Sample records for aldehyde dehydrogenase enzymes

  1. Five Fatty Aldehyde Dehydrogenase Enzymes from Marinobacter and Acinetobacter spp. and Structural Insights into the Aldehyde Binding Pocket

    PubMed Central

    Bertram, Jonathan H.; Mulliner, Kalene M.; Shi, Ke; Plunkett, Mary H.; Nixon, Peter; Serratore, Nicholas A.; Douglas, Christopher J.; Aihara, Hideki

    2017-01-01

    ABSTRACT Enzymes involved in lipid biosynthesis and metabolism play an important role in energy conversion and storage and in the function of structural components such as cell membranes. The fatty aldehyde dehydrogenase (FAldDH) plays a central function in the metabolism of lipid intermediates, oxidizing fatty aldehydes to the corresponding fatty acid and competing with pathways that would further reduce the fatty aldehydes to fatty alcohols or require the fatty aldehydes to produce alkanes. In this report, the genes for four putative FAldDH enzymes from Marinobacter aquaeolei VT8 and an additional enzyme from Acinetobacter baylyi were heterologously expressed in Escherichia coli and shown to display FAldDH activity. Five enzymes (Maqu_0438, Maqu_3316, Maqu_3410, Maqu_3572, and the enzyme reported under RefSeq accession no. WP_004927398) were found to act on aldehydes ranging from acetaldehyde to hexadecanal and also acted on the unsaturated long-chain palmitoleyl and oleyl aldehydes. A comparison of the specificities of these enzymes with various aldehydes is presented. Crystallization trials yielded diffraction-quality crystals of one particular FAldDH (Maqu_3316) from M. aquaeolei VT8. Crystals were independently treated with both the NAD+ cofactor and the aldehyde substrate decanal, revealing specific details of the likely substrate binding pocket for this class of enzymes. A likely model for how catalysis by the enzyme is accomplished is also provided. IMPORTANCE This study provides a comparison of multiple enzymes with the ability to oxidize fatty aldehydes to fatty acids and provides a likely picture of how the fatty aldehyde and NAD+ are bound to the enzyme to facilitate catalysis. Based on the information obtained from this structural analysis and comparisons of specificities for the five enzymes that were characterized, correlations to the potential roles played by specific residues within the structure may be drawn. PMID:28389542

  2. Five Fatty Aldehyde Dehydrogenase Enzymes from Marinobacter and Acinetobacter spp. and Structural Insights into the Aldehyde Binding Pocket

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bertram, Jonathan H.; Mulliner, Kalene M.; Shi, Ke

    ABSTRACT Enzymes involved in lipid biosynthesis and metabolism play an important role in energy conversion and storage and in the function of structural components such as cell membranes. The fatty aldehyde dehydrogenase (FAldDH) plays a central function in the metabolism of lipid intermediates, oxidizing fatty aldehydes to the corresponding fatty acid and competing with pathways that would further reduce the fatty aldehydes to fatty alcohols or require the fatty aldehydes to produce alkanes. In this report, the genes for four putative FAldDH enzymes fromMarinobacter aquaeoleiVT8 and an additional enzyme fromAcinetobacter baylyiwere heterologously expressed inEscherichia coliand shown to display FAldDH activity.more » Five enzymes (Maqu_0438, Maqu_3316, Maqu_3410, Maqu_3572, and the enzyme reported under RefSeq accession no.WP_004927398) were found to act on aldehydes ranging from acetaldehyde to hexadecanal and also acted on the unsaturated long-chain palmitoleyl and oleyl aldehydes. A comparison of the specificities of these enzymes with various aldehydes is presented. Crystallization trials yielded diffraction-quality crystals of one particular FAldDH (Maqu_3316) fromM. aquaeoleiVT8. Crystals were independently treated with both the NAD +cofactor and the aldehyde substrate decanal, revealing specific details of the likely substrate binding pocket for this class of enzymes. A likely model for how catalysis by the enzyme is accomplished is also provided. IMPORTANCEThis study provides a comparison of multiple enzymes with the ability to oxidize fatty aldehydes to fatty acids and provides a likely picture of how the fatty aldehyde and NAD +are bound to the enzyme to facilitate catalysis. Based on the information obtained from this structural analysis and comparisons of specificities for the five enzymes that were characterized, correlations to the potential roles played by specific residues within the structure may be drawn.« less

  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. Structural and Kinetic Properties of the Aldehyde Dehydrogenase NahF, a Broad Substrate Specificity Enzyme for Aldehyde Oxidation.

    PubMed

    Coitinho, Juliana B; Pereira, Mozart S; Costa, Débora M A; Guimarães, Samuel L; Araújo, Simara S; Hengge, Alvan C; Brandão, Tiago A S; Nagem, Ronaldo A P

    2016-09-27

    The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD(+) as a cofactor, the last reaction of the upper degradation pathway of naphthalene in Pseudomonas putida G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large kcat/Km values close to 10(6) M(-1) s(-1). The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows α/β folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 ofmore » 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.« less

  6. Aldehyde dehydrogenase 7A1 (ALDH7A1) is a novel enzyme involved in cellular defense against hyperosmotic stress.

    PubMed

    Brocker, Chad; Lassen, Natalie; Estey, Tia; Pappa, Aglaia; Cantore, Miriam; Orlova, Valeria V; Chavakis, Triantafyllos; Kavanagh, Kathryn L; Oppermann, Udo; Vasiliou, Vasilis

    2010-06-11

    Mammalian ALDH7A1 is homologous to plant ALDH7B1, an enzyme that protects against various forms of stress, such as salinity, dehydration, and osmotic stress. It is known that mutations in the human ALDH7A1 gene cause pyridoxine-dependent and folic acid-responsive seizures. Herein, we show for the first time that human ALDH7A1 protects against hyperosmotic stress by generating osmolytes and metabolizing toxic aldehydes. Human ALDH7A1 expression in Chinese hamster ovary cells attenuated osmotic stress-induced apoptosis caused by increased extracellular concentrations of sucrose or sodium chloride. Purified recombinant ALDH7A1 efficiently metabolized a number of aldehyde substrates, including the osmolyte precursor, betaine aldehyde, lipid peroxidation-derived aldehydes, and the intermediate lysine degradation product, alpha-aminoadipic semialdehyde. The crystal structure for ALDH7A1 supports the enzyme's substrate specificities. Tissue distribution studies in mice showed the highest expression of ALDH7A1 protein in liver, kidney, and brain, followed by pancreas and testes. ALDH7A1 protein was found in the cytosol, nucleus, and mitochondria, making it unique among the aldehyde dehydrogenase enzymes. Analysis of human and mouse cDNA sequences revealed mitochondrial and cytosolic transcripts that are differentially expressed in a tissue-specific manner in mice. In conclusion, ALDH7A1 is a novel aldehyde dehydrogenase expressed in multiple subcellular compartments that protects against hyperosmotic stress by generating osmolytes and metabolizing toxic aldehydes.

  7. Aldehyde Dehydrogenase 7A1 (ALDH7A1) Is a Novel Enzyme Involved in Cellular Defense against Hyperosmotic Stress*

    PubMed Central

    Brocker, Chad; Lassen, Natalie; Estey, Tia; Pappa, Aglaia; Cantore, Miriam; Orlova, Valeria V.; Chavakis, Triantafyllos; Kavanagh, Kathryn L.; Oppermann, Udo; Vasiliou, Vasilis

    2010-01-01

    Mammalian ALDH7A1 is homologous to plant ALDH7B1, an enzyme that protects against various forms of stress, such as salinity, dehydration, and osmotic stress. It is known that mutations in the human ALDH7A1 gene cause pyridoxine-dependent and folic acid-responsive seizures. Herein, we show for the first time that human ALDH7A1 protects against hyperosmotic stress by generating osmolytes and metabolizing toxic aldehydes. Human ALDH7A1 expression in Chinese hamster ovary cells attenuated osmotic stress-induced apoptosis caused by increased extracellular concentrations of sucrose or sodium chloride. Purified recombinant ALDH7A1 efficiently metabolized a number of aldehyde substrates, including the osmolyte precursor, betaine aldehyde, lipid peroxidation-derived aldehydes, and the intermediate lysine degradation product, α-aminoadipic semialdehyde. The crystal structure for ALDH7A1 supports the enzyme's substrate specificities. Tissue distribution studies in mice showed the highest expression of ALDH7A1 protein in liver, kidney, and brain, followed by pancreas and testes. ALDH7A1 protein was found in the cytosol, nucleus, and mitochondria, making it unique among the aldehyde dehydrogenase enzymes. Analysis of human and mouse cDNA sequences revealed mitochondrial and cytosolic transcripts that are differentially expressed in a tissue-specific manner in mice. In conclusion, ALDH7A1 is a novel aldehyde dehydrogenase expressed in multiple subcellular compartments that protects against hyperosmotic stress by generating osmolytes and metabolizing toxic aldehydes. PMID:20207735

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

    PubMed

    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

    2015-05-01

    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.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Halavaty, Andrei S.; Rich, Rebecca L.; Chen, Chao

    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 (NADmore » +) 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. Finally, 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.« less

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

    DOE PAGES

    Halavaty, Andrei S.; Rich, Rebecca L.; Chen, Chao; ...

    2015-04-25

    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 (NADmore » +) 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. Finally, 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.« less

  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

    2015-01-07

    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.

  12. 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

  13. Betaine aldehyde dehydrogenase isozymes of spinach

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 inmore » salinized leaves. The phenotype of a natural variant of isozyme number1 suggests that the enzyme is a dimer.« less

  14. 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

  15. 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

    2016-02-22

    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.

  16. Aldehyde Dehydrogenases in Arabidopsis thaliana: Biochemical Requirements, Metabolic Pathways, and Functional Analysis.

    PubMed

    Stiti, Naim; Missihoun, Tagnon D; Kotchoni, Simeon O; Kirch, Hans-Hubert; Bartels, Dorothea

    2011-01-01

    Aldehyde dehydrogenases (ALDHs) are a family of enzymes which catalyze the oxidation of reactive aldehydes to their corresponding carboxylic acids. Here we summarize molecular genetic and biochemical analyses of selected ArabidopsisALDH genes. Aldehyde molecules are very reactive and are involved in many metabolic processes but when they accumulate in excess they become toxic. Thus activity of aldehyde dehydrogenases is important in regulating the homeostasis of aldehydes. Overexpression of some ALDH genes demonstrated an improved abiotic stress tolerance. Despite the fact that several reports are available describing a role for specific ALDHs, their precise physiological roles are often still unclear. Therefore a number of genetic and biochemical tools have been generated to address the function with an emphasis on stress-related ALDHs. ALDHs exert their functions in different cellular compartments and often in a developmental and tissue specific manner. To investigate substrate specificity, catalytic efficiencies have been determined using a range of substrates varying in carbon chain length and degree of carbon oxidation. Mutational approaches identified amino acid residues critical for coenzyme usage and enzyme activities.

  17. Aldehyde Dehydrogenases in Arabidopsis thaliana: Biochemical Requirements, Metabolic Pathways, and Functional Analysis

    PubMed Central

    Stiti, Naim; Missihoun, Tagnon D.; Kotchoni, Simeon O.; Kirch, Hans-Hubert; Bartels, Dorothea

    2011-01-01

    Aldehyde dehydrogenases (ALDHs) are a family of enzymes which catalyze the oxidation of reactive aldehydes to their corresponding carboxylic acids. Here we summarize molecular genetic and biochemical analyses of selected Arabidopsis ALDH genes. Aldehyde molecules are very reactive and are involved in many metabolic processes but when they accumulate in excess they become toxic. Thus activity of aldehyde dehydrogenases is important in regulating the homeostasis of aldehydes. Overexpression of some ALDH genes demonstrated an improved abiotic stress tolerance. Despite the fact that several reports are available describing a role for specific ALDHs, their precise physiological roles are often still unclear. Therefore a number of genetic and biochemical tools have been generated to address the function with an emphasis on stress-related ALDHs. ALDHs exert their functions in different cellular compartments and often in a developmental and tissue specific manner. To investigate substrate specificity, catalytic efficiencies have been determined using a range of substrates varying in carbon chain length and degree of carbon oxidation. Mutational approaches identified amino acid residues critical for coenzyme usage and enzyme activities. PMID:22639603

  18. Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase.

    PubMed Central

    Modig, Tobias; Lidén, Gunnar; Taherzadeh, Mohammad J

    2002-01-01

    The kinetics of furfural inhibition of the enzymes alcohol dehydrogenase (ADH; EC 1.1.1.1), aldehyde dehydrogenase (AlDH; EC 1.2.1.5) and the pyruvate dehydrogenase (PDH) complex were studied in vitro. At a concentration of less than 2 mM furfural was found to decrease the activity of both PDH and AlDH by more than 90%, whereas the ADH activity decreased by less than 20% at the same concentration. Furfural inhibition of ADH and AlDH activities could be described well by a competitive inhibition model, whereas the inhibition of PDH was best described as non-competitive. The estimated K(m) value of AlDH for furfural was found to be about 5 microM, which was lower than that for acetaldehyde (10 microM). For ADH, however, the estimated K(m) value for furfural (1.2 mM) was higher than that for acetaldehyde (0.4 mM). The inhibition of the three enzymes by 5-hydroxymethylfurfural (HMF) was also measured. The inhibition caused by HMF of ADH was very similar to that caused by furfural. However, HMF did not inhibit either AlDH or PDH as severely as furfural. The inhibition effects on the three enzymes could well explain previously reported in vivo effects caused by furfural and HMF on the overall metabolism of Saccharomyces cerevisiae, suggesting a critical role of these enzymes in the observed inhibition. PMID:11964178

  19. AAV Gene Therapy for Alcoholism: Inhibition of Mitochondrial Aldehyde Dehydrogenase Enzyme Expression in Hepatoma Cells.

    PubMed

    Sanchez, Anamaria C; Li, Chengwen; Andrews, Barbara; Asenjo, Juan A; Samulski, R Jude

    2017-09-01

    Most ethanol is broken down in the liver in two steps by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH2) enzymes, which metabolize down ethanol into acetaldehyde and then acetate. Some individuals from the Asian population who carry a mutation in the aldehyde dehydrogenase gene (ALDH2*2) cannot metabolize acetaldehyde as efficiently, producing strong effects, including facial flushing, dizziness, hypotension, and palpitations. This results in an aversion to alcohol intake and protection against alcoholism. The large prevalence of this mutation in the human population strongly suggests that modulation of ALDH2 expression by genetic technologies could result in a similar phenotype. scAAV2 vectors encoding ALDH2 small hairpin RNA (shRNA) were utilized to validate this hypothesis by silencing ALDH2 gene expression in human cell lines. Human cell lines HEK-293 and HepG2 were transduced with scAAV2/shRNA, showing a reduction in ALDH2 RNA and protein expression with the two viral concentration assayed (1 × 10 4 and 1 × 10 5 vg/cell) at two different time points. In both cell lines, ALDH2 RNA levels were reduced by 90% and protein expression was inhibited by 90% and 52%, respectively, 5 days post infection. Transduced HepG2 VL17A cells (ADH+) exposed to ethanol resulted in a 50% increase in acetaldehyde levels. These results suggest that gene therapy could be a useful tool for the treatment of alcoholism by knocking down ALDH2 expression using shRNA technology delivered by AAV vectors.

  20. 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. © 2016 Authors; published by Portland Press Limited.

  1. The metabolism of ethanol-derived acetaldehyde by alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase (EC 1.2.1.3) in Drosophila melanogaster larvae.

    PubMed Central

    Heinstra, P W; Geer, B W; Seykens, D; Langevin, M

    1989-01-01

    Both aldehyde dehydrogenase (ALDH, EC 1.2.1.3) and the aldehyde dehydrogenase activity of alcohol dehydrogenase (ADH, EC 1.1.1.1) were found to coexist in Drosophila melanogaster larvae. The enzymes, however, showed different inhibition patterns with respect to pyrazole, cyanamide and disulphiram. ALDH-1 and ALDH-2 isoenzymes were detected in larvae by electrophoretic methods. Nonetheless, in tracer studies in vivo, more than 75% of the acetaldehyde converted to acetate by the ADH ethanol-degrading pathway appeared to be also catalysed by the ADH enzyme. The larval fat body probably was the major site of this pathway. Images Fig. 1. Fig. 2. PMID:2499314

  2. Heat-stable, FE-dependent alcohol dehydrogenase for aldehyde detoxification

    DOEpatents

    Elkins, James G.; Clarkson, Sonya

    2018-04-24

    The present invention relates to microorganisms and polypeptides for detoxifying aldehydes associated with industrial fermentations. In particular, a heat-stable, NADPH- and iron-dependent alcohol dehydrogenase was cloned from Thermoanaerobacter pseudethanolicus 39E and displayed activity against a number of aldehydes including inhibitory compounds that are produced during the dilute-acid pretreatment process of lignocellulosic biomass before fermentation to biofuels. Methods to use the microorganisms and polypeptides of the invention for improved conversion of bio mass to biofuel are provided as well as use of the enzyme in metabolic engineering strategies for producing longer-chain alcohols from sugars using thermophilic, fermentative microorganisms.

  3. Increased salivary aldehyde dehydrogenase 1 in non-reticular oral lichen planus.

    PubMed

    Mansourian, Arash; Shanbehzadeh, Najmeh; Kia, Seyed Javad; Moosavi, Mahdieh-Sadat

    2017-01-01

    Oral lichen planus is a potentially malignant disorder. One of the malignant transformation markers is cancer stem cells. One of the proposed marker for the detection of cancer stem cells's in head and neck cancer is aldehyde dehydrogenase. Recently it is shown that aldehyde dehydrogenase 1 expression in tissue samples is associated with oral lichen planus malignant transformation. This study evaluates salivary aldehyde dehydrogenase 1 in oral lichen planus. Thirty patients and 30 age and sex-matched healthy volunteers were recruited. Oral lichen planus was diagnosed based on the modified World Health Organization criteria. Subjects in the case group were divided into reticular and non-reticular forms. Unstimulated salivary samples were collected at 10-12 AM. Saliva concentrations of aldehyde dehydrogenase 1 were measured by ELISA. The differences between aldehyde dehydrogenase levels in the oral lichen planus group compared with the control group were not significant but aldehyde dehydrogenase in non-reticular oral lichen planus was significantly higher than that of the reticular form. This is a cross-sectional study, thus longitudinal studies in oral lichen planus may present similar or different results. The mechanism of malignant transformation in oral lichen planus is not defined. Previous analyses revealed that the aldehyde dehydrogenase 1 expression is significantly correlated with increased risk of transformation. This finding is consistent with our results because in the erosive and ulcerative forms of oral lichen planus, which have an increased risk of transformation, salivary aldehyde dehydrogenase 1 was overexpressed. A higher salivary aldehyde dehydrogenase level in non-reticular oral lichen planus can be a defensive mechanism against higher oxidative stress in these groups. Aldehyde dehydrogenase may be one of the malignant transformation markers in oral lichen planus. Further studies are needed for introducing aldehyde dehydrogenase as a prognostic

  4. Increased salivary aldehyde dehydrogenase 1 in non-reticular oral lichen planus*

    PubMed Central

    Mansourian, Arash; Shanbehzadeh, Najmeh; Kia, Seyed Javad; Moosavi, Mahdieh-Sadat

    2017-01-01

    Background Oral lichen planus is a potentially malignant disorder. One of the malignant transformation markers is cancer stem cells. One of the proposed marker for the detection of cancer stem cells's in head and neck cancer is aldehyde dehydrogenase. Recently it is shown that aldehyde dehydrogenase 1 expression in tissue samples is associated with oral lichen planus malignant transformation. Objective This study evaluates salivary aldehyde dehydrogenase 1 in oral lichen planus. Method Thirty patients and 30 age and sex-matched healthy volunteers were recruited. Oral lichen planus was diagnosed based on the modified World Health Organization criteria. Subjects in the case group were divided into reticular and non-reticular forms. Unstimulated salivary samples were collected at 10-12 AM. Saliva concentrations of aldehyde dehydrogenase 1 were measured by ELISA. Results The differences between aldehyde dehydrogenase levels in the oral lichen planus group compared with the control group were not significant but aldehyde dehydrogenase in non-reticular oral lichen planus was significantly higher than that of the reticular form. Limitations of the study This is a cross-sectional study, thus longitudinal studies in oral lichen planus may present similar or different results. Conclusions The mechanism of malignant transformation in oral lichen planus is not defined. Previous analyses revealed that the aldehyde dehydrogenase 1 expression is significantly correlated with increased risk of transformation. This finding is consistent with our results because in the erosive and ulcerative forms of oral lichen planus, which have an increased risk of transformation, salivary aldehyde dehydrogenase 1 was overexpressed. A higher salivary aldehyde dehydrogenase level in non-reticular oral lichen planus can be a defensive mechanism against higher oxidative stress in these groups. Aldehyde dehydrogenase may be one of the malignant transformation markers in oral lichen planus. Further

  5. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  7. 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...

  8. Guinea-pig liver testosterone 17 beta-dehydrogenase (NADP+) and aldehyde reductase exhibit benzene dihydrodiol dehydrogenase activity.

    PubMed Central

    Hara, A; Hayashibara, M; Nakayama, T; Hasebe, K; Usui, S; Sawada, H

    1985-01-01

    We have kinetically and immunologically demonstrated that testosterone 17 beta-dehydrogenase (NADP+) isoenzymes (EC 1.1.1.64) and aldehyde reductase (EC 1.1.1.2) from guinea-pig liver catalyse the oxidation of benzene dihydrodiol (trans-1,2-dihydroxycyclohexa-3,5-diene) to catechol. One isoenzyme of testosterone 17 beta-dehydrogenase, which has specificity for 5 beta-androstanes, oxidized benzene dihydrodiol at a 3-fold higher rate than 5 beta-dihydrotestosterone, and showed a more than 4-fold higher affinity for benzene dihydrodiol and Vmax. value than did another isoenzyme, which exhibits specificity for 5 alpha-androstanes, and aldehyde reductase. Immunoprecipitation of guinea-pig liver cytosol with antisera against the testosterone 17 beta-dehydrogenase isoenzymes and aldehyde reductase indicated that most of the benzene dihydrodiol dehydrogenase activity in the tissue is due to testosterone 17 beta-dehydrogenase. PMID:2983661

  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.

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

    PubMed Central

    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.

    2012-01-01

    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 outgenome-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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nakamura, Tomofumi; Fukuoka Institute of Health and Environmental Sciences, 39 Mukaizano, Dazaifu-shi, Fukuoka 818-0135; Ichinose, Hirofumi

    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 conservedmore » 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.« less

  12. Oligo(cis-1,4-isoprene) aldehyde-oxidizing dehydrogenases of the rubber-degrading bacterium Gordonia polyisoprenivorans VH2.

    PubMed

    Vivod, Robin; Oetermann, Sylvia; Hiessl, Sebastian; Gutsche, Stefanie; Remmers, Naomi; Meinert, Christina; Voigt, Birgit; Riedel, Katharina; Steinbüchel, Alexander

    2017-11-01

    The actinomycete Gordonia polyisoprenivorans strain VH2 is well-known for its ability to efficiently degrade and catabolize natural rubber [poly(cis-1,4-isoprene)]. Recently, a pathway for the catabolism of rubber by strain VH2 was postulated based on genomic data and the analysis of mutants (Hiessl et al. in Appl Environ Microbiol 78:2874-2887, 2012). To further elucidate the degradation pathway of poly(cis-1,4-isoprene), 2-dimensional-polyacrylamide gel electrophoresis was performed. The analysis of the identified protein spots by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry confirmed the postulated intracellular pathway suggesting a degradation of rubber via β-oxidation. In addition, other valuable information on rubber catabolism of G. polyisoprenivorans strain VH2 (e.g. oxidative stress response) was provided. Identified proteins, which were more abundant in cells grown with rubber than in cells grown with propionate, implied a putative long-chain acyl-CoA-dehydrogenase, a 3-ketoacyl-CoA-thiolase, and an aldehyde dehydrogenase. The amino acid sequence of the latter showed a high similarity towards geranial dehydrogenases. The expression of the corresponding gene was upregulated > 10-fold under poly(cis-1,4-isoprene)-degrading conditions. The putative geranial dehydrogenase and a homolog were purified and used for enzyme assays. Deletion mutants for five aldehyde dehydrogenases were generated, and growth with poly(cis-1,4-isoprene) was investigated. While none of the mutants had an altered phenotype regarding growth with poly(cis-1,4-isoprene) as sole carbon and energy source, purified aldehyde dehydrogenases were able to catalyze the oxidation of oligoisoprene aldehydes indicating an involvement in rubber degradation.

  13. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. 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

  15. Human Salivary Aldehyde Dehydrogenase: Purification, Kinetic Characterization and Effect of Ethanol, Hydrogen Peroxide and Sodium Dodecyl Sulfate on the Activity of the Enzyme.

    PubMed

    Alam, Md Fazle; Laskar, Amaj Ahmed; Choudhary, Hadi Hasan; Younus, Hina

    2016-09-01

    Human salivary aldehyde dehydrogenase (hsALDH) enzyme appears to be the first line of defense in the body against exogenous toxic aldehydes. However till date much work has not been done on this important member of the ALDH family. In this study, we have purified hsALDH to homogeneity by diethylaminoethyl-cellulose (DEAE-cellulose) ion-exchange chromatography in a single step. The molecular mass of the homodimeric enzyme was determined to be approximately 108 kDa. Four aromatic substrates; benzaldehyde, cinnamaldehyde, 2-naphthaldehyde and 6-methoxy-2-naphthaldehyde were used for determining the activity of pure hsALDH. K m values for these substrates were calculated to be 147.7, 5.31, 0.71 and 3.31 μM, respectively. The best substrates were found to be cinnamaldehyde and 2-naphthaldehyde since they exhibited high V max /K m values. 6-methoxy-2-naphthaldehyde substrate was used for further kinetic characterization of pure hsALDH. The pH and temperature optima of hsALDH were measured to be pH 8 and 45 °C, respectively. The pure enzyme is highly unstable at high temperatures. Ethanol, hydrogen peroxide and SDS activate hsALDH, therefore it is safe and beneficial to include them in mouthwashes and toothpastes in low concentrations.

  16. The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 gene encodes an aldehyde dehydrogenase involved in ferulic acid and sinapic acid biosynthesis.

    PubMed

    Nair, Ramesh B; Bastress, Kristen L; Ruegger, Max O; Denault, Jeff W; Chapple, Clint

    2004-02-01

    Recent research has significantly advanced our understanding of the phenylpropanoid pathway but has left in doubt the pathway by which sinapic acid is synthesized in plants. The reduced epidermal fluorescence1 (ref1) mutant of Arabidopsis thaliana accumulates only 10 to 30% of the sinapate esters found in wild-type plants. Positional cloning of the REF1 gene revealed that it encodes an aldehyde dehydrogenase, a member of a large class of NADP(+)-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. Consistent with this finding, extracts of ref1 leaves exhibit low sinapaldehyde dehydrogenase activity. These data indicate that REF1 encodes a sinapaldehyde dehydrogenase required for sinapic acid and sinapate ester biosynthesis. When expressed in Escherichia coli, REF1 was found to exhibit both sinapaldehyde and coniferaldehyde dehydrogenase activity, and further phenotypic analysis of ref1 mutant plants showed that they contain less cell wall-esterified ferulic acid. These findings suggest that both ferulic acid and sinapic acid are derived, at least in part, through oxidation of coniferaldehyde and sinapaldehyde. This route is directly opposite to the traditional representation of phenylpropanoid metabolism in which hydroxycinnamic acids are instead precursors of their corresponding aldehydes.

  17. Ultraviolet Radiation: Cellular Antioxidant Response and the Role of Ocular Aldehyde Dehydrogenase Enzymes

    PubMed Central

    Marchitti, Satori A.; Chen, Ying; Thompson, David C.; Vasiliou, Vasilis

    2011-01-01

    Solar ultraviolet radiation (UVR) exposes the human eye to near constant oxidative stress. Evidence suggests that UVR is the most important environmental insult leading to the development of a variety of ophthalmoheliosis disorders. UVR-induced reactive oxygen species are highly reactive with DNA, proteins and cellular membranes, resulting in cellular and tissue damage. Antioxidant defense systems present in ocular tissues function to combat reactive oxygen species and protect the eye from oxidative damage. Important enzymatic antioxidants are the superoxide dismutases, catalase, glutathione peroxidases, glutathione reductase and members of the aldehyde dehydrogenase (ALDH) superfamily. Glutathione, ascorbic and uric acids, α-tocopherol, NADPH and ferritin serve as small molecule, nonenzymatic antioxidants. Ocular tissues have high levels of these antioxidants which are essential for the maintenance of redox homeostasis in the eye and protection against oxidative damage. ALDH1A1 and ALDH3A1, present abundantly in the cornea and lens, have been shown to have unique roles in the defense against UVR and the downstream effects of oxidative stress. This review presents the properties and functions of ocular antioxidants that play critical roles in the cellular response to UVR exposure, including a focused discussion of the unique roles that the ALDH1A1 and ALDH3A1 enzymes have as multi-functional ocular antioxidants. PMID:21670692

  18. 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

  19. Contribution of aldehyde oxidizing enzymes on the metabolism of 3,4-dimethoxy-2-phenylethylamine to 3,4-dimethoxyphenylacetic acid by guinea pig liver slices.

    PubMed

    Panoutsopoulos, Georgios I

    2006-01-01

    3,4-Dimethoxy-2-phenylethylamine is catalyzed to its aldehyde derivative by monoamine oxidase B, but the subsequent oxidation into the corresponding acid has not yet been studied. Oxidation of aromatic aldehydes is catalyzed mainly by aldehyde dehydrogenase and aldehyde oxidase. The present study examines the metabolism of 3,4-dimethoxy-2-phenylethylamine in vitro and in freshly prepared and cryopreserved guinea pig liver slices and the relative contribution of different aldehyde-oxidizing enzymes was estimated by pharmacological means. 3,4-Dimethoxy-2- phenylethylamine was converted into the corresponding aldehyde when incubated with monoamine oxidase and further oxidized into the acid when incubated with both, monoamine oxidase and aldehyde oxidase. In freshly prepared and cryopreserved liver slices, 3,4-dimethoxyphenylacetic acid was the main metabolite of 3,4-dimethoxy-2- phenylethylamine. 3,4-Dimethoxyphenylacetic acid formation was inhibited by 85% from disulfiram (aldehyde dehydrogenase inhibitor) and by 75-80% from isovanillin (aldehyde oxidase inhibitor), whereas allopurinol (xanthine oxidase inhibitor) inhibited acid formation by only 25-30%. 3,4- Dimethoxy-2-phenylethylamine is oxidized mainly to its acid, via 3,4-dimethoxyphenylacetaldehyde, by aldehyde dehydrogenase and aldehyde oxidase with a lower contribution from xanthine oxidase.

  20. The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 Gene Encodes an Aldehyde Dehydrogenase Involved in Ferulic Acid and Sinapic Acid Biosynthesis

    PubMed Central

    Nair, Ramesh B.; Bastress, Kristen L.; Ruegger, Max O.; Denault, Jeff W.; Chapple, Clint

    2004-01-01

    Recent research has significantly advanced our understanding of the phenylpropanoid pathway but has left in doubt the pathway by which sinapic acid is synthesized in plants. The reduced epidermal fluorescence1 (ref1) mutant of Arabidopsis thaliana accumulates only 10 to 30% of the sinapate esters found in wild-type plants. Positional cloning of the REF1 gene revealed that it encodes an aldehyde dehydrogenase, a member of a large class of NADP+-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. Consistent with this finding, extracts of ref1 leaves exhibit low sinapaldehyde dehydrogenase activity. These data indicate that REF1 encodes a sinapaldehyde dehydrogenase required for sinapic acid and sinapate ester biosynthesis. When expressed in Escherichia coli, REF1 was found to exhibit both sinapaldehyde and coniferaldehyde dehydrogenase activity, and further phenotypic analysis of ref1 mutant plants showed that they contain less cell wall–esterified ferulic acid. These findings suggest that both ferulic acid and sinapic acid are derived, at least in part, through oxidation of coniferaldehyde and sinapaldehyde. This route is directly opposite to the traditional representation of phenylpropanoid metabolism in which hydroxycinnamic acids are instead precursors of their corresponding aldehydes. PMID:14729911

  1. 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...

