that two loops of high sequence variability at substrate access site could play an important role in modulating these substrate specificities. PMID:26616098

  4. Fatty Aldehyde and Fatty Alcohol Metabolism: Review and Importance for Epidermal Structure and Function

    PubMed Central

    Rizzo, William B.

    2014-01-01

    Normal fatty aldehyde and alcohol metabolism is essential for epidermal differentiation and function. Long-chain aldehydes are produced by catabolism of several lipids including fatty alcohols, sphingolipids, ether glycerolipids, isoprenoid alcohols and certain aliphatic lipids that undergo α- or ω-oxidation. The fatty aldehyde generated by these pathways is chiefly metabolized to fatty acid by fatty aldehyde dehydrogenase (FALDH, alternately known as ALDH3A2), which also functions to oxidize fatty alcohols as a component of the fatty alcohol:NAD oxidoreductase (FAO) enzyme complex. Genetic deficiency of FALDH/FAO in patients with Sjögren-Larsson syndrome (SLS) results in accumulation of fatty aldehydes, fatty alcohols and related lipids (ether glycerolipids, wax esters) in cultured keratinocytes. These biochemical changes are associated with abnormalities in formation of lamellar bodies in the stratum granulosum and impaired delivery of their precursor membranes to the stratum corneum (SC). The defective extracellular SC membranes are responsible for a leaky epidermal water barrier and ichthyosis. Although lamellar bodies appear to be the pathogenic target for abnormal fatty aldehyde/alcohol metabolism in SLS, the precise biochemical mechanisms are yet to be elucidated. Nevertheless, studies in SLS highlight the critical importance of FALDH and normal fatty aldehyde/alcohol metabolism for epidermal function. PMID:24036493

  5. Purification and Characterization of Glucose 6-Phosphate Dehydrogenase, 6-Phosphogluconate Dehydrogenase, and Glutathione Reductase from Rat Heart and Inhibition Effects of Furosemide, Digoxin, and Dopamine on the Enzymes Activities.

    PubMed

    Adem, Sevki; Ciftci, Mehmet

    2016-06-01

    The present study was aimed to investigate characterization and purification of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase from rat heart and the inhibitory effect of three drugs. The purification of the enzymes was performed using 2',5'-ADP sepharose 4B affinity material. The subunit and the natural molecular weights were analyzed by SDS-PAGE and gel filtration. Biochemical characteristics such as the optimum temperature, pH, stable pH, and salt concentration were examined for each enzyme. Types of product inhibition and Ki values with Km and Vmax values of the substrates and coenzymes were determined. According to the obtained Ki and IC50 values, furosemide, digoxin, and dopamine showed inhibitory effect on the enzyme activities at low millimolar concentrations in vitro conditions. Dopamine inhibited the activity of these enzymes as competitive, whereas furosemide and digoxin inhibited the activity of the enzyme as noncompetitive. PMID:26820767

  6. The intrinsically disordered protein LEA7 from Arabidopsis thaliana protects the isolated enzyme lactate dehydrogenase and enzymes in a soluble leaf proteome during freezing and drying.

    PubMed

    Popova, Antoaneta V; Rausch, Saskia; Hundertmark, Michaela; Gibon, Yves; Hincha, Dirk K

    2015-10-01

    The accumulation of Late Embryogenesis Abundant (LEA) proteins in plants is associated with tolerance against stresses such as freezing and desiccation. Two main functions have been attributed to LEA proteins: membrane stabilization and enzyme protection. We have hypothesized previously that LEA7 from Arabidopsis thaliana may stabilize membranes because it interacts with liposomes in the dry state. Here we show that LEA7, contrary to this expectation, did not stabilize liposomes during drying and rehydration. Instead, it partially preserved the activity of the enzyme lactate dehydrogenase (LDH) during drying and freezing. Fourier-transform infrared (FTIR) spectroscopy showed no evidence of aggregation of LDH in the dry or rehydrated state under conditions that lead to complete loss of activity. To approximate the complex influence of intracellular conditions on the protective effects of a LEA protein in a convenient in-vitro assay, we measured the activity of two Arabidopsis enzymes (glucose-6-P dehydrogenase and ADP-glucose pyrophosphorylase) in total soluble leaf protein extract (Arabidopsis soluble proteome, ASP) after drying and rehydration or freezing and thawing. LEA7 partially preserved the activity of both enzymes under these conditions, suggesting its role as an enzyme protectant in vivo. Further FTIR analyses indicated the partial reversibility of protein aggregation in the dry ASP during rehydration. Similarly, aggregation in the dry ASP was strongly reduced by LEA7. In addition, mixtures of LEA7 with sucrose or verbascose reduced aggregation more than the single additives, presumably through the effects of the protein on the H-bonding network of the sugar glasses. PMID:25988244

  7. Inhibition of Vibrio harveyi bioluminescence by cerulenin: In vivo evidence for covalent modification of the reductase enzyme involved in aldehyde synthesis

    SciTech Connect

    Byers, D.M. ); Meighen, E.A. )

    1989-07-01

    Bacterial bioluminescence is very sensitive to cerulenin, a fungal antibiotic which is known to inhibit fatty acid synthesis. When Vibrio harveyi cells pretreated with cerulenin were incubated with ({sup 3}H)myristic acid in vivo, acylation of the 57-kilodalton reductase subunit of the luminescence-specific fatty acid reductase complex was specifically inhibited. Light emission of wild-type V. harveyi was 20-fold less sensitive to cerulenin at low concentrations (10{mu}g/ml) than that of the dark mutant strain M17, which requires exogenous myristic acid for luminescence because of a defective transferase subunit. The sensitivity of myristic acid-stimulated luminescence in the mutant strain M17 exceeded that of phospholipid synthesis from ({sup 14}C)acetate, whereas uptake and incorporation of exogenous ({sup 14}C)myristic acid into phospholipids was increased by cerulenin. The reductase subunit could be labeled by incubating M17 cells with ({sup 3}H)tetrahydrocerulenin; this labeling was prevented by preincubation with either unlabeled cerulenin or myristic acid. Labeling of the reductase subunit with ({sup 3}H)tetrahydrocerulenin was also noted in an aldehyde-stimulated mutant (A16) but not in wild-type cells or in another aldehyde-stimulated mutant (M42) in which ({sup 3}H)myristoyl turnover at the reductase subunit was found to be defective. These results indicate that (i) cerulenin specifically and covalently inhibits the reductase component of aldehyde synthesis, (ii) this enzyme is partially protected from cerulenin inhibition in the wild-type strain in vivo, and (iii) two dark mutants which exhibit similar luminescence phenotypes (mutants A16 and M42) are blocked at different stages of fatty acid reduction.

  8. Structural and transcriptional analysis of plant genes encoding the bifunctional lysine ketoglutarate reductase saccharopine dehydrogenase enzyme

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The analysis of a wheat lysine ketoglutarate reductase – saccharopine dehydrogenase (LKR/SDH) gene and comparative structural and functional analyses among available plant genes provides new information on this important gene. Both the structure of the LKR/SDH gene and the immediately adjacent genes...

  9. Ethylene Glycol Monomethyl Ether–Induced Toxicity Is Mediated through the Inhibition of Flavoprotein Dehydrogenase Enzyme Family

    PubMed Central

    Takei, Makoto; Ando, Yosuke; Saitoh, Wataru; Tanimoto, Tomoe; Kiyosawa, Naoki; Manabe, Sunao; Sanbuissho, Atsushi; Okazaki, Osamu; Iwabuchi, Haruo; Yamoto, Takashi; Adam, Klaus-Peter; Weiel, James E.; Ryals, John A.; Milburn, Michael V.; Guo, Lining

    2010-01-01

    Ethylene glycol monomethyl ether (EGME) is a widely used industrial solvent known to cause adverse effects to human and other mammals. Organs with high metabolism and rapid cell division, such as testes, are especially sensitive to its actions. In order to gain mechanistic understanding of EGME-induced toxicity, an untargeted metabolomic analysis was performed in rats. Male rats were administrated with EGME at 30 and 100 mg/kg/day. At days 1, 4, and 14, serum, urine, liver, and testes were collected for analysis. Testicular injury was observed at day 14 of the 100 mg/kg/day group only. Nearly 1900 metabolites across the four matrices were profiled using liquid chromatography-mass spectrometry/mass spectrometry and gas chromatography-mass spectrometry. Statistical analysis indicated that the most significant metabolic perturbations initiated from the early time points by EGME were the inhibition of choline oxidation, branched-chain amino acid catabolism, and fatty acid β-oxidation pathways, leading to the accumulation of sarcosine, dimethylglycine, and various carnitine- and glycine-conjugated metabolites. Pathway mapping of these altered metabolites revealed that all the disrupted steps were catalyzed by enzymes in the primary flavoprotein dehydrogenase family, suggesting that inhibition of flavoprotein dehydrogenase–catalyzed reactions may represent the mode of action for EGME-induced toxicity. Similar urinary and serum metabolite signatures are known to be the hallmarks of multiple acyl-coenzyme A dehydrogenase deficiency in humans, a genetic disorder because of defects in primary flavoprotein dehydrogenase reactions. We postulate that disruption of key biochemical pathways utilizing flavoprotein dehydrogenases in conjugation with downstream metabolic perturbations collectively result in the EGME-induced tissue damage. PMID:20616209

  10. Going Beyond Common Drug Metabolizing Enzymes: Case Studies of Biotransformation Involving Aldehyde Oxidase, γ-Glutamyl Transpeptidase, Cathepsin B, Flavin-Containing Monooxygenase, and ADP-Ribosyltransferase.

    PubMed

    Fan, Peter W; Zhang, Donglu; Halladay, Jason S; Driscoll, James P; Khojasteh, S Cyrus

    2016-08-01

    The significant roles that cytochrome P450 (P450) and UDP-glucuronosyl transferase (UGT) enzymes play in drug discovery cannot be ignored, and these enzyme systems are commonly examined during drug optimization using liver microsomes or hepatocytes. At the same time, other drug-metabolizing enzymes have a role in the metabolism of drugs and can lead to challenges in drug optimization that could be mitigated if the contributions of these enzymes were better understood. We present examples (mostly from Genentech) of five different non-P450 and non-UGT enzymes that contribute to the metabolic clearance or bioactivation of drugs and drug candidates. Aldehyde oxidase mediates a unique amide hydrolysis of GDC-0834 (N-[3-[6-[4-[(2R)-1,4-dimethyl-3-oxopiperazin-2-yl]anilino]-4-methyl-5-oxopyrazin-2-yl]-2-methylphenyl]-4,5,6,7-tetrahydro-1-benzothiophene-2-carboxamide), leading to high clearance of the drug. Likewise, the rodent-specific ribose conjugation by ADP-ribosyltransferase leads to high clearance of an interleukin-2-inducible T-cell kinase inhibitor. Metabolic reactions by flavin-containing monooxygenases (FMO) are easily mistaken for P450-mediated metabolism such as oxidative defluorination of 4-fluoro-N-methylaniline by FMO. Gamma-glutamyl transpeptidase is involved in the initial hydrolysis of glutathione metabolites, leading to formation of proximate toxins and nephrotoxicity, as is observed with cisplatin in the clinic, or renal toxicity, as is observed with efavirenz in rodents. Finally, cathepsin B is a lysosomal enzyme that is highly expressed in human tumors and has been targeted to release potent cytotoxins, as in the case of brentuximab vedotin. These examples of non-P450- and non-UGT-mediated metabolism show that a more complete understanding of drug metabolizing enzymes allows for better insight into the fate of drugs and improved design strategies of molecules in drug discovery. PMID:27117704

  11. Physiological Studies of Methane- and Methanol-Oxidizing Bacteria: Comparison of a Primary Alcohol Dehydrogenase from Methylococcus capsulatus (Texas Strain) and Pseudomonas Species M27

    PubMed Central

    Patel, R. N.; Bose, H. R.; Mandy, W. J.; Hoare, D. S.

    1972-01-01

    A primary alcohol dehydrogenase has been purified from Methylococcus capsulatus (Texas strain). The purified enzyme catalyzes the oxidation of methanol and formaldehyde to formate; other primary alcohols are oxidized to their corresponding aldehydes. Ammonium ions are required for enzyme activity. The enzyme has a molecular weight of 120,000 daltons and consists of two 62,000 molecular-weight subunits which dissociate at acidic pH. The enzyme is similar to an alcohol dehydrogenase enzyme isolated from Pseudomonas sp. M27. Images PMID:5022170

  12. Structural Studies of the Final Enzyme in the alpha-Aminoadipate Pathway-Saccharopine Dehydrogenase from Saccharomyces cerevisiae

    SciTech Connect

    Burk,D.; Hwang, J.; Kwok, E.; Marrone, L.; Goodfellow, V.; Dmitrienko, G.; Berghuis, A.

    2007-01-01

    The 1.64 Angstroms structure of the apoenzyme form of saccharopine dehydrogenase (SDH) from Saccharomyces cerevisiae shows the enzyme to be composed of two domains with similar dinucleotide binding folds with a deep cleft at the interface. The structure reveals homology to alanine dehydrogense, despite low primary sequence similarity. A model of the ternary complex of SDH, NAD, and saccharopine identifies residues Lys77 and Glu122 as potentially important for substrate binding and/or catalysis, consistent with a proton shuttle mechanism. Furthermore, the model suggests that a conformational change is required for catalysis and that residues Lys99 and Asp281 may be instrumental in mediating this change. Analysis of the crystal structure in the context of other homologous enzymes from pathogenic fungi and human sources sheds light into the suitability of SDH as a target for antimicrobial drug development.

  13. Structural and Functional Insights into (S)-Ureidoglycolate Dehydrogenase, a Metabolic Branch Point Enzyme in Nitrogen Utilization

    PubMed Central

    Kim, Myung-Il; Shin, Inchul; Cho, Suhee; Lee, Jeehyun; Rhee, Sangkee

    2012-01-01

    Nitrogen metabolism is one of essential processes in living organisms. The catabolic pathways of nitrogenous compounds play a pivotal role in the storage and recovery of nitrogen. In Escherichia coli, two different, interconnecting metabolic routes drive nitrogen utilization through purine degradation metabolites. The enzyme (S)-ureidoglycolate dehydrogenase (AllD), which is a member of l-sulfolactate dehydrogenase-like family, converts (S)-ureidoglycolate, a key intermediate in the purine degradation pathway, to oxalurate in an NAD(P)-dependent manner. Therefore, AllD is a metabolic branch-point enzyme for nitrogen metabolism in E. coli. Here, we report crystal structures of AllD in its apo form, in a binary complex with NADH cofactor, and in a ternary complex with NADH and glyoxylate, a possible spontaneous degradation product of oxalurate. Structural analyses revealed that NADH in an extended conformation is bound to an NADH-binding fold with three distinct domains that differ from those of the canonical NADH-binding fold. We also characterized ligand-induced structural changes, as well as the binding mode of glyoxylate, in the active site near the NADH nicotinamide ring. Based on structural and kinetic analyses, we concluded that AllD selectively utilizes NAD+ as a cofactor, and further propose that His116 acts as a general catalytic base and that a hydride transfer is possible on the B-face of the nicotinamide ring of the cofactor. Other residues conserved in the active sites of this novel l-sulfolactate dehydrogenase-like family also play essential roles in catalysis. PMID:23284870

  14. Conformational Change Near the Redox Center of Dihydrolipoamide Dehydrogenase Induced by NAD(+) to Regulate the Enzyme Activity.

    PubMed

    Fukamichi, Tomoe; Nishimoto, Etsuko

    2015-05-01

    Dihydrolipoamide dehydrogenase (LipDH) transfers two electrons from dihydrolipoamide (DHL) to NAD(+) mediated by FAD. Since this reaction is the final step of a series of catalytic reaction of pyruvate dehydrogenase multi-enzyme complex (PDC), LipDH is a key enzyme to maintain the fluent metabolic flow. We reported here the conformational change near the redox center of LipDH induced by NAD(+) promoting the access of the DHL to FAD. The increase in the affinity of DHL to redox center was evidenced by the decrease in K M responding to the increase in the concentration of NAD(+) in Lineweaver-Burk plots. The fluorescence intensity of FAD transiently reduced by the addition of DHL was not recovered but rather reduced by the binding of NAD(+) with LipDH. The fluorescence decay lifetimes of FAD and Trp were prolonged in the presence of NAD(+) to show that FAD would be free from the electron transfer from the neighboring Tyrs and the resonance energy transfer efficiency between Trp and FAD lowered. These results consistently reveal that the conformation near the FAD and the surroundings would be so rearranged by NAD(+) to allow the easier access of DHL to the redox center of LipDH. PMID:25757537

  15. N-acylethanolamines as novel alcohol dehydrogenase 3 substrates.

    PubMed

    Ivkovic, Milena; Dempsey, Daniel R; Handa, Sumit; Hilton, Joshua H; Lowe, Edward W; Merkler, David J

    2011-02-15

    N-acylethanolamines (NAEs) are members of the fatty acid amide family. The NAEs have been proposed to serve as metabolic precursors to N-acylglycines (NAGs). The sequential oxidation of the NAEs by an alcohol dehydrogenase and an aldehyde dehydrogenase would yield the N-acylglycinals and/or the NAGs. Alcohol dehydrogenase 3 (ADH3) is one enzyme that might catalyze this reaction. To define a potential role for ADH3 in NAE catabolism, we synthesized a set of NAEs and evaluated these as ADH3 substrates. NAEs were oxidized by ADH3, yielding the N-acylglycinals as the product. The (V/K)(app) values for the NAEs included here were low relative to cinnamyl alcohol. Our data show that the NAEs can serve as alcohol dehydrogenase substrates. PMID:21144815

  16. Tryptophan in Alcoholism Treatment II:  Inhibition of the Rat Liver Mitochondrial Low Km Aldehyde Dehydrogenase Activity, Elevation of Blood Acetaldehyde Concentration and Induction of Aversion to Alcohol by Combined Administration of Tryptophan and Benserazide

    PubMed Central

    Badawy, Abdulla A.-B.; Bano, Samina; Steptoe, Alex

    2011-01-01

    Aims: The aims were to provide proofs of mechanism and principle by establishing the ability of the amino acid L-tryptophan (Trp) combined with the kynureninase inhibitor benserazide (BSZ) to inhibit the liver mitochondrial low Km aldehyde dehydrogenase (ALDH) activity after administration and in vivo and to induce aversion to alcohol. Methods: Trp, BSZ or both were administered to male Wistar rats and ALDH activity was determined both in vitro in liver homogenates and in vivo (by measuring acetaldehyde accumulation in blood after ethanol administration). Alcohol consumption was studied in an aversion model in rats and in alcohol-preferring C57 mice. Results: Combined administration of Trp + BSZ, but neither compound alone, produced a strong inhibition of ALDH activity and an increase in blood acetaldehyde concentration after ethanol, and induced aversion to alcohol in rats and decreased preference in mice. Another kynureninase inhibitor, carbidopa, induced aversion to alcohol by itself, which was reversed by Trp co-administration. Conclusions: The present results establish a prior art for the use of a combination of Trp plus BSZ in the treatment of alcoholism by aversion, which merits rapid clinical development. PMID:21896551

  17. Synergetic bitherapy in mice with xenografts of human prostate cancer using a methional mimic (METLICO) and an aldehyde dehydrogenase 3 inhibitor (MATE): systemic intraperitoneal (IP) and targeted intra-tumoral (IT) administration.

    PubMed

    Hiltbrand, E; Fournet, G; Giliberto, J-P; Paret, M-J; Chantepie, J; Morel, D; Goré, J; Reichert, U; Mehier, H; Rochedix, M-E; Quash, G

    2009-01-01

    An intraperitoneal (IP) monotherapy in nu/nu mice with subcutaneous xenografts of a human prostate epithelial cancer cell line:DU145 was undertaken with an aldehyde dehydrogenase 3 inhibitor MATE, that is a potent apoptogen on (DU145) in culture but not on their human prostate epithelial normal counterparts [13] . Tumour growth was slowed down but treatment had to be done 5days/week. To try to potentiate the action of MATE in vivo, a bitherapy was undertaken based on the synergetic apoptotic effect that had been observed previously in culture on DU145 treated with a methional mimic METLICO and DIMATE, an inhibitor of ALDH1 and ALDH3 [19]. The bitherapy with METLICO/MATE administered IP was as effective as the monotherapy with MATE alone by IP, but at a 2-fold lower dose of MATE and at a dose of METLICO that had no growth-inhibitory effect as a monotherapy . Hence there was definite synergism with bitherapy. To try to increase the efficacy of bitherapy, it was administered by the intra-tumoral (IT) route using the recently developed 20-bars-pressurized microinjection system from CERMA [16, 17]. IT administration of the bitherapy was indeed more effective than that by IP as regards tumour volumes are concerned. Histopathological analysis of IT-treated tumours confirmed that there were many necrotized zones but intact cells were still present. Approaches for treating a wider zone of tumour tissue by IT-bitherapy are discussed. PMID:19355878

  18. The preparation of nylon-tube-supported hexokinase and glucose 6-phosphate dehydrogenase and the use of the co-immobilized enzymes in the automated determination of glucose.

    PubMed Central

    Morris, D L; Campbell, J; Hornby, W E

    1975-01-01

    Triethyloxonium tetrafluoroborate was used to O-alkylate nylon-tube thus producing the imidate salt of the nylon which was further made to react with 1,6-diaminohexane. 2. Hexokinase (EC 2.7.1.1) and glucose 6-phosphate dehydrogenase (EC 1.1.1.49) were immobilized on the amino-substituted nylon tube through glutaraldeyde and bisimidates. 3. The effect of varying the conditions of O-alkylation and the amount of enzyme immobilized on the activity of nylon tube-hexokinase derivatives was determined. 4. The effect of varying the amount of enzyme immobilized on the activity of nylon-tube-glucose 6-phosphate dehydrogenase derivatives was determined. 5. The thermal stability of nylon-tube-hexokinase and nylon-tube-glucose 6-phosphate dehydrogenase derivatives was studied. 6. Different ratios of hexokinase and glucose 6-phosphate dehydrogenase were co-immobilized on nylon tube, and the rate of conversion of glucose into 6-phosphogluconolactone was compared with the individual activities of the immobilized enzymes. 7. Hexokinase and glucose 6-phosphate dehydrogenase co-immobilized on nylon tube were used in the automated analysis of glucose. PMID:1167161

  19. 20-alpha-Hydroxysteroid dehydrogenase from pseudopregnant rat ovary: obtention and characterization of a monoclonal antibody against the enzyme activity.

    PubMed

    De La Llosa-Hermier, M P; Nocart, M; Paly, J; Hermier, C

    1992-12-01

    The enzyme 20-alpha-hydroxysteroid dehydrogenase (20-alpha-HSD) was purified from pseudopregnant rat ovaries and used as antigen for the development of a monoclonal antibody by the hybridoma technique. Spleen cells of BALB/c mice immunized with purified 20-alpha-HSD were fused with SP2/0 mouse myeloma cells. Among the colonies of hybrid cells, one (designated mAb-HSD 11) was found to be secreting antibodies (IgM) able to inhibit 20-alpha-HSD activity. The antibody-secreting hybridome was amplified by ascitic fluid production and the monoclonal antibody purified by Bakerbond ABx procedure. Purified mAb-HSD 11 was able to inhibit 20-alpha-HSD activity in a dose-dependent manner. Studies of Michaelis constants of 20-alpha-HSD indicate that this monoclonal antibody increases the Km for 20-alpha-dihydroprogesterone and decreases the Vmax. PMID:1292619

  20. Diverse point mutations in the human glucose-6-phosphate dehydrogenase gene cause enzyme deficiency and mild or severe hemolytic anemia

    SciTech Connect

    Vulliamy, T.J.; D'Urso, M.; Battistuzzi, G.; Estrada, M.; Foulkes, N.S.; Martini, G.; Calabro, V.; Poggi, V.; Giordano, R.; Town, M.; Luzzatto, L.; Persico, M.G. )

    1988-07-01

    Glucose-6-phosphate dehydrogenase deficiency is a common genetic abnormality affecting an estimated 400 million people worldwide. Clinical and biochemical analyses have identified many variants exhibiting a range of phenotypes, which have been well characterized from the hematological point of view. However, until now, their precise molecular basis has remained unknown. The authors have cloned and sequenced seven mutant G6PD alleles. In the nondeficient polymorphic African variant G6PD A they have found a single point mutation. The other six mutants investigated were all associated with enzyme deficiency. The mutations observed show a striking predominance of C {yields} T transitions, with CG doublets involved in four of seven cases. Thus, diverse point mutations may account largely for the phenotypic heterogeneity of G6PD deficiency.

  1. Chloroplast NDH: A different enzyme with a structure similar to that of respiratory NADH dehydrogenase.

    PubMed

    Shikanai, Toshiharu

    2016-07-01

    Eleven genes encoding chloroplast NADH dehydrogenase-like (NDH) complex have been discovered in plastid genomes on the basis of their homology with genes encoding respiratory complex I. Despite this structural similarity, chloroplast NDH and its evolutionary origin NDH-1 in cyanobacteria accept electrons from ferredoxin (Fd), indicating that chloroplast NDH is an Fd-dependent plastoquinone (PQ) reductase rather than an NAD(P)H dehydrogenase. In Arabidopsis thaliana, chloroplast NDH interacts with photosystem I (PSI); this interaction is needed to stabilize NDH, especially under high light. On the basis of these distinct characters of chloroplast and cyanobacterial NDH, it can be distinguished as a photosynthetic NDH from respiratory complex I. In fact, chloroplast NDH forms part of the machinery of photosynthesis by mediating the minor pathway of PSI cyclic electron transport. Along with the antimycin A-sensitive main pathway of PSI cyclic electron transport, chloroplast NDH compensates the ATP/NADPH production ratio in the light reactions of photosynthesis. In this review, I revisit the original concept of chloroplast NDH on the basis of its similarity to respiratory complex I and thus introduce current progress in the field to researchers focusing on respiratory complex I. I summarize recent progress on the basis of structure and function. Finally, I introduce the results of our examination of the process of assembly of chloroplast NDH. Although the process requires many plant-specific non-subunit factors, the core processes of assembly are conserved between chloroplast NDH and respiratory complex I. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt. PMID:26519774

  2. Benzene toxicity: emphasis on cytosolic dihydrodiol dehydrogenases

    SciTech Connect

    Bolcsak, L.E.

    1982-01-01

    Blood dyscrasias such as leukopenia and anemia have been clearly identified as consequences of chronic benzene exposure. The metabolites, phenol, catechol, and hydroquinone produced inhibition of /sup 59/Fe uptake in mice which followed the same time course as that produced by benzene. The inhibitor of benzene oxidation, 3-amino-1,2,4-triazole, mitigated the inhibitory effects of benzene and phenol only. These data support the contention that benzene toxicity is mediated by a metabolite and suggest that the toxicity of phenol is a consequence of its metabolism to hydroquinone and that the route of metabolism to catechol may also contribute to the production of toxic metabolite(s). The properties of mouse liver cytosolic dihydrodiol dehydrogenases were examined. These enzymes catalyze the NADP/sup +/-dependent oxidation of trans-1,2-dihydro-1,2-dihydroxybenzene (BDD) to catechol, a possible toxic metabolite of benzene produced via this metabolic route. Four distinct dihydrodiol dehydrogenases (DD1, DD2, DD3, and DD4) were purified to apparent homogeneity as judged by SDS polyacrylamide gel electrophoresis and isoelectric focusing. DD1 appeared to be identical to the major ketone reductase and 17..beta..-hydroxysteroid dehydrogenase activity in the liver. DD2 exhibited aldehyde reductase activity. DD3 and DD4 oxidized 17..beta..-hydroxysteroids, but no carbonyl reductase activity was detected. These relationships between BDD dehydrogenases and carbonyl reductase and/or 17..beta..-hydroxysteroid dehydrogenase activities were supported by several lines of evidence.

  3. Antibodies to inactive conformations of glyceraldehyde-3-phosphate dehydrogenase inactivate the apo- and holoforms of the enzyme.

    PubMed

    Arutiunova, E I; Pleten, A P; Nagradova, N K; Muronetz, V I

    2006-06-01

    Polyclonal antibodies produced after the immunization of a rabbit with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus were used to isolate two types of antibodies interacting with different non-native forms of the antigen. Type I antibodies were purified using Sepharose-bound apo-GAPDH that was treated with glutaraldehyde to stabilize the enzyme in the tetrameric form. Type II antibodies were isolated using immobilized denatured monomers of the enzyme. It was shown that the type I antibodies bound to the native holo- and apoforms of the enzyme with the ratio of one antibody molecule per GAPDH tetramer. While interacting with the native holoenzyme, the type I antibodies induce a time-dependent decrease in its activity by 80-90%. In the case of the apoenzyme, the decrease in the activity constitutes only 25%, this indicating that only one subunit of the tetramer is inactivated. Differential scanning calorimetry experiments showed that the formation of the complex between both forms of the enzyme and the type I antibodies resulted in a shift of the maximum of the thermal capacity curves (T(m) value) to lower temperatures. The extremely stable holoenzyme was affected to the greatest extent, the shift of the T(m) value constituting approximately 20 degrees C. We assume that the formation of the complex between the holo- or apo-GAPDH and the type I antibody results in time-dependent conformational changes in the enzyme molecule. Thus, the antibodies induce the structural rearrangements yielding the conformation that is identical to the structure of the antigen used for the selection of the antibodies (i.e., inactive). The interaction of the antibodies with the apo-GAPDH results in the inactivation of the subunit directly bound to the antibody. Virtually complete inactivation of the holoenzyme by the antibodies is likely due to the transmission of the conformational changes through the intersubunit contacts. The type II antibodies, which

  4. Purification of glucose-6-phosphate dehydrogenase and glutathione reductase enzymes from the gill tissue of Lake Van fish and analyzing the effects of some chalcone derivatives on enzyme activities.

    PubMed

    Kuzu, Muslum; Aslan, Abdulselam; Ahmed, Ishtiaq; Comakli, Veysel; Demirdag, Ramazan; Uzun, Naim

    2016-04-01

    Glucose-6-phosphate dehydrogenase (G6PD) and glutathione reductase (GR) are metabolically quite important enzymes. Within this study, these two enzymes were purified for the first time from the gills of Lake Van fish. In the purifying process, ammonium sulfate precipitation and 2',5'-ADP Sepharose 4B affinity column chromatography techniques for glucose-6-phosphate dehydrogenase, temperature degradation and 2',5'-ADP Sepharose 4B affinity column chromatography for glutathione reductase enzyme were used. The control of the enzyme purity and determination of molecular weight were done with sodium dodecyl sulfate polyacrylamide gel electrophoresis. K M and V max values were determined with Lineweaver-Burk plot. Besides, the effects of some chalcone derivatives on the purified enzymes were analyzed. For the ones showing inhibition effect, % activity-[I] figures were drawn and IC50 values were determined. K i value was calculated by using Cheng-Prusoff equation. PMID:26676512

  5. Chromate reduction by rabbit liver aldehyde oxidase

    SciTech Connect

    Banks, R.B.; Cooke, R.T. Jr.

    1986-05-29

    Chromate was reduced during the oxidation of 1-methylnicotinamide chlorine by partially purified rabbit liver aldehyde oxidase. In addition to l-methylnicotinamide, several other electron donor substrates for aldehyde oxidase were able to support the enzymatic chromate reduction. The reduction required the presence of both enzyme and the electron donor substrate. The rate of the chromate reduction was retarded by inhibitors or aldehyde oxidase but was not affected by substrates or inhibitors of xanthine oxidase. These results are consistent with the involvement of aldehyde oxidase in the reduction of chromate by rabbit liver cytosolic enzyme preparations.

  6. Multiple forms of octopine dehydrogenase in Strombus luhuanus (mollusca, gastropoda, strombidae): genetic basis of polymorphism, properties of the enzymes, and relationship between the octopine dehydrogenase phenotype and the accumulation of anaerobic end products during exercise.

    PubMed

    Baldwin, J; England, W R

    1982-10-01

    Octopine dehydrogenase (ODH) is electrophoretically polymorphic in the gastropod mollusk Strombus luhuanus. The frequencies of the six electrophoretic phenotypes in the Heron Island population, together with the molecular weight values of 38,000 obtained for each of the three forms of the enzyme, demonstrate that the monomeric enzyme is encoded by three codominant alleles at a single locus. The purified allozymes are indistinguishable in terms of Km values for substrates, product inhibition by octopine and NAD, pH optima, and substrate inhibition by pyruvate. No statistically significant correlations were found between the ODH phenotype and the maximum activities of ODH or alanopine dehydrogenase, the capacity for anaerobic muscle work, or the accumulation of octopine or strombine/alanopine during exercise. It would appear that the ODH allozymes may be functionally equivalent both in vitro and in vivo. PMID:7181845

  7. Harnessing functional plasticity of enzymes: a fluorogenic probe for imaging 17beta-HSD10 dehydrogenase, an enzyme involved in Alzheimer's and Parkinson's diseases.

    PubMed

    Froemming, Mary K; Sames, Dalibor

    2007-11-21

    In this paper, we describe the development of a fluorogenic substrate for 17beta-hydroxysteroid-dehydrogenase type 10 (17beta-HSD10), which is a multifunctional metabolic enzyme fulfilling several metabolic roles (beta-oxidation of fatty acids, catabolism of isoleucine, and metabolism of steroids). In recent years, it has emerged as an important stress and pathological marker in neurons and glial cells (expression down-regulation in Parkinson's disease, up-regulation and association with beta-amyloid peptide in Alzheimer's disease). Through the iterative molecular design and chemical synthesis described herein, compound 1 was developed, which possesses all required properties for a selective optical reporter substrate: alcohol-ketone optical switching, the ability to function as a good enzyme substrate (expressed in kinetic parameters), cell permeability, and cell retention. Probe 1 provides a blue-to-green/yellow bright switch and enables non-invasive, real-time imaging of 17beta-HSD10 in live human cells. The selectivity of reporter 1 was established by the quantitative correlation of metabolic activity to protein expression in human kidney cell line HEK-293T. PMID:17958419

  8. GRE2 from Scheffersomyces stipitis as an aldehyde reductase contributes tolerance to aldehyde inhibitors derived from lignocellulosic biomass.

    PubMed

    Wang, Xu; Ma, Menggen; Liu, Z Lewis; Xiang, Quanju; Li, Xi; Liu, Na; Zhang, Xiaoping

    2016-08-01

    Scheffersomyces (Pichia) stipitis is one of the most promising yeasts for industrial bioethanol production from lignocellulosic biomass. S. stipitis is able to in situ detoxify aldehyde inhibitors (such as furfural and 5-hydroxymethylfurfural (HMF)) to less toxic corresponding alcohols. However, the reduction enzymes involved in this reaction remain largely unknown. In this study, we reported that an uncharacterized open reading frame PICST_72153 (putative GRE2) from S. stipitis was highly induced in response to furfural and HMF stresses. Overexpression of this gene in Saccharomyces cerevisiae improved yeast tolerance to furfural and HMF. GRE2 was identified as an aldehyde reductase which can reduce furfural to FM with either NADH or NADPH as the co-factor and reduce HMF to FDM with NADPH as the co-factor. This enzyme can also reduce multiple aldehydes to their corresponding alcohols. Amino acid sequence analysis indicated that it is a member of the subclass "intermediate" of the short-chain dehydrogenase/reductase (SDR) superfamily. Although GRE2 from S. stipitis is similar to GRE2 from S. cerevisiae in a three-dimensional structure, some differences were predicted. GRE2 from S. stipitis forms loops at D133-E137 and T143-N145 locations with two α-helices at E154-K157 and E252-A254 locations, different GRE2 from S. cerevisiae with an α-helix at D133-E137 and a β-sheet at T143-N145 locations, and two loops at E154-K157 and E252-A254 locations. This research provided guidelines for the study of other SDR enzymes from S. stipitis and other yeasts on tolerant mechanisms to aldehyde inhibitors derived from lignocellulosic biomass. PMID:27003269

  9. Enzymic assay of creatinine in serum and urine with creatinine iminohydrolase and glutamate dehydrogenase.

    PubMed

    Tanganelli, E; Prencipe, L; Bassi, D; Cambiaghi, S; Murador, E

    1982-07-01

    We describe an assay for creatinine in which it is converted by creatinine iminohydrolase (EC 3.5.4.21) into ammonia and N-methylhydantoin. The ammonia is subsequently assayed by use of alpha-ketoglutarate and glutamate dehydrogenase (EC 1.4.1.3). Use of NADPH as coenzyme eliminates all interferences from endogenous reactions. Endogenous ammonia in the sample is eliminated during a preincubation. The reaction reaches the endpoint in 15 min at working temperatures of 20-37 degrees C. No sample blank or reagent blank is needed. The standard curve is linear at least to 884 mumol (100 mg) of creatinine per liter. Average analytical recovery of creatinine in serum and urine is 99%. Within-run and between-run CVs are less than or equal to 2% and less than or equal to 6% for creatinine values of 335 mumol/L (38 mg/L) and 80 mumol/L (0 mg/L), respectively. Results by the described method (y) compare well with those by Jaffé's kinetic test (y = 1.01x -- 12.8), Berthelot/AutoAnalyzer method after treatment with immobilized creatinine iminohydrolase (y = 0.987x -- 13.2), Jaffé's test run on the SMA 12/60 (y = 1.011x -- 5.8), the Wahlefeld method (y = 1.014x -- 0.88), and Jaffé's test after deproteinization and absorption on fuller's earth (y = 0.985x -- 3.08). The method may be suitable for discrete, including centrifugal, automation. PMID:7083556

  10. Gene Expression Profiling of NF1-Associated and Sporadic Pilocytic Astrocytoma Identifies Aldehyde Dehydrogenase 1 Family, Member L1 (ALDH1L1) as an Underexpressed Candidate Biomarker in Aggressive Subtypes

    PubMed Central

    Rodriguez, Fausto J.; Giannini, Caterina; Asmann, Yan W.; Sharma, Mukesh K.; Perry, Arie; Tibbetts, Kathleen M.; Jenkins, Robert B.; Scheithauer, Bernd W.; Anant, Shrikant; Jenkins, Sarah; Eberhart, Charles G.; Sarkaria, Jann N.; Gutmann, David H.

    2009-01-01

    Pilocytic astrocytomas (PAs) are WHO grade I gliomas; they most often affect children and young adults and occur in patients with neurofibromatosis type 1 (NF-1). To identify genes that are differentially expressed in sporadic (S-PA) versus NF-1-associated PAs (NF1-PAs) and those that might reflect differences in clinical behavior, we performed gene expression profiling using Affymetrix U133 Plus2.0 GeneChip arrays in 36 S-PAs and 11 NF1-PAs. Thirteen genes were over-expressed and another 13 genes were under-expressed in NF1-associated PAs relative to S-PAs. Immunohistochemical studies performed on 103 tumors, representing 2 independently generated tissue microarrays, confirmed the differential expression of CUGBP2 (p = 0.0014), RANBP9 (p = 0.0075), ITGAV1 (p = 0.0001), and INFGR1 (p = 0.024) proteins. One of the underexpressed genes, aldehyde dehydrogenase 1 family, member L1 (ALDH1L1), was also reduced in clinically aggressive compared to typical PAs (p = 0.01) and in PAs with increased cellularity and necrosis. Furthermore, in an additional independent set of tumors, weak to absent ALDH1L1 expression was found in 13/18 (72%) clinically aggressive PAs, in 8/9 (89%) PAs with pilomyxoid features, in 7/10 (70%) PAs with anaplastic transformation and in 16/21 (76%) diffusely infiltrating astrocytomas of various grades. In summary, we have identified a molecular signature that distinguishes NF1-PA from S-PA, and found that ALDH1L1 underexpression is associated with aggressive histology and/or biological behavior. PMID:19018242

  11. Essential role of aldehyde dehydrogenase 1A3 (ALDH1A3) for the maintenance of non-small cell lung cancer stem cells is associated with the STAT3 pathway

    PubMed Central

    Shao, Chunli; Sullivan, James P.; Girard, Luc; Augustyn, Alexander; Yenerall, Paul; Rodriguez, Jaime; Liu, Hui; Behrens, Carmen; Shay, Jerry W.; Wistuba, Ignacio I.; Minna, John D.

    2014-01-01

    Purpose Lung cancer stem cells (CSCs) with elevated aldehyde dehydrogenase (ALDH) activity are self-renewing, clonogenic and tumorigenic. The purpose of our study is to elucidate the mechanisms by which lung CSCs are regulated. Experimental Design A genome-wide gene expression analysis was performed to identify genes differentially expressed in the ALDH+ vs. ALDH− cells. RT-PCR, western blot and Aldefluor assay were used to validate identified genes. To explore the function in CSCs we manipulated their expression followed by colony and tumor formation assays. Results We identified a subset of genes that were differentially expressed in common in ALDH+ cells, among which ALDH1A3 was the most upregulated gene in ALDH+ vs. ALDH− cells. ShRNA-mediated knockdown of ALDH1A3 in NSCLCs resulted in a dramatic reduction in ALDH activity, clonogenicity and tumorigenicity, indicating that ALDH1A3 is required for tumorigenic properties. By contrast, overexpression of ALDH1A3 by itself it was not sufficient to increase tumorigenicity. The ALDH+ cells also expressed more activated Signal Transducers and Activators of Transcription 3 (STAT3) than ALDH− cells. Inhibition of STAT3 or its activator EZH2 genetically or pharmacologically diminished the level of ALDH+ cells and clonogenicity. Unexpectedly, ALDH1A3 was highly expressed in female, never smokers, well differentiated tumors, or adenocarcinoma. ALDH1A3 low expression was associated with poor overall survival. Conclusion Our data show that ALDH1A3 is the predominant ALDH isozyme responsible for ALDH activity and tumorigenicity in most NSCLCs, and that inhibiting either ALDH1A3 or the STAT3 pathway are potential therapeutic strategies to eliminate the ALDH+ subpopulation in NSCLCs. PMID:24907115

  12. Novel mutations in 3-phosphoglycerate dehydrogenase (PHGDH) are distributed throughout the protein and result in altered enzyme kinetics.

    PubMed

    Tabatabaie, L; de Koning, T J; Geboers, A J J M; van den Berg, I E T; Berger, R; Klomp, L W J

    2009-05-01

    Three-phosphoglycerate dehydrogenase (3-PGDH) deficiency is a rare recessive inborn error in the biosynthesis of the amino acid L-serine characterized clinically by congenital microcephaly, psychomotor retardation, and intractable seizures. The biochemical abnormalities associated with this disorder are low concentrations of L-serine, D-serine, and glycine in cerebrospinal fluid (CSF). Only two missense mutations (p.V425M and p.V490M) have been identified in PHGDH, the gene encoding 3-PGDH, but it is currently unclear how these mutations in the carboxy-terminal regulatory domain of the protein affect enzyme function. We now describe five novel mutations in five patients with 3-PGDH deficiency; one frameshift mutation (p.G238fsX), and four missense mutations (p.R135W, p.V261M, p.A373T, and p.G377S). The missense mutations were located in the nucleotide binding and regulatory domains of 3-PGDH and did not affect steady-state expression, protein stability, and protein degradation rates. Patients' fibroblasts displayed a significant, but incomplete, reduction in maximal enzyme activities associated with all missense mutations. In transient overexpression studies in HEK293T cells, the p.A373T, p.V425M, and p.V490M mutations resulted in almost undetectable enzyme activities. Molecular modeling of the p.R135W and p.V261M mutations onto the partial crystal structure of 3-PGDH predicted that these mutations affect substrate and cofactor binding. This prediction was confirmed by the results of kinetic measurements in fibroblasts and transiently transfected HEK293T cells, which revealed a markedly decreased V(max) and an increase in K(m) values, respectively. Taken together, these data suggest that missense mutations associated with 3-PGDH deficiency either primarily affect substrate binding or result in very low residual enzymatic activity. PMID:19235232

  13. Microsphere coated substrate containing reactive aldehyde groups

    NASA Technical Reports Server (NTRS)

    Rembaum, Alan (Inventor); Yen, Richard C. K. (Inventor)

    1984-01-01

    A synthetic organic resin is coated with a continuous layer of contiguous, tangential, individual microspheres having a uniform diameter preferably between 100 Angstroms and 2000 Angstroms. The microspheres are an addition polymerized polymer of an unsaturated aldehyde containing 4 to 20 carbon atoms and are covalently bonded to the substrate by means of high energy radiation grafting. The microspheres contain reactive aldehyde groups and can form conjugates with proteins such as enzymes or other aldehyde reactive materials.

  14. Genetics Home Reference: lactate dehydrogenase deficiency

    MedlinePlus

    ... dehydrogenase-B pieces (subunits) of the lactate dehydrogenase enzyme. This enzyme is found throughout the body and is important ... cells. There are five different forms of this enzyme, each made up of four protein subunits. Various ...

  15. Plant Formate Dehydrogenase

    SciTech Connect

    John Markwell

    2005-01-10

    The research in this study identified formate dehydrogenase, an enzyme that plays a metabolic role on the periphery of one-carbon metabolism, has an unusual localization in Arabidopsis thaliana and that the enzyme has an unusual kinetic plasticity. These properties make it possible that this enzyme could be engineered to attempt to engineer plants with an improved photosynthetic efficiency. We have produced transgenic Arabidopsis and tobacco plants with increased expression of the formate dehydrogenase enzyme to initiate further studies.

  16. Oxidation of cinnamyl alcohols and aldehydes by a basic peroxidase from lignifying Zinnia elegans hypocotyls.

    PubMed

    Barceló, A R; Pomar, F

    2001-08-01

    The xylem of 26-day old Zinnia elegans hypocotyls synthesizes lignins derived from coniferyl alcohol and sinapyl alcohol with a G/S ratio of 43/57 in the aryl-glycerol-beta-aryl ether core, as revealed by thioacidolysis. Thioacidolysis of Z. elegans lignins also reveals the presence of coniferyl aldehyde end groups linked by beta-0-4 bonds. Both coniferyl and sinapyl alcohols, as well as coniferyl and sinapyl aldehyde, are substrates of a xylem cell wall-located strongly basic peroxidase, which is capable of oxidizing them in the absence and in the presence of hydrogen peroxide. This peroxidase shows a particular affinity for cinnamyl aldehydes with kappa(M) values in the mu(M) range, and some specificity for syringyl-type phenols. The affinity of this strongly basic peroxidase for cinnamyl alcohols and aldehydes is similar to that shown by the preceding enzymes in the lignin biosynthetic pathway (microsomal 5-hydroxylases and cinnamyl alcohol dehydrogenase), which also use cinnamyl alcohols and aldehydes as substrates, indicating that the one-way highway of construction of the lignin macromolecule has no metabolic "potholes" in which the lignin building blocks might accumulate. This fact suggests a high degree of metabolic plasticity for this basic peroxidase, which has been widely conserved during the evolution of vascular plants, making it one of the driving forces in the evolution of plant lignin heterogeneity. PMID:11430983

  17. Structure and function of mammalian aldehyde oxidases.

    PubMed

    Terao, Mineko; Romão, Maria João; Leimkühler, Silke; Bolis, Marco; Fratelli, Maddalena; Coelho, Catarina; Santos-Silva, Teresa; Garattini, Enrico

    2016-04-01

    Mammalian aldehyde oxidases (AOXs; EC1.2.3.1) are a group of conserved proteins belonging to the family of molybdo-flavoenzymes along with the structurally related xanthine dehydrogenase enzyme. AOXs are characterized by broad substrate specificity, oxidizing not only aromatic and aliphatic aldehydes into the corresponding carboxylic acids, but also hydroxylating a series of heteroaromatic rings. The number of AOX isoenzymes expressed in different vertebrate species is variable. The two extremes are represented by humans, which express a single enzyme (AOX1) in many organs and mice or rats which are characterized by tissue-specific expression of four isoforms (AOX1, AOX2, AOX3, and AOX4). In vertebrates each AOX isoenzyme is the product of a distinct gene consisting of 35 highly conserved exons. The extant species-specific complement of AOX isoenzymes is the result of a complex evolutionary process consisting of a first phase characterized by a series of asynchronous gene duplications and a second phase where the pseudogenization and gene deletion events prevail. In the last few years remarkable advances in the elucidation of the structural characteristics and the catalytic mechanisms of mammalian AOXs have been made thanks to the successful crystallization of human AOX1 and mouse AOX3. Much less is known about the physiological function and physiological substrates of human AOX1 and other mammalian AOX isoenzymes, although the importance of these proteins in xenobiotic metabolism is fairly well established and their relevance in drug development is increasing. This review article provides an overview and a discussion of the current knowledge on mammalian AOX. PMID:26920149

  18. Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase

    PubMed Central

    Hebbelmann, Inga; Selinski, Jennifer; Wehmeyer, Corinna; Goss, Tatjana; Voss, Ingo; Mulo, Paula; Kangasjärvi, Saijaliisa; Aro, Eva-Mari; Oelze, Marie-Luise; Dietz, Karl-Josef; Nunes-Nesi, Adriano; Do, Phuc T.; Fernie, Alisdair R.; Talla, Sai K.; Raghavendra, Agepati S.; Linke, Vera; Scheibe, Renate

    2012-01-01

    The nuclear-encoded chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme controlling the malate valve, to allow the indirect export of reducing equivalents. Arabidopsis thaliana (L.) Heynh. T-DNA insertion mutants of NADP-MDH were used to assess the role of the light-activated NADP-MDH in a typical C3 plant. Surprisingly, even when exposed to high-light conditions in short days, nadp-mdh knockout mutants were phenotypically indistinguishable from the wild type. The photosynthetic performance and typical antioxidative systems, such as the Beck–Halliwell–Asada pathway, were barely affected in the mutants in response to high-light treatment. The reactive oxygen species levels remained low, indicating the apparent absence of oxidative stress, in the mutants. Further analysis revealed a novel combination of compensatory mechanisms in order to maintain redox homeostasis in the nadp-mdh plants under high-light conditions, particularly an increase in the NTRC/2-Cys peroxiredoxin (Prx) system in chloroplasts. There were indications of adjustments in extra-chloroplastic components of photorespiration and proline levels, which all could dissipate excess reducing equivalents, sustain photosynthesis, and prevent photoinhibition in nadp-mdh knockout plants. Such metabolic flexibility suggests that the malate valve acts in concert with other NADPH-consuming reactions to maintain a balanced redox state during photosynthesis under high-light stress in wild-type plants. PMID:22140244

  19. Effects and Mechanism of Atmospheric-Pressure Dielectric Barrier Discharge Cold Plasma on Lactate Dehydrogenase (LDH) Enzyme

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Xu, Zimu; Shen, Jie; Li, Xu; Ding, Lili; Ma, Jie; Lan, Yan; Xia, Weidong; Cheng, Cheng; Sun, Qiang; Zhang, Zelong; Chu, Paul K.