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 screenedmore » 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.« less

  3. Different specificities of two aldehyde dehydrogenases from Saccharomyces cerevisiae var. boulardii.

    PubMed

    Datta, Suprama; Annapure, Uday S; Timson, David J

    2017-04-30

    Aldehyde dehydrogenases play crucial roles in the detoxification of exogenous and endogenous aldehydes by catalysing their oxidation to carboxylic acid counterparts. The present study reports characterization of two such isoenzymes from the yeast Saccharomyces cerevisiae var. boulardii (NCYC 3264), one mitochondrial (Ald4p) and one cytosolic (Ald6p). Both Ald4p and Ald6p were oligomeric in solution and demonstrated positive kinetic cooperativity towards aldehyde substrates. Wild-type Ald6p showed activity only with aliphatic aldehydes. Ald4p, on the contrary, showed activity with benzaldehyde along with a limited range of aliphatic aldehydes. Inspection of modelled structure of Ald6p revealed that a bulky amino acid residue (Met 177 , compared with the equivalent residue Leu 196 in Ald4p) might cause steric hindrance of cyclic substrates. Therefore, we hypothesized that specificities of the two isoenzymes towards aldehyde substrates were partly driven by steric hindrance in the active site. A variant of wild-type Ald6p with the Met 177 residue replaced by a valine was also characterized to address to the hypothesis. It showed an increased specificity range and a gain of activity towards cyclohexanecarboxaldehyde. It also demonstrated an increased thermal stability when compared with both the wild-types. These data suggest that steric bulk in the active site of yeast aldehyde dehydrogenases is partially responsible for controlling specificity. © 2017 The Author(s).

  4. 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

    2016-06-20

    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.

  5. Betaine Aldehyde Dehydrogenase expression during physiological cardiac hypertrophy induced by pregnancy.

    PubMed

    Rosas-Rodríguez, Jesús Alfredo; Soñanez-Organis, José Guadalupe; Godoy-Lugo, José Arquimides; Espinoza-Salazar, Juan Alberto; López-Jacobo, Cesar Jeravy; Stephens-Camacho, Norma Aurora; González-Ochoa, Guadalupe

    2017-08-26

    Betaine Aldehyde Dehydrogenase (betaine aldehyde: NAD(P) + oxidoreductase, (E.C. 1.2.1.8; BADH) catalyze the irreversible oxidation of betaine aldehyde (BA) to glycine betaine (GB) and is essential for polyamine catabolism, γ-aminobutyric acid synthesis, and carnitine biosynthesis. GB is an important osmolyte that regulates the homocysteine levels, contributing to a vascular risk factor reduction. In this sense, distinct investigations describe the physiological roles of GB, but there is a lack of information about the GB novo synthesis process and regulation during cardiac hypertrophy induced by pregnancy. In this work, the BADH mRNA expression, protein level, and activity were quantified in the left ventricle before, during, and after pregnancy. The mRNA expression, protein content and enzyme activity along with GB content of BADH increased 2.41, 1.95 and 1.65-fold respectively during late pregnancy compared to not pregnancy, and returned to basal levels at postpartum. Besides, the GB levels increased 1.53-fold during pregnancy and remain at postpartum. Our results demonstrate that physiological cardiac hypertrophy induced BADH mRNA expression and activity along with GB production, suggesting that BADH participates in the adaptation process of physiological cardiac hypertrophy during pregnancy, according to the described GB role in cellular osmoregulation, osmoprotection and reduction of vascular risk. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Salivary aldehyde dehydrogenase - temporal and population variability, correlations with drinking and smoking habits and activity towards aldehydes contained in food.

    PubMed

    Giebułtowicz, Joanna; Dziadek, Marta; Wroczyński, Piotr; Woźnicka, Katarzyna; Wojno, Barbara; Pietrzak, Monika; Wierzchowski, Jacek

    2010-01-01

    Fluorimetric method based on oxidation of the fluorogenic 6-methoxy-2-naphthaldehyde was applied to evaluate temporal and population variability of the specific activity of salivary aldehyde dehydrogenase (ALDH) and the degree of its inactivation in healthy human population. Analyzed was also its dependence on drinking and smoking habits, coffee consumption, and its sensitivity to N-acetylcysteine. Both the specific activity of salivary ALDH and the degree of its inactivation were highly variable during the day, with the highest activities recorded in the morning hours. The activities were also highly variable both intra- and interpersonally, and negatively correlated with age, and this correlation was stronger for the subgroup of volunteers declaring abstinence from alcohol and tobacco. Moderately positive correlations of salivary ALDH specific activity with alcohol consumption and tobacco smoking were also recorded (r(s) ~0.27; p=0.004 and r(s) =0.30; p=0.001, respectively). Moderate coffee consumption correlated positively with the inactivation of salivary ALDH, particularly in the subgroup of non-drinking and non-smoking volunteers. It was found that mechanical stimulation of the saliva flow increases the specific activity of salivary ALDH. The specific activity of the salivary ALDH was strongly and positively correlated with that of superoxide dismutase, and somewhat less with salivary peroxidase. The antioxidant-containing drug N-acetylcysteine increased activity of salivary ALDH presumably by preventing its inactivation in the oral cavity. Some food-related aldehydes, mainly cinnamic aldehyde and anisaldehyde, were excellent substrates of the salivary ALDH3A1 enzyme, while alkenals, particularly those with short chain, were characterized by lower affinity towards this enzyme but high catalytic constants. The protective role of salivary ALDH against aldehydes in food and those found in the cigarette smoke is discussed, as well as its participation in

  7. 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

  8. Inhibition of human alcohol and aldehyde dehydrogenases by cimetidine and assessment of its effects on ethanol metabolism.

    PubMed

    Lai, Ching-Long; Li, Yeung-Pin; Liu, Chiu-Ming; Hsieh, Hsiu-Shan; Yin, Shih-Jiun

    2013-02-25

    Previous studies have reported that cimetidine, an H2-receptor antagonist used to treat gastric and duodenal ulcers, can inhibit alcohol dehydrogenases (ADHs) and ethanol metabolism. Human alcohol dehydrogenases and aldehyde dehydrogenases (ALDHs), the 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 by cimetidine of alcohol oxidation by recombinant human ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2, ADH2, and ADH4, and aldehyde oxidation by ALDH1A1 and ALDH2 at pH 7.5 and a cytosolic NAD(+) concentration. Cimetidine acted as competitive or noncompetitive inhibitors for the ADH and ALDH isozymes/allozymes with near mM inhibition constants. The metabolic interactions between cimetidine and ethanol/acetaldehyde were assessed by computer simulation using the inhibition equations and the determined kinetic constants. At therapeutic drug levels (0.015 mM) and physiologically relevant concentrations of ethanol (10 mM) and acetaldehyde (10 μM) in target tissues, cimetidine could weakly inhibit (<5%) the activities of ADH1B2 and ADH1B3 in liver, ADH2 in liver and small intestine, ADH4 in stomach, and ALDH1A1 in the three tissues, but not significantly affect ADH1A, ADH1B1, ADH1C1/2, or ALDH2. At higher drug levels, which may accumulate in cells (0.2 mM), the activities of the weakly-inhibited enzymes may be decreased more significantly. The quantitative effects of cimetidine on metabolism of ethanol and other physiological substrates of ADHs need further investigation. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  9. [Distribution of genotypes of alcohol dehydrogenase 2 and aldehyde dehydrogenase 2 in Japanese twin children].

    PubMed

    Qu, W; Yamagata, Z; Wu, D; Zhang, B; Zhang, Y

    1999-03-01

    In order to prevent alcohol related deseases, this study investigated the distribution of the genes controlling alcohol metabolism in Japan's twin. Restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) technique was used to measure the control gene of alcohol metabolized enzymes and the genotypes of alcohol dehydrogenase 2 (ADH2) and aldehyde dehydrogenase 2 (ALDH2), which were distributed in Japan's twins. At the same time, according to the difference in genotypes, the sensitive individuals were screened from the study subjects. The distribution of ADH2 and ALDH2 genes were consistent with the Hardy-weinberg equation. The three genotypes of ADH2 gene were ADH2(1)/ADH2(1) (1.1%), ADH2(1)/ADH2(2) (44.6%) and ADH2(2)/ADH2(2) (54.3%). And those of ALDH2 gene were ALDH2(1)/ALDH2(1) (41.3%), ALDH2(1)/ALDH2(2) (39.1%) and ALDH2(2)/ALDH2(2) (19.6%). The frequency of ADH2 and ALDH2 genes was 0.255, 0.745 and 0.609, 0.391 respectively. Not only the distribution of genotypes of ADH2 and ALDH2 is known, but also the sensitive individuals are found, which can help prevent alcohol related disease.

  10. The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is necessary for ethanol production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum.

    PubMed

    Lo, Jonathan; Zheng, Tianyong; Hon, Shuen; Olson, Daniel G; Lynd, Lee R

    2015-04-01

    Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of the adhE gene in C. thermocellum and T. saccharolyticum. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In both adhE deletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. In T. saccharolyticum, the adhE deletion strain lost >85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in the T. saccharolyticum parent strain but did not increase activity in the adhE deletion strain, suggesting that ALDH activity was inhibited. In C. thermocellum, the adhE deletion strain lost >90% of ALDH and ADH activity in cell extracts. The C. thermocellum adhE deletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase. Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are bacteria that have been investigated for their ability to produce biofuels from plant biomass. They have been engineered to produce higher yields of ethanol, yet questions remain about the enzymes responsible for ethanol formation in these bacteria. The genomes of these bacteria encode multiple predicted aldehyde and alcohol dehydrogenases which could be

  11. Steady-state kinetic mechanism of the NADP+- and NAD+-dependent reactions catalysed by betaine aldehyde dehydrogenase from Pseudomonas aeruginosa.

    PubMed Central

    Velasco-García, R; González-Segura, L; Muñoz-Clares, R A

    2000-01-01

    Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible oxidation of betaine aldehyde to glycine betaine with the concomitant reduction of NAD(P)(+) to NADP(H). In Pseudomonas aeruginosa this reaction is a compulsory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. The kinetic mechanisms of the NAD(+)- and NADP(+)-dependent reactions were examined by steady-state kinetic methods and by dinucleotide binding experiments. The double-reciprocal patterns obtained for initial velocity with NAD(P)(+) and for product and dead-end inhibition establish that both mechanisms are steady-state random. However, quantitative analysis of the inhibitions, and comparison with binding data, suggest a preferred route of addition of substrates and release of products in which NAD(P)(+) binds first and NAD(P)H leaves last, particularly in the NADP(+)-dependent reaction. Abortive binding of the dinucleotides, or their analogue ADP, in the betaine aldehyde site was inferred from total substrate inhibition by the dinucleotides, and parabolic inhibition by NADH and ADP. A weak partial uncompetitive substrate inhibition by the aldehyde was observed only in the NADP(+)-dependent reaction. The kinetics of P. aeruginosa BADH is very similar to that of glucose-6-phosphate dehydrogenase, suggesting that both enzymes fulfil a similar amphibolic metabolic role when the bacteria grow in choline and when they grow in glucose. PMID:11104673

  12. Characterization of Two Distinct Structural Classes of Selective Aldehyde Dehydrogenase 1A1 Inhibitors

    DOE PAGES

    Morgan, Cynthia A.; Hurley, Thomas D.

    2015-01-29

    Aldehyde dehydrogenases (ALDH) catalyze the irreversible oxidation of aldehydes to their corresponding carboxylic acid. Alterations in ALDH1A1 activity are associated with such diverse diseases as cancer, Parkinson’s disease, obesity, and cataracts. Inhibitors of ALDH1A1 could aid in illuminating the role of this enzyme in disease processes. However, there are no commercially available selective inhibitors for ALDH1A1. Here we characterize two distinct chemical classes of inhibitors that are selective for human ALDH1A1 compared to eight other ALDH isoenzymes. The prototypical members of each structural class, CM026 and CM037, exhibit sub-micromolar inhibition constants, but have different mechanisms of inhibition. The crystal structuresmore » of these compounds bound to ALDH1A1 demonstrate that they bind within the aldehyde binding pocket of ALDH1A1 and exploit the presence of a unique Glycine residue to achieve their selectivity. Lastly, these two novel and selective ALDH1A1 inhibitors may serve as chemical tools to better understand the contributions of ALDH1A1 to normal biology and to disease states.« less

  13. Acute and chronic ethanol exposure differentially alters alcohol dehydrogenase and aldehyde dehydrogenase activity in the zebrafish liver.

    PubMed

    Tran, Steven; Nowicki, Magda; Chatterjee, Diptendu; Gerlai, Robert

    2015-01-02

    Chronic ethanol exposure paradigms have been successfully used in the past to induce behavioral and central nervous system related changes in zebrafish. However, it is currently unknown whether chronic ethanol exposure alters ethanol metabolism in adult zebrafish. In the current study we examine the effect of acute ethanol exposure on adult zebrafish behavioral responses, as well as alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activity in the liver. We then examine how two different chronic ethanol exposure paradigms (continuous and repeated ethanol exposure) alter behavioral responses and liver enzyme activity during a subsequent acute ethanol challenge. Acute ethanol exposure increased locomotor activity in a dose-dependent manner. ADH activity was shown to exhibit an inverted U-shaped curve and ALDH activity was decreased by ethanol exposure at all doses. During the acute ethanol challenge, animals that were continuously housed in ethanol exhibited a significantly reduced locomotor response and increased ADH activity, however, ALDH activity did not change. Zebrafish that were repeatedly exposed to ethanol demonstrated a small but significant attenuation of the locomotor response during the acute ethanol challenge but ADH and ALDH activity was similar to controls. Overall, we identified two different chronic ethanol exposure paradigms that differentially alter behavioral and physiological responses in zebrafish. We speculate that these two paradigms may allow dissociation of central nervous system-related and liver enzyme-dependent ethanol induced changes in zebrafish. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Immobilisation and characterisation of biocatalytic co-factor recycling enzymes, glucose dehydrogenase and NADH oxidase, on aldehyde functional ReSyn™ polymer microspheres.

    PubMed

    Twala, Busisiwe V; Sewell, B Trevor; Jordaan, Justin

    2012-05-10

    The use of enzymes in industrial applications is limited by their instability, cost and difficulty in their recovery and re-use. Immobilisation is a technique which has been shown to alleviate these limitations in biocatalysis. Here we describe the immobilisation of two biocatalytically relevant co-factor recycling enzymes, glucose dehydrogenase (GDH) and NADH oxidase (NOD) on aldehyde functional ReSyn™ polymer microspheres with varying functional group densities. The successful immobilisation of the enzymes on this new high capacity microsphere technology resulted in the maintenance of activity of ∼40% for GDH and a maximum of 15.4% for NOD. The microsphere variant with highest functional group density of ∼3500 μmol g⁻¹ displayed the highest specific activity for the immobilisation of both enzymes at 33.22 U mg⁻¹ and 6.75 U mg⁻¹ for GDH and NOD with respective loading capacities of 51% (0.51 mg mg⁻¹) and 129% (1.29 mg mg⁻¹). The immobilised GDH further displayed improved activity in the acidic pH range. Both enzymes displayed improved pH and thermal stability with the most pronounced thermal stability for GDH displayed on ReSyn™ A during temperature incubation at 65 °C with a 13.59 fold increase, and NOD with a 2.25-fold improvement at 45 °C on the same microsphere variant. An important finding is the suitability of the microspheres for stabilisation of the multimeric protein GDH. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Body odor aldehyde reduction by acetic acid bacterial extract including enzymes: alcohol dehydrogenase and aldehyde dehydrogenase.

    PubMed

    Yoshioka, N; Kurata, K; Takahashi, T; Ariizumi, M; Mori, T; Fujisawa, H; Kameyama, N; Okuyama, Y

    2018-06-13

    Body odor is mainly caused by secreted sweat. Although sweat is almost odorless immediately after secretion, decomposition or denaturation of components contained in sweat by bacteria on the skin surface contributes to unpleasant body odor. Body odor is due to various substances and aldehydes are primarily detected in body odor [1-4]. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  16. Fatty aldehyde dehydrogenases in Acinetobacter sp. strain HO1-N: role in hexadecanol metabolism.

    PubMed Central

    Singer, M E; Finnerty, W R

    1985-01-01

    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: a membrane-bound, NADP-dependent FALDH activity induced 5-, 15-, and 9-fold by growth on hexadecanol, dodecyl aldehyde, and hexadecane, respectively, and a constitutive, NAD-dependent, membrane-localized FALDH. The NADP-dependent FALDH exhibited apparent Km and Vmax values for decyl aldehyde of 5.0, 13.0, 18.0, and 18.3 microM and 537.0, 500.0, 25.0, and 38.0 nmol/min in hexadecane-, hexadecanol-, ethanol-, palmitate-grown cells, respectively. FALDH isozymes ald-a, ald-b, and ald-c were demonstrated by gel electrophoresis in extracts of hexadecane- and hexadecanol-grown cells. ald-a, ald-b, and ald-d were present in dodecyl aldehyde-grown cells, while palmitate-grown control cells contained ald-b and ald-d. 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. The oxidation of [3H]hexadecane byAld21 yielded the accumulation of 61% more fatty aldehyde than the wild type, while Ald24 accumulated 27% more fatty aldehyde, 95% more fatty alcohol, and 65% more wax ester than the wild type. 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

  17. 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

    2014-05-16

    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.

  18. The Activity of Class I-IV Alcohol Dehydrogenase Isoenzymes and Aldehyde Dehydrogenase in Bladder Cancer Cells.

    PubMed

    Orywal, Karolina; Jelski, Wojciech; Werel, Tadeusz; Szmitkowski, Maciej

    2018-01-02

    The aim of this study was to determine the differences in the activity of Alcohol Dehydrogenase (ADH) isoenzymes and Aldehyde Dehydrogenase (ALDH) in normal and cancerous bladder cells. Class III, IV of ADH and total ADH activity were measured by the photometric method and class I, II ADH and ALDH activity by the fluorometric method. Significantly higher total activity of ADH was found in both, low-grade and high-grade bladder cancer, in comparison to healthy tissues. The increased activity of total ADH in bladder cancer cells may be the cause of metabolic disorders in cancer cells, which may intensify carcinogenesis.

  19. Aromatic aldehydes at the active site of aldehyde oxidoreductase from Desulfovibrio gigas: reactivity and molecular details of the enzyme-substrate and enzyme-product interaction.

    PubMed

    Correia, Hugo D; Marangon, Jacopo; Brondino, Carlos D; Moura, Jose J G; Romão, Maria J; González, Pablo J; Santos-Silva, Teresa

    2015-03-01

    Desulfovibrio gigas aldehyde oxidoreductase (DgAOR) is a mononuclear molybdenum-containing enzyme from the xanthine oxidase (XO) family, a group of enzymes capable of catalyzing the oxidative hydroxylation of aldehydes and heterocyclic compounds. The kinetic studies reported in this work showed that DgAOR catalyzes the oxidative hydroxylation of aromatic aldehydes, but not heterocyclic compounds. NMR spectroscopy studies using (13)C-labeled benzaldehyde confirmed that DgAOR catalyzes the conversion of aldehydes to the respective carboxylic acids. Steady-state kinetics in solution showed that high concentrations of the aromatic aldehydes produce substrate inhibition and in the case of 3-phenyl propionaldehyde a suicide substrate behavior. Hydroxyl-substituted aromatic aldehydes present none of these behaviors but the kinetic parameters are largely affected by the position of the OH group. High-resolution crystallographic structures obtained from single crystals of active-DgAOR soaked with benzaldehyde showed that the side chains of Phe425 and Tyr535 are important for the stabilization of the substrate in the active site. On the other hand, the X-ray data of DgAOR soaked with trans-cinnamaldehyde showed a cinnamic acid molecule in the substrate channel. The X-ray data of DgAOR soaked with 3-phenyl propionaldehyde showed clearly how high substrate concentrations inactivate the enzyme by binding covalently at the surface of the enzyme and blocking the substrate channel. The different reactivity of DgAOR versus aldehyde oxidase and XO towards aromatic aldehydes and N-heterocyclic compounds is explained on the basis of the present kinetic and structural data.

  20. 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.

  1. 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.

  2. 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

  3. Kinetic and biophysical investigation of the inhibitory effect of caffeine on human salivary aldehyde dehydrogenase: Implications in oral health and chemotherapy

    NASA Astrophysics Data System (ADS)

    Laskar, Amaj Ahmed; Alam, Md Fazle; Ahmad, Mohammad; Younus, Hina

    2018-04-01

    Human salivary aldehyde dehydrogenase (hsALDH) is primarily a class 3 ALDH (ALDH3A1), and is an important antioxidant enzyme present in the saliva which maintains healthy oral cavity. It detoxifies toxic aldehydes into non-toxic carboxylic acids in the oral cavity. Reduced level of hsALDH activity is a risk factor for oral cancer development. It is involved in the resistance of certain chemotherapeutic drugs. Coffee has been reported to affect the activity of salivary ALDH. In this study, the effect of caffeine on the activity (dehydrogenase and esterase) of hsALDH was investigated. The binding of caffeine to hsALDH was studied using different biophysical methods and molecular docking analysis. Caffeine was found to inhibit both crude and purified hsALDH. The Km increased and the Vmax decreased showing a mixed type of inhibition. Caffeine decreased the nucleophilicity of the catalytic cysteine residue. It binds to the active site of ALDH3A1 by forming a complex through non-covalent interactions with some highly conserved amino acid residues. It partially alters the secondary structure of the enzyme. Therefore, it is very likely that caffeine binds and inhibits the activity of hsALDH by decreasing substrate binding affinity and the catalytic efficiency of the enzyme. The study indicates that oral intake of caffeine may have a harmful effect on the oral health and may increase the risk of carcinogenesis through the inhibition of this important enzyme. Further, the inactivation of oxazaphosphorine based chemotherapeutic drugs by ALDH3A1 may be prevented by using caffeine as an adjuvant during medication which is expected to increase the sensitivity of these drugs through its inhibitory effect on the enzyme.

  4. Fatty Aldehydes in Cyanobacteria Are a Metabolically Flexible Precursor for a Diversity of Biofuel Products

    PubMed Central

    Kaiser, Brett K.; Carleton, Michael; Hickman, Jason W.; Miller, Cameron; Lawson, David; Budde, Mark; Warrener, Paul; Paredes, Angel; Mullapudi, Srinivas; Navarro, Patricia; Cross, Fred; Roberts, James M.

    2013-01-01

    We describe how pathway engineering can be used to convert a single intermediate derived from lipid biosynthesis, fatty aldehydes, into a variety of biofuel precursors including alkanes, free fatty acids and wax esters. In cyanobacteria, long-chain acyl-ACPs can be reduced to fatty aldehydes, and then decarbonylated to alkanes. We discovered a cyanobacteria class-3 aldehyde-dehydrogenase, AldE, that was necessary and sufficient to instead oxidize fatty aldehyde precursors into fatty acids. Overexpression of enzymes in this pathway resulted in production of 50 to 100 fold more fatty acids than alkanes, and the fatty acids were secreted from the cell. Co-expression of acyl-ACP reductase, an alcohol-dehydrogenase and a wax-ester-synthase resulted in a third fate for fatty aldehydes: conversion to wax esters, which accumulated as intracellular lipid bodies. Conversion of acyl-ACP to fatty acids using endogenous cyanobacterial enzymes may allow biofuel production without transgenesis. PMID:23505484

  5. Cardiac-specific overexpression of aldehyde dehydrogenase 2 exacerbates cardiac remodeling in response to pressure overload.

    PubMed

    Dassanayaka, Sujith; Zheng, Yuting; Gibb, Andrew A; Cummins, Timothy D; McNally, Lindsey A; Brittian, Kenneth R; Jagatheesan, Ganapathy; Audam, Timothy N; Long, Bethany W; Brainard, Robert E; Jones, Steven P; Hill, Bradford G

    2018-06-01

    Pathological cardiac remodeling during heart failure is associated with higher levels of lipid peroxidation products and lower abundance of several aldehyde detoxification enzymes, including aldehyde dehydrogenase 2 (ALDH2). An emerging idea that could explain these findings concerns the role of electrophilic species in redox signaling, which may be important for adaptive responses to stress or injury. The purpose of this study was to determine whether genetically increasing ALDH2 activity affects pressure overload-induced cardiac dysfunction. Mice subjected to transverse aortic constriction (TAC) for 12 weeks developed myocardial hypertrophy and cardiac dysfunction, which were associated with diminished ALDH2 expression and activity. Cardiac-specific expression of the human ALDH2 gene in mice augmented myocardial ALDH2 activity but did not improve cardiac function in response to pressure overload. After 12 weeks of TAC, ALDH2 transgenic mice had larger hearts than their wild-type littermates and lower capillary density. These findings show that overexpression of ALDH2 augments the hypertrophic response to pressure overload and imply that downregulation of ALDH2 may be an adaptive response to certain forms of cardiac pathology. Copyright © 2018. Published by Elsevier B.V.

  6. 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. Copyright © 2015 John Wiley & Sons, Ltd.

  7. DB Dehydrogenase: an online integrated structural database on enzyme dehydrogenase.

    PubMed

    Nandy, Suman Kumar; Bhuyan, Rajabrata; Seal, Alpana

    2012-01-01

    Dehydrogenase enzymes are almost inevitable for metabolic processes. Shortage or malfunctioning of dehydrogenases often leads to several acute diseases like cancers, retinal diseases, diabetes mellitus, Alzheimer, hepatitis B & C etc. With advancement in modern-day research, huge amount of sequential, structural and functional data are generated everyday and widens the gap between structural attributes and its functional understanding. DB Dehydrogenase is an effort to relate the functionalities of dehydrogenase with its structures. It is a completely web-based structural database, covering almost all dehydrogenases [~150 enzyme classes, ~1200 entries from ~160 organisms] whose structures are known. It is created by extracting and integrating various online resources to provide the true and reliable data and implemented by MySQL relational database through user friendly web interfaces using CGI Perl. Flexible search options are there for data extraction and exploration. To summarize, sequence, structure, function of all dehydrogenases in one place along with the necessary option of cross-referencing; this database will be utile for researchers to carry out further work in this field. The database is available for free at http://www.bifku.in/DBD/

  8. Hydrogen Sulfide Ameliorates Homocysteine-Induced Cognitive Dysfunction by Inhibition of Reactive Aldehydes Involving Upregulation of ALDH2.

    PubMed

    Li, Min; Zhang, Ping; Wei, Hai-Jun; Li, Man-Hong; Zou, Wei; Li, Xiang; Gu, Hong-Feng; Tang, Xiao-Qing

    2017-04-01

    Homocysteine, a risk factor for Alzheimer's disease, induces cognitive dysfunction. Reactive aldehydes play an important role in cognitive dysfunction. Aldehyde-dehydrogenase 2 detoxifies reactive aldehydes. Hydrogen sulfide, a novel neuromodulator, has neuroprotective effects and regulates learning and memory. Our previous work confirmed that the disturbance of hydrogen sulfide synthesis is invovled in homocysteine-induced defects in learning and memory. Therefore, the present work was to explore whether hydrogen sulfide ameliorates homocysteine-generated cognitive dysfunction and to investigate whether its underlying mechanism is related to attenuating accumulation of reactive aldehydes by upregulation of aldehyde-dehydrogenase 2. The cognitive function of rats was assessed by the Morris water maze test and the novel object recognition test. The levels of malondialdehyde, 4-hydroxynonenal, and glutathione as well as the activity of aldehyde-dehydrogenase 2 were determined by enzyme linked immunosorbent assay; the expression of aldehyde-dehydrogenase 2 was detected by western blot. The behavior experiments, Morris water maze test and novel objects recognition test, showed that homocysteine induced deficiency in learning and memory in rats, and this deficiency was reversed by treatment of NaHS (a donor of hydrogen sulfide). We demonstrated that NaHS inhibited homocysteine-induced increases in generations of MDA and 4-HNE in the hippocampus of rats and that hydrogen sulfide reversed homocysteine-induced decreases in the level of glutathione as well as the activity and expression of aldehyde-dehydrogenase 2 in the hippocampus of rats. Hydrogen sulfide ameliorates homocysteine-induced impairment in cognitive function by decreasing accumulation of reactive aldehydes as a result of upregulations of glutathione and aldehyde-dehydrogenase 2. © The Author 2016. Published by Oxford University Press on behalf of CINP.

  9. Hydrogen Sulfide Ameliorates Homocysteine-Induced Cognitive Dysfunction by Inhibition of Reactive Aldehydes Involving Upregulation of ALDH2

    PubMed Central

    Li, Min; Zhang, Ping; Wei, Hai-jun; Li, Man-Hong; Li, Xiang; Gu, Hong-Feng

    2017-01-01

    Abstract Background: Homocysteine, a risk factor for Alzheimer’s disease, induces cognitive dysfunction. Reactive aldehydes play an important role in cognitive dysfunction. Aldehyde-dehydrogenase 2 detoxifies reactive aldehydes. Hydrogen sulfide, a novel neuromodulator, has neuroprotective effects and regulates learning and memory. Our previous work confirmed that the disturbance of hydrogen sulfide synthesis is invovled in homocysteine-induced defects in learning and memory. Therefore, the present work was to explore whether hydrogen sulfide ameliorates homocysteine-generated cognitive dysfunction and to investigate whether its underlying mechanism is related to attenuating accumulation of reactive aldehydes by upregulation of aldehyde-dehydrogenase 2. Methods: The cognitive function of rats was assessed by the Morris water maze test and the novel object recognition test. The levels of malondialdehyde, 4-hydroxynonenal, and glutathione as well as the activity of aldehyde-dehydrogenase 2 were determined by enzyme linked immunosorbent assay; the expression of aldehyde-dehydrogenase 2 was detected by western blot. Results: The behavior experiments, Morris water maze test and novel objects recognition test, showed that homocysteine induced deficiency in learning and memory in rats, and this deficiency was reversed by treatment of NaHS (a donor of hydrogen sulfide). We demonstrated that NaHS inhibited homocysteine-induced increases in generations of MDA and 4-HNE in the hippocampus of rats and that hydrogen sulfide reversed homocysteine-induced decreases in the level of glutathione as well as the activity and expression of aldehyde-dehydrogenase 2 in the hippocampus of rats. Conclusion: Hydrogen sulfide ameliorates homocysteine-induced impairment in cognitive function by decreasing accumulation of reactive aldehydes as a result of upregulations of glutathione and aldehyde-dehydrogenase 2. PMID:27988490

  10. RP-HPLC-fluorescence analysis of aliphatic aldehydes: application to aldehyde-generating enzymes HACL1 and SGPL1

    PubMed Central

    Mezzar, Serena; de Schryver, Evelyn; Van Veldhoven, Paul P.

    2014-01-01

    Long-chain aldehydes are commonly produced in various processes, such as peroxisomal α-oxidation of long-chain 3-methyl-branched and 2-hydroxy fatty acids and microsomal breakdown of phosphorylated sphingoid bases. The enzymes involved in the aldehyde-generating steps of these processes are 2-hydroxyacyl-CoA lyase (HACL1) and sphingosine-1-phosphate lyase (SGPL1), respectively. In the present work, nonradioactive assays for these enzymes were developed employing the Hantzsch reaction. Tridecanal (C13-al) and heptadecanal (C17-al) were selected as model compounds and cyclohexane-1,3-dione as 1,3-diketone, and the fluorescent derivatives were analyzed by reversed phase (RP)-HPLC. Assay mixture composition, as well as pH and heating, were optimized for C13-al and C17-al. Under optimized conditions, these aldehydes could be quantified in picomolar range and different long-chain aldehyde derivatives were well resolved with a linear gradient elution by RP-HPLC. Aldehydes generated by recombinant enzymes could easily be detected via this method. Moreover, the assay allowed to document activity or deficiency in tissue homogenates and fibroblast lysates without an extraction step. In conclusion, a simple, quick, and cheap assay for the study of HACL1 and SGPL1 activities was developed, without relying on expensive mass spectrometric detectors or radioactive substrates. PMID:24323699

  11. Aldehyde dehydrogenase polymorphism in North American, South American, and Mexican Indian populations.

    PubMed Central

    Goedde, H W; Agarwal, D P; Harada, S; Rothhammer, F; Whittaker, J O; Lisker, R

    1986-01-01

    While about 40% of the South American Indian populations (Atacameños, Mapuche, Shuara) were found to be deficient in aldehyde dehydrogenase isozyme I (ALDH2 or E2), preliminary investigations showed very low incidence of isozyme deficiency among North American natives (Sioux, Navajo) and Mexican Indians (mestizo). Possible implications of such trait differences on cross-cultural behavioral response to alcohol drinking are discussed. PMID:3953578

  12. Resolution and partial characterization of two aldehyde reductases of mammalian liver.

    PubMed

    Tulsiani, D R; Touster

    1977-04-25

    Investigation of NADP-dependent aldehyde reductase activity in mouse liver led to the finding that two distinct reductases are separable by DE52 ion exchange chromatography. Aldehyde reductase I (AR I) appears in the effluent, while aldehyde reductase II (AR II) is eluted with a salt gradient. By several procedures AR II was purified over 1100-fold from liver supernatant fraction, but AR I could be pruified only 107-fold because of its instability. The two enzymes are different in regard to pH optimum, substrate specificity, response to inhibitors, and reactivity with antibody to AR II. While both enzymes utilize aromatic aldehydes well, only AR II ACTS ON D-glucuronate, indicating that it is the aldyhyde reductase recently reported to be identical to NADP-L-gulonate dehydrogenase. The presence of two NADP-linked aldehyde reductases in liver has apparently not heretofore been reported.

  13. Aldehyde dehydrogenase 1a1 mediates a GABA synthesis pathway in midbrain dopaminergic neurons.

    PubMed

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

    2015-10-02

    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 co-release in dopamine neurons does not use 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 (EtOH) at concentrations seen in blood alcohol after binge drinking, 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. Copyright © 2015, American Association for the Advancement of Science.

  14. 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.

  15. Age-dependent neurodegeneration accompanying memory loss in transgenic mice defective in mitochondrial aldehyde dehydrogenase 2 activity.

    PubMed

    Ohsawa, Ikuroh; Nishimaki, Kiyomi; Murakami, Yayoi; Suzuki, Yuko; Ishikawa, Masahiro; Ohta, Shigeo

    2008-06-11

    Oxidative stress may underlie age-dependent memory loss and cognitive decline. Toxic aldehydes, including 4-hydroxy-2-nonenal (HNE), an end product of lipid peroxides, are known to accumulate in the brain in neurodegenerative disease. We have previously shown that mitochondrial aldehyde dehydrogenase 2 (ALDH2) detoxifies HNE by oxidizing its aldehyde group. To investigate the role of such toxic aldehydes, we produced transgenic mice, which expressed a dominant-negative form of ALDH2 in the brain. The mice had decreased ability to detoxify HNE in their cortical neurons and accelerated accumulation of HNE in the brain. Consequently, their lifespan was shortened and age-dependent neurodegeneration and hyperphosphorylation of tau were observed. Object recognition and Morris water maze tests revealed that the onset of cognitive impairment correlated with the degeneration, which was further accelerated by APOE (apolipoprotein E) knock-out; therefore, the accumulation of toxic aldehydes is by itself critical in the progression of neurodegenerative disease, which could be suppressed by ALDH2.

  16. Aldehyde dehydrogenase 3A1 activation prevents radiation-induced xerostomia by protecting salivary stem cells from toxic aldehydes

    PubMed Central

    Saiki, Julie P.; Cao, Hongbin; Van Wassenhove, Lauren D.; Viswanathan, Vignesh; Bloomstein, Joshua; Nambiar, Dhanya K.; Mattingly, Aaron J.; Jiang, Dadi; Chen, Che-Hong; Simmons, Amanda L.; Park, Hyun Shin; von Eyben, Rie; Kool, Eric T.; Sirjani, Davud; Knox, Sarah M.; Le, Quynh Thu; Mochly-Rosen, Daria

    2018-01-01

    Xerostomia (dry mouth) is the most common side effect of radiation therapy in patients with head and neck cancer and causes difficulty speaking and swallowing. Since aldehyde dehydrogenase 3A1 (ALDH3A1) is highly expressed in mouse salivary stem/progenitor cells (SSPCs), we sought to determine the role of ALDH3A1 in SSPCs using genetic loss-of-function and pharmacologic gain-of-function studies. Using DarkZone dye to measure intracellular aldehydes, we observed higher aldehyde accumulation in irradiated Aldh3a1−/− adult murine salisphere cells and in situ in whole murine embryonic salivary glands enriched in SSPCs compared with wild-type glands. To identify a safe ALDH3A1 activator for potential clinical testing, we screened a traditional Chinese medicine library and isolated d-limonene, commonly used as a food-flavoring agent, as a single constituent activator. ALDH3A1 activation by d-limonene significantly reduced aldehyde accumulation in SSPCs and whole embryonic glands, increased sphere-forming ability, decreased apoptosis, and improved submandibular gland structure and function in vivo after radiation. A phase 0 study in patients with salivary gland tumors showed effective delivery of d-limonene into human salivary glands following daily oral dosing. Given its safety and bioavailability, d-limonene may be a good clinical candidate for mitigating xerostomia in patients with head and neck cancer receiving radiation therapy. PMID:29794221

  17. YLL056C from Saccharomyces cerevisiae encodes a novel protein with aldehyde reductase activity.

    PubMed

    Wang, Han-Yu; Xiao, Di-Fan; Zhou, Chang; Wang, Lin-Lu; Wu, Lan; Lu, Ya-Ting; Xiang, Quan-Ju; Zhao, Ke; Li, Xi; Ma, Meng -Gen

    2017-06-01

    The short-chain dehydrogenase/reductase (SDR) family, the largest family in dehydrogenase/reductase superfamily, is divided into "classical," "extended," "intermediate," "divergent," "complex," and "atypical" groups. Recently, several open reading frames (ORFs) were characterized as intermediate SDR aldehyde reductase genes in Saccharomyces cerevisiae. However, no functional protein in the atypical group has been characterized in S. cerevisiae till now. Herein, we report that an uncharacterized ORF YLL056C from S. cerevisiae was significantly upregulated under high furfural (2-furaldehyde) or 5-(hydroxymethyl)-2-furaldehyde concentrations, and transcription factors Yap1p, Hsf1p, Pdr1/3p, Yrr1p, and Stb5p likely controlled its upregulated transcription. This ORF indeed encoded a protein (Yll056cp), which was grouped into the atypical subgroup 7 in the SDR family and localized to the cytoplasm. Enzyme activity assays showed that Yll056cp is not a quinone or ketone reductase but an NADH-dependent aldehyde reductase, which can reduce at least seven aldehyde compounds. This enzyme showed the best Vmax, Kcat, and Kcat/Km to glycolaldehyde, but the highest affinity (Km) to formaldehyde. The optimum pH and temperature of this enzyme was pH 6.5 for reduction of glycolaldehyde, furfural, formaldehyde, butyraldehyde, and propylaldehyde, and 30 °C for reduction of formaldehyde or 35 °C for reduction of glycolaldehyde, furfural, butyraldehyde, and propylaldehyde. Temperature and pH affected stability of this enzyme and this influence varied with aldehyde substrate. Metal ions, salts, and chemical protective additives, especially at high concentrations, had different influence on enzyme activities for reduction of different aldehydes. This research provided guidelines for study of more uncharacterized atypical SDR enzymes from S. cerevisiae and other organisms.

  18. Aldehyde Dehydrogenase 2 in Aplastic Anemia, Fanconi Anemia and Hematopoietic Stem Cells

    PubMed Central

    Van Wassenhove, Lauren D.; Mochly-Rosen, Daria; Weinberg, Kenneth I.

    2016-01-01

    Maintenance of the hematopoietic stem cell (HSC) compartment depends on the ability to metabolize exogenously and endogenously generated toxins, and to repair cellular damage caused by such toxins. Reactive aldehydes have been demonstrated to cause specific genotoxic injury, namely DNA interstrand cross-links. Aldehyde dehydrogenase 2 (ALDH2) is a member of a 19 isoenzyme ALDH family with different substrate specificities, subcellular localization, and patterns of expression. ALDH2 is localized in mitochondria and is essential for the metabolism of acetaldehyde, thereby placing it directly downstream of ethanol metabolism. Deficiency in ALDH2 expression and function are caused by a single nucleotide substitution and resulting amino acid change, called ALDH2*2. This genetic polymorphism affects 35–45% of East Asians (about ~560 million people), and causes the well-known Asian flushing syndrome, which results in disulfiram-like reactions after ethanol consumption. Recently, the ALDH2*2 genotype has been found to be associated with marrow failure, with both an increased risk of sporadic aplastic anemia and more rapid progression of Fanconi Anemia. This review discusses the unexpected interrelationship between aldehydes, ALDH2 and hematopoietic stem cell biology, and in particular its relationship to Fanconi anemia. PMID:27650066

  19. Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action

    DOE PAGES

    Huo, Lu; Davis, Ian; Liu, Fange; ...

    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 sp 3-to-sp 2 transition during enzyme-mediated oxidation.« less

  20. Aldehyde dehydrogenase 2 in aplastic anemia, Fanconi anemia and hematopoietic stem cells.

    PubMed

    Van Wassenhove, Lauren D; Mochly-Rosen, Daria; Weinberg, Kenneth I

    2016-09-01

    Maintenance of the hematopoietic stem cell (HSC) compartment depends on the ability to metabolize exogenously and endogenously generated toxins, and to repair cellular damage caused by such toxins. Reactive aldehydes have been demonstrated to cause specific genotoxic injury, namely DNA interstrand cross-links. Aldehyde dehydrogenase 2 (ALDH2) is a member of a 19 isoenzyme ALDH family with different substrate specificities, subcellular localization, and patterns of expression. ALDH2 is localized in mitochondria and is essential for the metabolism of acetaldehyde, thereby placing it directly downstream of ethanol metabolism. Deficiency in ALDH2 expression and function are caused by a single nucleotide substitution and resulting amino acid change, called ALDH2*2. This genetic polymorphism affects 35-45% of East Asians (about ~560 million people), and causes the well-known Asian flushing syndrome, which results in disulfiram-like reactions after ethanol consumption. Recently, the ALDH2*2 genotype has been found to be associated with marrow failure, with both an increased risk of sporadic aplastic anemia and more rapid progression of Fanconi anemia. This review discusses the unexpected interrelationship between aldehydes, ALDH2 and hematopoietic stem cell biology, and in particular its relationship to Fanconi anemia. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Hypoglycemic drugs induce antioxidant aldehyde dehydrogenase activity and remain high in patients with glycemic control in type 2 diabetes.

    PubMed

    Picazo, Alejandra; Jiménez-Osorio, Angélica S; Zúñiga-Mejía, Porfirio; Pedraza-Chaverri, José; Monroy, Adriana; Rodríguez-Arellano, M Eunice; Barrera-Oviedo, Diana

    2017-04-05

    The antioxidant system results essential to control and prevent lipid peroxidation due to stress damage in type 2 diabetes. An example is aldehyde dehydrogenase (ALDH), an enzyme that is involved in the detoxification of aldehydes formed during lipid peroxidation. This study was conducted to evaluate ALDH activity and to determine their association with hypoglycemic treatment in type 2 diabetes patients. The study population consisted of 422 Mexican subjects: a control group and type 2 diabetes patients. Type 2 diabetes patients were re-classified as those with or without hypoglycemic treatment and those with or without glycemic control (according to glycated hemoglobin (HbA1c)). Clinical parameters, antioxidant enzyme activities (ALDH, superoxide dismutase (SOD), catalase and glutathione peroxidase) and oxidative markers (reactive oxygen species and thiobarbituric acid reactive substances (TBARS)) were evaluated. The activity of antioxidant enzymes and oxidative stress markers were higher in type 2 diabetes patients with hypoglycemic treatment and without glycemic control than control group. The activity of ALDH and SOD remained high in type 2 diabetes patients with moderate glycemic control while only ALDH's remained high in type 2 diabetes patients with tight glycemic control. Increased ALDH and SOD activities were associated with hypoglycemic therapy. TBARS levels were associated with glycemic control. The persistence of high ALDH and SOD activities in type 2 diabetes patients with glycemic control may be to avoid a significant damage due to the increase in reactive oxygen species and TBARS. It is possible that this new oxidative status prevented the development the classical complications of diabetes. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Effect of pentachlorophenol and 2,6-dichloro-4-nitrophenol on the activity of cDNA-expressed human alcohol and aldehyde dehydrogenases.

    PubMed

    Kollock, Ronny; Rost, Katharina; Batke, Monika; Glatt, Hansruedi

    2009-12-15

    Pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP), potent inhibitors of phenol sulphotransferases, are frequently used in animal studies to elucidate the role of these enzymes in the biotransformation and toxicity of xenobiotics. An unexpected finding with 1-hydroxymethylpyrene--a strong decrease in the excretion of the corresponding carboxylic acid in rats concurrently treated with PCP-led us to suspect that this sulphotransferase inhibitor may also affect alcohol dehydrogenases (ADHs) and/or aldehyde dehydrogenases (ALDHs). Subsequently we investigated the influence of PCP and DCNP on the activity of cDNA-expressed human ADHs and ALDHs. PCP inhibited all four ADHs studied. The inhibition was strong for ADH3 (K(i) 1.4 microM, K(i)' 5.2 microM, mixed-type) and ADH2 (K(i) 3.7 microM, competitive), but moderate for ADH4 (K(i) 81 microM, competitive) and ADH1C (K(i)' 310 microM, uncompetitive). Activities of ALDH2 and ALDH3A1 were unaffected by PCP (used up to a concentration of 1 mM). In contrast, DCNP primarily inhibited ALDH2 (K(i)=K(i)' 7.4 microM, non-competitive), showed moderate competitive inhibition of ADH2 (K(i) 160 microM) and ADH4 (K(i) 710 microM), but did not affect the remaining enzymes (ADH1C, ADH3 and ALDH3A1). The study demonstrates that caution is required when using putative specific enzyme inhibitors in biotransformation studies.

  3. Preventive effects of Chlorella on skeletal muscle atrophy in muscle-specific mitochondrial aldehyde dehydrogenase 2 activity-deficient mice.

    PubMed

    Nakashima, Yuya; Ohsawa, Ikuroh; Nishimaki, Kiyomi; Kumamoto, Shoichiro; Maruyama, Isao; Suzuki, Yoshihiko; Ohta, Shigeo

    2014-10-11

    Oxidative stress is involved in age-related muscle atrophy, such as sarcopenia. Since Chlorella, a unicellular green alga, contains various antioxidant substances, we used a mouse model of enhanced oxidative stress to investigate whether Chlorella could prevent muscle atrophy. Aldehyde dehydrogenase 2 (ALDH2) is an anti-oxidative enzyme that detoxifies reactive aldehydes derived from lipid peroxides such as 4-hydroxy-2-nonenal (4-HNE). We therefore used transgenic mice expressing a dominant-negative form of ALDH2 (ALDH2*2 Tg mice) to selectively decrease ALDH2 activity in the muscles. To evaluate the effect of Chlorella, the mice were fed a Chlorella-supplemented diet (CSD) for 6 months. ALDH2*2 Tg mice exhibited small body size, muscle atrophy, decreased fat content, osteopenia, and kyphosis, accompanied by increased muscular 4-HNE levels. The CSD helped in recovery of body weight, enhanced oxidative stress, and increased levels of a muscle impairment marker, creatine phosphokinase (CPK) induced by ALDH2*2. Furthermore, histological and histochemical analyses revealed that the consumption of the CSD improved skeletal muscle atrophy and the activity of the mitochondrial cytochrome c oxidase. This study suggests that long-term consumption of Chlorella has the potential to prevent age-related muscle atrophy.

  4. Autoantibody against aldehyde dehydrogenase 2 could be a biomarker to monitor progression of Graves' orbitopathy.

    PubMed

    Cheng, Kai-Chun; Wu, Yu-Jen; Cheng, Kai-Hung; Cheng, Kai-Yuan; Chen, Kuo-Jen; Wu, Wen-Chuan; Lee, Po-Yen; Chang, Cheng-Hsien

    2018-06-01

    This study surveyed the novel autoantigens expressed in the orbital fat tissue of patients with Graves' orbitopathy (GO) and explored the possibility of the autoantibodies against novel autoantigens as biomarkers for GO. We used immuno-proteomic methods to survey novel autoantigens expressed in the orbit fat tissue of GO patients and confirmed by enzyme-linked immunosorbent assay (ELISA). One protein spot (aldehyde dehydrogenase 2 (ALDH2)) revealed high reactivity with the GO serum than did the healthy control serum and was further verified by ELISA. We found that the plasma anti-ALDH2 antibody level was increased in GO patients compared to healthy control donors. In addition, anti-ALDH2 antibody level was correlated with GO activity classified by clinical activity score(r = 0.588, p < 0.001, using Pearson's correlation). These increased levels of anti-ALDH2 antibody in GO serum suggested that ALDH2 could attribute target autoantigen in GO, and anti-ALDH2 autoantibody might serve as a biomarker for GO and help to predict disease activity.

  5. ALD5, PAD1, ATF1 and ATF2 facilitate the catabolism of coniferyl aldehyde, ferulic acid and p-coumaric acid in Saccharomyces cerevisiae

    PubMed Central

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

    2017-01-01

    The ability of Saccharomyces cerevisiae to catabolize phenolic compounds remains to be fully elucidated. Conversion of coniferyl aldehyde, ferulic acid and p-coumaric acid by S. cerevisiae under aerobic conditions was previously reported. A conversion pathway was also proposed. In the present study, possible enzymes involved in the reported conversion were investigated. Aldehyde dehydrogenase Ald5, phenylacrylic acid decarboxylase Pad1, and alcohol acetyltransferases Atf1 and Atf2, were hypothesised to be involved. Corresponding genes for the four enzymes were overexpressed in a S. cerevisiae strain named APT_1. The ability of APT_1 to tolerate and convert the three phenolic compounds was tested. APT_1 was also compared to strains B_CALD heterologously expressing coniferyl aldehyde dehydrogenase from Pseudomonas, and an ald5Δ strain, all previously reported. APT_1 exhibited the fastest conversion of coniferyl aldehyde, ferulic acid and p-coumaric acid. Using the intermediates and conversion products of each compound, the catabolic route of coniferyl aldehyde, ferulic acid and p-coumaric acid in S. cerevisiae was studied in greater detail. PMID:28205618

  6. Cloning, Expression, and Purification of Choline Dehydrogenase from the Moderate Halophile Halomonas elongata

    PubMed Central

    Gadda, Giovanni; McAllister-Wilkins, Elien Elizabeth

    2003-01-01

    Choline dehydrogenase (EC 1.1.99.1) catalyzes the four-electron oxidation of choline to glycine-betaine via a betaine-aldehyde intermediate. Such a reaction is of considerable interest for biotechnological applications in that transgenic plants engineered with bacterial glycine-betaine-synthesizing enzymes have been shown to have enhanced tolerance towards various environmental stresses, such as hypersalinity, freezing, and high temperatures. To date, choline dehydrogenase has been poorly characterized in its biochemical and kinetic properties, mainly because its purification has been hampered by instability of the enzyme in vitro. In the present report, we cloned and expressed in Escherichia coli the betA gene from the moderate halophile Halomonas elongata which codes for a hypothetical choline dehydrogenase. The recombinant enzyme was purified to more than 70% homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by treatment with 30 to 50% saturation of ammonium sulfate followed by column chromatography using DEAE-Sepharose. The purified enzyme showed similar substrate specificities with either choline or betaine-aldehyde as the substrate, as indicated by the apparent V/K values (where V is the maximal velocity and K is the Michaelis constant) of 0.9 and 0.6 μmol of O2 min−1 mg−1 mM−1 at pH 7 and 25°C, respectively. With 1 mM phenazine methosulfate as the primary electron acceptor, the apparent Vmax values for choline and betaine-aldehyde were 10.9 and 5.7 μmol of O2 min−1 mg−1, respectively. These Vmax values decreased four- to sevenfold when molecular oxygen was used as the electron acceptor. Altogether, the kinetic data are consistent with the conclusion that H. elongata betA codes for a choline dehydrogenase that can also act as an oxidase when electron acceptors other than molecular oxygen are not available. PMID:12676692

  7. Aldehyde Dehydrogenase Plays a Pivotal Role in the Maintenance of Stallion Sperm Motility.

    PubMed

    Gibb, Zamira; Lambourne, Sarah R; Curry, Benjamin J; Hall, Sally E; Aitken, Robert J

    2016-06-01

    Although stallion spermatozoa produce significant quantities of reactive oxygen species, a lag between 4-hydroxynonenal (4HNE) adduction and the loss of motility in stallion spermatozoa suggests the presence of a robust aldehyde detoxification mechanism. Because there is a paucity of studies characterizing the role of aldehyde dehydrogenase (ALDH) in sperm functionality, the aim of this study was to ascertain the relationship between 4HNE production and motility and ALDH expression by stallion spermatozoa. PCR analysis revealed the presence of the ALDH1A3, ALDH1B1, and ALDH2 isoforms in these cells. Strong correlations (P < 0.001) were found between ALDH expression and various motility parameters of stallion spermatozoa including the percentage of progressive (r = 0.79) and rapidly motile (r = 0.79) spermatozoa, whereas repeated measurements over 24 h revealed highly significant correlations among progressive motility loss, 4HNE accumulation, and ALDH expression (P ≤ 0.001). ALDH inhibition resulted in a spontaneous increase in 4HNE levels in viable cells (21.1 ± 5.8% vs. 42.6 ± 5.2%; P ≤ 0.05) and a corresponding decrease in total motility (41.7 ± 6.2% vs. 6.4 ± 2.6%; P ≤ 0.001) and progressive motility (17.0 ± 4.1% vs. 0.7 ± 0.4%; P ≤ 0.001) of stallion spermatozoa over 24 h. Similarly, inhibition of ALDH in 4HNE-challenged spermatozoa significantly reduced total motility over 4 h (35.4 ± 9.7% vs. 15.3 ± 5.1%, respectively; P ≤ 0.05). This study contributes valuable information about the role of the ALDH enzymes in the maintenance of stallion sperm functionality, with potential diagnostic and in vitro applications for assisted reproductive technologies. © 2016 by the Society for the Study of Reproduction, Inc.

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

    PubMed

    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.

  9. Kinetic properties of the human liver cytosolic aldehyde dehydrogenase for retinal isomers.

    PubMed

    Bhat, P V; Samaha, H

    1999-01-15

    Retinoic acid exerts pleiotropic effects by acting through two families of nuclear receptors, RAR and RXR. All-trans and 9-cis retinoic acid bind RARs, whereas 9-cis retinoic acid binds and activates only the RXRs. To understand the role of human liver cytosolic aldehyde dehydrogenase (ALDH1) in retinoic acid synthesis, we examined the ability of ALDH 1 to catalyze the oxidation of the naturally occurring retinal isomers. ALDH1 catalyzed the oxidation of all-trans, 9-cis, and 13-cis retinal with equal efficiency. However, the affinity to all-trans retinal (Km = 2.2 microM) was twofold higher than to 9-cis (Km = 5.5 microM) and 13-cis (Km = 4.6 microM) retinal. All-trans retinol was a potent inhibitor of ALDH1 activity, and inhibited all-trans retinal oxidation uncompetitively. Comparison of the kinetic properties of ALDH1 for retinal isomers with those of previously reported rat kidney retinal dehydrogenase showed distinct differences, suggesting that ALDH1 may play a different role in retinal metabolism in liver.

  10. The importance of alcohol dehydrogenase in regulation of ethanol metabolism in rat liver cells.

    PubMed Central

    Page, R A; Kitson, K E; Hardman, M J

    1991-01-01

    We used titration with the inhibitors tetramethylene sulphoxide and isobutyramide to assess quantitatively the importance of alcohol dehydrogenase in regulation of ethanol oxidation in rat hepatocytes. In hepatocytes isolated from starved rats the apparent Flux Control Coefficient (calculated assuming a single-substrate irreversible reaction with non-competitive inhibition) of alcohol dehydrogenase is 0.3-0.5. Adjustment of this coefficient to allow for alcohol dehydrogenase being a two-substrate reversible enzyme increases the value by 1.3-1.4-fold. The final value of the Flux Control Coefficient of 0.5-0.7 indicates that alcohol dehydrogenase is a major rate-determining enzyme, but that other factors also have a regulatory role. In hepatocytes from fed rats the Flux Control Coefficient for alcohol dehydrogenase decreases with increasing acetaldehyde concentration. This suggests that, as acetaldehyde concentrations rise, control of the pathway shifts from alcohol dehydrogenase to other enzymes, particularly aldehyde dehydrogenase. There is not a single rate-determining step for the ethanol metabolism pathway and control is shared among several steps. PMID:1898355

  11. Aldehyde dehydrogenase expression in Metaphire posthuma as a bioindicator to monitor heavy metal pollution in soil.

    PubMed

    Panday, Raju; Bhatt, Padam Shekhar; Bhattarai, Tribikram; Shakya, Kumudini; Sreerama, Lakshmaiah

    2016-11-21

    Soil contamination and associated pollution plays a detrimental role in soil flora and fauna. Soil is processed and remodeled by subterranean earthworms, accordingly are referred to as soil chemical engineers. These worms, besides processing carbon and nitrogen, serve as minors for processing metals. In heavy metal contaminated soils, they accumulate heavy metals, which in turn cause altered gene expression, including aldehyde dehydrogenase (ALDH) enzymes. This study explores the possibility of ALDH expression in earthworms as a novel biomarker for the heavy metal contamination of soil. Earthworms cultured in contaminated soils accumulated significantly higher levels of Pb and Cd. Similarly, significantly higher levels of ALDH enzyme activities were observed in earthworms cultured in soils contaminated with Pb and Cd. The ALDH activity was found to be highest in worms cultured in 5 ppm heavy metal contaminated soils. Although, ALDH activities decreased as the heavy metal concentration in soil increased, they were significantly higher when compared to control worms cultured in uncontaminated soils. The accumulation of heavy metal in earthworms measured after 28 days decreased as the heavy metal concentration in soil increased. Levels of ALDH expression correlated with total Pb and Cd concentration in the earthworm tissue. This study showed that the ALDH activity in earthworms could potentially be used as a biomarker to show heavy metal pollution in soil.

  12. Bioactivation to an aldehyde metabolite--possible role in the onset of toxicity induced by the anti-HIV drug abacavir.

    PubMed

    Grilo, Nádia M; Charneira, Catarina; Pereira, Sofia A; Monteiro, Emília C; Marques, M Matilde; Antunes, Alexandra M M

    2014-01-30

    Aldehydes are highly reactive molecules, which can be generated during numerous physiological processes, including the biotransformation of drugs. Several non-P450 enzymes participate in their metabolism albeit alcohol dehydrogenase and aldehyde dehydrogenase are the ones most frequently involved in this process. Endogenous and exogenous aldehydes have been strongly implicated in multiple human pathologies. Their ability to react with biomacromolecules (e.g. proteins) yielding covalent adducts is suggested to be the common primary mechanism underlying the toxicity of these reactive species. Abacavir is one of the options for combined anti-HIV therapy. Although individual susceptibilities to adverse effects differ among patients, abacavir is associated with idiosyncratic hypersensitivity drug reactions and an increased risk of cardiac dysfunction. This review highlights the current knowledge on abacavir metabolism and discusses the potential role of bioactivation to an aldehyde metabolite, capable of forming protein adducts, in the onset of abacavir-induced toxic outcomes. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  13. Cloning and molecular characterization of the betaine aldehyde dehydrogenase involved in the biosynthesis of glycine betaine in white shrimp (Litopenaeus vannamei).

    PubMed

    Delgado-Gaytán, María F; Rosas-Rodríguez, Jesús A; Yepiz-Plascencia, Gloria; Figueroa-Soto, Ciria G; Valenzuela-Soto, Elisa M

    2017-10-01

    The enzyme betaine aldehyde dehydrogenase (BADH) catalyzes the irreversible oxidation of betaine aldehyde to glycine betaine (GB), a very efficient osmolyte accumulated during osmotic stress. In this study, we determined the nucleotide sequence of the cDNA for the BADH from the white shrimp Litopenaeus vannamei (LvBADH). The cDNA was 1882 bp long, with a complete open reading frame of 1524 bp, encoding 507 amino acids with a predicted molecular mass of 54.15 kDa and a pI of 5.4. The predicted LvBADH amino acid sequence shares a high degree of identity with marine invertebrate BADHs. Catalytic residues (C-298, E-264 and N-167) and the decapeptide VTLELGGKSP involved in nucleotide binding and highly conserved in BADHs were identified in the amino acid sequence. Phylogenetic analyses classified LvBADH in a clade that includes ALDH9 sequences from marine invertebrates. Molecular modeling of LvBADH revealed that the protein has amino acid residues and sequence motifs essential for the function of the ALDH9 family of enzymes. LvBADH modeling showed three potential monovalent cation binding sites, one site is located in an intra-subunit cavity; other in an inter-subunit cavity and a third in a central-cavity of the protein. The results show that LvBADH shares a high degree of identity with BADH sequences from marine invertebrates and enzymes that belong to the ALDH9 family. Our findings suggest that the LvBADH has molecular mechanisms of regulation similar to those of other BADHs belonging to the ALDH9 family, and that BADH might be playing a role in the osmoregulation capacity of L. vannamei. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. A bacterial aromatic aldehyde dehydrogenase critical for the efficient catabolism of syringaldehyde.

    PubMed

    Kamimura, Naofumi; Goto, Takayuki; Takahashi, Kenji; Kasai, Daisuke; Otsuka, Yuichiro; Nakamura, Masaya; Katayama, Yoshihiro; Fukuda, Masao; Masai, Eiji

    2017-03-15

    Vanillin and syringaldehyde obtained from lignin are essential intermediates for the production of basic chemicals using microbial cell factories. However, in contrast to vanillin, the microbial conversion of syringaldehyde is poorly understood. Here, we identified an aromatic aldehyde dehydrogenase (ALDH) gene responsible for syringaldehyde catabolism from 20 putative ALDH genes of Sphingobium sp. strain SYK-6. All these genes were expressed in Escherichia coli, and nine gene products, including previously characterized BzaA, BzaB, and vanillin dehydrogenase (LigV), exhibited oxidation activities for syringaldehyde to produce syringate. Among these genes, SLG_28320 (desV) and ligV were most highly and constitutively transcribed in the SYK-6 cells. Disruption of desV in SYK-6 resulted in a significant reduction in growth on syringaldehyde and in syringaldehyde oxidation activity. Furthermore, a desV ligV double mutant almost completely lost its ability to grow on syringaldehyde. Purified DesV showed similar k cat /K m values for syringaldehyde (2100 s -1 ·mM -1 ) and vanillin (1700 s -1 ·mM -1 ), whereas LigV substantially preferred vanillin (8800 s -1 ·mM -1 ) over syringaldehyde (1.4 s -1 ·mM -1 ). These results clearly demonstrate that desV plays a major role in syringaldehyde catabolism. Phylogenetic analyses showed that DesV-like ALDHs formed a distinct phylogenetic cluster separated from the vanillin dehydrogenase cluster.

  15. A bacterial aromatic aldehyde dehydrogenase critical for the efficient catabolism of syringaldehyde

    PubMed Central

    Kamimura, Naofumi; Goto, Takayuki; Takahashi, Kenji; Kasai, Daisuke; Otsuka, Yuichiro; Nakamura, Masaya; Katayama, Yoshihiro; Fukuda, Masao; Masai, Eiji

    2017-01-01

    Vanillin and syringaldehyde obtained from lignin are essential intermediates for the production of basic chemicals using microbial cell factories. However, in contrast to vanillin, the microbial conversion of syringaldehyde is poorly understood. Here, we identified an aromatic aldehyde dehydrogenase (ALDH) gene responsible for syringaldehyde catabolism from 20 putative ALDH genes of Sphingobium sp. strain SYK-6. All these genes were expressed in Escherichia coli, and nine gene products, including previously characterized BzaA, BzaB, and vanillin dehydrogenase (LigV), exhibited oxidation activities for syringaldehyde to produce syringate. Among these genes, SLG_28320 (desV) and ligV were most highly and constitutively transcribed in the SYK-6 cells. Disruption of desV in SYK-6 resulted in a significant reduction in growth on syringaldehyde and in syringaldehyde oxidation activity. Furthermore, a desV ligV double mutant almost completely lost its ability to grow on syringaldehyde. Purified DesV showed similar kcat/Km values for syringaldehyde (2100 s−1·mM−1) and vanillin (1700 s−1·mM−1), whereas LigV substantially preferred vanillin (8800 s−1·mM−1) over syringaldehyde (1.4 s−1·mM−1). These results clearly demonstrate that desV plays a major role in syringaldehyde catabolism. Phylogenetic analyses showed that DesV-like ALDHs formed a distinct phylogenetic cluster separated from the vanillin dehydrogenase cluster. PMID:28294121

  16. Role of a membrane-bound aldehyde dehydrogenase complex AldFGH in acetic acid fermentation with Acetobacter pasteurianus SKU1108.