    2015-05-01

    Proteins are carriers of biological functions and the effects of atmospheric-pressure non-thermal plasmas on proteins are important to applications such as sterilization and plasma-induced apoptosis of cancer cells. Herein, we report our detailed investigation of the effects of helium-oxygen non-thermal dielectric barrier discharge (DBD) plasmas on the inactivation of lactate dehydrogenase (LDH) enzyme solutions. Circular dichroism (CD) and dynamic light scattering (DLS) indicate that the loss of activity stems from plasma-induced modification of the secondary molecular structure as well as polymerization of the peptide chains. Raising the treatment intensity leads to a reduced alpha-helix content, increase in the percentage of the beta-sheet regions and random sequence, as well as gradually decreasing LDH activity. However, the structure of the LDH plasma-treated for 300 seconds exhibits a recovery trend after storage for 24 h and its activity also increases slightly. By comparing direct and indirect plasma treatments, plasma-induced LDH inactivation can be attributed to reactive species (RS) in the plasma, especially ones with a long lifetime including hydrogen peroxide, ozone, and nitrate ion which play the major role in the alteration of the macromolecular structure and molecular diameter in lieu of heat, UV radiation, and charged particles.

  20. Effects and Mechanism of Atmospheric-Pressure Dielectric Barrier Discharge Cold Plasma on Lactate Dehydrogenase (LDH) Enzyme

    PubMed Central

    Zhang, Hao; Xu, Zimu; Shen, Jie; Li, Xu; Ding, Lili; Ma, Jie; Lan, Yan; Xia, Weidong; Cheng, Cheng; Sun, Qiang; Zhang, Zelong; Chu, Paul K.

    2015-01-01

    Proteins are carriers of biological functions and the effects of atmospheric-pressure non-thermal plasmas on proteins are important to applications such as sterilization and plasma-induced apoptosis of cancer cells. Herein, we report our detailed investigation of the effects of helium-oxygen non-thermal dielectric barrier discharge (DBD) plasmas on the inactivation of lactate dehydrogenase (LDH) enzyme solutions. Circular dichroism (CD) and dynamic light scattering (DLS) indicate that the loss of activity stems from plasma-induced modification of the secondary molecular structure as well as polymerization of the peptide chains. Raising the treatment intensity leads to a reduced alpha-helix content, increase in the percentage of the beta-sheet regions and random sequence, as well as gradually decreasing LDH activity. However, the structure of the LDH plasma-treated for 300 seconds exhibits a recovery trend after storage for 24 h and its activity also increases slightly. By comparing direct and indirect plasma treatments, plasma-induced LDH inactivation can be attributed to reactive species (RS) in the plasma, especially ones with a long lifetime including hydrogen peroxide, ozone, and nitrate ion which play the major role in the alteration of the macromolecular structure and molecular diameter in lieu of heat, UV radiation, and charged particles. PMID:25992482

  1. Putative role of the malate valve enzyme NADP-malate dehydrogenase in H2O2 signalling in Arabidopsis.

    PubMed

    Heyno, Eiri; Innocenti, Gilles; Lemaire, Stéphane D; Issakidis-Bourguet, Emmanuelle; Krieger-Liszkay, Anja

    2014-04-19

    In photosynthetic organisms, sudden changes in light intensity perturb the photosynthetic electron flow and lead to an increased production of reactive oxygen species. At the same time, thioredoxins can sense the redox state of the chloroplast. According to our hypothesis, thioredoxins and related thiol reactive molecules downregulate the activity of H2O2-detoxifying enzymes, and thereby allow a transient oxidative burst that triggers the expression of H2O2 responsive genes. It has been shown recently that upon light stress, catalase activity was reversibly inhibited in Chlamydomonas reinhardtii in correlation with a transient increase in the level of H2O2. Here, it is shown that Arabidopsis thaliana mutants lacking the NADP-malate dehydrogenase have lost the reversible inactivation of catalase activity and the increase in H2O2 levels when exposed to high light. The mutants were slightly affected in growth and accumulated higher levels of NADPH in the chloroplast than the wild-type. We propose that the malate valve plays an essential role in the regulation of catalase activity and the accumulation of a H2O2 signal by transmitting the redox state of the chloroplast to other cell compartments. PMID:24591715

  2. Purification and characterization of 6-phosphogluconate dehydrogenase from the wing-polymorphic cricket, Gryllus firmus, and assessment of causes of morph-differences in enzyme activity.

    PubMed

    Zera, Anthony J; Wehrkamp, Cody; Schilder, Rudolf; Black, Christine; Gribben, Paul

    2014-01-01

    Considerable information exists on the physiological correlates of life history adaptation, while molecular data on this topic are rapidly accumulating. However, much less is known about the enzymological basis of life history adaptation in outbred populations. In the present study, we compared developmental profiles of fat body specific activity, kinetic constants of homogeneously purified and unpurified enzyme, and fat body enzyme concentration of the pentose-shunt enzyme, 6-phosphogluconate dehydrogenase (6PGDH, E.C.1.1.1.44) between the dispersing [long-winged, LW(f)] and flightless [short-winged, SW] genotypes of the cricket Gryllus firmus. Neither kcat nor the Michaelis constant for 6-phosphogluconate differed between 6PGDH from LW(f) versus SW morphs for either homogeneously purified or unpurified enzyme. Purified enzyme from the LW(f) morph exhibited reduced KM for NADP(+), but this was not observed for multiple KM(NADP+) estimates for unpurified enzyme. A polyclonal antibody was generated against 6PGDH which was used to develop a chemiluminescence assay to quantify 6PGDH concentration in fat body homogenates. Elevated enzyme concentration accounted for all of the elevated 6PGDH specific activity in the LW(f) morph during the juvenile and adult stages. Finally, activity of another pentose-shunt enzyme, glucose-6-phosphate dehydrogenase, strongly covaried with 6PGDH activity suggesting that variation in 6PGDH activity gives rise to variation in pentose shunt flux. This is one of the first life-history studies and one of the few studies of intraspecific enzyme adaptation to identify the relative importance of evolutionary change in enzyme concentration vs. kinetic constants to adaptive variation in enzyme activity in an outbred population. PMID:24726622

  3. Toward cell-free biofuel production: Stable immobilization of oligomeric enzymes.

    PubMed

    Grimaldi, J; Collins, C H; Belfort, G

    2014-01-01

    To overcome the main challenges facing alcohol-based biofuel production, we propose an alternate simplified biofuel production scheme based on a cell-free immobilized enzyme system. In this paper, we measured the activity of two tetrameric enzymes, a control enzyme with a colorimetric assay, β-galactosidase, and an alcohol-producing enzyme, alcohol dehydrogenase, immobilized on multiple surface curvatures and chemistries. Several solid supports including silica nanoparticles (convex), mesopourous silica (concave), diatomaceous earth (concave), and methacrylate (concave) were examined. High conversion rates and low protein leaching was achieved by covalent immobilization of both enzymes on methacrylate resin. Alcohol dehydrogenase (ADH) exhibited long-term stability and over 80% conversion of aldehyde to alcohol over 16 days of batch cycles. The complete reaction scheme for the conversion of acid to aldehyde to alcohol was demonstrated in vitro by immobilizing ADH with keto-acid decarboxylase free in solution. PMID:24449684

  4. Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei: characterization of the enzyme, cloning and sequencing of the gene, and expression in Escherichia coli.

    PubMed Central

    Zwickl, P; Fabry, S; Bogedain, C; Haas, A; Hensel, R

    1990-01-01

    The glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei (optimal growth temperature, 100 to 103 degrees C) was purified to homogeneity. This enzyme was strictly phosphate dependent, utilized either NAD+ or NADP+, and was insensitive to pentalenolactone like the enzyme from the methanogenic archaebacterium Methanothermus fervidus. The enzyme exhibited a considerable thermostability, with a 44-min half-life at 100 degrees C. The amino acid sequence of the glyceraldehyde-3-phosphate dehydrogenase from P. woesei was deduced from the nucleotide sequence of the coding gene. Compared with the enzyme homologs from mesophilic archaebacteria (Methanobacterium bryantii, Methanobacterium formicicum) and an extremely thermophilic archaebacterium (Methanothermus fervidus), the primary structure of the P. woesei enzyme exhibited a strikingly high proportion of aromatic amino acid residues and a low proportion of sulfur-containing residues. The coding gene of P. woesei was expressed at a high level in Escherichia coli, thus providing an ideal basis for detailed structural and functional studies of that enzyme. Images PMID:2165475

  5. Mitochondrial 3β-Hydroxysteroid Dehydrogenase Enzyme Activity Requires Reversible pH-dependent Conformational Change at the Intermembrane Space*

    PubMed Central

    Prasad, Manoj; Thomas, James L.; Whittal, Randy M.; Bose, Himangshu S.

    2012-01-01

    The inner mitochondrial membrane protein 3β-hydroxysteroid dehydrogenase 2 (3βHSD2) synthesizes progesterone and androstenedione through its dehydrogenase and isomerase activities. This bifunctionality requires 3βHSD2 to undergo a conformational change. Given its proximity to the proton pump, we hypothesized that pH influences 3βHSD2 conformation and thus activity. Circular dichroism (CD) showed that between pH 7.4 and 4.5, 3βHSD2 retained its primarily α-helical character with a decrease in α-helical content at lower pH values, whereas the β-sheet content remained unchanged throughout. Titrating the pH back to 7.4 restored the original conformation within 25 min. Metabolic conversion assays indicated peak 3βHSD2 activity at pH 4.5 with ∼2-fold more progesterone synthesized at pH 4.5 than at pH 3.5 and 7.4. Increasing the 3βHSD2 concentration from 1 to 40 μg resulted in a 7-fold increase in progesterone at pH 4.5, but no change at pH 7.4. Incubation with guanidinum hydrochloride (GdmHCl) showed a three-step cooperative unfolding of 3βHSD2 from pH 7.4 to 4.5, possibly due to the native state unfolding to the intermediate ion core state. With further decreases in pH, increasing concentrations of GdmHCl led to rapid two-step unfolding that may represent complete loss of structure. Between pH 4 and 5, the two intermediate states appeared stable. Stopped-flow kinetics showed slower unfolding at around pH 4, where the protein is in a pseudostable state. Based on our data, we conclude that at pH 4–5, 3βHSD2 takes on a molten globule conformation that promotes the dual functionality of the enzyme. PMID:22262841

  6. Aldehyde dehydrogenase and ATP binding cassette transporter G2 (ABCG2) functional assays isolate different populations of prostate stem cells where ABCG2 function selects for cells with increased stem cell activity

    PubMed Central

    2013-01-01

    Introduction High expression of aldehyde dehydrogenase1A1 (ALDH1A1) is observed in many organs and tumors and may identify benign and cancer stem cell populations. Methods In the current study, the stem cell characteristics were determined in cells isolated from human prostate cell lines and clinical prostate specimens based upon the ALDEFLUOR™ assay. Cells isolated based on the ALDEFLUOR™ assay were compared to cells isolated based on ATP binding cassette transporter G2 (ABCG2) activity using the side population assay. To test for stem cell characteristics of self-renewal and multipotency, cells with high and low ALDH1A1 activity, based on the ALDEFLUOR™ assay (ALDHHi and ALDHLow), were isolated from prostate clinical specimens and were recombined with rat urogenital sinus mesenchyme to induce prostate gland formation. Results The percentage of ALDHHi cells in prostate cell lines (RWPE-1, RWPE-2, CWR-R1, and DU-145) was 0.5 to 6%, similarly in non-tumor and tumor clinical specimens the percentage of ALDHHi cells was 0.6 to 4%. Recombinants using ALDHHi cells serially generated prostate tissue up to three generations with as few as 250 starting cells. Immunohistochemical analysis of the recombinants using ALDHHi cells contained prostatic glands frequently expressing androgen receptor (AR), p63, chromogranin A, ALDH1A1, ABCG2, and prostate specific antigen (PSA), compared to their ALDHLow counterparts. Inhibition of ALDH resulted in the reduction of sphere formation capabilities in the CWR-R1, but not in the RWPE-2 and DU-145, prostate cell lines. ABCG2 inhibition resulted in a more robust decrease of sphere formation in androgen sensitive cell lines, CWR-R1 and RWPE-2, but not androgen insensitive DU-145. ALDH1A1 expression was enriched in ALDHHi cells and non-side population cells. ABCG2 expression was only enriched in side population cells. Conclusions The percentage of ALDHHi cells in prostate cell lines and prostate tissue was consistently higher compared

  7. A stable intermediate in the thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase between a thermophilic and a mesophilic enzymes.

    PubMed

    Hayashi-Iwasaki, Y; Numata, K; Yamagishi, A; Yutani, K; Sakurai, M; Tanaka, N; Oshima, T

    1996-03-01

    The thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, enzymes was studied by CD spectrophotometry and differential scanning calorimetry (DSC). The enzyme is a homodimer with a subunit containing two structural domains. The DSC melting profile of the chimeric enzyme in 20 mM NaHCO3, pH 10.4, showed two endothermic peaks, whereas that of the T. thermophilus wild-type enzyme had one peak. The CD melting profiles of the chimeric enzyme under the same conditions as the DSC measurement, also indicated biphasic unfolding transition. Concentration dependence of the unfolding profile revealed that the first phase was protein concentration-independent, whereas the second transition was protein concentration-dependent. When cooled after the first transition, the intermediate was isolated, which showed only the second transition upon heating. These results indicated the existence of a stable dimeric intermediate followed by the further unfolding and dissociation in the thermal unfolding of the chimeric enzyme at pH 10-11. Because the portion derived from the mesophilic isopropylmalate dehydrogenase in the chimeric enzyme is located in the hinge region between two domains of the enzyme, it is probably responsible for weakening of the interdomain interaction and causing the decooperativity of two domains. The dimeric form of the intermediate suggested that the first unfolding transition corresponds to the unfolding of domain 1 containing the N- and C-termini of the enzyme, and the second to that of domain 2 containing the subunit interface. PMID:8868488

  8. Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action

    SciTech Connect

    Huo, Lu; Davis, Ian; Liu, Fange; Andi, Babak; Esaki, Shingo; Iwaki, Hiroaki; Hasegawa, Yoshie; Orville, Allen M.; Liu, Aimin

    2015-01-07

    Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NAD+-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp3-to-sp2 transition during enzyme-mediated oxidation.

  9. Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action

    DOE PAGESBeta

    Huo, Lu; Davis, Ian; Liu, Fange; Andi, Babak; Esaki, Shingo; Iwaki, Hiroaki; Hasegawa, Yoshie; Orville, Allen M.; Liu, Aimin

    2015-01-07

    Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacylmore » intermediate and an NAD+-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp3-to-sp2 transition during enzyme-mediated oxidation.« less

  10. Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action

    PubMed Central

    Huo, Lu; Davis, Ian; Liu, Fange; Andi, Babak; Esaki, Shingo; Iwaki, Hiroaki; Hasegawa, Yoshie; Orville, Allen M.; Liu, Aimin

    2015-01-01

    Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NAD+-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp3-to-sp2 transition during enzyme-mediated oxidation. PMID:25565451

  11. Alcohol dehydrogenases from olive (Olea europaea) fruit.

    PubMed

    Salas, J J; Sánchez, J

    1998-05-01

    Alcohol dehydrogenase activity was detected in extracts from the pericarp tissues of developing olive fruits using hexanal as the substrate. Total activity in the crude extract was 20-fold higher with NADPH than with NADH. Three discrete enzymes were resolved by means of a purification protocol involving ammonium sulfate fractionation followed by ion-exchange and affinity chromatography. One of the enzymes was NAD-dependent and displayed a high K(m) for hexanal (K(m) = 2.1 mM). Two NADP-dependent alcohol dehydrogenases were resolved, one showing a high K(m) for hexanal (K(m) = 1.9 mM) and the second with a lower K(m) for the same substrate (K(m) = 0.04 mM). The three enzymes have been partially purified and their kinetic parameters and specificities for various aldehydes determined. The involvement of these enzymes in the biogenesis of six carbon alcohols constituent of the aroma of olive oil is discussed. PMID:9621451

  12. Toward aldehyde and alkane production by removing aldehyde reductase activity in Escherichia coli

    PubMed Central

    Rodriguez, Gabriel M.; Atsumi, Shota

    2015-01-01

    Advances in synthetic biology and metabolic engineering have enabled the construction of novel biological routes to valuable chemicals using suitable microbial hosts. Aldehydes serve as chemical feedstocks in the synthesis of rubbers, plastics, and other larger molecules. Microbial production of alkanes is dependent on the formation of a fatty aldehyde intermediate which is converted to an alkane by an aldehyde deformylating oxygenase (ADO). However, microbial hosts such as Escherichia coli are plagued by many highly active endogenous aldehyde reductases (ALRs) that convert aldehydes to alcohols, which greatly complicates strain engineering for aldehyde and alkane production. It has been shown that the endogenous ALR activity outcompetes the ADO enzyme for fatty aldehyde substrate. The large degree of ALR redundancy coupled with an incomplete database of ALRs represents a significant obstacle in engineering E. coli for either aldehyde or alkane production. In this study, we identified 44 ALR candidates encoded in the E. coli genome using bioinformatics tools, and undertook a comprehensive screening by measuring the ability of these enzymes to produce isobutanol. From the pool of 44 candidates, we found five new ALRs using this screening method (YahK, DkgA, GldA, YbbO, and YghA). Combined deletions of all 13 known ALRs resulted in a 90–99% reduction in endogenous ALR activity for a wide range of aldehyde substrates (C2–C12). Elucidation of the ALRs found in E. coli could guide one in reducing competing alcohol formation during alkane or aldehyde production. PMID:25108218

  13. Multiple aldehyde reductases of human brain.

    PubMed

    Hoffman, P L; Wermuth, B; von Wartburg, J P

    1980-01-01

    Human brain contains four forms of aldehyde reducing enzymes. One major activity, designated AR3, has properties indicating its identity with the NADPH-dependent aldehyde reductase, EC 1.1.1.2. The other major form of human brain enzyme, AR1, which is also NADPH-dependent, reduces both aldehyde and ketone-containing substrates, including vitamin K3 (menadione) and daunorubicin, a cancer chemotherapeutic agent. This enzyme is very sensitive to inhibition by the flavonoids quercitrin and quercetine, and may be analogous to a daunorubicin reductase previously described in liver of other species. One minor form of human brain aldehyde reductase, AR2, demonstrates substrate specificity and inhibitor sensitivity which suggest its similarity to aldose reductases found in lens and other tissues of many species. This enzyme, which can also use NADH as cofactor to some extent, is the most active in reducing the aldehyde derivatives of the biogenic amines. The fourth human brain enzyme ("SSA reductase") differs from the other forms in its ability to use NADH as well as or better than NADPH as cofactor, and in its molecular weight, which is nearly twice that of the other forms. It is quite specific for succinic semialdehyde (SSA) as substrate, and was found to be significantly inhibited only by quercetine and quercitrin. AR3 can also reduce SSA, and both enzymes may contribute to the production of gamma-hydroxybutyric acid in vivo. These results indicate that the human brain aldehyde reductases can play relatively specific physiologic roles. PMID:7424738

  14. Structural Studies of Cinnamoyl-CoA Reductase and Cinnamyl-Alcohol Dehydrogenase, Key Enzymes of Monolignol Biosynthesis[C][W

    PubMed Central

    Pan, Haiyun; Zhou, Rui; Louie, Gordon V.; Mühlemann, Joëlle K.; Bomati, Erin K.; Bowman, Marianne E.; Dudareva, Natalia; Dixon, Richard A.; Noel, Joseph P.; Wang, Xiaoqiang

    2014-01-01

    The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates. PMID:25217505

  15. Electronic Structure Contributions to Reactivity in Xanthine Oxidase Family Enzymes

    PubMed Central

    Stein, Benjamin W.; Kirk, Martin L.

    2016-01-01

    We review the xanthine oxidase (XO) family of pyranopterin molybdenum enzymes with a specific emphasis on electronic structure contributions to reactivity. In addition to xanthine and aldehyde oxidoreductases, which catalyze the 2-electron oxidation of aromatic heterocycles and aldehyde substrates, this mini-review highlights recent work on the closely related carbon monoxide dehydrogenase (CODH) that catalyzes the oxidation of CO using a unique Mo-Cu heterobimetallic active site. A primary focus of this mini-review relates to how spectroscopy and computational methods have been used to develop an understanding of critical relationships between geometric structure, electronic structure, and catalytic function. PMID:25425163

  16. Biochemical Studies and Ligand-bound Structures of Biphenyl Dehydrogenase from Pandoraea pnomenusa Strain B-356 Reveal a Basis for Broad Specificity of the Enzyme*

    PubMed Central

    Dhindwal, Sonali; Patil, Dipak N.; Mohammadi, Mahmood; Sylvestre, Michel; Tomar, Shailly; Kumar, Pravindra

    2011-01-01

    Biphenyl dehydrogenase, a member of short-chain dehydrogenase/reductase enzymes, catalyzes the second step of the biphenyl/polychlorinated biphenyls catabolic pathway in bacteria. To understand the molecular basis for the broad substrate specificity of Pandoraea pnomenusa strain B-356 biphenyl dehydrogenase (BphBB-356), the crystal structures of the apo-enzyme, the binary complex with NAD+, and the ternary complexes with NAD+-2,3-dihydroxybiphenyl and NAD+-4,4′-dihydroxybiphenyl were determined at 2.2-, 2.5-, 2.4-, and 2.1-Å resolutions, respectively. A crystal structure representing an intermediate state of the enzyme was also obtained in which the substrate binding loop was ordered as compared with the apo and binary forms but it was displaced significantly with respect to the ternary structures. These five structures reveal that the substrate binding loop is highly mobile and that its conformation changes during ligand binding, starting from a disorganized loop in the apo state to a well organized loop structure in the ligand-bound form. Conformational changes are induced during ligand binding; forming a well defined cavity to accommodate a wide variety of substrates. This explains the biochemical data that shows BphBB-356 converts the dihydrodiol metabolites of 3,3′-dichlorobiphenyl, 2,4,4′-trichlorobiphenyl, and 2,6-dichlorobiphenyl to their respective dihydroxy metabolites. For the first time, a combination of structural, biochemical, and molecular docking studies of BphBB-356 elucidate the unique ability of the enzyme to transform the cis-dihydrodiols of double meta-, para-, and ortho-substituted chlorobiphenyls. PMID:21880718

  17. Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme)

    PubMed Central

    2005-01-01

    Human mitochondrial NAD(P)+-dependent malate dehydrogenase (decarboxylating) (malic enzyme) can be specifically and allosterically activated by fumarate. X-ray crystal structures have revealed conformational changes in the enzyme in the absence and in the presence of fumarate. Previous studies have indicated that fumarate is bound to the allosteric pocket via Arg67 and Arg91. Mutation of these residues almost abolishes the activating effect of fumarate. However, these amino acid residues are conserved in some enzymes that are not activated by fumarate, suggesting that there may be additional factors controlling the activation mechanism. In the present study, we tried to delineate the detailed molecular mechanism of activation of the enzyme by fumarate. Site-directed mutagenesis was used to replace Asp102, which is one of the charged amino acids in the fumarate binding pocket and is not conserved in other decarboxylating malate dehydrogenases. In order to explore the charge effect of this residue, Asp102 was replaced by alanine, glutamate or lysine. Our experimental data clearly indicate the importance of Asp102 for activation by fumarate. Mutation of Asp102 to Ala or Lys significantly attenuated the activating effect of fumarate on the enzyme. Kinetic parameters indicate that the effect of fumarate was mainly to decrease the Km values for malate, Mg2+ and NAD+, but it did not notably elevate kcat. The apparent substrate Km values were reduced by increasing concentrations of fumarate. Furthermore, the greatest effect of fumarate activation was apparent at low malate, Mg2+ or NAD+ concentrations. The Kact values were reduced with increasing concentrations of malate, Mg2+ and NAD+. The Asp102 mutants, however, are much less sensitive to regulation by fumarate. Mutation of Asp102 leads to the desensitization of the co-operative effect between fumarate and substrates of the enzyme. PMID:15989682

  18. Cloning, expression, purification and preliminary crystallographic analysis of the short-chain dehydrogenase enzymes WbmF, WbmG and WbmH from Bordetella bronchiseptica

    SciTech Connect

    Harmer, Nicholas J.; King, Jerry D.; Palmer, Colin M.; Preston, Andrew; Maskell, Duncan J.; Blundell, Tom L.

    2007-08-01

    The expression, purification, and crystallisation of the short-chain dehydrogenases WbmF, WbmG and WbmH from B. bronchiseptica are described. Native diffraction data to 1.5, 2.0, and 2.2 Å were obtained for the three proteins, together with complexes with nucleotides. The short-chain dehydrogenase enzymes WbmF, WbmG and WbmH from Bordetella bronchiseptica were cloned into Escherichia coli expression vectors, overexpressed and purified to homogeneity. Crystals of all three wild-type enzymes were obtained using vapour-diffusion crystallization with high-molecular-weight PEGs as a primary precipitant at alkaline pH. Some of the crystallization conditions permitted the soaking of crystals with cofactors and nucleotides or nucleotide sugars, which are possible substrate compounds, and further conditions provided co-complexes of two of the proteins with these compounds. The crystals diffracted to resolutions of between 1.50 and 2.40 Å at synchrotron X-ray sources. The synchrotron data obtained were sufficient to determine eight structures of the three enzymes in complex with a variety of cofactors and substrate molecules.