    PubMed

    Yakushi, Toshiharu; Fukunari, Seiya; Kodama, Tomohiro; Matsutani, Minenosuke; Nina, Shun; Kataoka, Naoya; Theeragool, Gunjana; Matsushita, Kazunobu

    2018-05-01

    Acetic acid fermentation is widely considered a consequence of ethanol oxidation by two membrane-bound enzymes-alcohol dehydrogenase and aldehyde dehydrogenase (ALDH)-of acetic acid bacteria. Here, we used a markerless gene disruption method to construct a mutant of the Acetobacter pasteurianus strain SKU1108 with a deletion in the aldH gene, which encodes the large catalytic subunit of a heterotrimeric ALDH complex (AldFGH), to examine the role of AldFGH in acetic acid fermentation. The ΔaldH strain grew less on ethanol-containing medium, i.e., acetic acid fermentation conditions, than the wild-type strain and significantly accumulated acetaldehyde in the culture medium. Unexpectedly, acetaldehyde oxidase activity levels of the intact ΔaldH cells and the ΔaldH cell membranes were similar to those of the wild-type strain, which might be attributed to an additional ALDH isozyme (AldSLC). The apparent K M values of the wild-type and ΔaldH membranes for acetaldehyde were similar to each other, when the cells were cultured in nonfermentation conditions, where ΔaldH cells grow as well as the wild-type cells. However, the membranes of the wild-type cells grown under fermentation conditions showed a 10-fold lower apparent K M value than those of the cells grown under nonfermentation conditions. Under fermentation conditions, transcriptional levels of a gene for AldSLC were 10-fold lower than those under nonfermentation conditions, whereas aldH transcript levels were not dramatically changed under the two conditions. We suggest that A. pasteurianus SKU1108 has two ALDHs, and the AldFGH complex is indispensable for acetic acid fermentation and is the major enzyme under fermentation conditions.

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

    USDA-ARS?s Scientific Manuscript database

    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. In vivo antitumor activity of 4-amino 4-methyl 2-pentyne 1-al, an inhibitor of aldehyde dehydrogenase.

    PubMed

    Quemener, V; Quash, G; Moulinoux, J P; Penlap, V; Ripoll, H; Havouis, R; Doutheau, A; Goré, J

    1989-01-01

    4-amino-4-methyl-2-pentyne-1-al (AMPAL), a new irreversible inhibitor of aldehyde dehydrogenase (ALDH) has been assayed for its in vitro and in vivo antitumor activity. In vitro, AMPAL inhibits the proliferation and the ALDH activity of L1210 and RBL5 cell lines. In vivo, AMPAL significantly increases the mean survival time of mice i.p. grafted with leukemia (L1210, P815, MBL2, EL4, RBL5 cell lines) or carcinoma cells (Krebs cell line), without haematopoetic toxicity. No carcinostatic effect was observed against the P388 leukemia and the 3LL Lewis lung carcinoma. A possible relationship between the ALDH isoenzyme activity of the tumor and its sensitivity to AMPAL is discussed in the light of previous reports concerning the role of aldehydes in cell growth control.

  19. Cloning, expression and characterization of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium strain BKM-F-1767

    PubMed Central

    2012-01-01

    Background The white-rot fungus Phanerochaete chrysosporium is among the small group of fungi that can degrade lignin to carbon dioxide while leaving the crystalline cellulose untouched. The efficient lignin oxidation system of this fungus requires cyclic redox reactions involving the reduction of aryl-aldehydes to the corresponding alcohols by aryl-alcohol dehydrogenase. However, the biochemical properties of this enzyme have not been extensively studied. These are of most interest for the design of metabolic engineering/synthetic biology strategies in the field of biotechnological applications of this enzyme. Results We report here the cloning of an aryl-alcohol dehydrogenase cDNA from the white-rot fungus Phanerochaete chrysosporium, its expression in Escherichia coli and the biochemical characterization of the encoded GST and His6 tagged protein. The purified recombinant enzyme showed optimal activity at 37°C and at pH 6.4 for the reduction of aryl- and linear aldehydes with NADPH as coenzyme. NADH could also be the electron donor, while having a higher Km (220 μM) compared to that of NADPH (39 μM). The purified recombinant enzyme was found to be active in the reduction of more than 20 different aryl- and linear aldehydes showing highest specificity for mono- and dimethoxylated Benzaldehyde at positions 3, 4, 3,4 and 3,5. The enzyme was also capable of oxidizing aryl-alcohols with NADP + at 30°C and an optimum pH of 10.3 but with 15 to 100-fold lower catalytic efficiency than for the reduction reaction. Conclusions In this work, we have characterized the biochemical properties of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. We show that this enzyme functions in the reductive sense under physiological conditions and that it displays relatively large substrate specificity with highest activity towards the natural compound Veratraldehyde. PMID:22742413

  20. In Vitro Characterization and Concerted Function of Three Core Enzymes of a Glycyl Radical Enzyme - Associated Bacterial Microcompartment.

    PubMed

    Zarzycki, Jan; Sutter, Markus; Cortina, Niña Socorro; Erb, Tobias J; Kerfeld, Cheryl A

    2017-02-16

    Many bacteria encode proteinaceous bacterial microcompartments (BMCs) that encapsulate sequential enzymatic reactions of diverse metabolic pathways. Well-characterized BMCs include carboxysomes for CO 2 -fixation, and propanediol- and ethanolamine-utilizing microcompartments that contain B 12 -dependent enzymes. Genes required to form BMCs are typically organized in gene clusters, which promoted their distribution across phyla by horizontal gene transfer. Recently, BMCs associated with glycyl radical enzymes (GREs) were discovered; these are widespread and comprise at least three functionally distinct types. Previously, we predicted one type of these GRE-associated microcompartments (GRMs) represents a B 12 -independent propanediol-utilizing BMC. Here we functionally and structurally characterize enzymes of the GRM of Rhodopseudomonas palustris BisB18 and demonstrate their concerted function in vitro. The GRM signature enzyme, the GRE, is a dedicated 1,2-propanediol dehydratase with a new type of intramolecular encapsulation peptide. It forms a complex with its activating enzyme and, in conjunction with an aldehyde dehydrogenase, converts 1,2-propanediol to propionyl-CoA. Notably, homologous GRMs are also encoded in pathogenic Escherichia coli strains. Our high-resolution crystal structures of the aldehyde dehydrogenase lead to a revised reaction mechanism. The successful in vitro reconstitution of a part of the GRM metabolism provides insights into the metabolic function and steps in the assembly of this BMC.

  1. In Vitro Characterization and Concerted Function of Three Core Enzymes of a Glycyl Radical Enzyme - Associated Bacterial Microcompartment

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zarzycki, Jan; Sutter, Markus; Cortina, Niña Socorro

    Many bacteria encode proteinaceous bacterial microcompartments (BMCs) that encapsulate sequential enzymatic reactions of diverse metabolic pathways. Well-characterized BMCs include carboxysomes for CO 2-fixation, and propanediol- and ethanolamine-utilizing microcompartments that contain B 12-dependent enzymes. Genes thus required to form BMCs are typically organized in gene clusters, which promoted their distribution across phyla by horizontal gene transfer. Recently, BMCs associated with glycyl radical enzymes (GREs) were discovered; these are widespread and comprise at least three functionally distinct types. Previously, we predicted one type of these GRE-associated microcompartments (GRMs) represents a B 12-independent propanediol-utilizing BMC. We functionally and structurally characterize enzymes of themore » GRM of Rhodopseudomonas palustris BisB18 and demonstrate their concerted function in vitro. The GRM signature enzyme, the GRE, is a dedicated 1,2-propanediol dehydratase with a new type of intramolecular encapsulation peptide. It forms a complex with its activating enzyme and, in conjunction with an aldehyde dehydrogenase, converts 1,2-propanediol to propionyl-CoA. Notably, homologous GRMs are also encoded in pathogenic Escherichia coli strains. Our high-resolution crystal structures of the aldehyde dehydrogenase lead to a revised reaction mechanism. The successful in vitro reconstitution of a part of the GRM metabolism provides insights into the metabolic function and steps in the assembly of this BMC.« less

  2. In Vitro Characterization and Concerted Function of Three Core Enzymes of a Glycyl Radical Enzyme - Associated Bacterial Microcompartment

    DOE PAGES

    Zarzycki, Jan; Sutter, Markus; Cortina, Niña Socorro; ...

    2017-02-16

    Many bacteria encode proteinaceous bacterial microcompartments (BMCs) that encapsulate sequential enzymatic reactions of diverse metabolic pathways. Well-characterized BMCs include carboxysomes for CO 2-fixation, and propanediol- and ethanolamine-utilizing microcompartments that contain B 12-dependent enzymes. Genes thus required to form BMCs are typically organized in gene clusters, which promoted their distribution across phyla by horizontal gene transfer. Recently, BMCs associated with glycyl radical enzymes (GREs) were discovered; these are widespread and comprise at least three functionally distinct types. Previously, we predicted one type of these GRE-associated microcompartments (GRMs) represents a B 12-independent propanediol-utilizing BMC. We functionally and structurally characterize enzymes of themore » GRM of Rhodopseudomonas palustris BisB18 and demonstrate their concerted function in vitro. The GRM signature enzyme, the GRE, is a dedicated 1,2-propanediol dehydratase with a new type of intramolecular encapsulation peptide. It forms a complex with its activating enzyme and, in conjunction with an aldehyde dehydrogenase, converts 1,2-propanediol to propionyl-CoA. Notably, homologous GRMs are also encoded in pathogenic Escherichia coli strains. Our high-resolution crystal structures of the aldehyde dehydrogenase lead to a revised reaction mechanism. The successful in vitro reconstitution of a part of the GRM metabolism provides insights into the metabolic function and steps in the assembly of this BMC.« less

  3. 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.

  4. Geraniol and Geranial Dehydrogenases Induced in Anaerobic Monoterpene Degradation by Castellaniella defragrans

    PubMed Central

    Lüddeke, Frauke; Wülfing, Annika; Timke, Markus; Germer, Frauke; Weber, Johanna; Dikfidan, Aytac; Rahnfeld, Tobias; Linder, Dietmar; Meyerdierks, Anke

    2012-01-01

    Castellaniella defragrans is a Betaproteobacterium capable of coupling the oxidation of monoterpenes with denitrification. Geraniol dehydrogenase (GeDH) activity was induced during growth with limonene in comparison to growth with acetate. The N-terminal sequence of the purified enzyme directed the cloning of the corresponding open reading frame (ORF), the first bacterial gene for a GeDH (geoA, for geraniol oxidation pathway). The C. defragrans geraniol dehydrogenase is a homodimeric enzyme that affiliates with the zinc-containing benzyl alcohol dehydrogenases in the superfamily of medium-chain-length dehydrogenases/reductases (MDR). The purified enzyme most efficiently catalyzes the oxidation of perillyl alcohol (kcat/Km = 2.02 × 106 M−1 s−1), followed by geraniol (kcat/Km = 1.57 × 106 M−1 s−1). Apparent Km values of <10 μM are consistent with an in vivo toxicity of geraniol above 5 μM. In the genetic vicinity of geoA is a putative aldehyde dehydrogenase that was named geoB and identified as a highly abundant protein during growth with phellandrene. Extracts of Escherichia coli expressing geoB demonstrated in vitro a geranial dehydrogenase (GaDH) activity. GaDH activity was independent of coenzyme A. The irreversible formation of geranic acid allows for a metabolic flux from β-myrcene via linalool, geraniol, and geranial to geranic acid. PMID:22286981

  5. Geraniol and geranial dehydrogenases induced in anaerobic monoterpene degradation by Castellaniella defragrans.

    PubMed

    Lüddeke, Frauke; Wülfing, Annika; Timke, Markus; Germer, Frauke; Weber, Johanna; Dikfidan, Aytac; Rahnfeld, Tobias; Linder, Dietmar; Meyerdierks, Anke; Harder, Jens

    2012-04-01

    Castellaniella defragrans is a Betaproteobacterium capable of coupling the oxidation of monoterpenes with denitrification. Geraniol dehydrogenase (GeDH) activity was induced during growth with limonene in comparison to growth with acetate. The N-terminal sequence of the purified enzyme directed the cloning of the corresponding open reading frame (ORF), the first bacterial gene for a GeDH (geoA, for geraniol oxidation pathway). The C. defragrans geraniol dehydrogenase is a homodimeric enzyme that affiliates with the zinc-containing benzyl alcohol dehydrogenases in the superfamily of medium-chain-length dehydrogenases/reductases (MDR). The purified enzyme most efficiently catalyzes the oxidation of perillyl alcohol (k(cat)/K(m) = 2.02 × 10(6) M(-1) s(-1)), followed by geraniol (k(cat)/K(m) = 1.57 × 10(6) M(-1) s(-1)). Apparent K(m) values of <10 μM are consistent with an in vivo toxicity of geraniol above 5 μM. In the genetic vicinity of geoA is a putative aldehyde dehydrogenase that was named geoB and identified as a highly abundant protein during growth with phellandrene. Extracts of Escherichia coli expressing geoB demonstrated in vitro a geranial dehydrogenase (GaDH) activity. GaDH activity was independent of coenzyme A. The irreversible formation of geranic acid allows for a metabolic flux from β-myrcene via linalool, geraniol, and geranial to geranic acid.

  6. Effects of cyanamide and clofibrate on the enzymes of ethanol oxydation and on ethanol consumption in the rat.

    PubMed

    Lamboeuf, Y; De Saint Blanquat, G

    1980-01-01

    The action of cyanamide and of clofibrate was studied on the voluntary drinking behaviour of alcohol intoxicated animals and also on the enzymes of ethyl oxydation. Both substances reduce alcohol consumption by about 35% and cause metabolic modifications: inhibition of aldehyde dehydrogenase and of catalase by cyanamide; activation of alcohol- and aldehyde-dehydrogenases by clofibrate. These effects are constantly found in most of the tissues of the rat. The results are discussed bearing in mind the relationships between alcohol metabolism, acetaldehyde metabolism, the toxicity of alcohol and the drinking behaviour.

  7. The aldehyde dehydrogenase, AldA, is essential for L-1,2-propanediol utilization in laboratory-evolved Escherichia coli.

    PubMed

    Aziz, Ramy K; Monk, Jonathan M; Andrews, Kathleen A; Nhan, Jenny; Khaw, Valerie L; Wong, Hesper; Palsson, Bernhard O; Charusanti, Pep

    2017-01-01

    Most Escherichia coli strains are naturally unable to grow on 1,2-propanediol (PDO) as a sole carbon source. Recently, however, a K-12 descendent E. coli strain was evolved to grow on 1,2-PDO, and it was hypothesized that this evolved ability was dependent on the aldehyde dehydrogenase, AldA, which is highly conserved among members of the family Enterobacteriacea. To test this hypothesis, we first performed computational model simulation, which confirmed the essentiality of the aldA gene for 1,2-PDO utilization by the evolved PDO-degrading E. coli. Next, we deleted the aldA gene from the evolved strain, and this deletion was sufficient to abolish the evolved phenotype. On re-introducing the gene on a plasmid, the evolved phenotype was restored. These findings provide experimental evidence for the computationally predicted role of AldA in 1,2-PDO utilization, and represent a good example of E. coli robustness, demonstrated by the bacterial deployment of a generalist enzyme (here AldA) in multiple pathways to survive carbon starvation and to grow on a non-native substrate when no native carbon source is available. Copyright © 2016 Elsevier GmbH. All rights reserved.

  8. Genome-wide identification and analysis of the aldehyde dehydrogenase (ALDH) gene superfamily in apple (Malus × domestica Borkh.).

    PubMed

    Li, Xiaoqin; Guo, Rongrong; Li, Jun; Singer, Stacy D; Zhang, Yucheng; Yin, Xiangjing; Zheng, Yi; Fan, Chonghui; Wang, Xiping

    2013-10-01

    Aldehyde dehydrogenases (ALDHs) represent a protein superfamily encoding NAD(P)(+)-dependent enzymes that oxidize a wide range of endogenous and exogenous aliphatic and aromatic aldehydes. In plants, they are involved in many biological processes and play a role in the response to environmental stress. In this study, a total of 39 ALDH genes from ten families were identified in the apple (Malus × domestica Borkh.) genome. Synteny analysis of the apple ALDH (MdALDH) genes indicated that segmental and tandem duplications, as well as whole genome duplications, have likely contributed to the expansion and evolution of these gene families in apple. Moreover, synteny analysis between apple and Arabidopsis demonstrated that several MdALDH genes were found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes appeared before the divergence of lineages that led to apple and Arabidopsis. In addition, phylogenetic analysis, as well as comparisons of exon-intron and protein structures, provided further insight into both their evolutionary relationships and their putative functions. Tissue-specific expression analysis of the MdALDH genes demonstrated diverse spatiotemporal expression patterns, while their expression profiles under abiotic stress and various hormone treatments indicated that many MdALDH genes were responsive to high salinity and drought, as well as different plant hormones. This genome-wide identification, as well as characterization of evolutionary relationships and expression profiles, of the apple MdALDH genes will not only be useful for the further analysis of ALDH genes and their roles in stress response, but may also aid in the future improvement of apple stress tolerance. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  9. A novel type of pathogen defense-related cinnamyl alcohol dehydrogenase.

    PubMed

    Logemann, E; Reinold, S; Somssich, I E; Hahlbrock, K

    1997-08-01

    We describe an aromatic alcohol dehydrogenase with properties indicating a novel type of function in the defense response of plants to pathogens. To obtain the enzyme free of contamination with possible isoforms, a parsley (Petroselinum crispum) cDNA comprising the entire coding region of the elicitor-responsive gene, ELI3, was expressed in Escherichia coli. In accord with large amino acid sequence similarities with established cinnamyl and benzyl alcohol dehydrogenases from other plants, the enzyme efficiently reduced various cinnamyl and benzyl aldehydes using NADPH as a co-substrate. Highest substrate affinities were observed for cinnamaldehyde, 4-coumaraldehyde and coniferaldehyde, whereas sinapaldehyde, one of the most efficient substrates of several previously analyzed cinnamyl alcohol dehydrogenases and a characteristic precursor molecule of angiosperm lignin, was not converted. A single form of ELI3 mRNA was strongly and rapidly induced in fungal elicitor-treated parsley cells. These results, together with earlier findings that the ELI3 gene is strongly activated both in elicitor-treated parsley cells and at fungal infection sites in parsley leaves, but not in lignifying tissue, suggest a specific role of this enzyme in pathogen defense-related phenylpropanoid metabolism.

  10. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. 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

  12. Aldehyde dehydrogenase inhibition blocks mucosal fibrosis in human and mouse ocular scarring

    PubMed Central

    Ahadome, Sarah D.; Abraham, David J.; Rayapureddi, Suryanarayana; Saw, Valerie P.; Saban, Daniel R.; Calder, Virginia L.; Norman, Jill T.; Ponticos, Markella; Daniels, Julie T.; Dart, John K.

    2016-01-01

    Mucous membrane pemphigoid (MMP) is a systemic mucosal scarring disease, commonly causing blindness, for which there is no antifibrotic therapy. Aldehyde dehydrogenase family 1 (ALDH1) is upregulated in both ocular MMP (OMMP) conjunctiva and cultured fibroblasts. Application of the ALDH metabolite, retinoic acid (RA), to normal human conjunctival fibroblasts in vitro induced a diseased phenotype. Conversely, application of ALDH inhibitors, including disulfiram, to OMMP fibroblasts in vitro restored their functionality to that of normal controls. ALDH1 is also upregulated in the mucosa of the mouse model of scarring allergic eye disease (AED), used here as a surrogate for OMMP, in which topical application of disulfiram decreased fibrosis in vivo. These data suggest that progressive scarring in OMMP results from ALDH/RA fibroblast autoregulation, that the ALDH1 subfamily has a central role in immune-mediated ocular mucosal scarring, and that ALDH inhibition with disulfiram is a potential and readily translatable antifibrotic therapy. PMID:27699226

  13. HEPATOCYTE EXPRESION OF TUMOR ASSOCIATED ALDEHYDE DEHYDROGENASE (ALDH-3) AND P21 RAS FOLLOWING DIETHYLNITROSAMINE (DEN) INITIATION AND CHRONIC EXPOSURE TO DI(2-ETHYLHEXYL) PHTHALATE (DHEP)

    EPA Science Inventory

    Phthalate esters such as di(2-ethylhexyl)phthalate (DEHP)either promote or inhibit rat liver tumorigenesis depending on the carcinogenesis protocol. In this study, we examined the expression of two histochemical markers, the tumor associated isozyme of aldehyde dehydrogenase (ALD...

  14. Lipoic acid protects gastric mucosa from ethanol-induced injury in rat through a mechanism involving aldehyde dehydrogenase 2 activation.

    PubMed

    Li, Jia-Hui; Ju, Gui-Xia; Jiang, Jun-Lin; Li, Nian-Sheng; Peng, Jun; Luo, Xiu-Ju

    2016-11-01

    Numerous studies demonstrate that reactive aldehydes are highly toxic and aldehyde dehydrogenase 2 (ALDH2)-mediated detoxification of reactive aldehydes is thought as an endogenous protective mechanism against reactive aldehydes-induced cell injury. This study aims to explore whether lipoic acid, a potential ALDH2 activator, is able to protect gastric mucosa from ethanol-induced injury through a mechanism involving clearance of reactive aldehydes. The rats received 60% of acidified ethanol through intragastric administration and held for 1 h to establish a mucosal injury model. Lipoic acid (10 or 30 mg/kg) or Alda-1 (a positive control, 10 mg/kg) was given 45 min before the ethanol treatment. The gastric tissues were collected for analysis of gastric ulcer index, cellular apoptosis, 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA) contents, and ALDH2 activity. The results showed that acute administration of ethanol led to an increase in gastric ulcer index, cellular apoptosis, 4-HNE and MDA contents concomitant with a decrease in ALDH2 activity; these phenomena were reversed by lipoic acid or Alda-1. The gastric protection of lipoic acid was attenuated in the presence of ALDH2 inhibitor. Based on these observations, we conclude that lipoic acid exerts the beneficial effects on ethanol-induced injury through a mechanism involving, at least in part, ALDH2 activation. As a dietary supplement or a medicine already in some countries, lipoic acid can be used to treat the ethanol - induced gastric mucosal injury. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. 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

  16. Molybdenum Incorporation in Tungsten Aldehyde Oxidoreductase Enzymes from Pyrococcus furiosus▿ †

    PubMed Central

    Sevcenco, Ana-Maria; Bevers, Loes E.; Pinkse, Martijn W. H.; Krijger, Gerard C.; Wolterbeek, Hubert T.; Verhaert, Peter D. E. M.; Hagen, Wilfred R.; Hagedoorn, Peter-Leon

    2010-01-01

    The hyperthermophilic archaeon Pyrococcus furiosus expresses five aldehyde oxidoreductase (AOR) enzymes, all containing a tungsto-bispterin cofactor. The growth of this organism is fully dependent on the presence of tungsten in the growth medium. Previous studies have suggested that molybdenum is not incorporated in the active site of these enzymes. Application of the radioisotope 99Mo in metal isotope native radioautography in gel electrophoresis (MIRAGE) technology to P. furiosus shows that molybdenum can in fact be incorporated in all five AOR enzymes. Mo(V) signals characteristic for molybdopterin were observed in formaldehyde oxidoreductase (FOR) in electron paramagnetic resonance (EPR)-monitored redox titrations. Our finding that the aldehyde oxidation activity of FOR and WOR5 (W-containing oxidoreductase 5) correlates only with the residual tungsten content suggests that the Mo-containing AORs are most likely inactive. An observed W/Mo antagonism is indicative of tungstate-dependent negative feedback of the expression of the tungstate/molybdate ABC transporter. An intracellular selection mechanism for tungstate and molybdate processing has to be present, since tungsten was found to be preferentially incorporated into the AORs even under conditions with comparable intracellular concentrations of tungstate and molybdate. Under the employed growth conditions of starch as the main carbon source in a rich medium, no tungsten- and/or molybdenum-associated proteins are detected in P. furiosus other than the high-affinity transporter, the proteins of the metallopterin insertion machinery, and the five W-AORs. PMID:20562313

  17. Aldehyde dehydrogenase induction in arsenic-exposed rat bladder epithelium.

    PubMed

    Huang, Ya-Chun; Yu, Hsin-Su; Chai, Chee-Yin

    2016-01-01

    Arsenic is widely distributed in the environment. Many human cancers, including urothelial carcinoma (UC), show a dose-dependent relationship with arsenic exposure in the south-west coast of Taiwan (also known as the blackfoot disease (BFD) areas). However, the molecular mechanisms of arsenic-mediated UC carcinogenesis has not yet been defined. In vivo study, the rat bladder epithelium were exposed with arsenic for 48 h. The proteins were extracted from untreated and arsenic-treated rat bladder cells and utilized two-dimensional gel electrophoresis and mass spectrometry. Selected peptides were extracted from the gel and identified by quadrupole-time of flight (Q-TOF) Ultima-Micromass spectra. The significantly difference expression of proteins in arsenic-treated groups as compared with untreated groups was confirmed by immunohistochemistry (IHC) and western blotting. We found that thirteen proteins were down-regulated and nine proteins were up-regulated in arsenic-treated rat bladder cells when compared with untreated groups. The IHC and western blotting results confirmed that aldehyde dehydrogenase (ALDH) protein was up-regulated in arsenic-treated rat bladder epithelium. Expression of ALDH protein was significantly higher in UC patients from BFD areas than those from non-BFD areas using IHC (p=0.018). In conclusion, the ALDH protein expression could be used as molecular markers for arsenic-induced transformation. Copyright © 2015 Elsevier GmbH. All rights reserved.

  18. 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.

  19. Complex inhibition of autophagy by mitochondrial aldehyde dehydrogenase shortens lifespan and exacerbates cardiac aging.

    PubMed

    Zhang, Yingmei; Wang, Cong; Zhou, Jingmin; Sun, Aijun; Hueckstaedt, Lindsay K; Ge, Junbo; Ren, Jun

    2017-08-01

    Autophagy, a conservative degradation process for long-lived and damaged proteins, participates in a cascade of biological processes including aging. A number of autophagy regulators have been identified. Here we demonstrated that mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme with the most common single point mutation in humans, governs cardiac aging through regulation of autophagy. Myocardial mechanical and autophagy properties were examined in young (4months) and old (26-28months) wild-type (WT) and global ALDH2 transgenic mice. ALDH2 overexpression shortened lifespan by 7.7% without affecting aging-associated changes in plasma metabolic profiles. Myocardial function was compromised with aging associated with cardiac hypertrophy, the effects were accentuated by ALDH2. Aging overtly suppressed autophagy and compromised autophagy flux, the effects were exacerbated by ALDH2. Aging dampened phosphorylation of JNK, Bcl-2, IKKβ, AMPK and TSC2 while promoting phosphorylation of mTOR, the effects of which were exaggerated by ALDH2. Co-immunoprecipitation revealed increased dissociation between Bcl-2 and Beclin-1 (result of decreased Bcl-2 phosphorylation) in aging, the effect of which was exacerbated with ALDH2. Chronic treatment of the autophagy inducer rapamycin alleviated aging-induced cardiac dysfunction in both WT and ALDH2 mice. Moreover, activation of JNK and inhibition of either Bcl-2 or IKKβ overtly attenuated ALDH2 activation-induced accentuation of cardiomyocyte aging. Examination of the otherwise elderly individuals revealed a positive correlation between cardiac function/geometry and ALDH2 gene mutation. Taken together, our data revealed that ALDH2 enzyme may suppress myocardial autophagy possibly through a complex JNK-Bcl-2 and IKKβ-AMPK-dependent mechanism en route to accentuation of myocardial remodeling and contractile dysfunction in aging. This article is part of a Special Issue entitled: Genetic and epigenetic control of heart failure

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 analysismore » 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.« less

  1. Badh2, Encoding Betaine Aldehyde Dehydrogenase, Inhibits the Biosynthesis of 2-Acetyl-1-Pyrroline, a Major Component in Rice Fragrance[W

    PubMed Central

    Chen, Saihua; Yang, Yi; Shi, Weiwei; Ji, Qing; He, Fei; Zhang, Ziding; Cheng, Zhukuan; Liu, Xiangnong; Xu, Mingliang

    2008-01-01

    In rice (Oryza sativa), the presence of a dominant Badh2 allele encoding betaine aldehyde dehydrogenase (BADH2) inhibits the synthesis of 2-acetyl-1-pyrroline (2AP), a potent flavor component in rice fragrance. By contrast, its two recessive alleles, badh2-E2 and badh2-E7, induce 2AP formation. Badh2 was found to be transcribed in all tissues tested except for roots, and the transcript was detected at higher abundance in young, healthy leaves than in other tissues. Multiple Badh2 transcript lengths were detected, and the complete, full-length Badh2 transcript was much less abundant than partial Badh2 transcripts. 2AP levels were significantly reduced in cauliflower mosaic virus 35S-driven transgenic lines expressing the complete, but not the partial, Badh2 coding sequences. In accordance, the intact, full-length BADH2 protein (503 residues) appeared exclusively in nonfragrant transgenic lines and rice varieties. These results indicate that the full-length BADH2 protein encoded by Badh2 renders rice nonfragrant by inhibiting 2AP biosynthesis. The BADH2 enzyme was predicted to contain three domains: NAD binding, substrate binding, and oligomerization domains. BADH2 was distributed throughout the cytoplasm, where it is predicted to catalyze the oxidization of betaine aldehyde, 4-aminobutyraldehyde (AB-ald), and 3-aminopropionaldehyde. The presence of null badh2 alleles resulted in AB-ald accumulation and enhanced 2AP biosynthesis. In summary, these data support the hypothesis that BADH2 inhibits 2AP biosynthesis by exhausting AB-ald, a presumed 2AP precursor. PMID:18599581

  2. [Importance of the 11β-hydroxysteroid dehydrogenase enzyme in clinical disorders].

    PubMed

    Feldman, Karolina; Likó, István; Nagy, Zsolt; Szappanos, Agnes; Grolmusz, Vince Kornél; Tóth, Miklós; Rácz, Károly; Patócs, Attila

    2013-02-24

    Glucocorticoids play an important role in the regulation of carbohydrate and amino acid metabolism, they modulate the function of the immune system, and contribute to stress response. Increased and decreased production of glucocorticoids causes specific diseases. In addition to systemic hypo- or hypercortisolism, alteration of local synthesis and metabolism of cortisol may result in tissue-specific hypo- or hypercortisolism. One of the key enzymes participating in the local synthesis and metabolism of cortisol is the 11β-hydroxysteroid dehydrogenase enzyme. Two isoforms, type 1 and type 2 enzymes are located in the endoplasmic reticulum and catalyze the interconversion of hormonally active cortisol and inactive cortisone. The type 1 enzyme mainly works as an activator, and it is responsible for the generation of cortisol from cortisone in liver, adipose tissue, brain and bone. The gene encoding this enzyme is located on chromosome 1. The authors review the physiological and pathophysiological processes related to the function of the type 1 11β-hydroxysteroid dehydrogenase enzyme. They summarize the potential significance of polymorphic variants of the enzyme in clinical diseases as well as knowledge related to inhibitors of enzyme activity. Although further studies are still needed, inhibition of the enzyme activity may prove to be an effective tool for the treatment of several diseases such as obesity, osteoporosis and type 2 diabetes.