  19. Affinity chromatography of nicotinamide–adenine dinucleotide-linked dehydrogenases on immobilized derivatives of the dinucleotide

    PubMed Central

    Barry, Standish; O'Carra, Pádraig

    1973-01-01

    1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD+ through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD+ (probably through the 8 position of the adenine residue) to a number of different spacer-arm–agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD+ derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD+. Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD+-binding site of this enzyme. Problems

  20. Crystal structure of L-sorbose dehydrogenase, a pyrroloquinoline quinone-dependent enzyme with homodimeric assembly, from Ketogulonicigenium vulgare.

    PubMed

    Han, Xiaodong; Xiong, Xianghua; Jiang, Dunquan; Chen, Sihan; Huang, Enyu; Zhang, Weicai; Liu, Xinqi

    2014-05-01

    The crystal structure of the L-sorbose dehydrogenase (SDH) from Ketogulonicigenium vulgare Y25 has been determined at 2.7 Å resolution using the molecular replacement method. The overall structure of SDH is similar to that of other quinoprotein dehydrogenases; consisting of an eight bladed β-propeller PQQ domain and protrusion loops. We identified a stable homodimer in crystal and demonstrated its existence in solution by sedimentation velocity measurement. By biochemical characterization of the SDH in vitro, using L-sorbose as substrate and cytochrome c551 as electron acceptor, we revealed cytochrome c551 acting as physiological primary electron acceptor for SDH. PMID:24557074

  1. Cytochromes P450 Catalyze the Reduction of α,β-Unsaturated Aldehydes

    PubMed Central

    Amunom, Immaculate; Dieter, Laura J.; Tamasi, Viola; Cai, Jan; Conklin, Daniel J.; Srivastava, Sanjay; Martin, Martha V.; Guengerich, F. Peter; Prough, Russell A.

    2011-01-01

    The metabolism of α,β-unsaturated aldehydes, e.g. 4-hydroxynonenal, involves oxidation to carboxylic acids, reduction to alcohols, and glutathionylation to eventually form mercapturide conjugates. Recently we demonstrated that P450s can oxidize aldehydes to carboxylic acids, a reaction previously thought to involve aldehyde dehydrogenase. When recombinant cytochrome P450 3A4 was incubated with 4-hydroxynonenal, O2, and NADPH, several products were produced, including 1,4-dihydroxynonene (DHN), 4-hydroxy-2-nonenoic acid (HNA), and an unknown metabolite. Several P450s catalyzed the reduction reaction in the order (human) P450 2B6 ≅ P450 3A4 > P450 1A2 > P450 2J2 > (mouse) P450 2c29. Other P450s did not catalyze the reduction reaction (human P450 2E1 & rabbit P450 2B4). Metabolism by isolated rat hepatocytes showed that HNA formation was inhibited by cyanamide, while DHN formation was not affected. Troleandomycin increased HNA production 1.6-fold while inhibiting DHN formation, suggesting that P450 3A11 is a major enzyme involved in rat hepatic clearance of 4-HNE. A fluorescent assay was developed using 9-anthracenealdehyde to measure both reactions. Feeding mice diet containing t-butylated hydroxyanisole increased the level of both activities with hepatic microsomal fractions, but not proportionally. Miconazole (0.5 mM) was a potent inhibitor of these microsomal reduction reactions, while phenytoin and α-naphthoflavone (both at 0.5 mM) were partial inhibitors, suggesting the role of multiple P450 enzymes. The oxidative metabolism of these aldehydes was inhibited >90% in an Ar or CO atmosphere, while the reductive reactions were not greatly affected. These results suggest that P450s are significant catalysts of reduction of α,β-unsaturated aldehydes in liver. PMID:21766881

  2. Ergot alkaloid biosynthesis in Aspergillus fumigatus: Conversion of chanoclavine-I aldehyde to festuclavine by the festuclavine synthase FgaFS in the presence of the old yellow enzyme FgaOx3.

    PubMed

    Wallwey, Christiane; Matuschek, Marco; Xie, Xiu-Lan; Li, Shu-Ming

    2010-08-01

    Ergot alkaloids are toxins and important pharmaceuticals which are produced biotechnologically on an industrial scale. A putative gene fgaFS has been identified in the biosynthetic gene cluster of fumigaclavine C, an ergot alkaloid of the clavine-type. The deduced gene product FgaFS comprises 290 amino acids with a molecular mass of about 32.1 kDa. The coding region of fgaFS consisting of three exons was amplified by PCR from a cDNA library of Aspergillus fumigatus, cloned into pQE70 and overexpressed in E. coli. The soluble monomeric His(6)-FgaFS was purified by affinity chromatography and used for enzyme assays. It has been shown that FgaFS is responsible for the conversion of chanoclavine-I aldehyde to festuclavine in the presence of the old yellow enzyme FgaOx3. The structure of festuclavine including the stereochemistry was unequivocally elucidated by NMR and MS analyses. Festuclavine formation was only observed when chanoclavine-I aldehyde was incubated with FgaOx3 and FgaFS simultaneously or as a tandem-reaction with a sequence of FgaOx3 before FgaFS. In the absence of FgaFS, two shunt products were formed and did not serve as substrates for FgaFS reaction. PMID:20526482

  3. Characterization of alcohol dehydrogenase 1 and 3 from Neurospora crassa FGSC2489.

    PubMed

    Park, Yong-Cheol; San, Ka-Yiu; Bennett, George N

    2007-08-01

    Alcohol dehydrogenase (ADH) is a key enzyme in the production and utilization of alcohols. Some also catalyze the formation of carboxylate esters from alcohols and aldehydes. The ADH1 and ADH3 genes of Neurospora crassa FGSC2489 were cloned and expressed in recombinant Escherichia coli to investigate their alcohol dehydrogenation and carboxylate ester formation abilities. Homology analysis and sequence alignment of amino acid sequence indicated that ADH1 and ADH3 of N. crassa contained a zinc-binding consensus sequence and a NAD(+)-binding motif and showed 54-75% identity with fungi ADHs. N. crassa ADH1 was expressed in E. coli to give a specific activity of 289 +/- 9 mU/mg using ethanol and NAD(+) as substrate and cofactor, respectively. Corresponding experiments on the expression and activity of ADH3 gave 4 mU/mg of specific activity. N. crassa ADH1 preferred primary alcohols containing C3-C8 carbons to secondary alcohols such as 2-propanol and 2-butanol. N. crassa ADH1 possessed 5.3 mU/mg of specific carboxylate ester-forming activity accumulating 0.4 mM of ethyl acetate in 18 h. Substrate specificity of various linear alcohols and aldehydes indicated that short chain-length alcohols and aldehydes were good substrates for carboxylate ester production. N. crassa ADH1 was a primary alcohol dehydrogenase using cofactor NAD(+) preferably and possessed carboxylate ester-forming activity with short chain alcohols and aldehydes. PMID:17516063

  4. Isolation and characterization of an inducible NAD-dependent butyraldehyde dehydrogenase from clostridium acetobutylicum

    SciTech Connect

    Schreiber, W.; Duerre, P.

    1996-12-31

    A NAD-dependent butyraldehyde dehydrogenase (BAD) has been purified from C. acetobutylicum DSM 792 and DSM 173 1. This key enzyme of butanol production, catalyzing the conversion of butyryl-CoA to butyraldehyde, was induced shortly before the onset of butanol production and proved to be oxygen-sensitive. A one step purification procedure on reactive green 19 allowed to purify the enzyme to homogeneity. The purified protein was found to be extremely unstable and could only partially be stabilized by addition of mercaptoethanol and storage below -20{degrees}C. The enzyme subunit had a molecular mass of 39.5 kDa. In the reverse reaction (butyryl-CoA-forming) the apparent pH optimum was 9.75 and Vmax was significantly higher with butyraldehyde and propionaldehyde than with acetaldehyde. BAD could also use NADP+, but NAD+ was the preferred coenzyme for the reverse reaction. The N-terminal amino acid sequence of the C. acetobutylicurn DSM 792 protein showed high homology to glyceraldehyde-3-phosphate dehydrogenases (GAP), especially to the protein of C. pasteurianum. Genomic libraries of C. acetobutylicum DSM 792 were screened by hybridization using PCR-generated heterologous probes encoding the gap gene of C. pasteurianum. Sequence analysis of the positive clones revealed high homology, but no identity to the N-terminal amino acid sequence of the butyraldehyde dehydrogenase. Thus, BAD from C. acetobutylicum is distinctly different from other reported aldehyde dehydrogenases with butyraldehyde dehydrogenase activity.

  5. Control of aldehyde synthesis in the luminous bacterium Beneckea harveyi.

    PubMed Central

    Ulitzur, S; Hastings, J W

    1979-01-01

    Some of the Beneckea harveyi dim aldehyde mutants, all of which emit light upon addition of exogenous long-chain aldehyde, also emit light when myristic acid is added. Analysis of these myristic acid-responsive mutants indicates that they are blocked before fatty acid formation, whereas another class of mutants, which respond only to aldehyde, appear to be defective in the enzyme(s) involved in the conversion of acid to aldehyde. Evidence is presented that this activity, designated myristic acid reductase, is coinduced with luciferase and is involved in the recycling of acid produced in the luciferase reaction, with specificity for the C14 compounds. PMID:311359

  6. Characterization of a NADH-Dependent Glutamate Dehydrogenase Mutant of Arabidopsis Demonstrates the Key Role of this Enzyme in Root Carbon and Nitrogen Metabolism[W

    PubMed Central

    Fontaine, Jean-Xavier; Tercé-Laforgue, Thérèse; Armengaud, Patrick; Clément, Gilles; Renou, Jean-Pierre; Pelletier, Sandra; Catterou, Manuella; Azzopardi, Marianne; Gibon, Yves; Lea, Peter J.; Hirel, Bertrand; Dubois, Frédéric

    2012-01-01

    The role of NADH-dependent glutamate dehydrogenase (GDH) was investigated by studying the physiological impact of a complete lack of enzyme activity in an Arabidopsis thaliana plant deficient in three genes encoding the enzyme. This study was conducted following the discovery that a third GDH gene is expressed in the mitochondria of the root companion cells, where all three active GDH enzyme proteins were shown to be present. A gdh1-2-3 triple mutant was constructed and exhibited major differences from the wild type in gene transcription and metabolite concentrations, and these differences appeared to originate in the roots. By placing the gdh triple mutant under continuous darkness for several days and comparing it to the wild type, the evidence strongly suggested that the main physiological function of NADH-GDH is to provide 2-oxoglutarate for the tricarboxylic acid cycle. The differences in key metabolites of the tricarboxylic acid cycle in the triple mutant versus the wild type indicated that, through metabolic processes operating mainly in roots, there was a strong impact on amino acid accumulation, in particular alanine, γ-aminobutyrate, and aspartate in both roots and leaves. These results are discussed in relation to the possible signaling and physiological functions of the enzyme at the interface of carbon and nitrogen metabolism. PMID:23054470

  7. Characterization of a NADH-dependent glutamate dehydrogenase mutant of Arabidopsis demonstrates the key role of this enzyme in root carbon and nitrogen metabolism.

    PubMed

    Fontaine, Jean-Xavier; Tercé-Laforgue, Thérèse; Armengaud, Patrick; Clément, Gilles; Renou, Jean-Pierre; Pelletier, Sandra; Catterou, Manuella; Azzopardi, Marianne; Gibon, Yves; Lea, Peter J; Hirel, Bertrand; Dubois, Frédéric

    2012-10-01

    The role of NADH-dependent glutamate dehydrogenase (GDH) was investigated by studying the physiological impact of a complete lack of enzyme activity in an Arabidopsis thaliana plant deficient in three genes encoding the enzyme. This study was conducted following the discovery that a third GDH gene is expressed in the mitochondria of the root companion cells, where all three active GDH enzyme proteins were shown to be present. A gdh1-2-3 triple mutant was constructed and exhibited major differences from the wild type in gene transcription and metabolite concentrations, and these differences appeared to originate in the roots. By placing the gdh triple mutant under continuous darkness for several days and comparing it to the wild type, the evidence strongly suggested that the main physiological function of NADH-GDH is to provide 2-oxoglutarate for the tricarboxylic acid cycle. The differences in key metabolites of the tricarboxylic acid cycle in the triple mutant versus the wild type indicated that, through metabolic processes operating mainly in roots, there was a strong impact on amino acid accumulation, in particular alanine, γ-aminobutyrate, and aspartate in both roots and leaves. These results are discussed in relation to the possible signaling and physiological functions of the enzyme at the interface of carbon and nitrogen metabolism. PMID:23054470

  8. Enzyme biosensor for androsterone based on 3α-hydroxysteroid dehydrogenase immobilized onto a carbon nanotubes/ionic liquid/NAD+ composite electrode.

    PubMed

    Mundaca, R A; Moreno-Guzmán, M; Eguílaz, M; Yáñez-Sedeño, P; Pingarrón, J M

    2012-09-15

    A 3α-hydrosteroid biosensor for androsterone determination has been prepared by immobilizing the enzyme 3α-hydroxysteroid dehydrogenase (3α-HSD) in a composite electrode platform constituted of a mixture of multi-walled carbon nanotubes (MWCNTs), octylpyridinium hexafluorophosphate (OPPF(6)) ionic liquid and NAD(+) cofactor. This configuration allowed the fast, sensitive and stable electrochemical detection of the NADH generated in the enzyme reaction. All the experimental variables involved in the preparation and performance of the enzyme biosensor were optimized. Amperometry in stirred solutions at +400 mV provided a linear calibration plot for androsterone in the 0.5-10 μM concentration range with a slope value more than 200-times higher than that previously reported. The detection limit achieved was 0.15 μM and a low value of the apparent Michaelis-Menten constant (K(app)(M)), 36.0 μM, similar to that reported for the enzyme in solution, was calculated. The 3α-HSD/MWCNTs/OPPF(6)/NAD(+) biosensor provided good results in the determination of androsterone in spiked human serum samples. PMID:22967613

  9. Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate.

    PubMed

    Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Sax, Martin; Brunskill, Andrew; Nemeria, Natalia; Jordan, Frank; Furey, William

    2004-03-01

    Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP). To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg(2+) has been determined at 2.1 A and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg(2+) complex. When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others. These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP. The observed hydrogen bonding pattern is not an invariant feature of ThDP-dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues. Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP. The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor. Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release. This study also provides additional insight into the role of water in enzyme inhibition and catalysis. PMID:14992577

  10. Nonlinear (amplified) relationship between nuclear occupancy by triiodothyronine and the appearance rate of hepatic alpha-glycerophosphate dehydrogenase and malic enzyme in the rat.

    PubMed Central

    Oppenheimer, J H; Coulombe, P; Schwartz, H L; Gutfeld, N W

    1978-01-01

    Three separate approaches were applied to examine the general relationship between R, the rate of induction of specific enzymes (mitochondrial alpha-glycero-phosphate dehydrogenase and cytosolic malic enzyme) and q, the fractional nuclear occupancy by triiodothyronine in male Sprague-Dawley rats. Daily 200-microgram injections of triiodothyronine per 10u g body wt for 7 days resulted in saturation of the hepatic nuclear sites and the achievement of an apparent new steady state of enzyme levels. The increase achieved over base-line hypothyroid levels was then compared with the increment over hypothyroid base line characteristic of intact euthyroid animals with 47% of nuclear sites occupied. The maximal theoretical reate of steady-state enzyme induction could be protected on the basis of the observed maximal increase in enzyme activity observed 1 day after the injection of graded doses of hormone and lambda, the known fractional rate of enzyme dissipation. The 24-h dose-response studies were used to generate R as a continuous function of q, both in hypothyroid as well as in euthyroid animals. This approach involved the numerical solution of an ordinary differential equation describing the rate of change of enzyme as a function of R, which was assumed to be uniquely related to q. Results of these analyses indicated that the ratio of the maximal rate of induction of enzyme at full occupancy to the rate of induction under euthyroid conditions assumes a value between 9.0 and 19.5, depending on the precise analytic and experimental approach applied. This value is far in excess of the theoretical ratio 2.13 which on would anticipate if R were linearly related to q and 47% of the nuclear sites occupied under physiological conditions. Thus, the signal for enzyme induction appears to undergo progressjive amplification with increasing nuclear occupancy. Moreover, the curve describing the relationship between R and q appears highly nonlinear throughout (concave upwards

  11. Anaerobic degradation of nitrilotriacetate (NTA) in a denitrifying bacterium: purification and characterization of the NTA dehydrogenase-nitrate reductase enzyme complex.

    PubMed Central

    Jenal-Wanner, U; Egli, T

    1993-01-01

    The initial step in the anoxic metabolism of nitrilotriacetate (NTA) was investigated in a denitrifying member of the gamma subgroup of the Proteobacteria. In membrane-free cell extracts, the first step of NTA oxidation was catalyzed by a protein complex consisting of two enzymes, NTA dehydrogenase (NTADH) and nitrate reductase (NtR). The products formed were iminodiacetate and glyoxylate. Electrons derived from the oxidation of NTA were transferred to nitrate only via the artificial dye phenazine methosulfate, and nitrate was stoichiometrically reduced to nitrite. NTADH activity could be measured only in the presence of NtrR and vice versa. The NTADH-NtrR enzyme complex was purified and characterized. NTADH and NtrR were both alpha 2 dimers and had molecular weights of 170,000 and 105,000, respectively. NTADH contained covalently bound flavin cofactor, and NtrR contained a type b cytochrome. Optimum NTA-oxidizing activity was achieved at a molar ratio of NTADH to NtrR of approximately 1:1. So far, NTA is the only known substrate for NTADH. This is the first report of a redox enzyme complex catalyzing the oxidation of a substrate and concomitantly reducing nitrate. Images PMID:8250558

  12. Molecular genetic analysis of the gene encoding the trifunctional enzyme MTHFD (methylenetetrahydrofolate-dehydrogenase, methenyltetrahydrofolate-cyclohydrolase, formyltetrahydrofolate synthetase) in patients with neural tube defects.

    PubMed

    Hol, F A; van der Put, N M; Geurds, M P; Heil, S G; Trijbels, F J; Hamel, B C; Mariman, E C; Blom, H J

    1998-02-01

    It is now well recognized that periconceptional folic acid or folic acid containing multivitamin supplementation reduces the risk of neural tube defects (NTDs). Recently we were able to show that homozygosity for a thermolabile variant of the enzyme methylenetetrahydrofolate reductase is associated with an increased risk for spina bifida in patients recruited from the Dutch population. However, this genetic risk factor could not account for all folic acid preventable NTDs. In an attempt to identify additional folate related enzymes that contribute to NTD etiology we now studied the methylenetetrahydrofolate dehydrogenase gene on chromosome 14q24 which encodes a single protein with three catalytic properties important in the folate metabolism. The cDNA sequence of 38 familial and 79 sporadic patients was screened for the presence of mutations by single strand conformation polymorphism (SSCP) analysis followed by sequencing. Two amino acid substitutions were identified. The first one (R293H) was detected in a patient with familial spina bifida and not in 300 control individuals. The mutation was inherited from the unaffected maternal grandmother and was also present in two younger brothers of the index patient, one of them displaying spina bifida occulta and the other being unaffected. The second change turned out to be an amino acid polymorphism (R653Q) that was present in both patients and controls with similar frequencies. Our results so far provide no evidence for a major role of the methylenetetrahydrofolate-dehydrogenase (MTHFD) gene in NTD etiology. However, the identification of a mutation in one family suggests that this gene can act as a risk factor for human NTD. PMID:9611072

  13. Characterization of a novel PQQ-dependent quinohemoprotein pyranose dehydrogenase from Coprinopsis cinerea classified into auxiliary activities family 12 in carbohydrate-active enzymes.

    PubMed

    Takeda, Kouta; Matsumura, Hirotoshi; Ishida, Takuya; Samejima, Masahiro; Ohno, Hiroyuki; Yoshida, Makoto; Igarashi, Kiyohiko; Nakamura, Nobuhumi

    2015-01-01

    The basidiomycete Coprinopsis cinerea contains a quinohemoprotein (CcPDH named as CcSDH in our previous paper), which is a new type of pyrroloquinoline-quinone (PQQ)-dependent pyranose dehydrogenase and is the first found among all eukaryotes. This enzyme has a three-domain structure consisting of an N-terminal heme b containing a cytochrome domain that is homologous to the cytochrome domain of cellobiose dehydrogenase (CDH; EC 1.1.99.18) from the wood-rotting basidiomycete Phanerochaete chrysosporium, a C-terminal family 1-type carbohydrate-binding module, and a novel central catalytic domain containing PQQ as a cofactor. Here, we describe the biochemical and electrochemical characterization of recombinant CcPDH. UV-vis and resonance Raman spectroscopic studies clearly reveal characteristics of a 6-coordinated low-spin heme b in both the ferric and ferrous states, as well as intramolecular electron transfer from the PQQ to heme b. Moreover, the formal potential of the heme was evaluated to be 130 mV vs. NHE by cyclic voltammetry. These results indicate that the cytochrome domain of CcPDH possesses similar biophysical properties to that in CDH. A comparison of the conformations of monosaccharides as substrates and the associated catalytic efficiency (kcat/Km) of CcPDH indicates that the enzyme prefers monosaccharides with equatorial C-2, C-3 hydroxyl groups and an axial C-4 hydroxyl group in the 1C4 chair conformation. Furthermore, a binding study shows a high binding affinity of CcPDH for cellulose, suggesting that CcPDH function is related to the enzymatic degradation of plant cell wall. PMID:25679509

  14. Characterization of a Novel PQQ-Dependent Quinohemoprotein Pyranose Dehydrogenase from Coprinopsis cinerea Classified into Auxiliary Activities Family 12 in Carbohydrate-Active Enzymes

    PubMed Central

    Takeda, Kouta; Matsumura, Hirotoshi; Ishida, Takuya; Samejima, Masahiro; Ohno, Hiroyuki; Yoshida, Makoto; Igarashi, Kiyohiko; Nakamura, Nobuhumi

    2015-01-01

    The basidiomycete Coprinopsis cinerea contains a quinohemoprotein (CcPDH named as CcSDH in our previous paper), which is a new type of pyrroloquinoline-quinone (PQQ)-dependent pyranose dehydrogenase and is the first found among all eukaryotes. This enzyme has a three-domain structure consisting of an N-terminal heme b containing a cytochrome domain that is homologous to the cytochrome domain of cellobiose dehydrogenase (CDH; EC 1.1.99.18) from the wood-rotting basidiomycete Phanerochaete chrysosporium, a C-terminal family 1-type carbohydrate-binding module, and a novel central catalytic domain containing PQQ as a cofactor. Here, we describe the biochemical and electrochemical characterization of recombinant CcPDH. UV-vis and resonance Raman spectroscopic studies clearly reveal characteristics of a 6-coordinated low-spin heme b in both the ferric and ferrous states, as well as intramolecular electron transfer from the PQQ to heme b. Moreover, the formal potential of the heme was evaluated to be 130 mV vs. NHE by cyclic voltammetry. These results indicate that the cytochrome domain of CcPDH possesses similar biophysical properties to that in CDH. A comparison of the conformations of monosaccharides as substrates and the associated catalytic efficiency (kcat/Km) of CcPDH indicates that the enzyme prefers monosaccharides with equatorial C-2, C-3 hydroxyl groups and an axial C-4 hydroxyl group in the 1C4 chair conformation. Furthermore, a binding study shows a high binding affinity of CcPDH for cellulose, suggesting that CcPDH function is related to the enzymatic degradation of plant cell wall. PMID:25679509

  15. Alcohol Dehydrogenase from Methylobacterium organophilum

    PubMed Central

    Wolf, H. J.; Hanson, R. S.

    1978-01-01

    The alcohol dehydrogenase from Methylobacterium organophilum, a facultative methane-oxidizing bacterium, has been purified to homogeneity as indicated by sodium dodecyl sulfate-gel electrophoresis. It has several properties in common with the alcohol dehydrogenases from other methylotrophic bacteria. The active enzyme is a dimeric protein, both subunits having molecular weights of about 62,000. The enzyme exhibits broad substrate specificity for primary alcohols and catalyzes the two-step oxidation of methanol to formate. The apparent Michaelis constants of the enzyme are 2.9 × 10−5 M for methanol and 8.2 × 10−5 M for formaldehyde. Activity of the purified enzyme is dependent on phenazine methosulfate. Certain characteristics of this enzyme distinguish it from the other alcohol dehydrogenases of other methylotrophic bacteria. Ammonia is not required for, but stimulates the activity of newly purified enzyme. An absolute dependence on ammonia develops after storage of the purified enzyme. Activity is not inhibited by phosphate. The fluorescence spectrum of the enzyme indicates that it and the cofactor associated with it may be chemically different from the alcohol dehydrogenases from other methylotrophic bacteria. The alcohol dehydrogenases of Hyphomicrobium WC-65, Pseudomonas methanica, Methylosinus trichosporium, and several facultative methylotrophs are serologically related to the enzyme purified in this study. The enzymes of Rhodopseudomonas acidophila and of organisms of the Methylococcus group did not cross-react with the antiserum prepared against the alcohol dehydrogenase of M. organophilum. Images PMID:80974

  16. Surface modifications for enhanced enzyme immobilization and improved electron transfer of PQQ-dependent glucose dehydrogenase anodes.

    PubMed

    Lopez, Ryan J; Babanova, Sofia; Artyushkova, Kateryna; Atanassov, Plamen

    2015-10-01

    Pyrroloquinoline quinone dependent soluble glucose dehydrogenase (PQQ-sGDH) enzymatic MWCNT electrodes were p roduced using 1-pyrenecarboxylic acid (PCA) activated through carbodiimide functionalization and 1-Pyrenebutyric acid N-hydroxysuccinimide ester (PBSE) as tethering agents. At 600 mV potential, the current density generated by the activated-PCA tethered PQQ-sGDH anode was significantly greater than the current density generated by the untethered PQQ-sGDH and PBSE tethered anodes, and performance was nearly identical to the performance of a covalently bound PQQ-sGDH anode. A technique for covalently bonding heme-b (hemin), a natural quinohemoprotein porphyrin redox cofactor, to carbon nanotubes modified with arylamine groups is reported. The resulting performance of the covalently bound hemin PQQ-sGDH anode is considerably higher than that of any other PQQ-sGDH anodes tested. PMID:26011132

  17. A novel glucose dehydrogenase from the white-rot fungus Pycnoporus cinnabarinus: production in Aspergillus niger and physicochemical characterization of the recombinant enzyme.

    PubMed

    Piumi, François; Levasseur, Anthony; Navarro, David; Zhou, Simeng; Mathieu, Yann; Ropartz, David; Ludwig, Roland; Faulds, Craig B; Record, Eric

    2014-12-01

    Data on glucose dehydrogenases (GDHs) are scarce and availability of these enzymes for application purposes is limited. This paper describes a new GDH from the fungus Pycnoporus cinnabarinus CIRM BRFM 137 that is the first reported GDH from a white-rot fungus belonging to the Basidiomycota. The enzyme was recombinantly produced in Aspergillus niger, a well-known fungal host producing an array of homologous or heterologous enzymes for industrial applications. The full-length gene that encodes GDH from P. cinnabarinus (PcGDH) consists of 2,425 bp and codes for a deduced protein of 620 amino acids with a calculated molecular mass of 62.5 kDa. The corresponding complementary DNA was cloned and placed under the control of the strong and constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. The signal peptide of the glucoamylase prepro sequence of A. niger was used to target PcGDH secretion into the culture medium, achieving a yield of 640 mg L(-1), which is tenfold higher than any other reported value. The recombinant PcGDH was purified twofold to homogeneity in a one-step procedure with a 41 % recovery using a Ni Sepharose column. The identity of the recombinant protein was further confirmed by immunodetection using western blot analysis and N-terminal sequencing. The molecular mass of the native PcGDH was 130 kDa, suggesting a homodimeric form. Optimal pH and temperature were found to be similar (5.5 and 60 °C, respectively) to those determined for the previously characterized GDH, i.e., from Glomerella cingulata. However PcGDH exhibits a lower catalytic efficiency of 67 M(-1) s(-1) toward glucose. This substrate is by far the preferred substrate, which constitutes an advantage over other sugar oxidases in the case of blood glucose monitoring. The substrate-binding domain of PcGDH turns out to be conserved as compared to other glucose-methanol-choline (GMCs) oxidoreductases. In addition, the ability of PcGDH to reduce oxidized quinones or radical

  18. Materials and methods for the alteration of enzyme and acetyl CoA levels in plants

    DOEpatents

    Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.; Schnable, Patrick S.; Wen, Tsui-Jung

    2009-04-28

    The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.alpha. subunit of pPDH, the E1.beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyruvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.alpha. pPDH, E1.beta. pPDH, E2 pPDH, mtPDH or ALDH.

  19. Materials and methods for the alteration of enzyme and acetyl CoA levels in plants

    DOEpatents

    Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.; Behal, Robert; Schnable, Patrick S.; Ke, Jinshan; Johnson, Jerry L.; Allred, Carolyn C.; Fatland, Beth; Lutziger, Isabelle; Wen, Tsui-Jung

    2004-07-20

    The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.sub..alpha. subunit of pPDH, the E1.sub..beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyurvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.sub..alpha. pPDH, E1.sub..beta. pPDH, E2 pPDH, mtPDH or ALDH.

  20. Materials and methods for the alteration of enzyme and acetyl CoA levels in plants

    DOEpatents

    Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.; Behal, Robert; Schnable, Patrick S.; Ke, Jinshan; Johnson, Jerry L.; Allred, Carolyn C.; Fatland, Beth; Lutziger, Isabelle; Wen, Tsui-Jung

    2005-09-13

    The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.alpha. subunit of pPDH, the E1.beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyruvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.alpha. pPDH, E1.beta. pPDH, E2 pPDH, mtPDH or ALDH.

  1. Direct Electrochemical Addressing of Immobilized Alcohol Dehydrogenase for the Heterogeneous Bioelectrocatalytic Reduction of Butyraldehyde to Butanol

    PubMed Central

    Schlager, S; Neugebauer, H; Haberbauer, M; Hinterberger, G; Sariciftci, N S

    2015-01-01

    Modified electrodes using immobilized alcohol dehydrogenase enzymes for the efficient electroreduction of butyraldehyde to butanol are presented as an important step for the utilization of CO2-reduction products. Alcohol dehydrogenase was immobilized, embedded in an alginate–silicate hybrid gel, on a carbon felt (CF) electrode. The application of this enzyme to the reduction of an aldehyde to an alcohol with the aid of the coenzyme nicotinamide adenine dinucleotide (NADH), in analogy to the final step in the natural reduction cascade of CO2 to alcohol, has been already reported. However, the use of such enzymatic reductions is limited because of the necessity of providing expensive NADH as a sacrificial electron and proton donor. Immobilization of such dehydrogenase enzymes on electrodes and direct pumping of electrons into the biocatalysts offers an easy and efficient way for the biochemical recycling of CO2 to valuable chemicals or alternative synthetic fuels. We report the direct electrochemical addressing of immobilized alcohol dehydrogenase for the reduction of butyraldehyde to butanol without consumption of NADH. The selective reduction of butyraldehyde to butanol occurs at room temperature, ambient pressure and neutral pH. Production of butanol was detected by using liquid-injection gas chromatography and was estimated to occur with Faradaic efficiencies of around 40 %. PMID:26113881

  2. Novel functions of the α-ketoglutarate dehydrogenase complex may mediate diverse oxidant-induced changes in mitochondrial enzymes associated with Alzheimer’s disease

    PubMed Central

    Shi, Qingli; Xu, Hui; Kleinman, Wayne A.; Gibson, Gary E.

    2011-01-01

    Measures in autopsied brains from Alzheimer’s Disease (AD) patients reveal a decrease in the activity of α-ketoglutarate dehydrogenase complex (KGDHC) and an increase in malate dehydrogenase (MDH) activity. The present experiments tested whether both changes could be caused by the common oxidant H2O2 and to probe the mechanism underlying these changes. Since the response to H2O2 is modified by the level of the E2k subunit of KGDHC, the interaction of MDH and KGDHC was studied in cells with varying levels of E2k. In cells with only 23% of normal E2k protein levels, one hour treatment with H2O2 decreased KGDHC and increased MDH activity as well as the mRNA level for both cytosolic and mitochondrial MDH. The increase in MDH did not occur in cells with 100% or 46% of normal E2k. Longer treatments with H2O2 inhibited the activity of both enzymes. Glutathione is a major regulator of cellular redox state and can modify enzyme activities. H2O2 converts reduced glutathione (GSH) to oxidized glutathione (GSSG), which reacts with protein thiols. Treatment of purified KGDHC with GSSG leads to glutathionylation of all three KGDHC subunits. Thus, cellular glutathione level was manipulated by two means to determine the effect on KGDHC and MDH activities. Both buthionine sulfoximine (BSO), which inhibits glutathione synthesis without altering redox state, and H2O2 diminished glutathione to a similar level after 24 hrs. However, H2O2, but not BSO, reduced KGDHC and MDH activities, and the reduction was greater in the E2k-23 line. These findings suggest that the E2k may mediate diverse responses of KGDHC and MDH to oxidants. In addition, the differential response of activities to BSO and H2O2 together with the in vitro interaction of KGDHC with GSSG suggests that glutathionylation is one possible mechanism underlying oxidative stress-induced inhibition of the TCA cycle enzymes. PMID:18206986

  3. A chemically modified carbon paste electrode with d-lactate dehydrogenase and alanine aminotranferase enzyme sequences for d-lactic acid analysis.

    PubMed

    Shu, H C; Wu, N P

    2001-04-12

    An amperometric biosensor was constructed for the analysis of d-lactic acid based on immobilizing d-lactate dehydrogenase(d-LDH), alanine aminotransferase (ALT), NAD(+), a redox polymer and polyethylenimine in carbon paste. The effect of addition of ALT in the paste, using enzyme sequences of ALT/d-LDH, was insignificant for d-lactic acid analysis. The responses of d-lactic acid in ALT/d-LDH paste electrode are the same as those in d-LDH paste electrode. However, the interference effect of pyruvate in the sample can be substantially reduced if sodium glutamate was applied in the carrier solution. When ALT immobilized in control porous glass as an immobilized enzyme reactor (IMER) was mounted in flow injection analysis system with the d-LDH paste electrode as detector for d-lactate analysis, the interference of the pyruvate can be significantly eliminated. The adverse effect of pyruvate in the samples for d-lactic acid analysis was reduced more effectively in ALT IMER with d-LDH electrode than in ALT/d-LDH electrode. PMID:18968259

  4. Synthesis of cinnamyl alcohol from cinnamaldehyde with Bacillus stearothermophilus alcohol dehydrogenase as the isolated enzyme and in recombinant E. coli cells.

    PubMed

    Pennacchio, Angela; Rossi, Mosè; Raia, Carlo A

    2013-07-01

    The synthesis of the aroma chemical cinnamyl alcohol (CMO) by means of enzymatic reduction of cinnamaldehyde (CMA) was investigated using NADH-dependent alcohol dehydrogenase from Bacillus stearothermophilus both as an isolated enzyme, and in recombinant Escherichia coli whole cells. The influence of parameters such as reaction time and cofactor, substrate, co-substrate 2-propanol and biocatalyst concentrations on the bioreduction reaction was investigated and an efficient and sustainable one-phase system developed. The reduction of CMA (0.5 g/L, 3.8 mmol/L) by the isolated enzyme occurred in 3 h at 50 °C with 97% conversion, and yielded high purity CMO (≥98%) with a yield of 88% and a productivity of 50 g/genzyme. The reduction of 12.5 g/L (94 mmol/L) CMA by whole cells in 6 h, at 37 °C and no requirement of external cofactor occurred with 97% conversion, 82% yield of 98% pure alcohol and a productivity of 34 mg/gwet cell weight. The results demonstrate the microbial system as a practical and efficient method for larger-scale synthesis of CMO. PMID:23686507

  5. Crystal structure of the γ-hydroxymuconic semialdehyde dehydrogenase from Pseudomonas sp. strainWBC-3, a key enzyme involved in para-Nitrophenol degradation

    PubMed Central

    2013-01-01

    Background para-Nitrophenol (PNP) is a highly toxic compound with threats to mammalian health. The pnpE-encoded γ-hydroxymuconic semialdehyde dehydrogenase catalyzes the reduction of γ-hydroxymuconic semialdehyde to maleylacetate in Pseudomonas sp. strain WBC-3, playing a key role in the catabolism of PNP to Krebs cycle intermediates. However, the catalyzing mechanism by PnpE has not been well understood. Results Here we report the crystal structures of the apo and NAD bound PnpE. In the PnpE-NAD complex structure, NAD is situated in a cleft of PnpE. The cofactor binding site is composed of two pockets. The adenosine and the first ribose group of NAD bind in one pocket and the nicotinamide ring in the other. Conclusions Six amino acids have interactions with the cofactor. They are C281, E247, Q210, W148, I146 and K172. Highly conserved residues C281 and E247 were identified to be critical for its catalytic activity. In addition, flexible docking studies of the enzyme-substrate system were performed to predict the interactions between PnpE and its substrate γ-hydroxymuconic semialdehyde. Amino acids that interact extensively with the substrate and stabilize the substrate in an orientation suitable for enzyme catalysis were identified. The importance of these residues for catalytic activity was confirmed by the relevant site-directed mutagenesis and their biochemical characterization. PMID:24252642

  6. Determination of Glutamate Dehydrogenase Activity and Its Kinetics in Mouse Tissues using Metabolic Mapping (Quantitative Enzyme Histochemistry)

    PubMed Central

    Botman, Dennis; Tigchelaar, Wikky

    2014-01-01

    Glutamate dehydrogenase (GDH) catalyses the reversible conversion of glutamate into α-ketoglutarate with the concomitant reduction of NAD(P)+ to NAD(P)H or vice versa. GDH activity is subject to complex allosteric regulation including substrate inhibition. To determine GDH kinetics in situ, we assessed the effects of various glutamate concentrations in combination with either the coenzyme NAD+ or NADP+ on GDH activity in mouse liver cryostat sections using metabolic mapping. NAD+-dependent GDH Vmax was 2.5-fold higher than NADP+-dependent Vmax, whereas the Km was similar, 1.92 mM versus 1.66 mM, when NAD+ or NADP+ was used, respectively. With either coenzyme, Vmax was determined at 10 mM glutamate and substrate inhibition was observed at higher glutamate concentrations with a Ki of 12.2 and 3.95 for NAD+ and NADP+ used as coenzyme, respectively. NAD+- and NADP+-dependent GDH activities were examined in various mouse tissues. GDH activity was highest in liver and much lower in other tissues. In all tissues, the highest activity was found when NAD+ was used as a coenzyme. In conclusion, GDH activity in mice is highest in the liver with NAD+ as a coenzyme and highest GDH activity was determined at a glutamate concentration of 10 mM. PMID:25124006

  7. DFT study of the active site of the XoxF-type natural, cerium-dependent methanol dehydrogenase enzyme.

    PubMed

    Bogart, Justin A; Lewis, Andrew J; Schelter, Eric J

    2015-01-19

    Rare-earth metal cations have recently been demonstrated to be essential co-factors for the growth of the methanotrophic bacterium Methylacidiphilum fumariolicum SolV. A crystal structure of the rare-earth-dependent methanol dehydrogenase (MDH) includes a cerium cation in the active site. Herein, the Ce-MDH active site has been analyzed through DFT calculations. The results show the stability of the Ce(III)-pyrroloquinoline quinone (PQQ) semiquinone configuration. Calculations on the active oxidized form of this complex indicate a 0.81 eV stabilization of the PQQ(0) LUMO at cerium versus calcium, supporting the observation that the cerium cation in the active site confers a competitive advantage to Methylacidiphilum fumariolicum SolV. Using reported aqueous electrochemical data, a semi-empirical correlation was established based on cerium(IV/III) redox potentials. The correlation allowed estimation of the cerium oxidation potential of +1.35 V versus saturated calomel electrode (SCE) in the active site. The results are expected to guide the design of functional model complexes and alcohol-oxidation catalysts based on lanthanide complexes of biologically relevant quinones. PMID:25421364

  8. Structural of the class II enzyme of human liver alcohol dehydrogenase: combined cDNA and protein sequence determination of the. pi. subunit

    SciTech Connect

    Hoeoeg, J.O.; von Bahr-Lindstroem, H.; Heden, L.O.; Holmquist, B.; Larsson, K.; Hempel, J.; Vallee, B.L.; Joernvall, H.

    1987-04-07

    The class II enzyme of human liver alcohol dehydrogenase was isolated, carboxymethylated, and cleaved with CNBr and proteolytic enzymes. Sequence analysis of peptides established structures corresponding to the ..pi.. subunit. Two segments from the C-terminal region unique to ..pi.. were selected for synthesis of oligodeoxyribonucleotide probes to screen a human liver cDNA library constructed in plasmid pT4. Sequence analysis of two identical hybridization-positive clones with cDNA inserts of about 2000 nucleotides gave the entire coding region of the ..pi.. subunit, a 61-nucleotide 5' noncoding region and a 741-nucleotide 3' noncoding region containing four possible polyadenylation sites. Translation of the coding region yields a 391-residue polypeptide, which in all regions except the C-terminal segment corresponds to the protein structure as determined directly by peptide analysis. With the class I numbering system, the exception concerns a residue exchange at position 368, the actual C-terminus which is Phe-374 by peptide data but a 12 residue extension by cDNA data, and possibly two further residue exchanges at positions 303 and 312. The size difference might indicate the existence of posttranslational modifications of the mature protein or, in combination with the residue exchanges, the existence of polymorphism at the locus for class II subunits. The ..pi.. subunit analyzed directly results in a 379-residue polypeptide and is the only class II size thus far known to occur in the mature protein. Comparison of the ..pi.. structure with those of the class I subunits (..cap alpha.., ..beta.., and ..gamma..) reveals a homology with extensive differences. Large variations in segments affecting relationships at the active site and the area of subunit interactions account for the significant alterations of enzymatic specificities and other properties that differentiate class II from class I enzymes.

  9. Enzyme

    MedlinePlus

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  10. An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

    SciTech Connect

    Cook, William J; Senkovich, Olga; Chattopadhyay, Debasish

    2009-06-08

    The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step. We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2{angstrom} resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in the substrate-free conformation

  11. The titanium binding protein of Rhodococcus ruber GIN1 (NCIMB 40340) is a cell-surface homolog of the cytosolic enzyme dihydrolipoamide dehydrogenase.

    PubMed

    Siegmann, Ari; Komarska, Avital; Betzalel, Yifaat; Brudo, Irene; Jindou, Sadanari; Mor, Gil; Fleminger, Gideon

    2009-01-01

    Rhodococcus ruber GIN1 (formally Rh. strain GIN1) was previously isolated on the basis of its strong adherence to coal fly ash (CFA) and titanium dioxide particles from CFA sedimentation ponds of an electrical power plant in Israel. The interaction of the bacterium with oxides has been shown to be mediated by a cell surface protein designated TiBP (titanium binding protein) involving primarily strong, non-electrostatic forces. In this work, we set forward to identify this unique exocellular protein. Sequence analysis of the purified protein by mass spectrometry (LC/MS/MS) following trypsinization revealed 11 peptides. All of them showed >90% amino acid residues identity with sequences of one of the orthologs (dldh1) of the cytosolic enzyme dihydrolipoamide dehydrogenase (DLDH), based on the genome sequence of Rhodococcus strain RHA1. This genome was selected as a reference since currently it is the only sequenced Rhodococcal genome. Altogether, these peptides covered over 25% of the 52 kDa protein molecule. N- and C-termini primers were prepared and used to sequence the paralog gene from Rh. ruber GIN1 after polymerase chain reaction (PCR) amplification. All 11 peptides showed 100% identity with the sequence of this gene. The homology of TiBP with the supposedly cytosolic DLDH raised the question of whether the exocellular TiBP possesses DLDH activity. Indeed, intact late logarithmic phase Rh. ruber GIN1 cells, previously shown to express TiBP, were found to possess such activity, while very low activity was associated with stationary phase cells which possess diminished TiBP expression on their surface. Further evidence for the exocellular location of TiBP/DLDH was achieved using specific anti-TiBP polyclonal antibodies by whole cell and protein enzyme-linked immunosorbent assay (ELISA), showing high reactivity of the logarithmic phase cell surface and substantially lower reactivity with the stationary phase cells. As expected, logarithmic phase spheroplasts were

  12. A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E

    DOE PAGESBeta

    Clarkson, Sonya M.; Hamilton-Brehm, Scott D.; Giannone, Richard J.; Engle, Nancy L.; Tschaplinski, Timothy J.; Hettich, Robert L.; Elkins, James G.

    2014-12-03

    Background: Chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, but also restricts growth via the release of furan aldehydes such as furfural and 5-hydroxymethylfurfural (5-HMF). The physiological effects of these inhibitors on thermophilic, fermentative bacteria is important to understand; especially as cellulolytic strains are being developed for consolidated bioprocessing (CBP) of lignocellulosic feedstocks. Identifying mechanisms for detoxification of aldehydes in naturally resistant strains such as Thermoanaerobacter spp. may also enable improvements in candidate CBP microorganisms. Results: T. pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30more » mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ. The proteomes of T. pseudethanolicus 39E grown in the presence or absence of 15 mM furfural were compared to identify upregulated enzymes potentially responsible for the observed reduction. A total of 225 proteins were differentially regulated in response to the 15 mM furfural treatment with 152 upregulated vs. 73 downregulated. Only 86 proteins exhibited a 2-fold change in abundance in either direction. Of these, 53 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least 2-fold by furfural and were targeted for further investigation: Teth39_1597, encodes a predicted butanol dehydrogenase (BdhA) and Teth39_1598, a predicted aldo/keto reductase (AKR). Both genes were cloned from T. pseudethanolicus 39E, with the respective enzymes overexpressed in E. coli and specific activities determined against a variety of aldehydes. BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. AKR also showed significant activity with NADPH, but only with four carbon butyr

  13. Dehydrogenase genes in the ectomycorrhizal fungus Tricholoma vaccinum: A role for Ald1 in mycorrhizal symbiosis.

    PubMed

    Henke, Catarina; Jung, Elke-Martina; Voit, Annekatrin; Kothe, Erika; Krause, Katrin

    2016-02-01

    Ectomycorrhizal symbiosis is important for forest ecosystem functioning with tree-fungal cooperation increasing performance and countering stress conditions. Aldehyde dehydrogenases (ALDHs) are key enzymes for detoxification and thus may play a role in stress response of the symbiotic association. With this focus, eight dehydrogenases, Ald1 through Ald7 and TyrA, of the ectomycorrhizal basidiomycete Tricholoma vaccinum were characterized and phylogenetically investigated. Functional analysis was performed through differential expression analysis by feeding different, environmentally important substances. A strong effect of indole-3-acetic acid (IAA) was identified, linking mycorrhiza formation and auxin signaling between the symbiosis partners. We investigated ald1 overexpressing strains for performance in mycorrhiza with the host tree spruce (Picea abies) and observed an increased width of the apoplast, accommodating the Hartig' net hyphae of the T. vaccinum over-expressing transformants. The results support a role for Ald1 in ectomycorrhiza formation and underline functional differentiation within fungal aldehyde dehydrogenases in the family 1 of ALDHs. PMID:26344933

  14. Mechanistic Insights from Reaction of α-Oxiranyl-Aldehydes with Cyanobacterial Aldehyde Deformylating Oxygenase

    PubMed Central

    Das, Debasis; Ellington, Benjamin; Paul, Bishwajit; Marsh, E. Neil G.

    2014-01-01

    The biosynthesis of long-chain aliphatic hydrocarbons, which are derived from fatty acids, is widespread in Nature. The last step in this pathway involves the decarbonylation of fatty aldehydes to the corresponding alkanes or alkenes. In cyanobacteria this is catalyzed by an aldehyde deformylating oxygenase. We have investigated the mechanism of this enzyme using substrates bearing an oxirane ring adjacent to the aldehyde carbon. The enzyme catalyzed the deformylation of these substrates to produce the corresponding oxiranes. Performing the reaction in D2O allowed the facial selectivity of proton addition to be examined by 1H-NMR spectroscopy. The proton is delivered with equal probability to either face of the oxirane ring, indicating the formation of an oxiranyl radical intermediate that is free to rotate during the reaction. Unexpectedly, the enzyme also catalyzes a side reaction in which oxiranyl-aldehydes undergo tandem deformylation to furnish alkanes two carbons shorter. We present evidence that this involves the rearrangement of the intermediate oxiranyl radical formed in the first step, resulting an aldehyde that is further deformylated in a second step. These observations provide support for a radical mechanism for deformylation and, furthermore, allow the lifetime of the radical intermediate to be estimated based on prior measurements of rate constants for the rearrangement of oxiranyl radicals. PMID:24313866

  15. Waste recycling by vermicomposting: Maturity and quality assessment via dehydrogenase enzyme activity, lignin, water soluble carbon, nitrogen, phosphorous and other indicators.

    PubMed

    Alidadi, Hossein; Hosseinzadeh, Ahmad; Najafpoor, Ali Asghar; Esmaili, Habibollah; Zanganeh, Jafar; Dolatabadi Takabi, Maryam; Piranloo, Fardin Ghasemy

    2016-11-01

    Present study aims to examine the dynamics of maturation and qualification indicators in various vermicompost treatments and selection of the best treatment along with best maturation time in this regard. In this empirical study, dynamics of chemical (pH, electrical conductivity (EC), total nitrogen (TN), phosphorous, lignin, water soluble carbon (WSC), C/N, NH4/NO3) and biological (dehydrogenase enzyme (DEH) and DEH/WSC) properties were investigated in four various treatments, including various ratios of compost produced from municipal solid waste (MSW) and carbonaceous materials (50:50, 70:30, 85:15 and 100:0) over 100 days. Results showed a significant fluctuation in EC, DEH and DEH/WSC proportions over the process. In addition, a noticeable increase was observed for the dynamics of TN, phosphorous and lignin. In contrast, the C/N, NH4/NO3 and WSC values gradually decreased during the process. Moreover, it was observed that the length of 75 days for the process is an appropriate time for maturation of all treatments. However, the first and second treatments resulted in better outcomes compared with the other types of treatments. From the point of view of quality obtained vermicompost was nitrogen enriched product in all treatments. Whereas, for the phosphorous elements this method is appropriate for the first treatment only. PMID:27472049

  16. Somatic-cell selection is a major determinant of the blood-cell phenotype in heterozygotes for glucose-6-phosphate dehydrogenase mutations causing severe enzyme deficiency.

    PubMed Central

    Filosa, S.; Giacometti, N.; Wangwei, C.; De Mattia, D.; Pagnini, D.; Alfinito, F.; Schettini, F.; Luzzatto, L.; Martini, G.