  3. Aldehyde dehydrogenase 3A1 protects airway epithelial cells from cigarette smoke-induced DNA damage and cytotoxicity.

    PubMed

    Jang, Jun-Ho; Bruse, Shannon; Liu, Yushi; Duffy, Veronica; Zhang, Chunyu; Oyamada, Nathaniel; Randell, Scott; Matsumoto, Akiko; Thompson, David C; Lin, Yong; Vasiliou, Vasilis; Tesfaigzi, Yohannes; Nyunoya, Toru

    2014-03-01

    Aldehyde dehydrogenase 3A1 (ALDH3A1), an ALDH superfamily member, catalyzes the oxidation of reactive aldehydes, highly toxic components of cigarette smoke (CS). Even so, the role of ALDH3A1 in CS-induced cytotoxicity and DNA damage has not been examined. Among all of the ALDH superfamily members, ALDH3A1 mRNA levels showed the greatest induction in response to CS extract (CSE) exposure of primary human bronchial epithelial cells (HBECs). ALDH3A1 protein accumulation was accompanied by increased ALDH enzymatic activity in CSE-exposed immortalized HBECs. The effects of overexpression or suppression of ALDH3A1 on CSE-induced cytotoxicity and DNA damage (γH2AX) were evaluated in cultured immortalized HBECs. Enforced expression of ALDH3A1 attenuated cytotoxicity and downregulated γH2AX. SiRNA-mediated suppression of ALDH3A1 blocked ALDH enzymatic activity and augmented cytotoxicity in CSE-exposed cells. Our results suggest that the availability of ALDH3A1 is important for cell survival against CSE in HBECs. Published by Elsevier Inc.

  4. 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.

  5. Aldehyde dehydrogenase inhibition as a pathogenic mechanism in Parkinson disease

    PubMed Central

    Fitzmaurice, Arthur G.; Rhodes, Shannon L.; Lulla, Aaron; Murphy, Niall P.; Lam, Hoa A.; O’Donnell, Kelley C.; Barnhill, Lisa; Casida, John E.; Cockburn, Myles; Sagasti, Alvaro; Stahl, Mark C.; Maidment, Nigel T.; Ritz, Beate; Bronstein, Jeff M.

    2013-01-01

    Parkinson disease (PD) is a neurodegenerative disorder particularly characterized by the loss of dopaminergic neurons in the substantia nigra. Pesticide exposure has been associated with PD occurrence, and we previously reported that the fungicide benomyl interferes with several cellular processes potentially relevant to PD pathogenesis. Here we propose that benomyl, via its bioactivated thiocarbamate sulfoxide metabolite, inhibits aldehyde dehydrogenase (ALDH), leading to accumulation of the reactive dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), preferential degeneration of dopaminergic neurons, and development of PD. This hypothesis is supported by multiple lines of evidence. (i) We previously showed in mice the metabolism of benomyl to S-methyl N-butylthiocarbamate sulfoxide, which inhibits ALDH at nanomolar levels. We report here that benomyl exposure in primary mesencephalic neurons (ii) inhibits ALDH and (iii) alters dopamine homeostasis. It induces selective dopaminergic neuronal damage (iv) in vitro in primary mesencephalic cultures and (v) in vivo in a zebrafish system. (vi) In vitro cell loss was attenuated by reducing DOPAL formation. (vii) In our epidemiology study, higher exposure to benomyl was associated with increased PD risk. This ALDH model for PD etiology may help explain the selective vulnerability of dopaminergic neurons in PD and provide a potential mechanism through which environmental toxicants contribute to PD pathogenesis. PMID:23267077

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

    PubMed

    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 the pathways/networks that contribute to pathophysiological outcomes. Here we interrogate primary dermal fibroblasts isolated from Gorlin syndrome patients (GDFs), who display a pronounced inducible tumorigenic response to radiation, in comparison to normal human dermal fibroblasts (NHDFs). Our approach exploits newly developed thiol reactive probes to define changes in protein thiol profiles in live cell studies, which minimizes artifacts associated with cell lysis. Redox probes revealed deficient expression of an apparent 55 kDa protein thiol in GDFs from independent Gorlin syndrome patients, compared with NHDFs. Proteomics tentatively identified this protein as aldehyde dehydrogenase 1A1 (ALDH1A1), a key enzyme regulating retinoic acid synthesis, and ALDH1A1 protein deficiency in GDFs was confirmed by Western blot. A number of additional protein thiol differences in GDFs were identified, including radiation responsive annexin family members and lamin A/C. Collectively, candidates identified in our study have plausible implications for radiation health effects and cancer susceptibility. © 2013 Wiley Periodicals, Inc.

  7. RNAi-directed downregulation of betaine aldehyde dehydrogenase 1 (OsBADH1) results in decreased stress tolerance and increased oxidative markers without affecting glycine betaine biosynthesis in rice (Oryza sativa).

    PubMed

    Tang, Wei; Sun, Jiaqi; Liu, Jia; Liu, Fangfang; Yan, Jun; Gou, Xiaojun; Lu, Bao-Rong; Liu, Yongsheng

    2014-11-01

    As an important osmoprotectant, glycine betaine (GB) plays an essential role in resistance to abiotic stress in a variety of organisms, including rice (Oryza sativa L.). However, GB content is too low to be detectable in rice, although rice genome possesses several orthologs coding for betaine aldehyde dehydrogenase (BADH) involved in plant GB biosynthesis. Rice BADH1 (OsBADH1) has been shown to be targeted to peroxisome and its overexpression resulted in increased GB biosynthesis and tolerance to abiotic stress. In this study, we demonstrated a pivotal role of OsBADH1 in stress tolerance without altering GB biosynthesis capacity, using the RNA interference (RNAi) technique. OsBADH1 was ubiquitously expressed in different organs, including roots, stems, leaves and flowers. Transgenic rice lines downregulating OsBADH1 exhibited remarkably reduced tolerance to NaCl, drought and cold stresses. The decrease of stress tolerance occurring in the OsBADH1-RNAi repression lines was associated with an elevated level of malondialdehyde content and hydrogen peroxidation. No GB accumulation was detected in transgene-positive and transgene-negative lines derived from heterozygous transgenic T0 plants. Moreover, transgenic OsBADH1-RNAi repression lines showed significantly reduced seed set and yield. In conclusion, the downregulation of OsBADH1, even though not causing any change of GB content, was accounted for the reduction of ability to dehydrogenate the accumulating metabolism-derived aldehydes and subsequently resulted in decreased stress tolerance and crop productivity. These results suggest that OsBADH1 possesses an enzyme activity to catalyze other aldehydes in addition to betaine aldehyde (the precursor of GB) and thus alleviate their toxic effects under abiotic stresses.

  8. Characterization of xanthine dehydrogenase and aldehyde oxidase of Marsupenaeus japonicus and their response to microbial pathogen.

    PubMed

    Okamura, Yo; Inada, Mari; Elshopakey, Gehad Elsaid; Itami, Toshiaki

    2018-05-16

    Reactive oxygen species (ROS) play key roles in many physiological processes. In particular, the sterilization mechanism of bacteria using ROS in macrophages is a very important function for biological defense. Xanthine dehydrogenase (XDH) and aldehyde oxidase (AOX), members of the molybdo-flavoenzyme subfamily, are known to generate ROS. Although these enzymes occur in many vertebrates, some insects, and plants, little research has been conducted on XDHs and AOXs in crustaceans. Here, we cloned the entire cDNA sequences of XDH (MjXDH: 4328 bp) and AOX (MjAOX: 4425 bp) from Marsupenaeus japonicus (kuruma shrimp) using reverse transcriptase-polymerase chain reaction (RT-PCR) and random amplification of cDNA ends (RACE). Quantitative real-time RT-PCR transcriptional analysis revealed that MjXDH mRNA is highly expressed in heart and stomach tissues, whereas MjAOX mRNA is highly expressed in the lymphoid organ and intestinal tissues. Furthermore, expression of MjAOX was determined to be up-regulated in the lymphoid organ in response to Vibrio penaeicida at 48 and 72 h after injection; in contrast, hydrogen peroxide (H 2 O 2 ) concentrations increased significantly at 6, 12, 48, and 72 h after injection with white spot syndrome virus (WSSV) and at 72 h after injection with V. penaeicida. To the best of our knowledge, this study is the first to have identified and cloned XDH and AOX from a crustacean species.

  9. Characteristics and expression patterns of the aldehyde dehydrogenase (ALDH) gene superfamily of foxtail millet (Setaria italica L.).

    PubMed

    Chen, Zhu; Chen, Ming; Xu, Zhao-shi; Li, Lian-cheng; Chen, Xue-ping; Ma, You-zhi

    2014-01-01

    Recent genomic sequencing of the foxtail millet, an abiotic, stress-tolerant crop, has provided a great opportunity for novel gene discovery and functional analysis of this popularly-grown grass. However, few stress-mediated gene families have been studied. Aldehyde dehydrogenases (ALDHs) comprise a gene superfamily encoding NAD (P) +-dependent enzymes that play the role of "aldehyde scavengers", which indirectly detoxify cellular ROS and reduce the effect of lipid peroxidation meditated cellular toxicity under various environmental stresses. In the current paper, we identified a total of 20 ALDH genes in the foxtail millet genome using a homology search and a phylogenetic analysis and grouped them into ten distinct families based on their amino acid sequence identity. Furthermore, evolutionary analysis of foxtail millet reveals that both tandem and segmental duplication contributed significantly to the expansion of its ALDH genes. The exon-intron structures of members of the same family in foxtail millet or the orthologous genes in rice display highly diverse distributions of their exonic and intronic regions. Also, synteny analysis shows that the majority of foxtail millet and rice ALDH gene homologs exist in the syntenic blocks between the two, implying that these ALDH genes arose before the divergence of cereals. Semi-quantitative and real-time quantitative PCR data reveals that a few SiALDH genes are expressed in an organ-specific manner and that the expression of a number of foxtail millet ALDH genes, such as, SiALDH7B1, SiALDH12A1 and SiALDH18B2 are up-regulated by osmotic stress, cold, H2O2, and phytohormone abscisic acid (ABA). Furthermore, the transformation of SiALDH2B2, SiALDH10A2, SiALDH5F1, SiALDH22A1, and SiALDH3E2 into Escherichia coli (E.coli) was able to improve their salt tolerance. Taken together, our results show that genome-wide identification characteristics and expression analyses provide unique opportunities for assessing the functional

  10. Development and validation of a rapid, aldehyde dehydrogenase bright–based cord blood potency assay

    PubMed Central

    Noldner, Pamela; Troy, Jesse D.; Cheatham, Lynn; Parrish, Amanda; Page, Kristin; Gentry, Tracy; Balber, Andrew E.; Kurtzberg, Joanne

    2016-01-01

    Banked, unrelated umbilical cord blood provides access to hematopoietic stem cell transplantation for patients lacking matched bone marrow donors, yet 10% to 15% of patients experience graft failure or delayed engraftment. This may be due, at least in part, to inadequate potency of the selected cord blood unit (CBU). CBU potency is typically assessed before cryopreservation, neglecting changes in potency occurring during freezing and thawing. Colony-forming units (CFUs) have been previously shown to predict CBU potency, defined as the ability to engraft in patients by day 42 posttransplant. However, the CFU assay is difficult to standardize and requires 2 weeks to perform. Consequently, we developed a rapid multiparameter flow cytometric CBU potency assay that enumerates cells expressing high levels of the enzyme aldehyde dehydrogenase (ALDH bright [ALDHbr]), along with viable CD45+ or CD34+ cell content. These measurements are made on a segment that was attached to a cryopreserved CBU. We validated the assay with prespecified criteria testing accuracy, specificity, repeatability, intermediate precision, and linearity. We then prospectively examined the correlations among ALDHbr, CD34+, and CFU content of 3908 segments over a 5-year period. ALDHbr (r = 0.78; 95% confidence interval [CI], 0.76-0.79), but not CD34+ (r = 0.25; 95% CI, 0.22-0.28), was strongly correlated with CFU content as well as ALDHbr content of the CBU. These results suggest that the ALDHbr segment assay (based on unit characteristics measured before release) is a reliable assessment of potency that allows rapid selection and release of CBUs from the cord blood bank to the transplant center for transplantation. PMID:26968535

  11. 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

  12. A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Clarkson, Sonya M.; Hamilton-Brehm, Scott D.; Giannone, Richard J.

    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. A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E

    DOE PAGES

    Clarkson, Sonya M.; Hamilton-Brehm, Scott D.; Giannone, Richard J.; ...

    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

  14. 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.

  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. Electronic structure contributions to reactivity in xanthine oxidase family enzymes.

    PubMed

    Stein, Benjamin W; Kirk, Martin L

    2015-03-01

    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 two-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.

  17. Aldehyde dehydrogenase-2 inhibition blocks remote preconditioning in experimental and human models.

    PubMed

    Contractor, Hussain; Støttrup, Nicolaj B; Cunnington, Colin; Manlhiot, Cedric; Diesch, Jonathan; Ormerod, Julian O M; Jensen, Rebekka; Bøtker, Hans Erik; Redington, Andrew; Schmidt, Michael R; Ashrafian, Houman; Kharbanda, Rajesh K

    2013-05-01

    Mitochondrial aldehyde dehydrogenase-2 (ALDH-2) is involved in preconditioning pathways, but its role in remote ischaemic preconditioning (rIPC) is unknown. We investigated its role in animal and human models of rIPC. (i) In a rabbit model of myocardial infarction, rIPC alone reduced infarct size [69 ± 5.8 % (n = 11) to 40 ± 6.5 % (n = 12), P = 0.019]. However, rIPC protection was lost after pre-treatment with the ALDH-2 inhibitor cyanamide (62 ± 7.6 % controls, n = 10, versus 61 ± 6.9 % rIPC after cyanamide, n = 10, P > 0.05). (ii) In a forearm plethysmography model of endothelial ischaemia-reperfusion injury, 24 individuals of Asian ethnic origin underwent combined rIPC and ischaemia-reperfusion (IR). 11 had wild-type (WT) enzyme and 13 carried the Glu504Lys (ALDH2*2) polymorphism (rendering ALDH-2 functionally inactive). In WT individuals, rIPC protected against impairment of response to acetylcholine (P = 0.9), but rIPC failed to protect carriers of Glu504Lys polymorphism (P = 0.004). (iii) In a second model of endothelial IR injury, 12 individuals participated in a double-blind placebo-controlled crossover study, receiving the ALDH-2 inhibitor disulfiram 600 mg od or placebo for 48 h prior to assessment of flow-mediated dilation (FMD) before and after combined rIPC and IR. With placebo, rIPC was effective with no difference in FMD before and after IR (6.18 ± 1.03 % and 4.76 ± 0.93 % P = 0.1), but disulfiram inhibited rIPC with a reduction in FMD after IR (7.87 ± 1.27 % and 3.05 ± 0.53 %, P = 0.001). This study demonstrates that ALDH-2 is involved in the rIPC pathway in three distinct rabbit and human models. This has potential implications for future clinical studies of remote conditioning.

  18. Toward aldehyde and alkane production by removing aldehyde reductase activity in Escherichia coli.

    PubMed

    Rodriguez, Gabriel M; Atsumi, Shota

    2014-09-01

    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. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  19. 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.

  20. Human placental indanol dehydrogenase: some properties of the microsomal enzyme.

    PubMed

    Kulkarni, A P; Strohm, B H; Houser, W H

    1985-06-01

    Indanol dehydrogenase activity of human placenta was examined in vitro. The enzyme, primarily localized in the particulate fractions of placenta, catalysed conversion of 1-indanol to 1-indanone in the presence of oxidized pyridine nucleotides. Both NAD+ and NADP+ supported the reaction with nearly equal efficiency.

  1. Characterization of human DHRS4: an inducible short-chain dehydrogenase/reductase enzyme with 3beta-hydroxysteroid dehydrogenase activity.

    PubMed

    Matsunaga, Toshiyuki; Endo, Satoshi; Maeda, Satoshi; Ishikura, Shuhei; Tajima, Kazuo; Tanaka, Nobutada; Nakamura, Kazuo T; Imamura, Yorishige; Hara, Akira

    2008-09-15

    Human DHRS4 is a peroxisomal member of the short-chain dehydrogenase/reductase superfamily, but its enzymatic properties, except for displaying NADP(H)-dependent retinol dehydrogenase/reductase activity, are unknown. We show that the human enzyme, a tetramer composed of 27kDa subunits, is inactivated at low temperature without dissociation into subunits. The cold inactivation was prevented by a mutation of Thr177 with the corresponding residue, Asn, in cold-stable pig DHRS4, where this residue is hydrogen-bonded to Asn165 in a substrate-binding loop of other subunit. Human DHRS4 reduced various aromatic ketones and alpha-dicarbonyl compounds including cytotoxic 9,10-phenanthrenequinone. The overexpression of the peroxisomal enzyme in cultured cells did not increase the cytotoxicity of 9,10-phenanthrenequinone. While its activity towards all-trans-retinal was low, human DHRS4 efficiently reduced 3-keto-C(19)/C(21)-steroids into 3beta-hydroxysteroids. The stereospecific conversion to 3beta-hydroxysteroids was observed in endothelial cells transfected with vectors expressing the enzyme. The mRNA for the enzyme was ubiquitously expressed in human tissues and several cancer cells, and the enzyme in HepG2 cells was induced by peroxisome-proliferator-activated receptor alpha ligands. The results suggest a novel mechanism of cold inactivation and role of the inducible human DHRS4 in 3beta-hydroxysteroid synthesis and xenobiotic carbonyl metabolism.

  2. Spectroscopic characterisation of interaction of ferulic acid with aldehyde dehydrogenase (ALDH).

    PubMed

    Kolawole, Ayodele O; Agaba, Ruth J; Oluwole, Matthew O

    2017-05-01

    Interaction of a pharmacological important phenolic, ferulic acid, with Aldehyde dehydrogenase (ALDH) at the simulative pH condition, was studied using spectroscopic approach. Ferulic acid caused a decrease in the fluorescence intensity formed from ALDH-ferulic acid complex resulting in mixed inhibition of ALDH activity (IC 50 =30.65μM). The intrinsic quenching was dynamic and induced altered conformation of ALDH and made the protein less compact but might not unfold it. ALDH has two binding sites for ferulic acid at saturating concentrations having association constant of 1.35×10 3 Lmol -1 and a dissociation constant of 9.7×10 7 Lmol -1 at 25°C indicating ALDH-ferulic acid complex formation is more favourable than its dissociation. The interaction was not spontaneous and endothermic and suggests the involvement of hydrophobic interactions with a FRET binding distance of 4.49nm. Change in pH near and far from isoelectric points of ferulic acid did not affect the bonding interaction. Using trehalose as viscosogen, the result from Stoke-Einstein hypothesis showed that ferulic acid-ALDH binding and dissociation equilibrium was diffusion controlled. These results clearly suggest the unique binding properties and lipophilicity influence of ferulic acid. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Direct Electrochemical Addressing of Immobilized Alcohol Dehydrogenase for the Heterogeneous Bioelectrocatalytic Reduction of Butyraldehyde to Butanol.

    PubMed

    Schlager, S; Neugebauer, H; Haberbauer, M; Hinterberger, G; Sariciftci, N S

    2015-03-01

    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 CO 2 -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 CO 2 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 CO 2 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 %.

  4. 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

  5. Development and validation of a rapid, aldehyde dehydrogenase bright-based cord blood potency assay.

    PubMed

    Shoulars, Kevin; Noldner, Pamela; Troy, Jesse D; Cheatham, Lynn; Parrish, Amanda; Page, Kristin; Gentry, Tracy; Balber, Andrew E; Kurtzberg, Joanne

    2016-05-12

    Banked, unrelated umbilical cord blood provides access to hematopoietic stem cell transplantation for patients lacking matched bone marrow donors, yet 10% to 15% of patients experience graft failure or delayed engraftment. This may be due, at least in part, to inadequate potency of the selected cord blood unit (CBU). CBU potency is typically assessed before cryopreservation, neglecting changes in potency occurring during freezing and thawing. Colony-forming units (CFUs) have been previously shown to predict CBU potency, defined as the ability to engraft in patients by day 42 posttransplant. However, the CFU assay is difficult to standardize and requires 2 weeks to perform. Consequently, we developed a rapid multiparameter flow cytometric CBU potency assay that enumerates cells expressing high levels of the enzyme aldehyde dehydrogenase (ALDH bright [ALDH(br)]), along with viable CD45(+) or CD34(+) cell content. These measurements are made on a segment that was attached to a cryopreserved CBU. We validated the assay with prespecified criteria testing accuracy, specificity, repeatability, intermediate precision, and linearity. We then prospectively examined the correlations among ALDH(br), CD34(+), and CFU content of 3908 segments over a 5-year period. ALDH(br) (r = 0.78; 95% confidence interval [CI], 0.76-0.79), but not CD34(+) (r = 0.25; 95% CI, 0.22-0.28), was strongly correlated with CFU content as well as ALDH(br) content of the CBU. These results suggest that the ALDH(br) segment assay (based on unit characteristics measured before release) is a reliable assessment of potency that allows rapid selection and release of CBUs from the cord blood bank to the transplant center for transplantation. © 2016 by The American Society of Hematology.

  6. High aldehyde dehydrogenase activity identifies cancer stem cells in human cervical cancer

    PubMed Central

    Liu, Shu-Yan; Zheng, Peng-Sheng

    2013-01-01

    High aldehyde dehydrogenase (ALDH) activity characterizes a subpopulation of cells with cancer stem cell (CSC) properties in several malignancies. To clarify whether ALDH can be used as a marker of cervical cancer stem cells (CCSCs), ALDHhigh and ALDHlow cells were sorted from 4 cervical cancer cell lines and 5 primary tumor xenografts and examined for CSC characteristics. Here, we demonstrate that cervical cancer cells with high ALDH activity fulfill the functional criteria for CSCs: (1) ALDHhigh cells, unlike ALDHlow cells, are highly tumorigenic in vivo; (2) ALDHhigh cells can give rise to both ALDHhigh and ALDHlow cells in vitro and in vivo, thereby establishing a cellular hierarchy; and (3) ALDHhigh cells have enhanced self-renewal and differentiation potentials. Additionally, ALDHhigh cervical cancer cells are more resistant to cisplatin treatment than ALDHlow cells. Finally, expression of the stem cell self-renewal-associated transcription factors OCT4, NANOG, KLF4 and BMI1 is elevated in ALDHhigh cervical cancer cells. Taken together, our data indicated that high ALDH activity may represent both a functional marker for CCSCs and a target for novel cervical cancer therapies. PMID:24318570

  7. 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

  8. An intact eight-membered water chain in drosophilid alcohol dehydrogenases is essential for optimal enzyme activity.

    PubMed

    Wuxiuer, Yimingjiang; Morgunova, Ekaterina; Cols, Neus; Popov, Alexander; Karshikoff, Andrey; Sylte, Ingebrigt; Gonzàlez-Duarte, Roser; Ladenstein, Rudolf; Winberg, Jan-Olof

    2012-08-01

    All drosophilid alcohol dehydrogenases contain an eight-member water chain connecting the active site with the solvent at the dimer interface. A similar water chain has also been shown to exist in other short-chain dehydrogenase/reductase (SDR) enzymes, including therapeutically important SDRs. The role of this water chain in the enzymatic reaction is unknown, but it has been proposed to be involved in a proton relay system. In the present study, a connecting link in the water chain was removed by mutating Thr114 to Val114 in Scaptodrosophila lebanonensis alcohol dehydrogenase (SlADH). This threonine is conserved in all drosophilid alcohol dehydrogenases but not in other SDRs. X-ray crystallography of the SlADH(T114V) mutant revealed a broken water chain, the overall 3D structure of the binary enzyme-NAD(+) complex was almost identical to the wild-type enzyme (SlADH(wt) ). As for the SlADH(wt) , steady-state kinetic studies revealed that catalysis by the SlADH(T114V) mutant was consistent with a compulsory ordered reaction mechanism where the co-enzyme binds to the free enzyme. The mutation caused a reduction of the k(on) velocity for NAD(+) and its binding strength to the enzyme, as well as the rate of hydride transfer (k) in the ternary enzyme-NAD(+) -alcohol complex. Furthermore, it increased the pK(a) value of the group in the binary enzyme-NAD(+) complex that regulates the k(on) velocity of alcohol and alcohol-competitive inhibitors. Overall, the results indicate that an intact water chain is essential for optimal enzyme activity and participates in a proton relay system during catalysis. © 2012 The Authors Journal compilation © 2012 FEBS.

  9. Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2 degrees Adh) as a bifunctional alcohol dehydrogenase--acetyl-CoA reductive thioesterase.

    PubMed Central

    Burdette, D; Zeikus, J G

    1994-01-01

    The purification and characterization of three enzymes involved in ethanol formation from acetyl-CoA in Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) is described. The secondary-alcohol dehydrogenase (2 degrees Adh) was determined to be a homotetramer of 40 kDa subunits (SDS/PAGE) with a molecular mass of 160 kDa. The 2 degrees Adh had a lower catalytic efficiency for the oxidation of 1 degree alcohols, including ethanol, than for the oxidation of secondary (2 degrees) alcohols or the reduction of ketones or aldehydes. This enzyme possesses a significant acetyl-CoA reductive thioesterase activity as determined by NADPH oxidation, thiol formation and ethanol production. The primary-alcohol dehydrogenase (1 degree Adh) was determined to be a homotetramer of 41.5 kDa (SDS/PAGE) subunits with a molecular mass of 170 kDa. The 1 degree Adh used both NAD(H) and NADP(H) and displayed higher catalytic efficiencies for NADP(+)-dependent ethanol oxidation and NADH-dependent acetaldehyde (identical to ethanal) reduction than for NADPH-dependent acetaldehyde reduction or NAD(+)-dependent ethanol oxidation. The NAD(H)-linked acetaldehyde dehydrogenase was a homotetramer (360 kDa) of identical subunits (100 kDa) that readily catalysed thioester cleavage and condensation. The 1 degree Adh was expressed at 5-20% of the level of the 2 degrees Adh throughout the growth cycle on glucose. The results suggest that the 2 degrees Adh primarily functions in ethanol production from acetyl-CoA and acetaldehyde, whereas the 1 degree Adh functions in ethanol consumption for nicotinamide-cofactor recycling. Images Figure 1 PMID:8068002

  10. Aldehyde Oxidase 4 Plays a Critical Role in Delaying Silique Senescence by Catalyzing Aldehyde Detoxification.

    PubMed

    Srivastava, Sudhakar; Brychkova, Galina; Yarmolinsky, Dmitry; Soltabayeva, Aigerim; Samani, Talya; Sagi, Moshe

    2017-04-01

    The Arabidopsis ( Arabidopsis thaliana ) aldehyde oxidases are a multigene family of four oxidases (AAO1-AAO4) that oxidize a variety of aldehydes, among them abscisic aldehyde, which is oxidized to the phytohormone abscisic acid. Toxic aldehydes are generated in plants both under normal conditions and in response to stress. The detoxification of such aldehydes by oxidation is attributed to aldehyde dehydrogenases but never to aldehyde oxidases. The feasibility of the detoxification of aldehydes in siliques via oxidation by AAO4 was demonstrated, first, by its ability to efficiently oxidize an array of aromatic and aliphatic aldehydes, including the reactive carbonyl species (RCS) acrolein, hydroxyl-2-nonenal, and malondialdehyde. Next, exogenous application of several aldehydes to siliques in AAO4 knockout (KO) Arabidopsis plants induced severe tissue damage and enhanced malondialdehyde levels and senescence symptoms, but not in wild-type siliques. Furthermore, abiotic stresses such as dark and ultraviolet C irradiation caused an increase in endogenous RCS and higher expression levels of senescence marker genes, leading to premature senescence of KO siliques, whereas RCS and senescence marker levels in wild-type siliques were hardly affected. Finally, in naturally senesced KO siliques, higher endogenous RCS levels were associated with enhanced senescence molecular markers, chlorophyll degradation, and earlier seed shattering compared with the wild type. The aldehyde-dependent differential generation of superoxide and hydrogen peroxide by AAO4 and the induction of AAO4 expression by hydrogen peroxide shown here suggest a self-amplification mechanism for detoxifying additional reactive aldehydes produced during stress. Taken together, our results indicate that AAO4 plays a critical role in delaying senescence in siliques by catalyzing aldehyde detoxification. © 2017 American Society of Plant Biologists. All Rights Reserved.

  11. Effects of Moderate Alcohol Consumption on Gene Expression Related to Colonic Inflammation and Antioxidant Enzymes in Rats

    PubMed Central

    Klarich, DawnKylee S.; Penprase, Jerrold; Cintora, Patricia; Medrano, Octavio; Erwin, Danielle; Brasser, Susan M.; Hong, Mee Young

    2017-01-01

    Excessive alcohol consumption is a risk factor associated with colorectal cancer; however, some studies have reported that moderate alcohol consumption may not contribute additional risk for developing colorectal cancer while others suggest that moderate alcohol consumption provides a protective effect that reduces colorectal cancer risk. The purpose of this study was to determine the effects of moderate voluntary alcohol (20% ethanol) intake on alternate days for 3 months in outbred Wistar rats on risk factors associated with colorectal cancer development. Colonic gene expression of cyclooxygenase-2, RelA, 8-oxoguanine DNA glycosylase 1, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase M1, and aldehyde dehydrogenase 2 were determined. Blood alcohol content, liver function enzyme activities, and 8-oxo-deoxyguanosine DNA adducts were also assessed. Alcohol-treated rats were found to have significantly lower 8-oxo-deoxyguanosine levels in blood, a marker of DNA damage. Alanine aminotransferase and lactate dehydrogenase were both significantly lower in the alcohol group. Moderate alcohol significantly decreased cyclooxygenase-2 gene expression, an inflammatory marker associated with colorectal cancer risk. The alcohol group had significantly increased glutathione-S-transferase M1 expression, an antioxidant enzyme that helps detoxify carcinogens, such as acetaldehyde, and significantly increased aldehyde dehydrogenase 2 expression, which allows for greater acetaldehyde clearance. Increased expression of glutathione-S-transferase M1 and aldehyde dehydrogenase 2 likely contributed to reduce mucosal damage that is caused by acetaldehyde accumulation. These results indicate that moderate alcohol may reduce the risk for colorectal cancer development, which was evidenced by reduced inflammation activity and lower DNA damage after alcohol exposure. PMID:28599714

  12. 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

  13. Amine dehydrogenases: efficient biocatalysts for the reductive amination of carbonyl compounds

    PubMed Central

    Mutti, Francesco G.

    2017-01-01

    Amines constitute the major targets for the production of a plethora of chemical compounds that have applications in the pharmaceutical, agrochemical and bulk chemical industries. However, the asymmetric synthesis of α-chiral amines with elevated catalytic efficiency and atom economy is still a very challenging synthetic problem. Here, we investigated the biocatalytic reductive amination of carbonyl compounds employing a rising class of enzymes for amine synthesis: amine dehydrogenases (AmDHs). The three AmDHs from this study – operating in tandem with a formate dehydrogenase from Candida boidinii (Cb-FDH) for the recycling of the nicotinamide coenzyme – performed the efficient amination of a range of diverse aromatic and aliphatic ketones and aldehydes with up to quantitative conversion and elevated turnover numbers (TONs). Moreover, the reductive amination of prochiral ketones proceeded with perfect stereoselectivity, always affording the (R)-configured amines with more than 99% enantiomeric excess. The most suitable amine dehydrogenase, the optimised catalyst loading and the required reaction time were determined for each substrate. The biocatalytic reductive amination with this dual-enzyme system (AmDH–Cb-FDH) possesses elevated atom efficiency as it utilizes the ammonium formate buffer as the source of both nitrogen and reducing equivalents. Inorganic carbonate is the sole by-product. PMID:28663713

  14. Amine dehydrogenases: efficient biocatalysts for the reductive amination of carbonyl compounds.

    PubMed

    Knaus, Tanja; Böhmer, Wesley; Mutti, Francesco G

    2017-01-21

    Amines constitute the major targets for the production of a plethora of chemical compounds that have applications in the pharmaceutical, agrochemical and bulk chemical industries. However, the asymmetric synthesis of α-chiral amines with elevated catalytic efficiency and atom economy is still a very challenging synthetic problem. Here, we investigated the biocatalytic reductive amination of carbonyl compounds employing a rising class of enzymes for amine synthesis: amine dehydrogenases (AmDHs). The three AmDHs from this study - operating in tandem with a formate dehydrogenase from Candida boidinii (Cb-FDH) for the recycling of the nicotinamide coenzyme - performed the efficient amination of a range of diverse aromatic and aliphatic ketones and aldehydes with up to quantitative conversion and elevated turnover numbers (TONs). Moreover, the reductive amination of prochiral ketones proceeded with perfect stereoselectivity, always affording the ( R )-configured amines with more than 99% enantiomeric excess. The most suitable amine dehydrogenase, the optimised catalyst loading and the required reaction time were determined for each substrate. The biocatalytic reductive amination with this dual-enzyme system (AmDH-Cb-FDH) possesses elevated atom efficiency as it utilizes the ammonium formate buffer as the source of both nitrogen and reducing equivalents. Inorganic carbonate is the sole by-product.