    1996-01-01

    X-chromosome inactivation in mammals is regarded as an essentially random process, but the resulting somatic-cell mosaicism creates the opportunity for cell selection. In most people with red-blood-cell glucose-6-phosphate dehydrogenase (G6PD) deficiency, the enzyme-deficient phenotype is only moderately expressed in nucleated cells. However, in a small subset of hemizygous males who suffer from chronic nonspherocytic hemolytic anemia, the underlying mutations (designated class I) cause more-severe G6PD deficiency, and this might provide an opportunity for selection in heterozygous females during development. In order to test this possibility we have analyzed four heterozygotes for class I G6PD mutations: two with G6PD Portici (1178G-->A) and two with G6PD Bari (1187C-->T). We found that in fractionated blood cell types (including erythroid, myeloid, and lymphoid cell lineages) there was a significant excess of G6PD-normal cells. The significant concordance that we have observed in the degree of imbalance in the different blood-cell lineages indicates that a selective mechanism is likely to operate at the level of pluripotent blood stem cells. Thus, it appears that severe G6PD deficiency affects adversely the proliferation or the survival of nucleated blood cells and that this phenotypic characteristic is critical during hematopoiesis. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8808605

  17. SIRT3 and SIRT5 Regulate the Enzyme Activity and Cardiolipin Binding of Very Long-Chain Acyl-CoA Dehydrogenase

    PubMed Central

    Zhang, Yuxun; Bharathi, Sivakama S.; Rardin, Matthew J.; Uppala, Radha; Verdin, Eric; Gibson, Bradford W.; Goetzman, Eric S.

    2015-01-01

    SIRT3 and SIRT5 have been shown to regulate mitochondrial fatty acid oxidation but the molecular mechanisms behind the regulation are lacking. Here, we demonstrate that SIRT3 and SIRT5 both target human very long-chain acyl-CoA dehydrogenase (VLCAD), a key fatty acid oxidation enzyme. SIRT3 deacetylates and SIRT5 desuccinylates K299 which serves to stabilize the essential FAD cofactor in the active site. Further, we show that VLCAD binds strongly to cardiolipin and isolated mitochondrial membranes via a domain near the C-terminus containing lysines K482, K492, and K507. Acetylation or succinylation of these residues eliminates binding of VLCAD to cardiolipin. SIRT3 deacetylates K507 while SIRT5 desuccinylates K482, K492, and K507. Sirtuin deacylation of recombinant VLCAD rescues membrane binding. Endogenous VLCAD from SIRT3 and SIRT5 knockout mouse liver shows reduced binding to cardiolipin. Thus, SIRT3 and SIRT5 promote fatty acid oxidation by converging upon VLCAD to promote its activity and membrane localization. Regulation of cardiolipin binding by reversible lysine acylation is a novel mechanism that is predicted to extrapolate to other metabolic proteins that localize to the inner mitochondrial membrane. PMID:25811481

  18. Identification of a novel operon in Lactococcus lactis encoding three enzymes for lactic acid synthesis: phosphofructokinase, pyruvate kinase, and lactate dehydrogenase.

    PubMed Central

    Llanos, R M; Harris, C J; Hillier, A J; Davidson, B E

    1993-01-01

    The discovery of a novel multicistronic operon that encodes phosphofructokinase, pyruvate kinase, and lactate dehydrogenase in the lactic acid bacterium Lactococcus lactis is reported. The three genes in the operon, designated pfk, pyk, and ldh, contain 340, 502, and 325 codons, respectively. The intergenic distances are 87 bp between pfk and pyk and 117 bp between pyk and ldh. Plasmids containing pfk and pyk conferred phosphofructokinase and pyruvate kinase activity, respectively, on their host. The identity of ldh was established previously by the same approach (R. M. Llanos, A. J. Hillier, and B. E. Davidson, J. Bacteriol. 174:6956-6964, 1992). Each of the genes is preceded by a potential ribosome binding site. The operon is expressed in a 4.1-kb transcript. The 5' end of the transcript was determined to be a G nucleotide positioned 81 bp upstream from the pfk start codon. The pattern of codon usage within the operon is highly biased, with 11 unused amino acid codons. This degree of bias suggests that the operon is highly expressed. The three proteins encoded on the operon are key enzymes in the Embden-Meyerhoff pathway, the central pathway of energy production and lactic acid synthesis in L. lactis. For this reason, we have called the operon the las (lactic acid synthesis) operon. Images PMID:8478320

  19. SIRT3 and SIRT5 regulate the enzyme activity and cardiolipin binding of very long-chain acyl-CoA dehydrogenase.

    PubMed

    Zhang, Yuxun; Bharathi, Sivakama S; Rardin, Matthew J; Uppala, Radha; Verdin, Eric; Gibson, Bradford W; Goetzman, Eric S

    2015-01-01

    SIRT3 and SIRT5 have been shown to regulate mitochondrial fatty acid oxidation but the molecular mechanisms behind the regulation are lacking. Here, we demonstrate that SIRT3 and SIRT5 both target human very long-chain acyl-CoA dehydrogenase (VLCAD), a key fatty acid oxidation enzyme. SIRT3 deacetylates and SIRT5 desuccinylates K299 which serves to stabilize the essential FAD cofactor in the active site. Further, we show that VLCAD binds strongly to cardiolipin and isolated mitochondrial membranes via a domain near the C-terminus containing lysines K482, K492, and K507. Acetylation or succinylation of these residues eliminates binding of VLCAD to cardiolipin. SIRT3 deacetylates K507 while SIRT5 desuccinylates K482, K492, and K507. Sirtuin deacylation of recombinant VLCAD rescues membrane binding. Endogenous VLCAD from SIRT3 and SIRT5 knockout mouse liver shows reduced binding to cardiolipin. Thus, SIRT3 and SIRT5 promote fatty acid oxidation by converging upon VLCAD to promote its activity and membrane localization. Regulation of cardiolipin binding by reversible lysine acylation is a novel mechanism that is predicted to extrapolate to other metabolic proteins that localize to the inner mitochondrial membrane. PMID:25811481

  20. Biofuel cells based on direct enzyme-electrode contacts using PQQ-dependent glucose dehydrogenase/bilirubin oxidase and modified carbon nanotube materials.

    PubMed

    Scherbahn, V; Putze, M T; Dietzel, B; Heinlein, T; Schneider, J J; Lisdat, F

    2014-11-15

    Two types of carbon nanotube electrodes (1) buckypaper (BP) and (2) vertically aligned carbon nanotubes (vaCNT) have been used for elaboration of glucose/O2 enzymatic fuel cells exploiting direct electron transfer. For the anode pyrroloquinoline quinone dependent glucose dehydrogenase ((PQQ)GDH) has been immobilized on [poly(3-aminobenzoic acid-co-2-methoxyaniline-5-sulfonic acid), PABMSA]-modified electrodes. For the cathode bilirubin oxidase (BOD) has been immobilized on PQQ-modified electrodes. PABMSA and PQQ act as promoter for enzyme bioelectrocatalysis. The voltammetric characterization of each electrode shows current densities in the range of 0.7-1.3 mA/cm(2). The BP-based fuel cell exhibits maximal power density of about 107 µW/cm(2) (at 490 mV). The vaCNT-based fuel cell achieves a maximal power density of 122 µW/cm(2) (at 540 mV). Even after three days and several runs of load a power density over 110 µW/cm(2) is retained with the second system (10mM glucose). Due to a better power exhibition and an enhanced stability of the vaCNT-based fuel cells they have been studied in human serum samples and a maximal power density of 41 µW/cm(2) (390 mV) can be achieved. PMID:24967753

  1. Somatic-cell selection is a major determinant of the blood-cell phenotype in heterozygotes for glucose-6-phosphate dehydrogenase mutations causing severe enzyme deficiency

    SciTech Connect

    Filosa, S.; Giacometti, N.; Wangwei, C.; Martini, G.

    1996-10-01

    X-chromosome inactivation in mammals is regarded as an essentially random process, but the resulting somatic-cell mosaicism creates the opportunity for cell selection. In most people with red-blood-cell glucose-6-phosphate dehydrogenase (G6PD) deficiency, the enzyme-deficient phenotype is only moderately expressed in nucleated cells. However, in a small subset of hemizygous males who suffer from chronic nonspherocytic hemolytic anemia, the underlying mutations (designated class I) cause more-severe G6PD deficiency, and this might provide an opportunity for selection in heterozygous females during development. In order to test this possibility we have analyzed four heterozygotes for class I G6PD mutations: two with G6PD Portici (1178G{r_arrow}A) and two with G6PD Bari (1187C{r_arrow}T). We found that in fractionated blood cell types (including erythroid, myeloid, and lymphoid cell lineages) there was a significant excess of G6PD-normal cells. The significant concordance that we have observed in the degree of imbalance in the different blood-cell lineages indicates that a selective mechanism is likely to operate at the level of pluripotent blood stem cells. Thus, it appears that severe G6PD deficiency affects adversely the proliferation or the survival of nucleated blood cells and that this phenotypic characteristic is critical during hematopoiesis. 65 refs., 6 figs., 3 tabs.

  2. Expression profiles of cortisol-inactivating enzyme, 11β-hydroxysteroid dehydrogenase-2, in human epidermal tumors and its role in keratinocyte proliferation.

    PubMed

    Terao, Mika; Itoi, Saori; Murota, Hiroyuki; Katayama, Ichiro

    2013-02-01

    The enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) catalyzes the interconversion between hormonally active cortisol and inactive cortisone within cells. There are two isozymes: 11β-HSD1 activates cortisol from cortisone and 11β-HSD2 inactivates cortisol to cortisone. 11β-HSD1 was recently discovered in skin, and we subsequently found that the enzyme negatively regulates keratinocyte proliferation. We verified 11β-HSD1 and 11β-HSD2 expression in benign and malignant skin tumors and investigated the role of 11β-HSD in skin tumor pathogenesis. Randomly selected formalin-fixed sections of skin lesions of seborrheic keratosis (SK), squamous cell carcinoma (SCC), and basal cell carcinoma (BCC) were stained with 11β-HSD1 and 11β-HSD2 antibodies, and 11β-HSD expression was also evaluated in murine epidermis in which hyperproliferation was induced by 12-O-tetradecanoylphorbol-13 acetate (TPA). We observed that 11β-HSD1 expression was decreased in all SK, SCC, and BCC lesions compared with unaffected skin. Conversely, 11β-HSD2 expression was increased in SK and BCC but not in SCC. Overexpression of 11β-HSD2 in keratinocytes increased cell proliferation. In the murine model, 11β-HSD1 expression was decreased in TPA-treated hyperproliferative skin. Our findings suggest that 11β-HSD1 expression is decreased in keratinocyte proliferative conditions, and 11β-HSD2 expression is increased in basal cell proliferating conditions, such as BCC and SK. Assessing 11β-HSD1 and 11β-HSD2 expression could be a useful tool for diagnosing and characterizing skin tumors. PMID:23362866

  3. Molecular cloning and characterization of a steroidogenic enzyme, 17β-hydroxysteroid dehydrogenase type 14, from the stony coral Euphyllia ancora (Cnidaria, Anthozoa).

    PubMed

    Shikina, Shinya; Chung, Yi-Jou; Chiu, Yi-Ling; Huang, Yi-Jie; Lee, Yan-Horn; Chang, Ching-Fong

    2016-03-01

    Sex steroids play a fundamental role not only in reproduction but also in various other biological processes in vertebrates. Although the presence of sex steroids has been confirmed in cnidarians (e.g., coral, sea anemone, jellyfish, and hydra), which are basal metazoans, only a few studies to date have characterized steroidogenesis-related genes in cnidarians. Based on a transcriptomic analysis of the stony coral Euphyllia ancora, we identified the steroidogenic enzyme 17β-hydroxysteroid dehydrogenase type 14 (17beta-hsd 14), an oxidative enzyme that catalyzes the NAD(+)-dependent inactivation of estrogen/androgen (estradiol to estrone and testosterone to androstenedione) in mammals. Phylogenetic analysis showed that E. ancora 17beta-Hsd 14 (Ea17beta-Hsd 14) clusters with other animal 17beta-HSD 14s but not with other members of the 17beta-HSD family. Subsequent quantitative RT-PCR analysis revealed a lack of correlation of Ea17beta-hsd 14 transcript levels with the coral's reproductive cycle. In addition, Ea17beta-hsd 14 transcript and protein were detected in all tissues examined, such as the tentacles, mesenterial filaments, and gonads, at similar levels in both sexes, as determined by quantitative RT-PCR analysis and Western blotting with an anti-Ea17beta-Hsd 14 antibody. Immunohistochemical analysis revealed that Ea17beta-Hsd 14 is mainly distributed in the endodermal regions of the polyps, but the protein was also observed in all tissues examined. These results suggest that Ea17beta-Hsd 14 is involved in important functions that commonly occur in endodermal cells or has multiple functions in different tissues. Our data provide information for comparison with advanced animals as well as insight into the evolution of steroidogenesis-related genes in metazoans. PMID:26868454

  4. Chemoenzymatic Fc Glycosylation via Engineered Aldehyde Tags

    PubMed Central

    2014-01-01

    Glycoproteins with chemically defined glycosylation sites and structures are important biopharmaceutical targets and critical tools for glycobiology. One approach toward constructing such molecules involves chemical glycosylation of aldehyde-tagged proteins. Here, we report the installation of a genetically encoded aldehyde tag at the internal glycosylation site of the crystallizable fragment (Fc) of IgG1. We replaced the natural Fc N-glycosylation sequon with a five amino-acid sequence that was efficiently converted by recombinant formylglycine generating enzyme in vitro, thereby introducing aldehyde groups for subsequent chemical elaboration. Oxime-linked glycoconjugates were synthesized by conjugating aminooxy N-acetylglucosamine to the modified Fc followed by enzymatic transfer of complex N-glycans from corresponding glycan oxazolines by an EndoS-derived glycosynthase. In this manner we generated specific Fc glycoforms without relying on natural protein glycosylation machineries. PMID:24702330

  5. Purification, crystallization and preliminary crystallographic study of a recombinant plant aminoaldehyde dehydrogenase from Pisum sativum

    PubMed Central

    Tylichová, Martina; Briozzo, Pierre; Kopečný, David; Ferrero, Julien; Moréra, Solange; Joly, Nathalie; Snégaroff, Jacques; Šebela, Marek

    2008-01-01

    Aminoaldehydes are products of polyamine degradation and are known to be reactive metabolites that are toxic to living cells at high concentrations. These compounds are catabolized by aminoaldehyde dehydrogenases, which are enzymes that contain a nicotinamide adenine dinucleotide coenzyme. Amino­aldehyde dehydrogenase from Pisum sativum was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop method. A complete data set was collected to 2.8 Å resolution at 100 K. Crystals belong to the monoclinic space group P21, with unit-cell parameters a = 86.4, b = 216.6, c = 205.4 Å, β = 98.1°. Molecular replacement was performed and led to the identification of six dimers per asymmetric unit. PMID:18259056

  6. Human oestrogenic 17beta-hydroxysteroid dehydrogenase specificity: enzyme regulation through an NADPH-dependent substrate inhibition towards the highly specific oestrone reduction.

    PubMed Central

    Gangloff, A; Garneau, A; Huang, Y W; Yang, F; Lin, S X

    2001-01-01

    Human oestrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1) catalyses the final step in the biosynthesis of all active oestrogens. Here we report the steady-state kinetics for 17beta-HSD1 at 37 degrees C and pH 7.5, using a homogeneous enzyme preparation with oestrone, dehydroepiandrosterone (DHEA) or dihydrotestosterone (DHT) as substrate and NADP(H) as the cofactor. Kinetic studies made over a wide range of oestrone concentrations (10 nM-10 microM) revealed a typical substrate-inhibition phenomenon. Data analysis using the substrate-inhibition equation v=V.[s]/[K(m)+[s](1+[s]/K(i))] gave a K(m) of 0.07+/-0.01 microM, a k(cat) (for the dimer) of 1.5+/-0.1 s(-1), a specificity of 21 microM(-1) x s(-1) and a K(i) of 1.3 microM. When NADH was used instead of NADPH, substrate inhibition was no longer observed and the kinetic constants were significantly modified to 0.42+/-0.07 microM for the K(m), 0.8+/-0.04 s(-1) for the k(cat) and 1.9 microM(-1) x s(-1) for the specificity. The modification of an amino acid in the cofactor-binding site (Leu36Asp) eliminated the substrate inhibition observed in the presence of NADPH, confirming the NADPH-dependence of the phenomenon. The possible formation of an enzyme-NADP(+)-oestrone dead-end complex during the substrate-inhibition process is supported by the competitive inhibition of oestradiol oxidation by oestrone. Kinetic studies performed with either DHEA (K(m)=24+/-4 microM; k(cat)=0.47+/-0.06 s(-1); specificity=0.002 microM(-1) x s(-1)) or DHT (K(m)=26+/-6 microM; k(cat)=0.2+/-0.02 s(-1); specificity=0.0008 microM(-1) x s(-1)) in the presence of NADP(H) resulted in low specificities and no substrate inhibition. Taken together, our results demonstrate that the high specificity of 17beta-HSD1 towards oestrone is coupled with an NADPH-dependent substrate inhibition, suggesting that both the specificity and the enzyme control are provided for the cognate substrate. PMID:11336660

  7. The dilemma of the gender assignment in a Portuguese adolescent with disorder of sex development due to 17β-hydroxysteroid-dehydrogenase type 3 enzyme deficiency

    PubMed Central

    Castro-Correia, Cíntia; Mira-Coelho, Alda; Monteiro, Bessa; Monteiro, Joaquim; Hughes, Ieuan; Fontoura, Manuel

    2014-01-01

    Summary The development of male internal and external genitalia in an XY fetus requires a complex interplay of many critical genes, enzymes, and cofactors. The enzyme 17β-hydroxysteroid-dehydrogenase type 3 (17βHSD3) is present almost exclusively in the testicles and converts Delta 4-androstenodione (Δ4) to testosterone. A deficiency in this enzyme is rare and is a frequently misdiagnosed autosomal recessive cause of 46,XY, disorder of sex development. The case report is of a 15-year-old adolescent, who was raised according to female gender. At puberty, the adolescent had a severe virilization and primary amenorrhea. The physical examination showed a male phenotype with micropenis and blind vagina. The Tanner stage was A3B1P4, nonpalpable gonads. The karyotype revealed 46,XY. The endocrinology study revealed: testosterone=2.38 ng/ml, Δ4>10.00 ng/ml, and low testosterone/Δ4 ratio=0.23. Magnetic resonance imaging of the abdominal–pelvic showed the presence of testicles in inguinal canal, seminal vesicle, prostate, micropenis, and absence of uterus and vagina. The genetic study confirmed the mutation p.Glu215Asp on HSD17B3 gene in homozygosity. The dilemma of sex reassignment was seriously considered when the diagnosis was made. During all procedures the patient was accompanied by a child psychiatrist/psychologist. The teenager desired to continue being a female, so gonadectomy was performed. Estrogen therapy and surgical procedure to change external genitalia was carried out. In this case, there was a severe virilization at puberty. It is speculated to be due to a partial activity of 17βHSD3 in the testicles and/or extratesticular ability to convert Δ4 to testosterone by 17βHSD5. Prenatal exposure of the brain to androgens has increasingly been put forward as a critical factor in gender identity development, but in this case the social factor was more important for the gender assignment. Learning points In this case, we highlight the late diagnosis

  8. An enzyme flow immunoassay that uses beta-galactosidase as the label and a cellobiose dehydrogenase biosensor as the label detector.

    PubMed

    Burestedt, E; Nistor, C; Schagerlöf, U; Emnéus, J

    2000-09-01

    The aim was to develop a fast generic enzyme flow immunoassay (EFIA) using a beta-galactosidase (beta-GAL) label in combination with colorimetric detection as well as with a new amperometric biosensor as the label detector. The amperometric biosensor was previously developed within the group for the determination of diphenols in surface water samples. Antigen (Ag, analyte), tracer (Ag*, antigen labeled with beta-GAL), and antibody (Ab) were incubated off-line. After the equilibrium was reached, the sample was introduced into the flow system. The antibody complexes, AgAb and Ag*Ab, were trapped in a protein G column while the free unbound tracer was eluted and detected by an amperometric biosensor downstream after substrate reaction. The enzyme label beta-GAL converted the substrate 4-aminophenyl-beta-D-galactopyranoside (4-APG) into 4-aminophenol (4-AP), which subsequently was detected by a cellobiose dehydrogenase (CDH) modified solid graphite electrode. 4-AP was first oxidized at the electrode surface at +300 mV vs Ag/AgCl, and the formed 4-imino quinone (4-IQ) was reduced back to 4-AP by the CDH in the presence of cellobiose. By combining the EFIA with the CDH biosensor, the overall signal of one tracer molecule is amplified at two occasions, i.e., one enzyme label converts the substrate into many 4-AP molecules, and second these are further amplified by the CDH biosensor. The optimum conditions for the EFIA in terms of the molar ratio between tracer and beta-GAL, temperature, flow rate, etc., was investigated with colorimetric detection, using 2-nitrophenyl-beta-D-galactopyranoside (2-NPG) as the beta-GAL substrate. The performance of both the colorimetric and CDH biosensor detection was investigated and both methods were applied for determination of the model compound atrazine in spiked surface water samples. Detection limits of 0.056 +/- 0.008 and 0.038 +/- 0.007 microg L(-1) and IC50 values of 2.04 +/- 0.294 and 0.42 +/- 0.08 microg L(-1) were obtained for

  9. The asymmetric distribution of enzymic activity between the six subunits of bovine liver glutamate dehydrogenase. Use of D- and L-glutamyl alpha-chloromethyl ketones (4-amino-6-chloro-5-oxohexanoic acid.

    PubMed Central

    Rasool, C G; Nicolaidis, S; Akhtar, M

    1976-01-01

    A method for the preparation of D- and L-glutamyl alpha-chloromethyl ketones (4-amino-6-chloro-5-oxohexanoic acid) is described. These chloromethyl ketones irreversibly inactivated bovine glutamate dehydrogenase, whereas several other related compounds had no adverse effect on the activity of the enzyme. The inactivation process was shown to be due to the modification of lysine-126. The time-courses for the inactivation and the incorporation of radioactivity from tritiated L-glutamyl alpha-chloromethyl ketone into the glutamate dehydrogenase were biphasic. The results were interpreted to suggest the involvement of 'negative co-operative' interactions in the reactivity of lysine-126. From the cumulative evidence it is argued that the first subunit of the enzyme, which takes part in catalysis, makes the largest, and the last the smallest, contribution to the overall catalysis. It is emphasized that three of the six subunits of the enzyme may possess as much as 80% of the total activity of bovine glutamate dehydrogenase. PMID:10889

  10. A low-molecular-mass protein from Methylococcus capsulatus (Bath) is responsible for the regulation of formaldehyde dehydrogenase activity in vitro.

    PubMed

    Tate, S; Dalton, H

    1999-01-01

    An 8.6 kDa protein, which the authors call a modifin, has been purified from Methylococcus capsulatus (Bath) and has been shown to alter the substrate specificity and kinetics of NAD+-linked formaldehyde dehydrogenase (FDH) isolated from the same organism. Purification methods for both the modifin and FDH are presented which reliably produced pure protein for further analysis. Analysis of the molecular mass and N-terminal sequence of both FDH and the modifin indicate that they are unique proteins and show no similarity to alcohol or aldehyde dehydrogenase enzymes isolated from methylotrophic bacteria. Substrate specificity studies demonstrated that FDH oxidized formaldehyde exclusively in the presence of the modifin; a diverse range of aldehydes and alcohols were oxidized by FDH in the absence of the modifin. No formaldehyde oxidation was detected in the absence of the modifin. Attempts to replace the modifin with glutathione or high concentrations of methanol to stimulate formaldehyde oxidation failed. With acetaldehyde as substrate, FDH showed standard Michaelis-Menten kinetics; interaction of FDH with the modifin using formaldehyde as substrate altered the kinetics of the reaction to sigmoidal. Kinetic analysis during turnover experiments indicated that the FDH may be associated with bound formaldehyde following enzyme isolation and that NAD may also be associated with the enzyme but in a form that is less tightly bound than found with the methanol dehydrogenase from Bacillus methanolicus. Data are presented which indicate that the modifin may play an important role in regulating formaldehyde concentration in vivo. PMID:10206695

  11. Identification of biotransformation enzymes in the antennae of codling moth Cydia pomonella.

    PubMed

    Huang, Xinglong; Liu, Lu; Su, Xiaoji; Feng, Jinian

    2016-04-10

    Biotransformation enzymes are found in insect antennae and play a critical role in degrading xenobiotics and odorants. In Cydia pomonella, we identified 26 biotransformation enzymes. Among these enzymes, twelve carboxylesterases (CXEs), two aldehyde oxidases (AOXs) and six alcohol dehydrogenases (ADs) were predominantly expressed in antennae. Each of the CpomCXEs presents a conserved catalytic triad "Ser-His-Glu", which is the structural characteristic of known insect CXEs. CpomAOXs present two redox centers, a FAD-binding domain and a molybdenum cofactor/substrate-binding domain. The antennal CpomADs are from two protein families, short-chain dehydrogenases/reducetases (SDRs) and medium-chain dehydrogenases/reducetases (MDRs). Putative catalytic active domain and cofactor binding domain were found in these CpomADs. Potential functions of these enzymes were determined by phylogenetic analysis. The results showed that these enzymes share close relationship with odorant degrading enzymes (ODEs) and resistance-associated enzymes of other insect species. Because of commonly observed roles of insect antennal biotransformation enzymes, we suggest antennal biotransformation enzymes presented here are candidate that involved in degradation of odorants and xenobiotics within antennae of C. pomonella. PMID:26778204

  12. The aromatic alcohol dehydrogenases in Pseudomonas putida N.C.I.B. 9869 grown on 3,5-xylenol and p-cresol.

    PubMed Central

    Keat, M J; Hopper, D J

    1978-01-01

    Whole cells of Pseudomonas putida N.C.I.B 9869, when grown on either 3,5-xylenol or p-cresol, oxidized both m- and p-hydroxybenzyl alcohols. Two distinct NAD+-dependent m-hydroxybenzyl alcohol dehydrogenases were purified from cells grown on 3,5-xylenol. Each is active with a range of aromatic alcohols, including both m- and p-hydroxybenzyl alcohol, but differ in their relative rates with the various substrates. An NAD+-dependent alcohol dehydrogenase was also partially purified from p-cresol grown cells. This too was active with m- and p-hydroxybenzyl alcohol and other aromatic alcohols, but was not identical with either of the other two dehydrogenases. All three enzymes were unstable, but were stabilized by dithiothreitol and all were inhibited with p-chloromercuribenzoate. All were specific for NAD+ and each was shown to catalyse conversion of alcohol into aldehyde. PMID:743216

  13. Effects of aldehydes on the growth and lipid accumulation of oleaginous yeast Trichosporon fermentans.

    PubMed

    Huang, Chao; Wu, Hong; Liu, Qiu-ping; Li, Yuan-yuan; Zong, Min-hua

    2011-05-11

    The effects of five representative aldehydes in lignocellulosic hydrolysates on the growth and the lipid accumulation of oleaginous yeast Trichosporon fermentans were investigated for the first time. There was no relationship between the hydrophobicity and the toxicity of aldehyde, and 5-hydroxymethylfurfural was less toxic than aromatic aldehydes and furfural. Binary combination of aromatic aldehydes caused a synergistic inhibitory effect, but combination of furan and aromatic aldehydes reduced the inhibition instead. A longer lag phase was found due to the presence of aldehydes and the decrease of sugar consumption rate, but more xylose was utilized by T. fermentans in the presence of aldehydes, especially at their low concentrations. The variation of malic enzyme activity was not related to the delay of lipid accumulation. Furthermore, the inhibition of aldehydes on cell growth was more dependent on inoculum size, temperature, and initial pH than that on lipid content. PMID:21443267

  14. Diagnosing Clostridium difficile-associated diarrhea using enzyme immunoassay: the clinical significance of toxin negativity in glutamate dehydrogenase-positive patients

    PubMed Central

    Yuhashi, Kazuhito; Yagihara, Yuka; Misawa, Yoshiki; Sato, Tomoaki; Saito, Ryoichi; Okugawa, Shu; Moriya, Kyoji

    2016-01-01

    Purpose The enzyme immunoassay (EIA) has lower sensitivity for Clostridium difficile toxins A and B than the polymerase chain reaction in the diagnosis of C. difficile-associated diarrhea (CDAD). Furthermore, toxin positivity with EIA performed on C. difficile isolates from stool cultures may be observed even in patients with EIA glutamate dehydrogenase (GDH)-positive and toxin-negative stool specimens. It is unclear whether such patients should be treated as having CDAD. Methods The present study retrospectively compared patient characteristics, treatment, and diarrhea duration among three groups of patients who underwent stool EIA testing for CDAD diagnosis: a toxin-positive stool group (positive stool group; n=39); a toxin-negative stool/toxin-positive isolate group (discrepant negative/positive group, n=14); and a dual toxin-negative stool and isolate group (dual negative group, n=15). All cases included were confirmed to be GDH positive on EIA test. Results Patients’ backgrounds and comorbidities were not significantly different among three groups. No difference was observed among the three groups with regard to antimicrobial drug use before diarrhea onset. Treatment was received by 82.1% of the positive stool group compared to 7.1% of the discrepant positive/negative group and 0% of the dual negative group, while mean diarrhea duration was 10.6 days compared to 7.9 days (P=0.6006) and 3.4 days (P=0.0312), respectively. Conclusion Even without treatment, patients with toxin-negative stool specimens had shorter diarrhea duration than those with toxin-positive stool specimens even with toxin-positive isolates. These findings may suggest a limited need for CDAD treatment for GDH-positive patients and toxin-negative stool specimens. PMID:27313472

  15. Population screening for glucose-6-phosphate dehydrogenase deficiencies in Isabel Province, Solomon Islands, using a modified enzyme assay on filter paper dried bloodspots

    PubMed Central

    2010-01-01

    Background Glucose-6-phosphate dehydrogenase deficiency poses a significant impediment to primaquine use for the elimination of liver stage infection with Plasmodium vivax and for gametocyte clearance, because of the risk of life-threatening haemolytic anaemia that can occur in G6PD deficient patients. Although a range of methods for screening G6PD deficiency have been described, almost all require skilled personnel, expensive laboratory equipment, freshly collected blood, and are time consuming; factors that render them unsuitable for mass-screening purposes. Methods A published WST8/1-methoxy PMS method was adapted to assay G6PD activity in a 96-well format using dried blood spots, and used it to undertake population screening within a malaria survey undertaken in Isabel Province, Solomon Islands. The assay results were compared to a biochemical test and a recently marketed rapid diagnostic test. Results Comparative testing with biochemical and rapid diagnostic test indicated that results obtained by filter paper assay were accurate providing that blood spots were assayed within 5 days when stored at ambient temperature and 10 days when stored at 4 degrees. Screening of 8541 people from 41 villages in Isabel Province, Solomon Islands revealed the prevalence of G6PD deficiency as defined by enzyme activity < 30% of normal control was 20.3% and a prevalence of severe deficiency that would predispose to primaquine-induced hemolysis (WHO Class I-II) of 6.9%. Conclusions The assay enabled simple and quick semi-quantitative population screening in a malaria-endemic region. The study indicated a high prevalence of G6PD deficiency in Isabel Province and highlights the critical need to consider G6PD deficiency in the context of P. vivax malaria elimination strategies in Solomon Islands, particularly in light of the potential role of primaquine mass drug administration. PMID:20684792

  16. Overexpression of Lactobacillus casei D-hydroxyisocaproic acid dehydrogenase in cheddar cheese.

    PubMed

    Broadbent, Jeffery R; Gummalla, Sanjay; Hughes, Joanne E; Johnson, Mark E; Rankin, Scott A; Drake, Mary Anne

    2004-08-01

    Metabolism of aromatic amino acids by lactic acid bacteria is an important source of off-flavor compounds in Cheddar cheese. Previous work has shown that alpha-keto acids produced from Trp, Tyr, and Phe by aminotransferase enzymes are chemically labile and may degrade spontaneously into a variety of off-flavor compounds. However, dairy lactobacilli can convert unstable alpha-keto acids to more-stable alpha-hydroxy acids via the action of alpha-keto acid dehydrogenases such as d-hydroxyisocaproic acid dehydrogenase. To further characterize the role of this enzyme in cheese flavor, the Lactobacillus casei d-hydroxyisocaproic acid dehydrogenase gene was cloned into the high-copy-number vector pTRKH2 and transformed into L. casei ATCC 334. Enzyme assays confirmed that alpha-keto acid dehydrogenase activity was significantly higher in pTRKH2:dhic transformants than in wild-type cells. Reduced-fat Cheddar cheeses were made with Lactococcus lactis starter only, starter plus L. casei ATCC 334, and starter plus L. casei ATCC 334 transformed with pTRKH2:dhic. After 3 months of aging, the cheese chemistry and flavor attributes were evaluated instrumentally by gas chromatography-mass spectrometry and by descriptive sensory analysis. The culture system used significantly affected the concentrations of various ketones, aldehydes, alcohols, and esters and one sulfur compound in cheese. Results further indicated that enhanced expression of d-hydroxyisocaproic acid dehydrogenase suppressed spontaneous degradation of alpha-keto acids, but sensory work indicated that this effect retarded cheese flavor development. PMID:15294819

  17. Alcohol dehydrogenases in Acinetobacter sp. strain HO1-N: role in hexadecanse and hexadecanol metabolism

    SciTech Connect

    Singer, M.E.; Finnerty, W.R.

    1985-12-01

    Multiple alcohol dehydrogenases (ADH) were demonstrated in Acinetobacter sp. strain HO1-N. ADH-A and ADH-B were distinguished on the basis of electrophoretic mobility, pyridine nucleotide cofactor requirement, and substrate specificity. ADH-A is a soluble, NAD-linked, inducible ethanol dehydrogenase (EDH). An ethanol-negative mutant (Eth1) was isolated which contained 6.5% of wild-type EDH activity and was deficient in ADH-A. Eth1 exhibited normal growth on hexadecane and hexadecanol. A second ethanol-negative mutant (Eth3) was acetaldehyde dehydrogenase (ALDH) deficient, having 12.5% of wild-type ALDH activity. Eth3 had threefold-higher EDH activity than the wild-type strain. ALDH is a soluble, NAD-linked, ethanol-inducible enzyme. Eth3 exhibited normal growth on hexadecane, hexadecanol, and fatty aldehyde. ADH-B is soluble, constitutive, NADP-linked ADH which was active with medium-chain-length alcohols. Hexadecanol dehydrogenase (HDH), a soluble and membrane-bound, NAD-linked ADH, was induced 5- to 11-fold by growth on hexadecane or hexadecanol. HDH was distinct from ADH-A and ADH-B. NAD-linked HDH appears to possess a functional role in hexadecane and hexadecanol dissimilation.

  18. The First Mammalian Aldehyde Oxidase Crystal Structure

    PubMed Central

    Coelho, Catarina; Mahro, Martin; Trincão, José; Carvalho, Alexandra T. P.; Ramos, Maria João; Terao, Mineko; Garattini, Enrico; Leimkühler, Silke; Romão, Maria João

    2012-01-01

    Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9 Å. This is the first mammalian AOX whose structure has been solved. The structure provides important insights into the protein active center and further evidence on the catalytic differences characterizing AOX and xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity. PMID:23019336

  19. A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E

    SciTech Connect

    Clarkson, Sonya M.; Hamilton-Brehm, Scott D.; Giannone, Richard J.; Engle, Nancy L.; Tschaplinski, Timothy J.; Hettich, Robert L.; Elkins, James G.

    2014-12-03

    Background: Chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, but also restricts growth via the release of furan aldehydes such as furfural and 5-hydroxymethylfurfural (5-HMF). The physiological effects of these inhibitors on thermophilic, fermentative bacteria is important to understand; especially as cellulolytic strains are being developed for consolidated bioprocessing (CBP) of lignocellulosic feedstocks. Identifying mechanisms for detoxification of aldehydes in naturally resistant strains such as Thermoanaerobacter spp. may also enable improvements in candidate CBP microorganisms. Results: T. pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30 mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ. The proteomes of T. pseudethanolicus 39E grown in the presence or absence of 15 mM furfural were compared to identify upregulated enzymes potentially responsible for the observed reduction. A total of 225 proteins were differentially regulated in response to the 15 mM furfural treatment with 152 upregulated vs. 73 downregulated. Only 86 proteins exhibited a 2-fold change in abundance in either direction. Of these, 53 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least 2-fold by furfural and were targeted for further investigation: Teth39_1597, encodes a predicted butanol dehydrogenase (BdhA) and Teth39_1598, a predicted aldo/keto reductase (AKR). Both genes were cloned from T. pseudethanolicus 39E, with the respective enzymes overexpressed in E. coli and specific activities determined against a variety of aldehydes. BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. AKR also showed significant activity with NADPH

  20. Enzymes of Glycerol and Glyceraldehyde Metabolism in Mouse Liver: Effects of Caloric Restriction and Age on Activities

    PubMed Central

    Hagopian, Kevork; Ramsey, Jon J.; Weindruch, Richard

    2008-01-01

    Synopsis The influence of caloric restriction on hepatic glyceraldehyde and glycerol metabolizing enzyme activities of young and old mice were studied. Glycerol kinase and cytoplasmic glycerol-3-phosphate dehydrogenase activities were increased in both young and old CR mice when compared to controls, while triokinase increased only in old CR mice. Aldehyde dehydrogenase and aldehyde reductase activities in both young and old CR were unchanged by CR. Mitochondrial glycerol-3-phosphate dehydrogenase showed a trend towards an increased activity in old CR mice, while a trend towards a decreased activity in alcohol dehydrogenase was observed in both young and old CR mice. Serum glycerol levels decreased in young and old CR mice. Therefore, increases in glycerol kinase and glycerol-3-phosphate dehydrogenase were associated with a decrease in fasting blood glycerol levels in CR animals. A prominent role for triokinase in glyceraldehyde metabolism with CR was also observed. The results indicate that long-term CR induces sustained increases in the capacity for gluconeogenesis from glycerol. PMID:18429748

  1. Examining the anti-candidal activity of 10 selected Indian herbs and investigating the effect of Lawsonia inermis extract on germ tube formation, protease, phospholipase, and aspartate dehydrogenase enzyme activity in Candida albicans

    PubMed Central

    Ravichandran, Sripathy; Muthuraman, Sundararaman

    2016-01-01

    Objective: The objective of the study is to identify potential anti-candidal agents from natural resources and elucidate the effect of Lawsonia inermis extract on major virulent factors of Candida albicans. Materials and Methods: Plants, the most abundant and readily available resource of diverse bioactives, were chosen for the anti-candidal screening study. Ten different plants that were proven to have antimicrobial activity but not explored much for anti-candidal activity were chosen for this study. Ethyl acetate extract of these plant leaves were tested for the anti-candidal activity. Extracts with good anti-candidal activity were further screened for its effect in C. albicans germ tube formation and enzyme (protease, phospholipase, and aspartate dehydrogenase) activity. Results: Among 10 plants screened, L. inermis extract showed complete inhibition of C. albicans. On further evaluation, this extract completely inhibited C. albicans germ tube formation in serum until the end of incubation period (3 h). This extract also exhibited dose-dependent inhibitory activity against two major virulent enzymes of C. albicans, proteases (27–33%) and phospholipases (44.5%). In addition to it, this extract completely inhibited both the isoforms of constitutive candidal enzyme aspartate dehydrogenase, thereby affecting amino acid biosynthesis. Conclusion: Thus, this study confirms the anti-candidal potential of L. inermis and hence can be considered further for development of anti-candidal drug. PMID:26997722

  2. Saturation transfer difference NMR studies on substrates and inhibitors of succinic semialdehyde dehydrogenases

    SciTech Connect

    Jaeger, Martin Rothacker, Boris; Ilg, Thomas

    2008-08-01

    Saturation transfer difference (STD) NMR experiments on Escherichia coli and Drosophila melanogaster succinic semialdehyde dehydrogenase (SSADH, EC1.2.1.24) suggest that only the aldehyde forms and not the gem-diol forms of the specific substrate succinic semialdehyde (SSA), of selected aldehyde substrates, and of the inhibitor 3-tolualdehyde bind to these enzymes. Site-directed mutagenesis of the active site cysteine311 to alanine in D. melanogaster SSADH leads to an inactive product binding both SSA aldehyde and gem-diol. Thus, the residue cysteine311 is crucial for their discrimination. STD experiments on SSADH and NAD{sup +}/NADP{sup +} indicate differential affinity in agreement with the respective cosubstrate properties. Epitope mapping by STD points to a strong interaction of the NAD{sup +}/NADP{sup +} adenine H2 proton with SSADH. Adenine H8, nicotinamide H2, H4, and H6 also show STD signals. Saturation transfer to the ribose moieties is limited to the anomeric protons of E. coli SSADH suggesting that the NAD{sup +}/NADP{sup +} adenine and nicotinamide, but not the ribose moieties are important for the binding of the coenzymes.

  3. Aldehyde Reduction by Cytochrome P450

    PubMed Central

    Amunom, Immaculate; Srivastava, Sanjay; Prough, Russell A.

    2011-01-01

    This protocol describes the procedure for measuring the relative rates of metabolism of the α,β-unsaturated aldehydes, 9-anthracene aldehyde (9-AA) and 4-hydroxy-trans-2-nonenal (4-HNE); specifically the aldehyde reduction reactions of cytochrome P450s (CYPs). These assays can be performed using either liver microsomal or other tissue fractions, spherosome preparations of recombinant CYPs, or recombinant CYPs from other sources. The method used here to study the reduction of a model α,β-unsaturated aldehyde, 9-AA, by CYPs was adapted from the assay used to investigate 9-anthracene oxidation as reported by Marini et al. (Marini et al., 2003). For experiments measuring reduction of the endogenous aldehyde, 4-HNE, the substrate was incubated with CYP in the presence of oxygen and NADPH and the metabolites were separated by High Pressure Liquid Chromatograpy (HPLC), using an adaptation of the method of Srivastava et al. (Srivastava et al., 2010). For study of 9-AA and 4-HNE reduction, the first step involves incubation of the substrate with the CYP in appropriate media, followed by quantification of metabolites through either spectrofluorimetry or analysis by HPLC coupled with a radiometric assay, respectively. Metabolite identification can be achieved by HPLC GC-mass spectrometric analysis. Inhibitors of cytochrome P450 function can be utilized to show the role of the hemoprotein or other enzymes in these reduction reactions. The reduction reactions for CYP’s were not inhibited by either anaerobiosis or inclusion of CO in the gaseous phase of the reaction mixture. These character of these reactions are similar to those reported for some cytochrome P450-catalyzed azo reduction reactions. PMID:21553396

  4. Michael hydratase alcohol dehydrogenase or just alcohol dehydrogenase?

    PubMed Central

    2014-01-01

    The Michael hydratase – alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans was previously identified as a bi-functional enzyme performing a hydration of α,β-unsaturated ketones and subsequent oxidation of the formed alcohols. The investigations of the bi-functionality were based on a spectrophotometric assay and an activity staining in a native gel of the dehydrogenase. New insights in the recently discovered organocatalytic Michael addition of water led to the conclusion that the previously performed experiments to identify MhyADH as a bi-functional enzyme and their results need to be reconsidered and the reliability of the methodology used needs to be critically evaluated. PMID:24949265

  5. Common catabolic enzyme patterns in a microplankton community of the Humboldt Current System off northern and central-south Chile: Malate dehydrogenase activity as an index of water-column metabolism in an oxygen minimum zone

    NASA Astrophysics Data System (ADS)

    González, R. R.; Quiñones, R. A.

    2009-07-01

    An extensive subsurface oxygen minimum zone off northern and central-south Chile, associated with the Peru-Chile undercurrent, has important effects on the metabolism of the organisms inhabiting therein. Planktonic species deal with the hypoxic and anoxic environments by relying on biochemical as well as physiological processes related to their anaerobic metabolisms. Here we characterize, for the first time, the potential enzymatic activities involved in the aerobic and anaerobic energy production pathways of microplanktonic organisms (<100 μm), their relationship, and this relationship's association with the oxygen concentration and microplanktonic biomass in the oxygen minimum zone and adjacent areas of the Humboldt Current System water column. Our results demonstrate significant potential enzymatic activity of catabolic pathways in the oxygen minimum zone. Malate dehydrogenase had the highest oxidizing activity of nicotinamide adenine dinucleotide (reduced form) in the batch of catabolic enzymatic activities assayed, including potential pyruvate oxidoreductases activity, the electron transport system, and dissimilatory nitrate reductase. Malate dehydrogenase correlated significantly with almost all the enzymes analyzed within and above the oxygen minimum zone, and also with the oxygen concentration and microplankton biomass in the water column of the Humboldt Current System, especially in the oxygen minimum zone off Iquique. These results suggest a possible specific pattern for the catabolic activity of the microplanktonic realm associated with the oxygen minimum zone spread along the Humboldt Current System off Chile. We hypothesize that malate dehydrogenase activity could be an appropriate indicator of microplankton catabolism in the oxygen minimum zone and adjacent areas.

  6. Characterization of enzymes in the oxidation of 1,2-propanediol to D: -(-)-lactic acid by Gluconobacter oxydans DSM 2003.

    PubMed

    Wei, Liujing; Yang, Xuepeng; Gao, Keliang; Lin, Jinping; Yang, Shengli; Hua, Qiang; Wei, Dongzhi

    2010-09-01

    Although Gluconobacter oxydans can convert 1,2-propanediol to D: -(-)-lactic acid, the enzyme(s) responsible for the conversion has remain unknown. In this study, the membrane-bound alcohol dehydrogenase (ADH) of Gluconobacter oxydans DSM 2003 was purified and confirmed to be essential for the process of D: -(-)-lactic acid production by gene knockout and complementation studies. A 25 percent decrease in D: -(-)-lactic acid production was found for the aldehyde dehydrogenase (ALDH) deficient strain of G. oxydans DSM 2003, indicating that this enzyme is involved in the reaction but not necessary. It is the first report that reveals the function of ADH and ALDH in the biooxidation of 1,2-propanediol to D: -(-)-lactic acid by G. oxydans DSM 2003. PMID:20300886

  7. Aldehyde-stabilized cryopreservation.

    PubMed

    McIntyre, Robert L; Fahy, Gregory M

    2015-12-01

    We describe here a new cryobiological and neurobiological technique, aldehyde-stabilized cryopreservation (ASC), which demonstrates the relevance and utility of advanced cryopreservation science for the neurobiological research community. ASC is a new brain-banking technique designed to facilitate neuroanatomic research such as connectomics research, and has the unique ability to combine stable long term ice-free sample storage with excellent anatomical resolution. To demonstrate the feasibility of ASC, we perfuse-fixed rabbit and pig brains with a glutaraldehyde-based fixative, then slowly perfused increasing concentrations of ethylene glycol over several hours in a manner similar to techniques used for whole organ cryopreservation. Once 65% w/v ethylene glycol was reached, we vitrified brains at -135 °C for indefinite long-term storage. Vitrified brains were rewarmed and the cryoprotectant removed either by perfusion or gradual diffusion from brain slices. We evaluated ASC-processed brains by electron microscopy of multiple regions across the whole brain and by Focused Ion Beam Milling and Scanning Electron Microscopy (FIB-SEM) imaging of selected brain volumes. Preservation was uniformly excellent: processes were easily traceable and synapses were crisp in both species. Aldehyde-stabilized cryopreservation has many advantages over other brain-banking techniques: chemicals are delivered via perfusion, which enables easy scaling to brains of any size; vitrification ensures that the ultrastructure of the brain will not degrade even over very long storage times; and the cryoprotectant can be removed, yielding a perfusable aldehyde-preserved brain which is suitable for a wide variety of brain assays. PMID:26408851

  8. Human microsomal carbonyl reducing enzymes in the metabolism of xenobiotics: well-known and promising members of the SDR superfamily.

    PubMed

    Skarydová, Lucie; Wsól, Vladimír

    2012-05-01

    The best known, most widely studied enzyme system in phase I biotransformation is cytochrome P450 (CYP), which participates in the metabolism of roughly 9 of 10 drugs in use today. The main biotransformation isoforms of CYP are associated with the membrane of the endoplasmatic reticulum (ER). Other enzymes that are also active in phase I biotransformation are carbonyl reducing enzymes. Much is known about the role of cytosolic forms of carbonyl reducing enzymes in the metabolism of xenobiotics, but their microsomal forms have been mostly poorly studied. The only well-known microsomal carbonyl reducing enzyme taking part in the biotransformation of xenobiotics is 11β-hydroxysteroid dehydrogenase 1, a member of the short-chain dehydrogenase/reductase superfamily. Physiological roles of microsomal carbonyl reducing enzymes are better known than their participation in the metabolism of xenobiotics. This review is a summary of the fragmentary information known about the roles of the microsomal forms. Besides 11β-hydroxysteroid dehydrogenase 1, it has been reported, so far, that retinol dehydrogenase 12 participates only in the detoxification of unsaturated aldehydes formed upon oxidative stress. Another promising group of microsomal biotransformation carbonyl reducing enzymes are some members of 17β-hydroxysteroid dehydrogenases. Generally, it is clear that this area is, overall, quite unexplored, but carbonyl reducing enzymes located in the ER have proven very interesting. The study of these enzymes could shed new light on the metabolism of several clinically used drugs or they could become an important target in connection with some diseases. PMID:22181347

  9. Growth on D-arabitol of a mutant strain of Escherichia coli K12 using a novel dehydrogenase and enzymes related to L-1,2-propanediol and D-xylose metabolism.