  15. Aldehyde Oxidase 4 Plays a Critical Role in Delaying Silique Senescence by Catalyzing Aldehyde Detoxification1[OPEN

    PubMed Central

    Yarmolinsky, Dmitry; Soltabayeva, Aigerim; Samani, Talya

    2017-01-01

    The Arabidopsis (Arabidopsis thaliana) aldehyde oxidases are a multigene family of four oxidases (AAO1–AAO4) that oxidize a variety of aldehydes, among them abscisic aldehyde, which is oxidized to the phytohormone abscisic acid. Toxic aldehydes are generated in plants both under normal conditions and in response to stress. The detoxification of such aldehydes by oxidation is attributed to aldehyde dehydrogenases but never to aldehyde oxidases. The feasibility of the detoxification of aldehydes in siliques via oxidation by AAO4 was demonstrated, first, by its ability to efficiently oxidize an array of aromatic and aliphatic aldehydes, including the reactive carbonyl species (RCS) acrolein, hydroxyl-2-nonenal, and malondialdehyde. Next, exogenous application of several aldehydes to siliques in AAO4 knockout (KO) Arabidopsis plants induced severe tissue damage and enhanced malondialdehyde levels and senescence symptoms, but not in wild-type siliques. Furthermore, abiotic stresses such as dark and ultraviolet C irradiation caused an increase in endogenous RCS and higher expression levels of senescence marker genes, leading to premature senescence of KO siliques, whereas RCS and senescence marker levels in wild-type siliques were hardly affected. Finally, in naturally senesced KO siliques, higher endogenous RCS levels were associated with enhanced senescence molecular markers, chlorophyll degradation, and earlier seed shattering compared with the wild type. The aldehyde-dependent differential generation of superoxide and hydrogen peroxide by AAO4 and the induction of AAO4 expression by hydrogen peroxide shown here suggest a self-amplification mechanism for detoxifying additional reactive aldehydes produced during stress. Taken together, our results indicate that AAO4 plays a critical role in delaying senescence in siliques by catalyzing aldehyde detoxification. PMID:28188272

  16. Genetics Home Reference: hereditary xanthinuria

    MedlinePlus

    ... xanthine dehydrogenase, described above, and another enzyme called aldehyde oxidase. Mutations in the MOCOS gene prevent xanthine dehydrogenase and aldehyde oxidase from being turned on (activated). The loss ...

  17. Inhibition of human alcohol and aldehyde dehydrogenases by acetaminophen: Assessment of the effects on first-pass metabolism of ethanol.

    PubMed

    Lee, Yung-Pin; Liao, Jian-Tong; Cheng, Ya-Wen; Wu, Ting-Lun; Lee, Shou-Lun; Liu, Jong-Kang; Yin, Shih-Jiun

    2013-11-01

    Acetaminophen is one of the most widely used over-the-counter analgesic, antipyretic medications. Use of acetaminophen and alcohol are commonly associated. Previous studies showed that acetaminophen might affect bioavailability of ethanol by inhibiting gastric alcohol dehydrogenase (ADH). However, potential inhibitions by acetaminophen of first-pass metabolism (FPM) of ethanol, catalyzed by the human ADH family and by relevant aldehyde dehydrogenase (ALDH) isozymes, remain undefined. ADH and ALDH both exhibit racially distinct allozymes and tissue-specific distribution of isozymes, and are principal enzymes responsible for ethanol metabolism in humans. In this study, we investigated acetaminophen inhibition of ethanol oxidation with recombinant human ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2, ADH2, and ADH4, and inhibition of acetaldehyde oxidation with recombinant human ALDH1A1 and ALDH2. The investigations were done at near physiological pH 7.5 and with a cytoplasmic coenzyme concentration of 0.5 mM NAD(+). Acetaminophen acted as a noncompetitive inhibitor for ADH enzymes, with the slope inhibition constants (Kis) ranging from 0.90 mM (ADH2) to 20 mM (ADH1A), and the intercept inhibition constants (Kii) ranging from 1.4 mM (ADH1C allozymes) to 19 mM (ADH1A). Acetaminophen exhibited noncompetitive inhibition for ALDH2 (Kis = 3.0 mM and Kii = 2.2 mM), but competitive inhibition for ALDH1A1 (Kis = 0.96 mM). The metabolic interactions between acetaminophen and ethanol/acetaldehyde were assessed by computer simulation using inhibition equations and the determined kinetic constants. At therapeutic to subtoxic plasma levels of acetaminophen (i.e., 0.2-0.5 mM) and physiologically relevant concentrations of ethanol (10 mM) and acetaldehyde (10 μm) in target tissues, acetaminophen could inhibit ADH1C allozymes (12-26%) and ADH2 (14-28%) in the liver and small intestine, ADH4 (15-31%) in the stomach, and ALDH1A1 (16-33%) and ALDH2 (8.3-19%) in all 3 tissues. The

  18. YKL071W from Saccharomyces cerevisiae encodes a novel aldehyde reductase for detoxification of glycolaldehyde and furfural derived from lignocellulose.

    PubMed

    Wang, Hanyu; Ouyang, Yidan; Zhou, Chang; Xiao, Difan; Guo, Yaping; Wu, Lan; Li, Xi; Gu, Yunfu; Xiang, Quanju; Zhao, Ke; Yu, Xiumei; Zou, Likou; Ma, Menggen

    2017-12-01

    Aldehydes generated as by-products during the pretreatment of lignocellulose are the key inhibitors to Saccharomyces cerevisiae, which is considered as the most promising microorganism for industrial production of biofuel, xylitol as well as other special chemicals from lignocellulose. S. cerevisiae has the inherent ability to in situ detoxify aldehydes to corresponding alcohols by multiple aldehyde reductases. Herein, we report that an uncharacterized open reading frame YKL071W from S. cerevisiae encodes a novel "classical" short-chain dehydrogenase/reductase (SDR) protein with NADH-dependent enzymatic activities for reduction of furfural (FF), glycolaldehyde (GA), formaldehyde (FA), and benzaldehyde (BZA). This enzyme showed much better specific activities for reduction of GA and FF than FA and BZA, and displayed much higher Km and Kcat/Km but lower Vmax and Kcat for reduction of GA than FF. For this enzyme, the optimum pH was 5.5 and 6.0 for reduction of GA and FF, and the optimum temperature was 30 °C for reduction of GA and FF. Both pH and temperature affected stability of this enzyme in a similar trend for reduction of GA and FF. Cu 2+ , Zn 2+ , Ni 2+ , and Fe 3+ had severe inhibition effects on enzyme activities of Ykl071wp for reduction of GA and FF. Transcription of YKL071W in S. cerevisiae was significantly upregulated under GA and FF stress conditions, and its transcription is most probably regulated by transcription factor genes of YAP1, CAD1, PDR3, and STB5. This research provides guidelines to identify more uncharacterized genes with reductase activities for detoxification of aldehydes derived from lignocellulose in S. cerevisiae.

  19. Cloning and molecular evolution of the aldehyde dehydrogenase 2 gene (Aldh2) in bats (Chiroptera).

    PubMed

    Chen, Yao; Shen, Bin; Zhang, Junpeng; Jones, Gareth; He, Guimei

    2013-02-01

    Old World fruit bats (Pteropodidae) and New World fruit bats (Phyllostomidae) ingest significant quantities of ethanol while foraging. Mitochondrial aldehyde dehydrogenase (ALDH2, encoded by the Aldh2 gene) plays an important role in ethanol metabolism. To test whether the Aldh2 gene has undergone adaptive evolution in frugivorous and nectarivorous bats in relation to ethanol elimination, we sequenced part of the coding region of the gene (1,143 bp, ~73 % coverage) in 14 bat species, including three Old World fruit bats and two New World fruit bats. Our results showed that the Aldh2 coding sequences are highly conserved across all bat species we examined, and no evidence of positive selection was detected in the ancestral branches leading to Old World fruit bats and New World fruit bats. Further research is needed to determine whether other genes involved in ethanol metabolism have been the targets of positive selection in frugivorous and nectarivorous bats.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. Biochemical Properties of Purified Human Retinol Dehydrogenase 12 (RDH12): Catalytic Efficiency toward Retinoids and C9 Aldehydes and Effects of Cellular Retinol-Binding Protein Type I (CRBPI) and Cellular Retinaldehyde-Binding Protein (CRALBP) on the Oxidation and Reduction of Retinoids†

    PubMed Central

    Belyaeva, Olga V.; Korkina, Olga V.; Stetsenko, Anton V.; Kim, Tom; Nelson, Peter S.; Kedishvili, Natalia Y.

    2008-01-01

    Retinol dehydrogenase 12 (RDH12) is a novel member of the short-chain dehydrogenase/reductase superfamily of proteins that was recently linked to Leber’s congenital amaurosis 3 (LCA). We report the first biochemical characterization of purified human RDH12 and analysis of its expression in human tissues. RDH12 exhibits ~2000-fold lower Km values for NADP+ and NADPH than for NAD+ and NADH and recognizes both retinoids and lipid peroxidation products (C9 aldehydes) as substrates. The kcat values of RDH12 for retinaldehydes and C9 aldehydes are similar, but the Km values are, in general, lower for retinoids. The enzyme exhibits the highest catalytic efficiency for all-trans-retinal (kcat/Km ~900 min−1 μM−1), followed by 11-cis-retinal (450 min−1 mM−1) and 9-cis-retinal (100 min−1 mM−1). Analysis of RDH12 activity toward retinoids in the presence of cellular retinol-binding protein (CRBP) type I or cellular retinaldehyde-binding protein (CRALBP) suggests that RDH12 utilizes the unbound forms of all-trans- and 11-cis-retinoids. As a result, the widely expressed CRBPI, which binds all-trans-retinol with much higher affinity than all-trans-retinaldehyde, restricts the oxidation of all-trans-retinol by RDH12, but has little effect on the reduction of all-trans-retinaldehyde, and CRALBP inhibits the reduction of 11-cis-retinal stronger than the oxidation of 11-cis-retinol, in accord with its higher affinity for 11-cis-retinal. Together, the tissue distribution of RDH12 and its catalytic properties suggest that, in most tissues, RDH12 primarily contributes to the reduction of all-trans-retinaldehyde; however, at saturating concentrations of peroxidic aldehydes in the cells undergoing oxidative stress, for example, photoreceptors, RDH12 might also play a role in detoxification of lipid peroxidation products. PMID:15865448

  5. Thermostable NADP+-Dependent Medium-Chain Alcohol Dehydrogenase from Acinetobacter sp. Strain M-1: Purification and Characterization and Gene Expression in Escherichia coli

    PubMed Central

    Tani, Akio; Sakai, Yasuyoshi; Ishige, Takeru; Kato, Nobuo

    2000-01-01

    NADPH-dependent alkylaldehyde reducing enzyme, which was greatly induced by n-hexadecane, from Acinetobacter sp. strain M-1 was purified and characterized. The purified enzyme had molecular masses of 40 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160 kDa as determined by gel filtration chromatography. The enzyme, which was shown to be highly thermostable, was most active toward n-heptanal and could use n-alkylaldehydes ranging from C2 to C14 and several substituted benzaldehydes, including the industrially important compounds cinnamyl aldehyde and anisaldehyde, as substrates. The alrA gene coding for this enzyme was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the alrA gene exhibited homology to the amino acid sequences of zinc-containing alcohol dehydrogenases from various sources. The gene could be highly expressed in Escherichia coli, and the product was purified to homogeneity by simpler procedures from the recombinant than from the original host. Our results show that this enzyme can be used for industrial bioconversion of useful alcohols and aldehydes. PMID:11097895

  6. Enzyme-dependent fluorescence recovery of NADH after photobleaching to assess dehydrogenase activity of isolated perfused hearts

    NASA Astrophysics Data System (ADS)

    Moreno, Angel; Kuzmiak-Glancy, Sarah; Jaimes, Rafael; Kay, Matthew W.

    2017-03-01

    Reduction of NAD+ by dehydrogenase enzymes to form NADH is a key component of cellular metabolism. In cellular preparations and isolated mitochondria suspensions, enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) of NADH has been shown to be an effective approach for measuring the rate of NADH production to assess dehydrogenase enzyme activity. Our objective was to demonstrate how dehydrogenase activity could be assessed within the myocardium of perfused hearts using NADH ED-FRAP. This was accomplished using a combination of high intensity UV pulses to photobleach epicardial NADH. Replenishment of epicardial NADH fluorescence was then imaged using low intensity UV illumination. NADH ED-FRAP parameters were optimized to deliver 23.8 mJ of photobleaching light energy at a pulse width of 6 msec and a duty cycle of 50%. These parameters provided repeatable measurements of NADH production rate during multiple metabolic perturbations, including changes in perfusate temperature, electromechanical uncoupling, and acute ischemia/reperfusion injury. NADH production rate was significantly higher in every perturbation where the energy demand was either higher or uncompromised. We also found that NADH production rate remained significantly impaired after 10 min of reperfusion after global ischemia. Overall, our results indicate that myocardial NADH ED-FRAP is a useful optical non-destructive approach for assessing dehydrogenase activity.

  7. 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

  8. Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues.

    PubMed

    Molenaar, Remco J; Khurshed, Mohammed; Hira, Vashendriya V V; Van Noorden, Cornelis J F

    2018-05-26

    Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation. We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P) + and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase's activity. The formazan's absorbance is therefore a direct measure of the dehydrogenase's activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays. Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an

  9. 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.

  10. Origin and dispersal of atypical aldehyde dehydrogenase ALDH2487Lys.

    PubMed

    Luo, Huai-Rong; Wu, Gui-Sheng; Pakstis, Andrew J; Tong, Li; Oota, Hiroki; Kidd, Kenneth K; Zhang, Ya-Ping

    2009-04-15

    The East Asian respond with a marked facial flushing and mild to moderate symptoms of intoxication after drinking the amounts of alcohol that has no detectable effect on European. The alcohol sensitivity in Orientals is due to a delayed oxidation of acetaldehyde by an atypical aldehyde dehydrogenase ALDH2487Lys, which is resulted from a structural mutation in gene ALDH2. The atypical ALDH2487Lys allele has been associated with various phenotypic statuses, such as protective against alcohol dependence and the risk of alcohol-related digestive tract cancers. Here, we have examined this SNP, adjacent four non-coding SNPs, and one downstream STRP on ALDH2 gene, in total of 1072 unrelated healthy individuals from 14 Chinese populations and 130 Indian individuals. Five major haplotypes based on five SNPs across the ALDH2 gene 40 kb were found in all East Asian populations. The frequencies of the ancestral haplotype GCCTG and the East Asian special haplotype GCCTA containing the atypical ALDH2487Lys allele were 44.8% and 14.9%, respectively. The frequency of the atypical ALDH2487Lys allele or the East Asian specific haplotype GCCTA is high in Yunnan, South coastal, east coastal of China, and decreased gradually toward inland China, West, Northwest and North China. Combined with demographic history in East Asian, our results showed that the presence of ALDH2487Lys allele in peripheral regions of China might be the results of historical migration events from China to these regions. The origin of ALDH2487Lys could be possibly traced back to ancient Pai-Yuei tribe in South China.

  11. Enzyme dynamics and hydrogen tunnelling in a thermophilic alcohol dehydrogenase

    NASA Astrophysics Data System (ADS)

    Kohen, Amnon; Cannio, Raffaele; Bartolucci, Simonetta; Klinman, Judith P.; Klinman, Judith P.

    1999-06-01

    Biological catalysts (enzymes) speed up reactions by many orders of magnitude using fundamental physical processes to increase chemical reactivity. Hydrogen tunnelling has increasingly been found to contribute to enzyme reactions at room temperature. Tunnelling is the phenomenon by which a particle transfers through a reaction barrier as a result of its wave-like property. In reactions involving small molecules, the relative importance of tunnelling increases as the temperature is reduced. We have now investigated whether hydrogen tunnelling occurs at elevated temperatures in a biological system that functions physiologically under such conditions. Using a thermophilic alcohol dehydrogenase (ADH), we find that hydrogen tunnelling makes a significant contribution at 65°C this is analogous to previous findings with mesophilic ADH at 25°C ( ref. 5). Contrary to predictions for tunnelling through a rigid barrier, the tunnelling with the thermophilic ADH decreases at and below room temperature. These findings provide experimental evidence for a role of thermally excited enzyme fluctuations in modulating enzyme-catalysed bond cleavage.

  12. The prognostic impact of cancer stem-like cell biomarker aldehyde dehydrogenase-1 (ALDH1) in ovarian cancer: A meta-analysis.

    PubMed

    Ruscito, Ilary; Darb-Esfahani, Silvia; Kulbe, Hagen; Bellati, Filippo; Zizzari, Ilaria Grazia; Rahimi Koshkaki, Hassan; Napoletano, Chiara; Caserta, Donatella; Rughetti, Aurelia; Kessler, Mirjana; Sehouli, Jalid; Nuti, Marianna; Braicu, Elena Ioana

    2018-05-10

    To investigate the association of cancer stem cell biomarker aldehyde dehydrogenase-1 (ALDH1) with ovarian cancer patients' prognosis and clinico-pathological characteristics. The electronic searches were performed in January 2018 through the databases PubMed, MEDLINE and Scopus by searching the terms: "ovarian cancer" AND "immunohistochemistry" AND ["aldehyde dehydrogenase-1" OR "ALDH1" OR "cancer stem cell"]. Studies evaluating the impact of ALDH1 expression on ovarian cancer survival and clinico-pathological variables were selected. 233 studies were retrieved. Thirteen studies including 1885 patients met all selection criteria. ALDH1-high expression was found to be significantly associated with poor 5-year OS (OR = 3.46; 95% CI: 1.61-7.42; P = 0.001, random effects model) and 5-year PFS (OR = 2.14; 95% CI: 1.11-4.13; P = 0.02, random effects model) in ovarian cancer patients. No correlation between ALDH1 expression and tumor histology (OR = 0.60; 95% CI: 0.36-1.02; P = 0.06, random effects model), FIGO Stage (OR = 0.65; 95% CI: 0.33-1.30; P = 0.22, random effects model), tumor grading (OR = 0.76; 95% CI: 0.40-1.45; P = 0.41, random effects model) lymph nodal status (OR = 2.05; 95% CI: 0.81-5.18; P = 0.13, random effects model) or patients' age at diagnosis (OR = 0.83; 95% CI: 0.54-1.29; P = 0.41, fixed effects model) was identified. Basing on the available evidence, this meta-analysis showed that high levels of ALDH1 expression correlate with worse OS and PFS in ovarian cancer patients. Copyright © 2018. Published by Elsevier Inc.

  13. Arabidopsis aldehyde dehydrogenase 10 family members confer salt tolerance through putrescine-derived 4-aminobutyrate (GABA) production.

    PubMed

    Zarei, Adel; Trobacher, Christopher P; Shelp, Barry J

    2016-10-11

    Polyamines represent a potential source of 4-aminobutyrate (GABA) in plants exposed to abiotic stress. Terminal catabolism of putrescine in Arabidopsis thaliana involves amine oxidase and the production of 4-aminobutanal, which is a substrate for NAD + -dependent aminoaldehyde dehydrogenase (AMADH). Here, two AMADH homologs were chosen (AtALDH10A8 and AtALDH10A9) as candidates for encoding 4-aminobutanal dehydrogenase activity for GABA synthesis. The two genes were cloned and soluble recombinant proteins were produced in Escherichia coli. The pH optima for activity and catalytic efficiency of recombinant AtALDH10A8 with 3-aminopropanal as substrate was 10.5 and 8.5, respectively, whereas the optima for AtALDH10A9 were approximately 9.5. Maximal activity and catalytic efficiency were obtained with NAD + and 3-aminopropanal, followed by 4-aminobutanal; negligible activity was obtained with betaine aldehyde. NAD + reduction was accompanied by the production of GABA and β-alanine, respectively, with 4-aminobutanal and 3-aminopropanal as substrates. Transient co-expression systems using Arabidopsis cell suspension protoplasts or onion epidermal cells and several organelle markers revealed that AtALDH10A9 was peroxisomal, but AtALDH10A8 was cytosolic, although the N-terminal 140 amino acid sequence of AtALDH10A8 localized to the plastid. Root growth of single loss-of-function mutants was more sensitive to salinity than wild-type plants, and this was accompanied by reduced GABA accumulation.

  14. Characterization of an aldolase-dehydrogenase complex from the cholesterol degradation pathway of Mycobacterium tuberculosis.

    PubMed

    Carere, Jason; McKenna, Sarah E; Kimber, Matthew S; Seah, Stephen Y K

    2013-05-21

    HsaF and HsaG are an aldolase and dehydrogenase from the cholesterol degradation pathway of Mycobacterium tuberculosis. HsaF could be heterologously expressed and purified as a soluble dimer, but the enzyme was inactive in the absence of HsaG. HsaF catalyzes the aldol cleavage of 4-hydroxy-2-oxoacids to produce pyruvate and an aldehyde. The enzyme requires divalent metals for activity, with a preference for Mn(2+). The Km values for 4-hydroxy-2-oxoacids were about 20-fold lower than observed for the aldolase homologue, BphI from the polychlorinated biphenyl degradation pathway. Acetaldehyde and propionaldehyde were channeled directly to the dehydrogenase, HsaG, without export to the bulk solvent where they were transformed to acyl-CoA in an NAD(+) and coenzyme A dependent reaction. HsaG is able to utilize aldehydes up to five carbons in length as substrates, with similar catalytic efficiencies. The HsaF-HsaG complex was crystallized and its structure was determined to a resolution of 1.93 Å. Substitution of serine 41 in HsaG with isoleucine or aspartate resulted in about 35-fold increase in Km for CoA but only 4-fold increase in Km dephospho-CoA, suggesting that this residue interacts with the 3'-ribose phosphate of CoA. A second protein annotated as a 4-hydroxy-2-oxopentanoic acid aldolase in M. tuberculosis (MhpE, Rv3469c) was expressed and purified, but was found to lack aldolase activity. Instead this enzyme was found to possess oxaloacetate decarboxylase activity, consistent with the conservation (with the 4-hydroxy-2-oxoacid aldolases) of residues involved in pyruvate enolate stabilization.

  15. Nitrite reductase activity of rat and human xanthine oxidase, xanthine dehydrogenase, and aldehyde oxidase: evaluation of their contribution to NO formation in vivo.

    PubMed

    Maia, Luisa B; Pereira, Vânia; Mira, Lurdes; Moura, José J G

    2015-01-27

    Nitrite is presently considered a NO "storage form" that can be made available, through its one-electron reduction, to maintain NO formation under hypoxia/anoxia. The molybdoenzymes xanthine oxidase/dehydrogenase (XO/XD) and aldehyde oxidase (AO) are two of the most promising mammalian nitrite reductases, and in this work, we characterized NO formation by rat and human XO/XD and AO. This is the first characterization of human enzymes, and our results support the employment of rat liver enzymes as suitable models of the human counterparts. A comprehensive kinetic characterization of the effect of pH on XO and AO-catalyzed nitrite reduction showed that the enzyme's specificity constant for nitrite increase 8-fold, while the Km(NO2(-)) decrease 6-fold, when the pH decreases from 7.4 to 6.3. These results demonstrate that the ability of XO/AO to trigger NO formation would be greatly enhanced under the acidic conditions characteristic of ischemia. The dioxygen inhibition was quantified, and the Ki(O2) values found (24.3-48.8 μM) suggest that in vivo NO formation would be fine-tuned by dioxygen availability. The potential in vivo relative physiological relevance of XO/XD/AO-dependent pathways of NO formation was evaluated using HepG2 and HMEC cell lines subjected to hypoxia. NO formation by the cells was found to be pH-, nitrite-, and dioxygen-dependent, and the relative contribution of XO/XD plus AO was found to be as high as 50%. Collectively, our results supported the possibility that XO/XD and AO can contribute to NO generation under hypoxia inside a living human cell. Furthermore, the molecular mechanism of XO/AO-catalyzed nitrite reduction was revised.

  16. [Enzyme kinetic glucose determination by the glucose dehydrogenase method. Enzyme kinetic substrate determination using competitive inhibitors, II (author's transl)].

    PubMed

    Müller-Matthesius, R

    1975-05-01

    The sensitivity of enzyme kinetic substrate determinations can be improved with the aid of competitive inhibitors. As an example, the determination of glucose dehydrogenase in the presence of potassium thiocyanate is described. The method has the advantage of rapid operation with satisfactory precision.

  17. Glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase: a unique bifunctional enzyme from Plasmodium falciparum.

    PubMed

    Jortzik, Esther; Mailu, Boniface M; Preuss, Janina; Fischer, Marina; Bode, Lars; Rahlfs, Stefan; Becker, Katja

    2011-06-15

    The survival of malaria parasites in human RBCs (red blood cells) depends on the pentose phosphate pathway, both in Plasmodium falciparum and its human host. G6PD (glucose-6-phosphate dehydrogenase) deficiency, the most common human enzyme deficiency, leads to a lack of NADPH in erythrocytes, and protects from malaria. In P. falciparum, G6PD is combined with the second enzyme of the pentose phosphate pathway to create a unique bifunctional enzyme named GluPho (glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase). In the present paper, we report for the first time the cloning, heterologous overexpression, purification and kinetic characterization of both enzymatic activities of full-length PfGluPho (P. falciparum GluPho), and demonstrate striking structural and functional differences with the human enzymes. Detailed kinetic analyses indicate that PfGluPho functions on the basis of a rapid equilibrium random Bi Bi mechanism, where the binding of the second substrate depends on the first substrate. We furthermore show that PfGluPho is inhibited by S-glutathionylation. The availability of recombinant PfGluPho and the major differences to hG6PD (human G6PD) facilitate studies on PfGluPho as an excellent drug target candidate in the search for new antimalarial drugs.

  18. Pharmacological activation of aldehyde dehydrogenase 2 promotes osteoblast differentiation via bone morphogenetic protein-2 and induces bone anabolic effect

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mittal, Monika; Pal, Subhashis; China, Shyamsundar

    Aldehyde dehydrogenases (ALDHs) are a family of enzymes involved in detoxifying aldehydes. Previously, we reported that an ALDH inhibitor, disulfiram caused bone loss in rats and among ALDHs, osteoblast expressed only ALDH2. Loss-of-function mutation in ALDH2 gene is reported to cause bone loss in humans which suggested its importance in skeletal homeostasis. We thus studied whether activating ALDH2 by N-(1, 3-benzodioxol-5-ylmethyl)-2, 6-dichlorobenzamide (alda-1) had osteogenic effect. We found that alda-1 increased and acetaldehyde decreased the differentiation of rat primary osteoblasts and expressions of ALDH2 and bone morphogenetic protein-2 (BMP-2). Silencing ALDH2 in osteoblasts abolished the alda-1 effects. Further, alda-1 attenuatedmore » the acetaldehyde-induced lipid-peroxidation and oxidative stress. BMP-2 is essential for bone regeneration and alda-1 increased its expression in osteoblasts. We then showed that alda-1 (40 mg/kg dose) augmented bone regeneration at the fracture site with concomitant increase in BMP-2 protein compared with control. The osteogenic dose (40 mg/kg) of alda-1 attained a bone marrow concentration that was stimulatory for osteoblast differentiation, suggesting that the tissue concentration of alda-1 matched its pharmacologic effect. In addition, alda-1 promoted modeling-directed bone growth and peak bone mass achievement, and increased bone mass in adult rats which reiterated its osteogenic effect. In osteopenic ovariectomized (OVX) rats, alda-1 reversed trabecular osteopenia with attendant increase in serum osteogenic marker (procollagen type I N-terminal peptide) and decrease in oxidative stress. Alda-1 has no effect on liver and kidney function. We conclude that activating ALDH2 by alda-1 had an osteoanabolic effect involving increased osteoblastic BMP-2 production and decreased OVX-induced oxidative stress. - Highlights: • Alda-1 induced osteoblast differentiation that involved upregulation of ALDH2 and BMP-2 • Alda

  19. Cloning of a cDNA encoding rat aldehyde dehydrogenase with high activity for retinal oxidation.

    PubMed

    Bhat, P V; Labrecque, J; Boutin, J M; Lacroix, A; Yoshida, A

    1995-12-12

    Retinoic acid (RA), an important regulator of cell differentiation, is biosynthesized from retinol via retinal by a two-step oxidation process. We previously reported the purification and partial amino acid (aa) sequence of a rat kidney aldehyde dehydrogenase (ALDH) isozyme that catalyzed the oxidation of 9-cis and all-trans retinal to corresponding RA with high efficiency [Labrecque et al. Biochem. J. 305 (1995) 681-684]. A rat kidney cDNA library was screened using a 291-bp PCR product generated from total kidney RNA using a pair of oligodeoxyribonucleotide primers matched with the aa sequence. The full-length rat kidney ALDH cDNA contains a 2315-bp (501 aa) open reading frame (ORF). The aa sequence of rat kidney ALDH is 89, 96 and 87% identical to that of the rat cytosolic ALDH, the mouse cytosolic ALDH and human cytosolic ALDH, respectively. Northern blot and RT-PCR-mediated analysis demonstrated that rat kidney ALDH is strongly expressed in kidney, lung, testis, intestine, stomach and trachea, but weakly in the liver.

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

    DOE PAGES

    Wright, Aaron T.; Magnaldo, Thierry; Sontag, Ryan L.; ...

    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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wright, Aaron T.; Magnaldo, Thierry; Sontag, Ryan L.

    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

  2. Enzymatic oxidation of 2-phenylethylamine to phenylacetic acid and 2-phenylethanol with special reference to the metabolism of its intermediate phenylacetaldehyde.

    PubMed

    Panoutsopoulos, Georgios I; Kouretas, Demetrios; Gounaris, Elias G; Beedham, Christine

    2004-12-01

    2-phenylethylamine is an endogenous constituent of the human brain and is implicated in cerebral transmission. This bioactive amine is also present in certain foodstuffs such as chocolate, cheese and wine and may cause undesirable side effects in susceptible individuals. Metabolism of 2-phenylethylamine to phenylacetaldehyde is catalysed by monoamine oxidase B but the oxidation to its acid is usually ascribed to aldehyde dehydrogenase and the contribution of aldehyde oxidase and xanthine oxidase, if any, is ignored. The objective of this study was to elucidate the role of the molybdenum hydroxylases, aldehyde oxidase and xanthine oxidase, in the metabolism of phenylacetaldehyde derived from its parent biogenic amine. Treatments of 2-phenylethylamine with monoamine oxidase were carried out for the production of phenylacetaldehyde, as well as treatments of synthetic or enzymatic-generated phenylacetaldehyde with aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase. The results indicated that phenylacetaldehyde is metabolised mainly to phenylacetic acid with lower concentrations of 2-phenylethanol by all three oxidising enzymes. Aldehyde dehydrogenase was the predominant enzyme involved in phenylacetaldehyde oxidation and thus it has a major role in 2-phenylethylamine metabolism with aldehyde oxidase playing a less prominent role. Xanthine oxidase does not contribute to the oxidation of phenylacetaldehyde due to low amounts being present in guinea pig. Thus aldehyde dehydrogenase is not the only enzyme oxidising xenobiotic and endobiotic aldehydes and the role of aldehyde oxidase in such reactions should not be ignored.

  3. 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

  4. Histamine H4-Receptors Inhibit Mast Cell Renin Release in Ischemia/Reperfusion via Protein Kinase Cε-Dependent Aldehyde Dehydrogenase Type-2 Activation

    PubMed Central

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

    2014-01-01

    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

  5. Direct Comparison of the Enzymatic Characteristics and Superoxide Production of the Four Aldehyde Oxidase Enzymes Present in Mouse.