    PubMed

    Wu, T T

    1976-06-01

    Escherichia coli K12 cannot grow on D-arabitol, L-arabitol, ribitol or xylitol (Reiner, 1975). Using a mutant of E. coli K12 (strain 3; Sridhara et al., 1969) that can grow on L-1,2-propanediol, a second-stage mutant was isolated which can utilize D-arabitol as sole source of carbon and energy for growth. D-Arabitol is probably transported into the bacteria by the same system as that used for the transport of L-1,2-propanediol. The second-stage mutant constitutively synthesizes a new dehydrogenase, which is not present in the parent strain 3. This enzyme, whose native substrate may be D-galactose, apparently dehydrogenates D-arabitol to D-xylulose, and its structural gene is located at 68.5 +/- 1 min on the E. coli genetic map. D-Xylulose is subsequently catabolized by the enzymes of the D-xylose metabolic pathway. PMID:181526

  10. Enzymes involved in vinyl acetate decomposition by Pseudomonas fluorescens PCM 2123 strain.

    PubMed

    Szczyrba, Elżbieta; Greń, Izabela; Bartelmus, Grażyna

    2014-03-01

    Esterases are widely used in food processing industry, but there is little information concerning enzymes involved in decompositions of esters contributing to pollution of environment. Vinyl acetate (an ester of vinyl alcohol and acetic acid) is a representative of volatile organic compounds (VOCs) in decomposition, of which hydrolyses and oxidoreductases are mainly involved. Their activities under periodically changing conditions of environment are essential for the removal of dangerous VOCs. Esterase and alcohol/aldehyde dehydrogenase activities were determined in crude cell extract from Pseudomonas fluorescens PMC 2123 after vinyl acetate induction. All examined enzymes exhibit their highest activity at 30-35 °C and pH 7.0-7.5. Esterase preferably hydrolyzed ester bonds with short fatty chains without plain differences for C2 or C4. Comparison of Km values for alcohol and aldehyde dehydrogenases for acetaldehyde suggested that this metabolite was preferentially oxidized than reduced. Activity of alcohol dehydrogenase reducing acetaldehyde to ethanol suggested that one mechanism of defense against the elevated concentration of toxic acetaldehyde could be its temporary reduction to ethanol. Esterase activity was inhibited by phenylmethanesulfonyl fluoride, while β-mercaptoethanol, dithiothreitol, and ethylenediaminetetraacetic acid had no inhibitor effect. From among metal ions, only Mg(2+) and Fe(2+) stimulated the cleavage of ester bond. PMID:23913099

  11. Binding and Channeling of Alternative Substrates in the Enzyme DmpFG: a Molecular Dynamics Study

    PubMed Central

    Smith, Natalie E.; Vrielink, Alice; Attwood, Paul V.; Corry, Ben

    2014-01-01

    DmpFG is a bifunctional enzyme comprised of an aldolase subunit, DmpG, and a dehydrogenase subunit, DmpF. The aldehyde intermediate produced by the aldolase is channeled directly through a buried molecular channel in the protein structure from the aldolase to the dehydrogenase active site. In this study, we have investigated the binding of a series of progressively larger substrates to the aldolase, DmpG, using molecular dynamics. All substrates investigated are easily accommodated within the active site, binding with free energy values comparable to the physiological substrate 4-hydroxy-2-ketovalerate. Subsequently, umbrella sampling was utilized to obtain free energy surfaces for the aldehyde intermediates (which would be generated from the aldolase reaction on each of these substrates) to move through the channel to the dehydrogenase DmpF. Small substrates were channeled with limited barriers in an energetically feasible process. We show that the barriers preventing bulky intermediates such as benzaldehyde from moving through the wild-type protein can be removed by selective mutation of channel-lining residues, demonstrating the potential for tailoring this enzyme to allow its use for the synthesis of specific chemical products. Furthermore, positions of transient escape routes in this flexible channel were determined. PMID:24739167

  12. Structural and kinetic characterization of recombinant 2-hydroxymuconate semialdehyde dehydrogenase from Pseudomonas putida G7.

    PubMed

    Araújo, Simara Semíramis de; Neves, Cíntia Mara Leal; Guimarães, Samuel Leite; Whitman, Christian P; Johnson, William H; Aparicio, Ricardo; Nagem, Ronaldo Alves Pinto

    2015-08-01

    The first enzyme in the oxalocrotonate branch of the naphthalene-degradation lower pathway in Pseudomonas putida G7 is NahI, a 2-hydroxymuconate semialdehyde dehydrogenase which converts 2-hydroxymuconate semialdehyde to 2-hydroxymuconate in the presence of NAD(+). NahI is in family 8 (ALDH8) of the NAD(P)(+)-dependent aldehyde dehydrogenase superfamily. In this work, we report the cloning, expression, purification and preliminary structural and kinetic characterization of the recombinant NahI. The nahI gene was subcloned into a T7 expression vector and the enzyme was overexpressed in Escherichia coli ArcticExpress as a hexa-histidine-tagged fusion protein. After purification by affinity and size-exclusion chromatography, dynamic light scattering and small-angle X-ray scattering experiments were conducted to analyze the oligomeric state and the overall shape of the enzyme in solution. The protein is a tetramer in solution and has nearly perfect 222 point group symmetry. Protein stability and secondary structure content were evaluated by a circular dichroism spectroscopy assay under different thermal conditions. Furthermore, kinetic assays were conducted and, for the first time, KM (1.3±0.3μM) and kcat (0.9s(-1)) values were determined at presumed NAD(+) saturation. NahI is highly specific for its biological substrate and has no activity with salicylaldehyde, another intermediate in the naphthalene-degradation pathway. PMID:26032336

  13. Mechanism of Substrate and Inhibitor Binding of Rhodobacter Capsulatus Xanthine Dehydrogenase

    SciTech Connect

    Dietzel, U.; Kuper, J; Doebbler, J; Schulte, A; Truglio, J; Leimkuhler, S; Kisker, C

    2009-01-01

    Rhodobacter capsulatus xanthine dehydrogenase (XDH) is an (ae)2 heterotetrameric cytoplasmic enzyme that resembles eukaryotic xanthine oxidoreductases in respect to both amino acid sequence and structural fold. To obtain a detailed understanding of the mechanism of substrate and inhibitor binding at the active site, we solved crystal structures of R. capsulatus XDH in the presence of its substrates hypoxanthine, xanthine, and the inhibitor pterin-6-aldehyde using either the inactive desulfo form of the enzyme or an active site mutant (EB232Q) to prevent substrate turnover. The hypoxanthine- and xanthine-bound structures reveal the orientation of both substrates at the active site and show the importance of residue GluB-232 for substrate positioning. The oxygen atom at the C-6 position of both substrates is oriented toward ArgB-310 in the active site. Thus the substrates bind in an orientation opposite to the one seen in the structure of the reduced enzyme with the inhibitor oxypurinol. The tightness of the substrates in the active site suggests that the intermediate products must exit the binding pocket to allow first the attack of the C-2, followed by oxidation of the C-8 atom to form the final product uric acid. Structural studies of pterin-6-aldehyde, a potent inhibitor of R. capsulatus XDH, contribute further to the understanding of the relative positioning of inhibitors and substrates in the binding pocket. Steady state kinetics reveal a competitive inhibition pattern with a Ki of 103.57 {+-} 18.96 nm for pterin-6-aldehyde.

  14. Catalytic and Molecular Properties of the Quinohemoprotein Tetrahydrofurfuryl Alcohol Dehydrogenase from Ralstonia eutropha Strain Bo

    PubMed Central

    Zarnt, Grit; Schräder, Thomas; Andreesen, Jan R.

    2001-01-01

    The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparent kcat/Km and Ki values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a kcat/Km value of 788 × 104 M−1 s−1. The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases. PMID:11222593

  15. The NAD(P)H-utilizing glutamate dehydrogenase of Bacteroides thetaiotaomicron belongs to enzyme family I, and its activity is affected by trans-acting gene(s) positioned downstream of gdhA.

    PubMed Central

    Baggio, L; Morrison, M

    1996-01-01

    Previous studies have suggested that regulation of the enzymes of ammonia assimilation in human colonic Bacteroides species is coordinated differently than in other eubacteria. The gene encoding an NAD(P)H-dependent glutamate dehydrogenase (gdhA) in Bacteroides thetaiotaomicron was cloned and expressed in Escherichia coli by mutant complementation from the recombinant plasmid pANS100. Examination of the predicted GdhA amino acid sequence revealed that this enzyme possesses motifs typical of the family I-type hexameric GDH proteins. Northern blot analysis with a gdhA-specific probe indicated that a single transcript with an electrophoretic mobility of approximately 1.6 kb was produced in both B. thetaiotaomicron and E. coli gdhA+ transformants. Although gdhA transcription was unaffected, no GdhA enzyme activity could be detected in E. coli transformants when smaller DNA fragments from pANS100, which contained the entire gdhA gene, were analyzed. Enzyme activity was restored if these E. coli strains were cotransformed with a second plasmid, which contained a 3-kb segment of DNA located downstream of the gdhA coding region. Frameshift mutagenesis within the DNA downstream of gdhA in pANS100 also resulted in the loss of GdhA enzyme activity. Collectively, these results are interpreted as evidence for the role of an additional gene product(s) in modulating the activity of GDH enzyme activity. Insertional mutagenesis experiments which led to disruption of the gdhA gene on the B. thetaiotaomicron chromosome indicated that gdhA mutants were not glutamate auxotrophs, but attempts to isolate similar mutants with insertion mutations in the region downstream of the gdhA gene were unsuccessful. PMID:8955404

  16. Metal organic frameworks for enzyme immobilization in biofuel cells

    NASA Astrophysics Data System (ADS)

    Bodell, JaDee

    enzymes: nicotinamide adenine dinucleotide (NAD)-dependent alcohol and aldehyde dehydrogenases, and pyrroloquinoline quinone (PQQ)-dependent alcohol and aldehyde dehydrogenases, as well as flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase. Tb-meso MOF was shown to immobilize PQQ-dependent enzymes through ? stacking interactions of the heme in the enzyme and the triazine molecules in the ligand of the MOF. However, the PQQ-dependent dehydrogenases did not have enough catalytic activity present to be measured electrochemically. Finally, ZIF-90 was synthesized under aqueous conditions in the presence of FAD-dependent glucose dehydrogenase (GDH) which led to size selective sheltering of FAD-GDH. FAD-GDH had activity an order of magnitude larger than any of the alcohol dehydrogenases, which provided sufficient catalytic activity to measure electrochemically. The FAD-GDH bound within ZIF-90 was used to build a full biofuel cell resulting in an open circuit voltage of 708 +/- 16 mV and a maximum power density of 2.75 +/- 0.40 microW/cm2.

  17. Fundamental molecular differences between alcohol dehydrogenase classes.

    PubMed Central

    Danielsson, O; Atrian, S; Luque, T; Hjelmqvist, L; Gonzàlez-Duarte, R; Jörnvall, H

    1994-01-01

    Two types of alcohol dehydrogenase in separate protein families are the "medium-chain" zinc enzymes (including the classical liver and yeast forms) and the "short-chain" enzymes (including the insect form). Although the medium-chain family has been characterized in prokaryotes and many eukaryotes (fungi, plants, cephalopods, and vertebrates), insects have seemed to possess only the short-chain enzyme. We have now also characterized a medium-chain alcohol dehydrogenase in Drosophila. The enzyme is identical to insect octanol dehydrogenase. It is a typical class III alcohol dehydrogenase, similar to the corresponding human form (70% residue identity), with mostly the same residues involved in substrate and coenzyme interactions. Changes that do occur are conservative, but Phe-51 is of functional interest in relation to decreased coenzyme binding and increased overall activity. Extra residues versus the human enzyme near position 250 affect the coenzyme-binding domain. Enzymatic properties are similar--i.e., very low activity toward ethanol (Km beyond measurement) and high selectivity for formaldehyde/glutathione (S-hydroxymethylglutathione; kcat/Km = 160,000 min-1.mM-1). Between the present class III and the ethanol-active class I enzymes, however, patterns of variability differ greatly, highlighting fundamentally separate molecular properties of these two alcohol dehydrogenases, with class III resembling enzymes in general and class I showing high variation. The gene coding for the Drosophila class III enzyme produces an mRNA of about 1.36 kb that is present at all developmental stages of the fly, compatible with the constitutive nature of the vertebrate enzyme. Taken together, the results bridge a previously apparent gap in the distribution of medium-chain alcohol dehydrogenases and establish a strictly conserved class III enzyme, consistent with an important role for this enzyme in cellular metabolism. Images PMID:8197167

  18. Alcohol, Aldehydes, Adducts and Airways.

    PubMed

    Sapkota, Muna; Wyatt, Todd A

    2015-01-01

    Drinking alcohol and smoking cigarettes results in the formation of reactive aldehydes in the lung, which are capable of forming adducts with several proteins and DNA. Acetaldehyde and malondialdehyde are the major aldehydes generated in high levels in the lung of subjects with alcohol use disorder who smoke cigarettes. In addition to the above aldehydes, several other aldehydes like 4-hydroxynonenal, formaldehyde and acrolein are also detected in the lung due to exposure to toxic gases, vapors and chemicals. These aldehydes react with nucleophilic targets in cells such as DNA, lipids and proteins to form both stable and unstable adducts. This adduction may disturb cellular functions as well as damage proteins, nucleic acids and lipids. Among several adducts formed in the lung, malondialdehyde DNA (MDA-DNA) adduct and hybrid malondialdehyde-acetaldehyde (MAA) protein adducts have been shown to initiate several pathological conditions in the lung. MDA-DNA adducts are pre-mutagenic in mammalian cells and induce frame shift and base-pair substitution mutations, whereas MAA protein adducts have been shown to induce inflammation and inhibit wound healing. This review provides an insight into different reactive aldehyde adducts and their role in the pathogenesis of lung disease. PMID:26556381

  19. MASS SPECTROMETRY OF FATTY ALDEHYDES

    PubMed Central

    Berdyshev, Evgeny V.

    2011-01-01

    Fatty aldehydes are important components of the cellular lipidome. Significant interest has been developed towards the analysis of the short chain α,β-unsaturated and hydroxylated aldehydes formed as a result of oxidation of polyunsaturated fatty acids. Multiple gas chromatography-mass spectrometry (GC/MS) and subsequently liquid chromatography-mass spectrometry (LC/MS) approaches have been developed to identify and quantify short-chain as well as long-chain fatty aldehydes. Due to the ability to non-enzymaticaly form Schiff bases with amino groups of proteins, lipids, and with DNA guanidine, free aldehydes are viewed as a marker or metric of fatty acid oxidation and not the part of intracellular signaling pathways which has significantly limited the overall attention this group of molecules have received. This review provides an overview of current GC/MS and LC/MS approaches of fatty aldehyde analysis as well as discusses technical challenges standing in the way of free fatty aldehyde quantitation. PMID:21930240

  20. Alcohol, Aldehydes, Adducts and Airways

    PubMed Central

    Sapkota, Muna; Wyatt, Todd A.

    2015-01-01

    Drinking alcohol and smoking cigarettes results in the formation of reactive aldehydes in the lung, which are capable of forming adducts with several proteins and DNA. Acetaldehyde and malondialdehyde are the major aldehydes generated in high levels in the lung of subjects with alcohol use disorder who smoke cigarettes. In addition to the above aldehydes, several other aldehydes like 4-hydroxynonenal, formaldehyde and acrolein are also detected in the lung due to exposure to toxic gases, vapors and chemicals. These aldehydes react with nucleophilic targets in cells such as DNA, lipids and proteins to form both stable and unstable adducts. This adduction may disturb cellular functions as well as damage proteins, nucleic acids and lipids. Among several adducts formed in the lung, malondialdehyde DNA (MDA-DNA) adduct and hybrid malondialdehyde-acetaldehyde (MAA) protein adducts have been shown to initiate several pathological conditions in the lung. MDA-DNA adducts are pre-mutagenic in mammalian cells and induce frame shift and base-pair substitution mutations, whereas MAA protein adducts have been shown to induce inflammation and inhibit wound healing. This review provides an insight into different reactive aldehyde adducts and their role in the pathogenesis of lung disease. PMID:26556381

  1. Cellobiose dehydrogenase in cellulose degradation

    SciTech Connect

    Eriksson, L.; Igarashi, Kiyohiko; Samejima, Masahiro

    1996-10-01

    Cellobiose dehydrogenase is produced by a variety of fungi. Although it was already discovered during the 70`s, it`s role in cellulose and lignin degradation is yet ambiguous. The enzyme contains both heme and FAD as prosthetic groups, and seems to have a domain specifically designed to bind the enzyme to cellulose. It`s affinity to amorphous cellulose is higher than to crystalline cellulose. We will report on the binding behavior of the enzyme, its usefulness in elucidation of cellulose structures and also, possibilities for applications such as its use in measuring individual and synergistic mechanisms for cellulose degradation by endo- and exo-glucanases.

  2. Purification and properties of S-hydroxymethylglutathione dehydrogenase of Paecilomyces variotii no. 5, a formaldehyde-degrading fungus

    PubMed Central

    2012-01-01

    S-hydroxymethylglutathione dehydrogenase from Paecilomyces variotii No. 5 strain (NBRC 109023), isolated as a formaldehyde-degrading fungus, was purified by a procedure that included ammonium sulfate precipitation, DEAE-Sepharose and hydroxyapatite chromatography and isoelectrofocusing. Approximately 122-fold purification was achieved with a yield of 10.5%. The enzyme preparation was homogeneous as judged by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the purified enzyme was estimated to be 49 kDa by SDS-PAGE and gel filtration, suggesting that it is a monomer. Enzyme activity was optimal at pH 8.0 and was stable in the range of pH 7.0–10. The optimum temperature for activity was 40°C and the enzyme was stable up to 40°C. The isoelectric point was pH 5.8. Substrate specificity was very high for formaldehyde. Besides formaldehyde, the only aldehyde or alcohol tested that served as a substrate was pyruvaldehyde. Enzyme activity was enhanced by several divalent cations such as Mn2+ (179%), Ba2+ (132%), and Ca2+ (112%) but was completely inhibited by Ni2+, Fe3+, Hg2+, p-chloromercuribenzoate (PCMB) and cuprizone. Inactivation of the enzyme by sulfhydryl reagents (Hg2+ and PCMB) indicated that the sulfhydryl group of the enzyme is essential for catalytic activity. PMID:22731626

  3. Phylogeny and structure of the cinnamyl alcohol dehydrogenase gene family in Brachypodium distachyon.

    PubMed

    Bukh, Christian; Nord-Larsen, Pia Haugaard; Rasmussen, Søren K

    2012-10-01

    Cinnamyl alcohol dehydrogenase (CAD) catalyses the final step of the monolignol biosynthesis, the conversion of cinnamyl aldehydes to alcohols, using NADPH as a cofactor. Seven members of the CAD gene family were identified in the genome of Brachypodium distachyon and five of these were isolated and cloned from genomic DNA. Semi-quantitative reverse-transcription PCR revealed differential expression of the cloned genes, with BdCAD5 being expressed in all tissues and highest in root and stem while BdCAD3 was only expressed in stem and spikes. A phylogenetic analysis of CAD-like proteins placed BdCAD5 on the same branch as bona fide CAD proteins from maize (ZmCAD2), rice (OsCAD2), sorghum (SbCAD2) and Arabidopsis (AtCAD4, 5). The predicted three-dimensional structures of both BdCAD3 and BdCAD5 resemble that of AtCAD5. However, the amino-acid residues in the substrate-binding domains of BdCAD3 and BdCAD5 are distributed symmetrically and BdCAD3 is similar to that of poplar sinapyl alcohol dehydrogenase (PotSAD). BdCAD3 and BdCAD5 expressed and purified from Escherichia coli both showed a temperature optimum of about 50 °C and molar weight of 49 kDa. The optimal pH for the reduction of coniferyl aldehyde were pH 5.2 and 6.2 and the pH for the oxidation of coniferyl alcohol were pH 8 and 9.5, for BdCAD3 and BdCAD5 respectively. Kinetic parameters for conversion of coniferyl aldehyde and coniferyl alcohol showed that BdCAD5 was clearly the most efficient enzyme of the two. These data suggest that BdCAD5 is the main CAD enzyme for lignin biosynthesis and that BdCAD3 has a different role in Brachypodium. All CAD enzymes are cytosolic except for BdCAD4, which has a putative chloroplast signal peptide adding to the diversity of CAD functions. PMID:23028019

  4. Structural and kinetic characterization of recombinant 2-hydroxymuconate semialdehyde dehydrogenase from Pseudomonas putida G7

    PubMed Central

    de Araújo, Simara Semíramis; Neves, Cíntia Mara Leal; Guimarães, Samuel Leite; Whitman, Christian P.; Johnson, William H.; Aparicio, Ricardo; Nagem, Ronaldo Alves Pinto

    2016-01-01

    The first enzyme in the oxalocrotonate branch of the naphthalene-degradation lower pathway in Pseudomonas putida G7 is NahI, a 2-hydroxymuconate semialdehyde dehydrogenase required for conversion of 2-hydroxymuconate semialdehyde to 2-hydroxymuconate in the presence of NAD+. NahI is in one family of the NAD(P)+-dependent aldehyde dehydrogenase superfamily (ALDH8). In this work, we report the cloning, expression, purification and preliminary structural and kinetic characterization of the recombinant NahI. The nahI gene was subcloned into a T7 expression vector and the enzyme was overexpressed in Escherichia coli ArcticExpress at 12 ºC as an N-terminal hexa-histidine-tagged fusion protein (6xHis-NahI). After the soluble protein was purified by affinity and size-exclusion chromatography, dynamic light scattering and small-angle X-ray scattering experiments were conducted to analyze the oligomeric state and the overall shape of the enzyme in solution. The protein is a tetramer in solution and has nearly perfect 222 point group symmetry. Protein stability and secondary structure content were also evaluated by a circular dichroism spectroscopy assay under different thermal conditions. Furthermore, kinetic assays were conducted for the recombinant enzyme and, for the first time, KM (1.3 ± 0.3 μM) and kcat (0.9 s−1) values were determined for this enzyme (at presumed NAD+ saturation). NahI is highly specific for its biological substrate (2-hydroxymuconate semialdehyde) and has no activity with salicylaldehyde, another intermediate in the naphthalene-degradation pathway. PMID:26032336

  5. Kinetic and chemical analyses of the cytokinin dehydrogenase-catalysed reaction: correlations with the crystal structure

    PubMed Central

    Popelková, Hana; Fraaije, Marco W.; Novák, Ondřej; Frébortová, Jitka; Bilyeu, Kristin D.; Frébort, Ivo

    2006-01-01

    CKX (cytokinin dehydrogenase) is a flavoprotein that cleaves cytokinins to adenine and the corresponding side-chain aldehyde using a quinone-type electron acceptor. In the present study, reactions of maize (Zea mays) CKX with five different substrates (N6-isopentenyladenine, trans-zeatin, kinetin, p-topolin and N-methyl-isopentenyladenine) were studied. By using stopped-flow analysis of the reductive half-reaction, spectral intermediates were observed indicative of the transient formation of a binary enzyme–product complex between the cytokinin imine and the reduced enzyme. The reduction rate was high for isoprenoid cytokinins that showed formation of a charge-transfer complex of reduced enzyme with bound cytokinin imine. For the other cytokinins, flavin reduction was slow and no charge-transfer intermediates were observed. The binary complex of reduced enzyme and imine product intermediate decays relatively slowly to form an unbound product, cytokinin imine, which accumulates in the reaction mixture. The imine product only very slowly hydrolyses to adenine and an aldehyde derived from the cytokinin N6 side-chain. Mixing of the substrate-reduced enzyme with Cu2+/imidazole as an electron acceptor to monitor the oxidative half-reaction revealed a high rate of electron transfer for this type of electron acceptor when using N6-isopentenyladenine. The stability of the cytokinin imine products allowed their fragmentation analysis and structure assessment by Q-TOF (quadrupole–time-of-flight) MS/MS. Correlations of the kinetic data with the known crystal structure are discussed for reactions with different cytokinins. PMID:16686601

  6. The cystathionine-β-synthase domains on the guanosine 5''-monophosphate reductase and inosine 5'-monophosphate dehydrogenase enzymes from Leishmania regulate enzymatic activity in response to guanylate and adenylate nucleotide levels.

    PubMed

    Smith, Sabrina; Boitz, Jan; Chidambaram, Ehzilan Subramanian; Chatterjee, Abhishek; Ait-Tihyaty, Maria; Ullman, Buddy; Jardim, Armando

    2016-06-01

    The Leishmania guanosine 5'-monophosphate reductase (GMPR) and inosine 5'-monophosphate dehydrogenase (IMPDH) are purine metabolic enzymes that function maintaining the cellular adenylate and guanylate nucleotide. Interestingly, both enzymes contain a cystathionine-β-synthase domain (CBS). To investigate this metabolic regulation, the Leishmania GMPR was cloned and shown to be sufficient to complement the guaC (GMPR), but not the guaB (IMPDH), mutation in Escherichia coli. Kinetic studies confirmed that the Leishmania GMPR catalyzed a strict NADPH-dependent reductive deamination of GMP to produce IMP. Addition of GTP or high levels of GMP induced a marked increase in activity without altering the Km values for the substrates. In contrast, the binding of ATP decreased the GMPR activity and increased the GMP Km value 10-fold. These kinetic changes were correlated with changes in the GMPR quaternary structure, induced by the binding of GMP, GTP, or ATP to the GMPR CBS domain. The capacity of these CBS domains to mediate the catalytic activity of the IMPDH and GMPR provides a regulatory mechanism for balancing the intracellular adenylate and guanylate pools. PMID:26853689

  7. Characterization of a transient intermediate formed in the liver alcohol dehydrogenase catalyzed reduction of 3-hydroxy-4-nitrobenzaldehyde

    SciTech Connect

    MacGibbon, A.K.H.; Koerber, S.C.; Pease, K.; Dunn, M.F.

    1987-06-02

    The compounds 3-hydroxy-4-nitrobenzaldehyde and 3-hydroxy-4-nitrobenzyl alcohol are introduced as new chromophoric substrates for probing the catalytic mechanism of horse liver alcohol dehydrogenase (LADH). Ionization of the phenolic hydroxyl group shifts the spectrum of the aldehyde from 360 to 433 nm (pK/sub a/ = 6.0), whereas the spectrum of the alcohol shifts from 350 to 417 nm (pK/sub a/ = 6.9). Rapid-scanning, stopped-flow (RSSF) studies at alkaline pH show that the LADH-catalyzed interconversion of these compounds occurs via the formation of an enzyme-bound intermediate with a blue-shifted spectrum. When reaction is limited to a single turnover of enzyme sites, the formation and decay of the intermediate when aldehyde reacts with enzyme-bound reduced nicotinamide adenine dinucleotide E(NADH) are characterized by two relaxations. Detailed stopped-flow kinetic studies were carried out to investigate the disappearance of aldehyde and NADH, the formation and decay of the intermediate, the displacement of Auramine O by substrate, and /sup 2/H kinetic isotope effects. It was found that (1) NADH oxidation takes p