    PubMed

    Kücükgöze, Gökhan; Terao, Mineko; Garattini, Enrico; Leimkühler, Silke

    2017-08-01

    Aldehyde oxidases (AOXs) are molybdoflavoenzymes with an important role in the metabolism and detoxification of heterocyclic compounds and aliphatic as well as aromatic aldehydes. The enzymes use oxygen as the terminal electron acceptor and produce reduced oxygen species during turnover. Four different enzymes, mAOX1, mAOX3, mAOX4, and mAOX2, which are the products of distinct genes, are present in the mouse. A direct and simultaneous comparison of the enzymatic properties and characteristics of the four enzymes has never been performed. In this report, the four catalytically active mAOX enzymes were purified after heterologous expression in Escherichia coli The kinetic parameters of the four mouse AOX enzymes were determined and compared with the use of six predicted substrates of physiologic and toxicological interest, i.e., retinaldehyde, N 1 -methylnicotinamide, pyridoxal, vanillin, 4-(dimethylamino)cinnamaldehyde ( p- DMAC), and salicylaldehyde. While retinaldehyde, vanillin, p- DMAC, and salycilaldehyde are efficient substrates for the four mouse AOX enzymes, N 1 -methylnicotinamide is not a substrate of mAOX1 or mAOX4, and pyridoxal is not metabolized by any of the purified enzymes. Overall, mAOX1, mAOX2, mAOX3, and mAOX4 are characterized by significantly different K M and k cat values for the active substrates. The four mouse AOXs are also characterized by quantitative differences in their ability to produce superoxide radicals. With respect to this last point, mAOX2 is the enzyme generating the largest rate of superoxide radicals of around 40% in relation to moles of substrate converted, and mAOX1, the homolog to the human enzyme, produces a rate of approximately 30% of superoxide radicals with the same substrate. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  6. Differential signatures of bacterial and mammalian IMP dehydrogenase enzymes.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, R.; Evans, G.; Rotella, F.

    1999-06-01

    IMP dehydrogenase (IMPDH) is an essential enzyme of de novo guanine nucleotide synthesis. IMPDH inhibitors have clinical utility as antiviral, anticancer or immunosuppressive agents. The essential nature of this enzyme suggests its therapeutic applications may be extended to the development of antimicrobial agents. Bacterial IMPDH enzymes show bio- chemical and kinetic characteristics that are different than the mammalian IMPDH enzymes, suggesting IMPDH may be an attractive target for the development of antimicrobial agents. We suggest that the biochemical and kinetic differences between bacterial and mammalian enzymes are a consequence of the variance of specific, identifiable amino acid residues. Identification ofmore » these residues or combination of residues that impart this mammalian or bacterial enzyme signature is a prerequisite for the rational identification of agents that specifically target the bacterial enzyme. We used sequence alignments of IMPDH proteins to identify sequence signatures associated with bacterial or eukaryotic IMPDH enzymes. These selections were further refined to discern those likely to have a role in catalysis using information derived from the bacterial and mammalian IMPDH crystal structures and site-specific mutagenesis. Candidate bacterial sequence signatures identified by this process include regions involved in subunit interactions, the active site flap and the NAD binding region. Analysis of sequence alignments in these regions indicates a pattern of catalytic residues conserved in all enzymes and a secondary pattern of amino acid conservation associated with the major phylogenetic groups. Elucidation of the basis for this mammalian/bacterial IMPDH signature will provide insight into the catalytic mechanism of this enzyme and the foundation for the development of highly specific inhibitors.« less

  7. Proteomic Analysis of Mitochondria-Enriched Fraction Isolated from the Frontal Cortex and Hippocampus of Apolipoprotein E Knockout Mice Treated with Alda-1, an Activator of Mitochondrial Aldehyde Dehydrogenase (ALDH2).

    PubMed

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

    2017-02-17

    The role of different genotypes of apolipoprotein E (apoE) in the etiology of Alzheimer's disease is widely recognized. It has been shown that altered functioning of apoE may promote 4-hydroxynonenal modification of mitochondrial proteins, which may result in mitochondrial dysfunction, aggravation of oxidative stress, and neurodegeneration. Mitochondrial aldehyde dehydrogenase (ALDH2) is an enzyme considered to perform protective function in mitochondria by the detoxification of the end products of lipid peroxidation, such as 4-hydroxynonenal and other reactive aldehydes. The goal of our study was to apply a differential proteomics approach in concert with molecular and morphological techniques to elucidate the changes in the frontal cortex and hippocampus of apolipoprotein E knockout (apoE -/- ) mice upon treatment with Alda-1-a small molecular weight activator of ALDH2. Despite the lack of significant morphological changes in the brain of apoE -/- mice as compared to age-matched wild type animals, the proteomic and molecular approach revealed many changes in the expression of genes and proteins, indicating the impairment of energy metabolism, neuroplasticity, and neurogenesis in brains of apoE -/- mice. Importantly, prolonged treatment of apoE -/- mice with Alda-1 led to the beneficial changes in the expression of genes and proteins related to neuroplasticity and mitochondrial function. The pattern of alterations implies mitoprotective action of Alda-1, however, the accurate functional consequences of the revealed changes require further research.

  8. 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

  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. Degradation of glyceraldehyde-3-phosphate dehydrogenase triggered by 4-hydroxy-2-nonenal and 4-hydroxy-2-hexenal.

    PubMed

    Tsuchiya, Yukihiro; Yamaguchi, Mitsune; Chikuma, Toshiyuki; Hojo, Hiroshi

    2005-06-15

    Lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE) may be responsible for various pathophysiological events under oxidative stress, since they injure cellular components such as proteins and DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a key enzyme of glycolysis and has been reported to be a multifunctional enzyme, is one of the enzymes inhibited by HNE. Previous studies showed that GAPDH is degraded when incubated with acetylleucine chloromethyl ketone (ALCK), resulting in the liberation of a 23-kDa fragment. In this study, we examined whether GAPDH incubated with HNE or other aldehydes of lipid peroxidation products are degraded similarly to that with ALCK. The U937 cell extract was incubated with these aldehydes at 37 degrees C and analyzed by Western blotting using anti-GAPDH antibodies. Incubation with HNE or 4-hydroxy-2-hexenal (HHE) decreased GAPDH activity and GAPDH protein level, and increased the 23-kDa fragment, in time- and dose-dependent manners, but that with other aldehydes did not. Gel filtration using the Superose 6 showed that the GAPDH-degrading activity was eluted in higher molecular fractions than proteasome activity. The enzyme activity was detected at the basic range of pH and inhibited by serine protease inhibitors, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, but not by other protease inhibitors including a proteasome inhibitor, MG-132, and a tripeptidyl peptidase II (TPP II) inhibitor, AAF-CMK. These results suggest that GAPDH modified by HNE and HHE is degraded by a giant serine protease, releasing the 23-kDa fragment, not by proteasome or TPP II.

  11. 5-ethynyl-2(1H)-pyrimidinone: aldehyde oxidase-activation to 5-ethynyluracil, a mechanism-based inactivator of dihydropyrimidine dehydrogenase.

    PubMed

    Porter, D J; Harrington, J A; Almond, M R; Lowen, G T; Zimmerman, T P; Spector, T

    1994-03-29

    5-Ethynyluracil is a potent mechanism-based inactivator of dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) in vitro (Porter et al., J Biol Chem 267: 5236-5242, 1992) and in vivo (Spector et al., Biochem Pharmacol, 46: 2243-2248, 1993. 5-Ethynyl-2(1H)-pyrimidinone was rapidly oxidized to 5-ethynyluracil by aldehyde oxidase. The substrate efficiency (kcat/Km) was 60-fold greater than that for N-methylnicotinamide. In contrast, xanthine oxidase oxidized 5-ethynyl-2(1H)-pyrimidinone to 5-ethynyluracil with a substrate efficiency that was only 0.02% that of xanthine. Because 5-ethynyl-2(1H)-pyrimidinone did not itself inactivate purified DPD in vitro and aldehyde oxidase is predominately found in liver, we hypothesized that 5-ethynyl-2(1H)-pyrimidinone could be a liver-specific inactivator of DPD. We found that 5-ethynyl-2(1H)-pyrimidinone administered orally to rats at 2 micrograms/kg inactivated DPD in all tissues studied. Although 5-ethynyl-2(1H)-pyrimidinone produced slightly less inactivation than 5-ethynyluracil, the two compounds showed fairly similar patterns of inactivation of DPD in these tissues. At doses of 20 micrograms/kg, however, 5-ethynyl-2-pyrimidinone and 5-ethynyluracil produced equivalent inactivation of DPD. Thus, 5-ethynyl-2(1H)-pyrimidinone appeared to be an efficient, but not highly liver-selective prodrug of 5-ethynyluracil.

  12. Initial steps of the peroxidase-catalyzed polymerization of coniferyl alcohol and/or sinapyl aldehyde: capillary zone electrophoresis study of pH effect.

    PubMed

    Fournand, David; Cathala, Bernard; Lapierre, Catherine

    2003-01-01

    Capillary zone electrophoresis has been used to monitor the first steps of the dehydrogenative polymerization of coniferyl alcohol, sinapyl aldehyde, or a mixture of both, catalyzed by the horseradish peroxidase (HRP)-H(2)O(2) system. When coniferyl alcohol was the unique HRP substrate, three major dimers were observed (beta-5, beta-beta, and beta-O-4 interunit linkages) and their initial formation velocity as well as their relative abundance varied with pH. The beta-O-4 interunit linkage was thus slightly favored at lower pH values. In contrast, sinapyl aldehyde turned out to be a very poor substrate for HRP except in basic conditions (pH 8). The major dimer observed was the beta,beta'-di-sinapyl aldehyde, a red-brown exhibiting compound which might partly participate in the red coloration usually observed in cinnamyl alcohol dehydrogenase-deficient angiosperms. Finally, when a mixture of coniferyl alcohol and sinapyl aldehyde was used, it looked as if sinapyl aldehyde became a very good substrate for HRP. Indeed, coniferyl alcohol turned out to serve as a redox mediator (i.e. "shuttle oxidant") for the sinapyl aldehyde incorporation in the lignin-like polymer. This means that in particular conditions the specificity of oxidative enzymes might not hinder the incorporation of poor substrates into the growing lignin polymer.

  13. Biochemical characterization of a recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius.

    PubMed

    Pennacchio, Angela; Giordano, Assunta; Pucci, Biagio; Rossi, Mosè; Raia, Carlo A

    2010-03-01

    The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75 degrees C and a 30-min half-inactivation temperature of ~90 degrees C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and alpha-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of alpha-methyl and alpha-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.

  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. 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. © 2016 Wiley Periodicals, Inc.

  16. Microsphere coated substrate containing reactive aldehyde groups

    NASA Technical Reports Server (NTRS)

    Yen, Richard C. K. (Inventor); Rembaum, Alan (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.

  17. The 2-oxoacid dehydrogenase multi-enzyme complex of the archaeon Thermoplasma acidophilum - recombinant expression, assembly and characterization.

    PubMed

    Heath, Caroline; Posner, Mareike G; Aass, Hans C; Upadhyay, Abhishek; Scott, David J; Hough, David W; Danson, Michael J

    2007-10-01

    The aerobic archaea possess four closely spaced, adjacent genes that encode proteins showing significant sequence identities with the bacterial and eukaryal components comprising the 2-oxoacid dehydrogenase multi-enzyme complexes. However, catalytic activities of such complexes have never been detected in the archaea, although 2-oxoacid ferredoxin oxidoreductases that catalyze the equivalent metabolic reactions are present. In the current paper, we clone and express the four genes from the thermophilic archaeon, Thermoplasma acidophilum, and demonstrate that the recombinant enzymes are active and assemble into a large (M(r) = 5 x 10(6)) multi-enzyme complex. The post-translational incorporation of lipoic acid into the transacylase component of the complex is demonstrated, as is the assembly of this enzyme into a 24-mer core to which the other components bind to give the functional multi-enzyme system. This assembled complex is shown to catalyze the oxidative decarboxylation of branched-chain 2-oxoacids and pyruvate to their corresponding acyl-CoA derivatives. Our data constitute the first proof that the archaea possess a functional 2-oxoacid dehydrogenase complex.

  18. High Aldehyde Dehydrogenase Activity Identifies a Subset of Human Mesenchymal Stromal Cells with Vascular Regenerative Potential.

    PubMed

    Sherman, Stephen E; Kuljanin, Miljan; Cooper, Tyler T; Putman, David M; Lajoie, Gilles A; Hess, David A

    2017-06-01

    During culture expansion, multipotent mesenchymal stromal cells (MSCs) differentially express aldehyde dehydrogenase (ALDH), an intracellular detoxification enzyme that protects long-lived cells against oxidative stress. Thus, MSC selection based on ALDH-activity may be used to reduce heterogeneity and distinguish MSC subsets with improved regenerative potency. After expansion of human bone marrow-derived MSCs, cell progeny was purified based on low versus high ALDH-activity (ALDH hi ) by fluorescence-activated cell sorting, and each subset was compared for multipotent stromal and provascular regenerative functions. Both ALDH l ° and ALDH hi MSC subsets demonstrated similar expression of stromal cell (>95% CD73 + , CD90 + , CD105 + ) and pericyte (>95% CD146 + ) surface markers and showed multipotent differentiation into bone, cartilage, and adipose cells in vitro. Conditioned media (CDM) generated by ALDH hi MSCs demonstrated a potent proliferative and prosurvival effect on human microvascular endothelial cells (HMVECs) under serum-free conditions and augmented HMVEC tube-forming capacity in growth factor-reduced matrices. After subcutaneous transplantation within directed in vivo angiogenesis assay implants into immunodeficient mice, ALDH hi MSC or CDM produced by ALDH hi MSC significantly augmented murine vascular cell recruitment and perfused vessel infiltration compared with ALDH l ° MSC. Although both subsets demonstrated strikingly similar mRNA expression patterns, quantitative proteomic analyses performed on subset-specific CDM revealed the ALDH hi MSC subset uniquely secreted multiple proangiogenic cytokines (vascular endothelial growth factor beta, platelet derived growth factor alpha, and angiogenin) and actively produced multiple factors with chemoattractant (transforming growth factor-β, C-X-C motif chemokine ligand 1, 2, and 3 (GRO), C-C motif chemokine ligand 5 (RANTES), monocyte chemotactic protein 1 (MCP-1), interleukin [IL]-6, IL-8) and matrix

  19. Demonstration of 3 alpha(17 beta)-hydroxysteroid dehydrogenase distinct from 3 alpha-hydroxysteroid dehydrogenase in hamster liver.

    PubMed Central

    Ohmura, M; Hara, A; Nakagawa, M; Sawada, H

    1990-01-01

    NAD(+)-linked and NADP(+)-linked 3 alpha-hydroxysteroid dehydrogenases were purified to homogeneity from hamster liver cytosol. The two monomeric enzymes, although having similar molecular masses of 38,000, differed from each other in pI values, activation energy and heat stability. The two proteins also gave different fragmentation patterns by gel electrophoresis after digestion with protease. The NADP(+)-linked enzyme catalysed the oxidoreduction of various 3 alpha-hydroxysteroids, whereas the NAD(+)-linked enzyme oxidized the 3 alpha-hydroxy group of pregnanes and some bile acids, and the 17 beta-hydroxy group of testosterone and androstanes. The thermal stabilities of the 3 alpha- and 17 beta-hydroxysteroid dehydrogenase activities of the NAD(+)-linked enzyme were identical, and the two enzyme activities were inhibited by mixing 17 beta- and 3 alpha-hydroxysteroid substrates, respectively. Medroxyprogesterone acetate, hexoestrol and 3 beta-hydroxysteroids competitively inhibited 3 alpha- and 17 beta-hydroxysteroid dehydrogenase activities of the enzyme. These results show that hamster liver contains a 3 alpha(17 beta)-hydroxysteroid dehydrogenase structurally and functionally distinct from 3 alpha-hydroxysteroid dehydrogenase. Images Fig. 1. Fig. 2. PMID:2317205

  20. RDH13L, an enzyme responsible for the aldehyde-alcohol redox coupling reaction (AL-OL coupling reaction) to supply 11-cis retinal in the carp cone retinoid cycle.

    PubMed

    Sato, Shinya; Miyazono, Sadaharu; Tachibanaki, Shuji; Kawamura, Satoru

    2015-01-30

    Cone photoreceptors require effective pigment regeneration mechanisms to maintain their sensitivity in the light. Our previous studies in carp cones suggested the presence of an unconventional and very effective mechanism to produce 11-cis retinal, the necessary component in pigment regeneration. In this reaction (aldehyde-alcohol redox coupling reaction, AL-OL coupling reaction), formation of 11-cis retinal, i.e. oxidation of 11-cis retinol is coupled to reduction of an aldehyde at a 1:1 molar ratio without exogenous NADP(H) which is usually required in this kind of reaction. Here, we identified carp retinol dehydrogenase 13-like (RDH13L) as an enzyme catalyzing the AL-OL coupling reaction. RDH13L was partially purified from purified carp cones, identified as a candidate protein, and its AL-OL coupling activity was confirmed using recombinant RDH13L. We further examined the substrate specificity, subcellular localization, and expression level of RDH13L. Based on these results, we concluded that RDH13L contributes to a significant part, but not all, of the AL-OL coupling activity in carp cones. RDH13L contained tightly bound NADP(+) which presumably functions as a cofactor in the reaction. Mouse RDH14, a mouse homolog of carp RDH13L, also showed the AL-OL coupling activity. Interestingly, although carp cone membranes, carp RDH13L and mouse RDH14 all showed the coupling activity at 15-37 °C, they also showed a conventional NADP(+)-dependent 11-cis retinol oxidation activity above 25 °C without addition of aldehydes. This dual mechanism of 11-cis retinal synthesis attained by carp RDH13L and mouse RDH14 probably contribute to effective pigment regeneration in cones that function in the light. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Empirical evaluation of a virtual laboratory approach to teach lactate dehydrogenase enzyme kinetics.

    PubMed

    Booth, Christine; Cheluvappa, Rajkumar; Bellinson, Zack; Maguire, Danni; Zimitat, Craig; Abraham, Joyce; Eri, Rajaraman

    2016-06-01

    Personalised instruction is increasingly recognised as crucial for efficacious learning today. Our seminal work delineates and elaborates on the principles, development and implementation of a specially-designed adaptive, virtual laboratory. We strived to teach laboratory skills associated with lactate dehydrogenase (LDH) enzyme kinetics to 2nd-year biochemistry students using our adaptive learning platform. Pertinent specific aims were to:(1)design/implement a web-based lesson to teach lactate dehydrogenase(LDH) enzyme kinetics to 2nd-year biochemistry students(2)determine its efficacious in improving students' comprehension of enzyme kinetics(3)assess their perception of its usefulness/manageability(vLab versus Conventional Tutorial). Our tools were designed using HTML5 technology. We hosted the program on an adaptive e-learning platform (AeLP). Provisions were made to interactively impart informed laboratory skills associated with measuring LDH enzyme kinetics. A series of e-learning methods were created. Tutorials were generated for interactive teaching and assessment. The learning outcomes herein were on par with that from a conventional classroom tutorial. Student feedback showed that the majority of students found the vLab learning experience "valuable"; and the vLab format/interface "well-designed". However, there were a few technical issues with the 1st roll-out of the platform. Our pioneering effort resulted in productive learning with the vLab, with parity with that from a conventional tutorial. Our contingent discussion emphasises not only the cornerstone advantages, but also the shortcomings of the AeLP method utilised. We conclude with an astute analysis of possible extensions and applications of our methodology.

  2. Interaction of the SPG21 protein ACP33/maspardin with the aldehyde dehydrogenase ALDH16A1

    PubMed Central

    2017-01-01

    Mast syndrome (SPG21) is an autosomal-recessive complicated form of hereditary spastic paraplegia characterized by dementia, thin corpus callosum, white matter abnormalities, and cerebellar and extrapyramidal signs in addition to spastic paraparesis. A nucleotide insertion resulting in premature truncation of the SPG21 gene product acidic cluster protein 33 (ACP33)/maspardin underlies this disorder, likely causing loss of protein function. However, little is known about the function of maspardin. Here, we report that maspardin localizes prominently to cytoplasm as well as to membranes, possibly at trans-Golgi network/late endosomal compartments. Immunoprecipitation of maspardin with identification of coprecipitating proteins by mass spectrometry revealed the aldehyde dehydrogenase ALDH16A1 as an interacting protein. This interaction was confirmed using overexpressed proteins as well as by fusion protein pull down experiments, and these proteins colocalized in cells. Further studies of the function of ALDH16A1 and the role of the maspardin–ALDH16A1 interaction in neuronal cells may clarify the cellular pathogenesis of Mast syndrome. PMID:19184135

  3. Alcohol and aldehyde dehydrogenase polymorphisms and a new strategy for prevention and screening for cancer in the upper aerodigestive tract in East Asians.

    PubMed

    Yokoyama, Akira; Omori, Tai; Yokoyama, Tetsuji

    2010-01-01

    The ethanol in alcoholic beverages and the acetaldehyde associated with alcohol consumption are Group 1 human carcinogens (WHO, International Agency for Research on Cancer). The combination of alcohol consumption, tobacco smoking, the inactive heterozygous aldehyde dehydrogenase-2 genotype (ALDH2*1/*2) and the less-active homozygous alcohol dehydrogenase-1B genotype (ADH1B*1/*1) increases the risk of squamous cell carcinoma (SCC) in the upper aerodigestive tract (UADT) in a multiplicative fashion in East Asians. In addition to being exposed to locally high levels of ethanol, the UADT is exposed to a very high concentration of acetaldehyde from a variety of sources, including that as an ingredient of alcoholic beverages per se and that found in tobacco smoke; acetaldehyde is also produced by salivary microorganisms and mucosal enzymes and is present as blood acetaldehyde. The inefficient degradation of acetaldehyde by weakly expressed ALDH2 in the UADT may be cri! tical to the local accumulation of acetaldehyde, especially in ALDH2*1/*2 carriers. ADH1B*1/*1 carriers tend to experience less intense alcohol flushing and are highly susceptible to heavy drinking and alcoholism. Heavy drinking by persons with the less-active ADH1B*1/*1 leads to longer exposure of the UADT to salivary ethanol and acetaldehyde. The ALDH2*1/*2 genotype is a very strong predictor of synchronous and metachronous multiple SCCs in the UADT. High red cell mean corpuscular volume (MCV), esophageal dysplasia, and melanosis in the UADT, all of which are frequently found in ALDH2*1/*2 drinkers, are useful for identifying high-risk individuals. We invented a simple flushing questionnaire that enables prediction of the ALDH2 phenotype. New health appraisal models that include ALDH2 genotype, the simple flushing questionnaire, or MCV are powerful tools for devising a new strategy for prevention and screening for UADT cancer in East Asians.

  4. Limonoate dehydrogenase from Arthrobacter globiformis: the native enzyme and its N-terminal sequence.

    PubMed

    Suhayda, C G; Omura, M; Hasegawa, S

    1995-09-01

    Bitter limonoids in citrus juice lower the quality and value of commercial juices. Limonoate dehydrogenase converts the precursor of bitter limonin, limonoate A-ring lactone, to nonbitter 17-dehydrolimonoate A-ring lactone. This enzyme was isolated from Arthrobacter globiformis cells by a combination of ammonium sulfate fractionation, Cibacron Blue affinity chromatography and DEAE ion exchange HPLC. Using this protocol a 428-fold purification of the enzyme was obtained. Gel filtration HPLC indicated a M(r) of 118,000 for the native enzyme. SDS-PAGE indicated an individual subunit M(r) of 31,000. N-Terminal sequencing of the protein provided a sequence of the first 16 amino acid residues. Since LDH activity in citrus is very low, cloning the gene for this bacterial enzyme into citrus trees should enhance the natural debittering mechanism in citrus fruit.

  5. Metabolism of MRX-I, a novel antibacterial oxazolidinone, in humans: the oxidative ring opening of 2,3-Dihydropyridin-4-one catalyzed by non-P450 enzymes.

    PubMed

    Meng, Jian; Zhong, Dafang; Li, Liang; Yuan, Zhengyu; Yuan, Hong; Xie, Cen; Zhou, Jialan; Li, Chen; Gordeev, Mikhail Fedorovich; Liu, Jinqian; Chen, Xiaoyan

    2015-05-01

    MRX-I is an analog of linezolid containing a 2,3-dihydropyridin-4-one (DHPO) ring rather than a morpholine ring. Our objectives were to characterize the major metabolic pathways of MRX-I in humans and clarify the mechanism underlying the oxidative ring opening of DHPO. After an oral dose of MRX-I (600 mg), nine metabolites were identified in humans. The principal metabolic pathway proposed involved the DHPO ring opening, generating the main metabolites in the plasma and urine: the hydroxyethyl amino propionic acid metabolite MRX445-1 and the carboxymethyl amino propionic acid metabolite MRX459. An in vitro phenotyping study demonstrated that multiple non-cytochrome P450 enzymes are involved in the formation of MRX445-1 and MRX459, including flavin-containing monooxygenase 5, short-chain dehydrogenase/reductase, aldehyde ketone reductase, and aldehyde dehydrogenase (ALDH). H2 (18)O experiments revealed that two (18)O atoms are incorporated into MRX445-1, one in the carboxyethyl group and the other in the hydroxyl group, and three (18)O atoms are incorporated into MRX459, two in the carboxymethyl group and one in the hydroxyl group. Based on these results, the mechanism proposed for the DHPO ring opening involves the metabolism of MRX-I via FMO5-mediated Baeyer-Villiger oxidation to an enol lactone, hydrolysis to an enol, and enol-aldehyde tautomerism to an aldehyde. The aldehyde is reduced by short-chain dehydrogenase/reductase, aldehyde ketone reductase, ALDH to MRX445-1, or oxidized by ALDH to MRX459. Our study suggests that few clinical adverse drug-drug interactions should be anticipated between MRX-I and cytochrome P450 inhibitors or inducers. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  7. Optimizing Immobilized Enzyme Performance in Cell-Free Environments to Produce Liquid Fuels

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Belfort, Georges; Grimaldi, Joseph J.

    2015-01-27

    Limitations on biofuel production using cell culture (Escherichia coli, Clostridium, Saccharomyces cerevisiae, brown microalgae, blue-green algae and others) include low product (alcohol) concentrations (≤0.2 vol%) due to feedback inhibition, instability of cells, and lack of economical product recovery processes. To overcome these challenges, an alternate simplified biofuel production scheme was tested based on a cell-free immobilized enzyme system. Using this cell free system, we were able to obtain about 2.6 times higher concentrations of iso-butanol using our non-optimized system as compared with live cell systems. This process involved two steps: (i) converts acid to aldehyde using keto-acid decarboxylase (KdcA), andmore » (ii) produces alcohol from aldehyde using alcohol dehydrogenase (ADH) with a cofactor (NADH) conversion from inexpensive formate using a third enzyme, formate dehydrogenase (FDH). To increase stability and conversion efficiency with easy separations, the first two enzymes were immobilized onto methacrylate resin. Fusion proteins of labile KdcA (fKdcA) were expressed to stabilize the covalently immobilized KdcA. Covalently immobilized ADH exhibited long-term stability and efficient conversion of aldehyde to alcohol over multiple batch cycles without fusions. High conversion rates and low protein leaching were achieved by covalent immobilization of enzymes on methacrylate resin. The complete reaction scheme was demonstrated by immobilizing both ADH and fKdcA and using FDH free in solution. The new system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.5 % (v/v). Further increases in titer will require continuous removal of the isobutanol using our novel brush membrane system that exhibits a 1.5 fold increase in the separation factor of isobutanol from water versus that obtained for commercial silicone rubber membranes. These bio-inspired brush membranes are based

  8. IMP dehydrogenase. II. Purification and properties of the enzyme from Yoshida sarcoma ascites tumor cells.

    PubMed

    Okada, M; Shimura, K; Shiraki, H; Nakagawa, H

    1983-11-01

    The preceding paper showed that IMP dehydrogenase [IMP:NAD+ oxidoreductase, EC 1.2.1.14] tended to form a precipitable complex(es) through ionic and hydrophobic interactions. On the basis of these observations, a method was developed for purification of IMP dehydrogenase from Yoshida sarcoma ascites cells. On SDS-polyacrylamide gel electrophoresis, the purified preparation (1.19 U/mg protein) appeared homogeneous and its minimum molecular weight was estimated to be 68K daltons. Amino acid analyses indicated a subunit molecular weight of 68,042. Molecular sieve chromatography in the presence of 10% (NH4)2SO4 showed that the molecular weight of the native enzyme was 127K daltons. These values indicate that the native enzyme is composed of two identical subunits. However, the purified enzyme gave 4 protein bands on polyacrylamide gel electrophoresis under non-denaturing conditions, and appeared as a single fraction in the vicinity of the void volume on Ultrogel AcA 34 column chromatography at low salt concentration, indicating that its molecular weight exceeded 200K daltons. These findings indicate that the enzyme tends to aggregate owing to its own physicochemical characteristics. The Km values for IMP and NAD were calculated to be 12 and 25 microM, respectively, and the Ki values for XMP, GMP, and AMP to be 109, 130, and 854 microM, respectively. The purified enzyme showed full activity in the presence of K+, and K+ could be partially replaced by Na+. PCMB inactivated the enzyme, but the activity was completely restored by the addition of DTT. Cl-IMP also inactivated the enzyme and IMP prevented this inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Enzyme markers in inbred rat strains: genetics of new markers and strain profiles.

    PubMed

    Adams, M; Baverstock, P R; Watts, C H; Gutman, G A

    1984-08-01

    Twenty-six inbred strains of the laboratory rat (Rattus norvegicus) were examined for electrophoretic variation at an estimated 97 genetic loci. In addition to previously documented markers, variation was observed for the enzymes aconitase, aldehyde dehydrogenase, and alkaline phosphatase. The genetic basis of these markers (Acon-1, Ahd-2, and Akp-1) was confirmed. Linkage analysis between 35 pairwise comparisons revealed that the markers Fh-1 and Pep-3 are linked. The strain profiles of the 25 inbred strains at 11 electrophoretic markers are given.

  10. Reassessment of the transhydrogenase/malate shunt pathway in Clostridium thermocellum ATCC 27405 through kinetic characterization of malic enzyme and malate dehydrogenase.

    PubMed

    Taillefer, M; Rydzak, T; Levin, D B; Oresnik, I J; Sparling, R

    2015-04-01

    Clostridium thermocellum produces ethanol as one of its major end products from direct fermentation of cellulosic biomass. Therefore, it is viewed as an attractive model for the production of biofuels via consolidated bioprocessing. However, a better understanding of the metabolic pathways, along with their putative regulation, could lead to improved strategies for increasing the production of ethanol. In the absence of an annotated pyruvate kinase in the genome, alternate means of generating pyruvate have been sought. Previous proteomic and transcriptomic work detected high levels of a malate dehydrogenase and malic enzyme, which may be used as part of a malate shunt for the generation of pyruvate from phosphoenolpyruvate. The purification and characterization of the malate dehydrogenase and malic enzyme are described in order to elucidate their putative roles in malate shunt and their potential role in C. thermocellum metabolism. The malate dehydrogenase catalyzed the reduction of oxaloacetate to malate utilizing NADH or NADPH with a kcat of 45.8 s(-1) or 14.9 s(-1), respectively, resulting in a 12-fold increase in catalytic efficiency when using NADH over NADPH. The malic enzyme displayed reversible malate decarboxylation activity with a kcat of 520.8 s(-1). The malic enzyme used NADP(+) as a cofactor along with NH4 (+) and Mn(2+) as activators. Pyrophosphate was found to be a potent inhibitor of malic enzyme activity, with a Ki of 0.036 mM. We propose a putative regulatory mechanism of the malate shunt by pyrophosphate and NH4 (+) based on the characterization of the malate dehydrogenase and malic enzyme. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. 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.

  12. Biogenic Aldehydes as Therapeutic Targets for Cardiovascular Disease

    PubMed Central

    Nelson, Margaret-Ann M; Baba, Shahid P; Andersonc, Ethan J

    2017-01-01

    Aldehydes are continuously formed in biological systems through enzyme-dependent and spontaneous oxidation of lipids, glucose, and primary amines. These highly reactive, biogenic electrophiles can become toxic via covalent modification of proteins, lipids and DNA. Thus, agents that scavenge aldehydes through conjugation have therapeutic value for a number of major cardiovascular diseases. Several commonly-prescribed drugs (e.g., hydralazine) have been shown to have potent aldehyde-conjugating properties which may contribute to their beneficial effects. Herein, we briefly describe the major sources and toxicities of biogenic aldehydes in cardiovascular system, and provide an overview of drugs that are known to have aldehyde-conjugating effects. Some compounds of phytochemical origin, and histidyl-dipeptides with emerging therapeutic value in this area are also discussed. PMID:28528297

  13. Alcohol and aldehyde dehydrogenase gene polymorphisms and oropharyngolaryngeal, esophageal and stomach cancers in Japanese alcoholics.

    PubMed

    Yokoyama, A; Muramatsu, T; Omori, T; Yokoyama, T; Matsushita, S; Higuchi, S; Maruyama, K; Ishii, H

    2001-03-01

    Alcohol dehydrogenase-2 (ADH2) and aldehyde dehydrogenase-2 (ALDH2) gene polymorphisms play roles in ethanol metabolism, drinking behavior and esophageal carcinogenesis in Japanese; however, the combined influence of ADH2 and ALDH2 genotypes on other aerodigestive tract cancers have not been investigated. ADH2/ALDH2 genotyping was performed on lymphocyte DNA samples from Japanese alcoholic men (526 cancer-free; 159 with solitary or multiple aerodigestive tract cancers, including 33 oropharyngolaryngeal, 112 esophageal, 38 stomach and 22 multiple primary cancers in two or three organs). After adjustment for age, drinking and smoking habits, and ADH2/ALDH2 genotypes, the presence of either ADH2*1/2*1 or ALDH2*1/2*2 significantly increased the risk for oropharyngolaryngeal cancer [odds ratios (ORs), 6.68 with ADH2*1/2*1 and 18.52 with ALDH2*1/2*2] and esophageal cancer (ORs, 2.64 and 13.50, respectively). For patients with both ADH2*1/2*1 and ALDH2*1/2*2, the risks for oropharyngolaryngeal and esophageal cancers were enhanced in a multiplicative fashion (OR = 121.77 and 40.40, respectively). A positive association with ALDH2*1/2*2 alone was observed for stomach cancer patients who also had oropharyngolaryngeal and/or esophageal cancer (OR = 110.58), but it was not observed for those with stomach cancer alone. Furthermore, in the presence of ALDH2*1/2*2, the risks for multiple intra-esophageal cancers (OR = 3.43) and for esophageal cancer with oropharyngolaryngeal and/or stomach cancer (OR = 3.95) were higher than the risks for solitary intra-esophageal cancer and for esophageal cancer alone, but these tendencies were not observed for ADH2*1/2*1 genotype. Alcoholics' population attributable risks due to ADH2/ALDH2 polymorphisms were estimated to be 82.0% for oropharyngolaryngeal cancer and 63.9% for esophageal cancer.

  14. Aldehyde dehydrogenase 2 polymorphism for development to hepatocellular carcinoma in East Asian alcoholic liver cirrhosis.

    PubMed

    Abe, Hiroshi; Aida, Yuta; Seki, Nobuyoshi; Sugita, Tomonori; Tomita, Yoichi; Nagano, Tomohisa; Itagaki, Munenori; Sutoh, Satoshi; Nagatsuma, Keisuke; Itoh, Kyoko; Matsuura, Tomokazu; Aizawa, Yoshio

    2015-09-01

    We aimed to clarify the influences of aldehyde dehydrogenase 2 (ALDH2), alcohol dehydrogenase 1B (ADH1B) polymorphisms, and ethanol consumption profile to hepatocellular carcinoma (HCC) development in alcoholic liver cirrhosis without chronic hepatitis B and C virus infection (non-B non-C). Of 236 freshly diagnosed non-B non-C alcoholic liver cirrhosis patients, 67 were diagnosed as HCC and the remaining 169 as not having HCC. The relationship between the genetic polymorphisms and development to HCC were evaluated in well-matched patients with HCC (HCC group, n = 67) and without HCC (non-HCC group, n = 67) using propensity scores in age, sex, and prevalence of diabetes mellitus. Daily amount of ethanol consumption was significantly lower (P = 0.005), and consumptive period was significantly longer (P = 0.003) in HCC group than non-HCC group. Of 134 well-matched patients, 113 (84.3%) had ALDH2*1/*1 genotype and 21 (15.7%) had ALDH2*1/*2 genotype. In HCC development, consumptive long period (P = 0.007) and carrying ALDH2*1/*2 genotype (P = 0.026) were identified as significant factors independently participated, while there was no relation to ADH1B polymorphism. In addition, consumptive period was significantly longer in HCC group than non-HCC group in ALDH2*1/*1 genotype patients (P = 0.0005), while there was no difference in profile of ethanol consumption in ALDH2*1/*2 genotype patients. Among HCC group, daily (P = 3.78 × 10(-6) ) and cumulative amount (P = 4.89 × 10(-6) ) of ethanol consumption were significantly higher in ALDH2*1/*1 genotype patients than ALDH2*1/*2 genotype patients. In alcoholic liver cirrhosis, investigations of ALDH2 polymorphism and ethanol consumption profile are useful for prediction of HCC development. © 2015 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

  15. 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

  16. Biogenic Aldehydes as Therapeutic Targets for Cardiovascular Disease.

    PubMed

    Nelson, Margaret-Ann M; Baba, Shahid P; Anderson, Ethan J

    2017-04-01

    Aldehydes are continuously formed in biological systems through enzyme-dependent and spontaneous oxidation of lipids, glucose, and primary amines. These highly reactive, biogenic electrophiles can become toxic via covalent modification of proteins, lipids and DNA. Thus, agents that scavenge aldehydes through conjugation have therapeutic value for a number of major cardiovascular diseases. Several commonly-prescribed drugs (e.g., hydralazine) have been shown to have potent aldehyde-conjugating properties which may contribute to their beneficial effects. Herein, we briefly describe the major sources and toxicities of biogenic aldehydes in cardiovascular system, and provide an overview of drugs that are known to have aldehyde-conjugating effects. Some compounds of phytochemical origin, and histidyl-dipeptides with emerging therapeutic value in this area are also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Formate Dehydrogenase of Clostridium thermoaceticum: Incorporation of Selenium-75, and the Effects of Selenite, Molybdate, and Tungstate on the Enzyme

    PubMed Central

    Andreesen, Jan R.; Ljungdahl, Lars G.

    1973-01-01

    The formation of the nicotinamide adenine dinucleotide phosphate-dependent formate dehydrogenase in Clostridium thermoaceticum is stimulated by the presence of molybdate and selenite in the growth medium. The highest formate dehydrogenase activity was obtained with 2.5 × 10−4 M Na2MoO4 and 5 × 10−5 Na2SeO3. Tungstate but not vanadate could replace molybdate and stimulate the formation of formate dehydrogenase. Tungstate stimulated activity more than molybdate, and in combination with molybdate the stimulation of formation of formate dehydrogenase was additive. Formate dehydrogenase was isolated from cells grown in the presence of Na275SeO2, and a correlation was observed between bound 75Se and enzyme activity. PMID:4147651

  18. Bifunctional isocitrate-homoisocitrate dehydrogenase: a missing link in the evolution of beta-decarboxylating dehydrogenase.

    PubMed

    Miyazaki, Kentaro

    2005-05-27

    Beta-decarboxylating dehydrogenases comprise 3-isopropylmalate dehydrogenase, isocitrate dehydrogenase, and homoisocitrate dehydrogenase. They share a high degree of amino acid sequence identity and occupy equivalent positions in the amino acid biosynthetic pathways for leucine, glutamate, and lysine, respectively. Therefore, not only the enzymes but also the whole pathways should have evolved from a common ancestral pathway. In Pyrococcus horikoshii, only one pathway of the three has been identified in the genomic sequence, and PH1722 is the sole beta-decarboxylating dehydrogenase gene. The organism does not require leucine, glutamate, or lysine for growth; the single pathway might play multiple (i.e., ancestral) roles in amino acid biosynthesis. The PH1722 gene was cloned and expressed in Escherichia coli and the substrate specificity of the recombinant enzyme was investigated. It exhibited activities on isocitrate and homoisocitrate at near equal efficiency, but not on 3-isopropylmalate. PH1722 is thus a novel, bifunctional beta-decarboxylating dehydrogenase, which likely plays a dual role in glutamate and lysine biosynthesis in vivo.

  19. Glutamate Dehydrogenase from Apodachlya (Oomycetes) 1

    PubMed Central

    Price, Jeffrey S.; Gleason, Frank H.

    1972-01-01

    A glutamate dehydrogenase specific for nicotinamide-adenine-dinucleotide has been purified 50-fold from Apodachlya brachynema (Leptomitales). Certain physical, chemical, and kinetic properties of this enzyme have been studied, particularly specificity for coenzymes and substrates. With glucose as the sole carbon source, the synthesis of glutamate dehydrogenase was repressed, whereas glutamate, proline, alanine, or ornithine plus aspartate as sole carbon sources induced synthesis of the enzyme. These data indicate that the function of this enzyme is primarily degradative, although there is no evidence for a nicotinamide-adenine-dinucleotide-phosphate-specific biosynthetic glutamate dehydrogenase in Apodachlya. PMID:16657902

  20. Crystal structure of human aldehyde dehydrogenase 1A3 complexed with NAD+ and retinoic acid

    PubMed Central

    Moretti, Andrea; Li, Jianfeng; Donini, Stefano; Sobol, Robert W.; Rizzi, Menico; Garavaglia, Silvia

    2016-01-01

    The aldehyde dehydrogenase family 1 member A3 (ALDH1A3) catalyzes the oxidation of retinal to the pleiotropic factor retinoic acid using NAD+. The level of ALDHs enzymatic activity has been used as a cancer stem cell marker and seems to correlate with tumour aggressiveness. Elevated ALDH1A3 expression in mesenchymal glioma stem cells highlights the potential of this isozyme as a prognosis marker and drug target. Here we report the first crystal structure of human ALDH1A3 complexed with NAD+ and the product all-trans retinoic acid (REA). The tetrameric ALDH1A3 folds into a three domain-based architecture highly conserved along the ALDHs family. The structural analysis revealed two different and coupled conformations for NAD+ and REA that we propose to represent two snapshots along the catalytic cycle. Indeed, the isoprenic moiety of REA points either toward the active site cysteine, or moves away adopting the product release conformation. Although ALDH1A3 shares high sequence identity with other members of the ALDH1A family, our structural analysis revealed few peculiar residues in the 1A3 isozyme active site. Our data provide information into the ALDH1As catalytic process and can be used for the structure-based design of selective inhibitors of potential medical interest. PMID:27759097

  1. Structural and functional comparison of two human liver dihydrodiol dehydrogenases associated with 3 alpha-hydroxysteroid dehydrogenase activity.

    PubMed Central

    Deyashiki, Y; Taniguchi, H; Amano, T; Nakayama, T; Hara, A; Sawada, H

    1992-01-01

    Two monomeric dihydrodiol dehydrogenases with pI values of 5.4 and 7.6 were co-purified with androsterone dehydrogenase activity to homogeneity from human liver. The two enzymes differed from each other on peptide mapping and in their heat-stabilities; with respect to the latter the dihydrodiol dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase activities of the respective enzymes were similarly inactivated. The pI 5.4 enzyme was equally active towards trans- and cis-benzene dihydrodiols, and towards (S)- and (R)-forms of indan-1-ol and 1,2,3,4-tetrahydronaphth-1-ol and oxidized the 3 alpha-hydroxy group of C19-, C21- and C24-steroids, whereas the pI 7.6 enzyme showed high specificity for trans-benzene dihydrodiol, (S)-forms of the alicyclic alcohols and C19- and C21-steroids. Although the two enzymes reduced various xenobiotic carbonyl compounds and the 3-oxo group of C19- and C21-steroids, and were A-specific in the hydrogen transfer from NADPH, only the pI 5.4 enzyme showed reductase activity towards 7 alpha-hydroxy-5 beta-cholestan-3-one and dehydrolithocholic acid. The affinity of the two enzymes for the steroidal substrates was higher than that for the xenobiotic substrates. The two enzymes also showed different susceptibilities to the inhibition by anti-inflammatory drugs and bile acids. Whereas the pI-5.4 enzyme was highly sensitive to anti-inflammatory steroids, showing mixed-type inhibitions with respect to indan-1-ol and androsterone, the pI 7.6 enzyme was inhibited more potently by non-steroidal anti-inflammatory drugs and bile acids than by the steroidal drugs, and the inhibitions were all competitive. These structural and functional differences suggest that the two enzymes are 3 alpha-hydroxysteroid dehydrogenase isoenzymes. Images Fig. 2. PMID:1554355

  2. 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

  3. Stringency of substrate specificity of Escherichia coli malate dehydrogenase.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Boernke, W. E.; Millard, C. S.; Stevens, P. W.

    1995-09-10

    Malate dehydrogenase and lactate dehydrogenase are members of the structurally and functionally homologous family of 2-ketoacid dehydrogenases. Both enzymes display high specificity for their respective keto substrates, oxaloacetate and pyruvate. Closer analysis of their specificity, however, reveals that the specificity of malate dehydrogenase is much stricter and less malleable than that of lactate dehydrogenase. Site-specific mutagenesis of the two enzymes in an attempt to reverse their specificity has met with contrary results. Conversion of a specific active-site glutamine to arginine in lactate dehydrogenase from Bacillus stearothermophilus generated an enzyme that displayed activity toward oxaloacetate equal to that of the nativemore » enzyme toward pyruvate (H. M. Wilks et al. (1988) Science 242, 1541-1544). We have constructed a series of mutants in the mobile, active site loop of the Escherichia coli malate dehydrogenase that incorporate the complementary change, conversion of arginine 81 to glutamine, to evaluate the role of charge distribution and conformational flexibility within this loop in defining the substrate specificity of these enzymes. Mutants incorporating the change R81Q all had reversed specificity, displaying much higher activity toward pyruvate than to the natural substrate, oxaloacetate. In contrast to the mutated lactate dehydrogenase, these reversed-specificity mutants were much less active than the native enzyme. Secondary mutations within the loop of the E. coli enzyme (A80N, A80P, A80P/M85E/D86T) had either no or only moderately beneficial effects on the activity of the mutant enzyme toward pyruvate. The mutation A80P, which can be expected to reduce the overall flexibility of the loop, modestly improved activity toward pyruvate. The possible physiological relevance of the stringent specificity of malate dehydrogenase was investigated. In normal strains of E. coli, fermentative metabolism was not affected by expression of the

  4. Deciphering the Origin, Evolution, and Physiological Function of the Subtelomeric Aryl-Alcohol Dehydrogenase Gene Family in the Yeast Saccharomyces cerevisiae.

    PubMed

    Yang, Dong-Dong; de Billerbeck, Gustavo M; Zhang, Jin-Jing; Rosenzweig, Frank; Francois, Jean-Marie

    2018-01-01

    Homology searches indicate that Saccharomyces cerevisiae strain BY4741 contains seven redundant genes that encode putative aryl-alcohol dehydrogenases (AAD). Yeast AAD genes are located in subtelomeric regions of different chromosomes, and their functional role(s) remain enigmatic. Here, we show that two of these genes, AAD4 and AAD14 , encode functional enzymes that reduce aliphatic and aryl-aldehydes concomitant with the oxidation of cofactor NADPH, and that Aad4p and Aad14p exhibit different substrate preference patterns. Other yeast AAD genes are undergoing pseudogenization. The 5' sequence of AAD15 has been deleted from the genome. Repair of an AAD3 missense mutation at the catalytically essential Tyr 73 residue did not result in a functional enzyme. However, ancestral-state reconstruction by fusing Aad6 with Aad16 and by N-terminal repair of Aad10 restores NADPH-dependent aryl-alcohol dehydrogenase activities. Phylogenetic analysis indicates that AAD genes are narrowly distributed in wood-saprophyte fungi and in yeast that occupy lignocellulosic niches. Because yeast AAD genes exhibit activity on veratraldehyde, cinnamaldehyde, and vanillin, they could serve to detoxify aryl-aldehydes released during lignin degradation. However, none of these compounds induce yeast AAD gene expression, and Aad activities do not relieve aryl-aldehyde growth inhibition. Our data suggest an ancestral role for AAD genes in lignin degradation that is degenerating as a result of yeast's domestication and use in brewing, baking, and other industrial applications. IMPORTANCE Functional characterization of hypothetical genes remains one of the chief tasks of the postgenomic era. Although the first Saccharomyces cerevisiae genome sequence was published over 20 years ago, 22% of its estimated 6,603 open reading frames (ORFs) remain unverified. One outstanding example of this category of genes is the enigmatic seven-member AAD family. Here, we demonstrate that proteins encoded by two

  5. Deciphering the Origin, Evolution, and Physiological Function of the Subtelomeric Aryl-Alcohol Dehydrogenase Gene Family in the Yeast Saccharomyces cerevisiae

    PubMed Central

    de Billerbeck, Gustavo M.; Zhang, Jin-jing; Rosenzweig, Frank

    2017-01-01

    ABSTRACT Homology searches indicate that Saccharomyces cerevisiae strain BY4741 contains seven redundant genes that encode putative aryl-alcohol dehydrogenases (AAD). Yeast AAD genes are located in subtelomeric regions of different chromosomes, and their functional role(s) remain enigmatic. Here, we show that two of these genes, AAD4 and AAD14, encode functional enzymes that reduce aliphatic and aryl-aldehydes concomitant with the oxidation of cofactor NADPH, and that Aad4p and Aad14p exhibit different substrate preference patterns. Other yeast AAD genes are undergoing pseudogenization. The 5′ sequence of AAD15 has been deleted from the genome. Repair of an AAD3 missense mutation at the catalytically essential Tyr73 residue did not result in a functional enzyme. However, ancestral-state reconstruction by fusing Aad6 with Aad16 and by N-terminal repair of Aad10 restores NADPH-dependent aryl-alcohol dehydrogenase activities. Phylogenetic analysis indicates that AAD genes are narrowly distributed in wood-saprophyte fungi and in yeast that occupy lignocellulosic niches. Because yeast AAD genes exhibit activity on veratraldehyde, cinnamaldehyde, and vanillin, they could serve to detoxify aryl-aldehydes released during lignin degradation. However, none of these compounds induce yeast AAD gene expression, and Aad activities do not relieve aryl-aldehyde growth inhibition. Our data suggest an ancestral role for AAD genes in lignin degradation that is degenerating as a result of yeast's domestication and use in brewing, baking, and other industrial applications. IMPORTANCE Functional characterization of hypothetical genes remains one of the chief tasks of the postgenomic era. Although the first Saccharomyces cerevisiae genome sequence was published over 20 years ago, 22% of its estimated 6,603 open reading frames (ORFs) remain unverified. One outstanding example of this category of genes is the enigmatic seven-member AAD family. Here, we demonstrate that proteins encoded

  6. Effect of peptide aldehydes with IL-1 beta converting enzyme inhibitory properties on IL-1 alpha and IL-1 beta production in vitro.

    PubMed

    Németh, K; Patthy, M; Fauszt, I; Széll, E; Székely, J I; Bajusz, S

    1995-12-01

    Tripeptide and pentapeptide aldehydes as substrate-base inhibitors of cysteine proteases were designed in our laboratory for the inhibition of interleukin-1 beta converting enzyme (ICE), a recently described cysteine protease responsible for the processing of IL-1 beta. The biological effectivity of the peptide aldehydes was studied in THP-1 cells and human whole blood. The released and cell-associated IL-1 alpha and IL-1 beta levels were determined by ELISA from the supernatants and cell lysates, respectively. The total IL-1 like bioactivity was assayed by the D10 G4.1 cell proliferation method. The tripeptide aldehyde (Z-Val-His-Asp-H) and pentapeptide aldehyde (Eoc-Ala-Tyr-Val-Ala-Asp-H) significantly reduced IL-1 beta levels in the supernatants in relatively high concentrations (10-100 microM), but the IL-1 alpha release was unaffected by these peptides. However, a considerable decrease in the cell-associated IL-1 beta and IL-1 alpha levels was observed. N-terminal extension of the tripeptide aldehyde yielded even more potent inhibitors. Amino acid substitution at the P2 position did not cause considerable changes in the inhibitory activity. The peptide aldehydes suppressed the IL-1 beta production in a reversible manner, whereas dexamethasone, a glucocorticoid, had a prolonged inhibitory effect. The inhibitory effect of these peptides and that of dexamethasone appeared to be additive. These findings indicate that these peptide aldehydes might be used as IL-beta inhibitory agents in experimental models in which IL-1 beta is a key mediator or ICE is implicated.

  7. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Knockdown of Both Mitochondrial Isocitrate Dehydrogenase Enzymes In Pancreatic Beta Cells Inhibits Insulin Secretion

    PubMed Central

    MacDonald, Michael J.; Brown, Laura J.; Longacre, Melissa J.; Stoker, Scott W.; Kendrick, Mindy A.; Hasan, Noaman M.

    2013-01-01

    Background There are three isocitrate dehydrogenases (IDHs) in the pancreatic insulin cell; IDH1 (cytosolic) and IDH2 (mitochondrial) use NADP(H). IDH3 is mitochondrial, uses NAD(H) and was believed to be the IDH that supports the citric acid cycle. Methods With shRNAs targeting mRNAs for these enzymes we generated cell lines from INS-1 832/13 cells with severe (80%–90%) knockdown of the mitochondrial IDHs separately and together in the same cell line. Results With knockdown of both mitochondrial IDH’s mRNA, enzyme activity and protein level, but not with knockdown of one mitochondrial IDH, glucose- and BCH (an allosteric activator of glutamate dehydrogenase)-plus-glutamine-stimulated insulin release were inhibited. Cellular levels of citrate, α-ketoglutarate, malate and ATP were altered in patterns consistent with blockage at the mitochondrial IDH reactions. We were able to generate only 50% knockdown of Idh1 mRNA in multiple cell lines (without inhibition of insulin release) possibly because greater knockdown of IDH1 was not compatible with cell line survival. Conclusions The mitochondrial IDHs are redundant for insulin secretion. When both enzymes are severely knocked down, their low activities (possibly assisted by transport of IDH products and other metabolic intermediates from the cytosol into mitochondria) are sufficient for cell growth, but inadequate for insulin secretion when the requirement for intermediates is certainly more rapid. The results also indicate that IDH2 can support the citric acid cycle. General Significance As almost all mammalian cells possess substantial amounts of all three IDH enzymes, the biological principles suggested by these results are probably extrapolatable to many tissues. PMID:23876293

  9. Geraniol dehydrogenase, the key enzyme in biosynthesis of the alarm pheromone, from the astigmatid mite Carpoglyphus lactis (Acari: Carpoglyphidae).

    PubMed

    Noge, Koji; Kato, Makiko; Mori, Naoki; Kataoka, Michihiko; Tanaka, Chihiro; Yamasue, Yuji; Nishida, Ritsuo; Kuwahara, Yasumasa

    2008-06-01

    Geraniol dehydrogenase (GeDH), which plays an important role in the biosynthesis of neral, an alarm pheromone, was purified from the astigmatid mite Carpoglyphus lactis. The enzyme was obtained in an apparently homogeneous and active form after 1879-fold purification through seven steps of chromatography. Car. lactis GeDH was determined to be a monomer in its active form with a relative molecular mass of 42 800, which is a unique subunit structure in comparison with already established alcohol dehydrogenases. Car. lactis GeDH oxidized geraniol into geranial in the presence of NAD+. NADP+ was ineffective as a cofactor, suggesting that Car. lactis GeDH is an NAD+-dependent alcohol dehydrogenase. The optimal pH and temperature for geraniol oxidation were determined to be pH 9.0 and 25 degrees C, respectively. The Km values for geraniol and NAD+ were 51.0 microm and 59.5 microm, respectively. Car. lactis GeDH was shown to selectively oxidize geraniol, whereas its geometrical isomer, nerol, was inert as a substrate. The high specificity for geraniol suggests that Car. lactis GeDH specializes in the alarm pheromone biosynthesis of Car. lactis. Car. lactis GeDH is composed of 378 amino acids. Structurally, Car. lactis GeDH showed homology with zinc-dependent alcohol dehydrogenases found in mammals and a mosquito (36.6-37.6% identical), and the enzyme was considered to be a member of the medium-chain dehydrogenase/reductase family, in view of the highly conserved sequences of zinc-binding and NAD+-binding sites. Phylogenetic analyses indicate that Car. lactis GeDH could be categorized as a new class, different from other established alcohol dehydrogenases.

  10. ALDH1A3, the Major Aldehyde Dehydrogenase Isoform in Human Cholangiocarcinoma Cells, Affects Prognosis and Gemcitabine Resistance in Cholangiocarcinoma Patients.

    PubMed

    Chen, Ming-Huang; Weng, Jing-Jie; Cheng, Chi-Tung; Wu, Ren-Chin; Huang, Shih-Chiang; Wu, Chiao-En; Chung, Yi-Hsiu; Liu, Chun-Yu; Chang, Mu-Hsin; Chen, Ming-Han; Chiang, Kun-Chun; Yeh, Ta-Sen; Su, Yeu; Yeh, Chun-Nan

    2016-08-15

    Intrahepatic cholangiocarcinoma is a fatal primary liver cancer resulting from diagnosis at an advanced stage. Understanding the mechanisms of drug resistance and metastasis of cholangiocarcinoma may improve the disease prognosis. Enhanced aldehyde dehydrogenase (ALDH) activity is suggested to be associated with increased drug resistance and the metastasis. This study aims to investigate the roles of the ALDH isoforms in cholangiocarcinoma. Aldefluor assays, RT-PCR, and Western blot analysis were used to identify the major ALDH isoforms contributing to Aldefluor activity in human cholangiocarcinoma cell lines. We manipulated isoform expression in HuCCT1 cells to elucidate the role of ALDH1A3 in the malignant progression of these cells. Finally, we used immunohistochemical staining to evaluate the clinical significance of ALDH1A3 in 77 hepatectomized cholangiocarcinoma patients and an additional 31 patients with advanced cholangiocarcinoma who received gemcitabine-based therapy. ALDH(high) cholangiocarcinoma cells not only migrated faster but were more resistant to gemcitabine. Among the 19 ALDH isoforms studied, ALDH1A3 was found to be the main contributor to Aldefluor activity. In addition, we also found that knockdown of ALDH1A3 expression in HuCCT1 cells markedly reduced not only their sensitivity to gemcitabine, which might be attributed to a decreased expression of ribonucleotide reductase M1, but also their migration. Most importantly, this enzyme was also identified as an independent poor prognostic factor for patients with intrahepatic cholangiocarcinoma, as well as a prognostic biomarker of gemcitabine-treated patients. ALDH1A3 plays an important role in enhancing malignant behavior of cholangiocarcinoma and serves as a new therapeutic target. Clin Cancer Res; 22(16); 4225-35. ©2016 AACR. ©2016 American Association for Cancer Research.

  11. Alcohol and aldehyde dehydrogenase gene polymorphisms influence susceptibility to esophageal cancer in Japanese alcoholics.

    PubMed

    Yokoyama, A; Muramatsu, T; Omori, T; Matsushita, S; Yoshimizu, H; Higuchi, S; Yokoyama, T; Maruyama, K; Ishii, H

    1999-11-01

    Studies have consistently demonstrated that inactive aldehyde dehydrogenase-2 (ALDH2), encoded by ALDH2*1/2*2, is closely associated with alcohol-related carcinogenesis. Recently, the contributions of alcohol dehydrogenase-2 (ADH2) polymorphism to alcoholism, esophageal cancer, and the flushing response have also been described. To determine the effects of ALDH2 and ADH2 genotypes in genetically based cancer susceptibility, lymphocyte DNA samples from 668 Japanese alcoholic men more than 40 years of age (91 with and 577 without esophageal cancer) were genotyped and the results were expressed as odds ratios (ORs). This study also tested 82 of the alcoholics with esophageal cancer to determine whether cancer susceptibility is associated with patients' responses to simple questions about current or former flushing after drinking a glass of beer. The frequencies of ADH2*1/2*1 and ALDH2*1/2*2 were significantly higher in alcoholics with, than in those without, esophageal cancer (0.473 vs. 0.289 and 0.560 vs. 0.099, respectively). After adjustment for drinking and smoking, the analysis showed significantly increased cancer risk for alcoholics with either ADH2*1/2*I (OR = 2.03) or ALDH2*1/2*2 (OR = 12.76). For those having ADH2*1/2*1 combined with ALDH2*1/2*2, the esophageal cancer risk was enhanced in a multiplicative fashion (OR = 27.66). Responses to flushing questions showed that only 47.8% of the ALDH2*1/2*2 heterozygotes with ADH2*1/ 2*1, compared with 92.3% of those with ALDH2*1/2*2 and the ADH2*2 allele, reported current or former flushing. Genotyping showed that for alcoholics who reported ever flushing, the questionnaire was 71.4% correct in identifying ALDH2*1/2*2 and 87.9% correct in identifying ALDH2*1/2*1. Japanese alcoholics can be divided into cancer susceptibility groups on the basis of their combined ADH2 and ALDH2 genotypes. The flushing questionnaire can predict high risk ALDH2*1/2*2 fairly accurately in persons with ADH2*2 allele, but a reliable

  12. Structural studies of cinnamoyl-CoA reductase and cinnamyl-alcohol dehydrogenase, key enzymes of monolignol biosynthesis.

    PubMed

    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

    2014-09-01

    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. © 2014 American Society of Plant Biologists. All rights reserved.

  13. Vasodilatory effect of nitroglycerin in Japanese subjects with different aldehyde dehydrogenase 2 (ALDH2) genotypes.

    PubMed

    Miura, Takeshi; Nishinaka, Toru; Terada, Tomoyuki; Yonezawa, Kazuya

    2017-10-01

    The functional genetic polymorphism of aldehyde dehydrogenase 2 (ALDH2) influences the enzymatic activities of its wild type (Glu504 encoded by ALDH2*1) and mutant type (Lys504 encoded by ALDH2*2) proteins. The enzymatic activities of mutant-type ALDH2 are limited compared with those of the wild type. ALDH2 has been suggested as a critical factor for nitroglycerin-mediated vasodilation by some human studies and in vitro studies. Currently, there is no research on direct observations of the vasodilatory effect of nitroglycerin sublingual tablets, which is the generally used dosage form. In the present study, the contribution of ALDH2 to the vasodilatory effect of nitroglycerin sublingual tablets was investigated among three genotype groups (ALDH2*1/*1, ALDH2*1/*2, and ALDH2*2/*2) in Japanese. The results by direct assessments of in vivo nitroglycerin-mediated dilation showed no apparent difference in vasodilation among all genotypes of ALDH2. Furthermore, to analyze the effect of other factors (age and flow-mediated dilation), multiple regression analysis and Pearson's correlation coefficient analysis were carried out. These analyses also indicated that the genotypes of ALDH2 were not related to the degree of vasodilation. These results suggest the existence of other predominant pathway(s) for nitroglycerin biotransformation, at least with regard to clinical nitroglycerin (e.g., a sublingual tablet) in Japanese subjects. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. A spontaneously immortalized Schwann cell line from aldose reductase-deficient mice as a useful tool for studying polyol pathway and aldehyde metabolism.

    PubMed

    Niimi, Naoko; Yako, Hideji; Takaku, Shizuka; Kato, Hiroshi; Matsumoto, Takafumi; Nishito, Yasumasa; Watabe, Kazuhiko; Ogasawara, Saori; Mizukami, Hiroki; Yagihashi, Soroku; Chung, Sookja K; Sango, Kazunori

    2018-03-01

    The increased glucose flux into the polyol pathway via aldose reductase (AR) is recognized as a major contributing factor for the pathogenesis of diabetic neuropathy, whereas little is known about the functional significance of AR in the peripheral nervous system. Spontaneously immortalized Schwann cell lines established from long-term cultures of AR-deficient and normal C57BL/6 mouse dorsal root ganglia and peripheral nerves can be useful tools for studying the physiological and pathological roles of AR. These cell lines, designated as immortalized knockout AR Schwann cells 1 (IKARS1) and 1970C3, respectively, demonstrated distinctive Schwann cell phenotypes, such as spindle-shaped morphology and immunoreactivity to S100, p75 neurotrophin receptor, and vimentin, and extracellular release of neurotrophic factors. Conditioned media obtained from these cells promoted neuronal survival and neurite outgrowth of cultured adult mouse dorsal root ganglia neurons. Microarray and real-time RT-PCR analyses revealed significantly down-regulated mRNA expression of polyol pathway-related enzymes, sorbitol dehydrogenase and ketohexokinase, in IKARS1 cells compared with those in 1970C3 cells. In contrast, significantly up-regulated mRNA expression of aldo-keto reductases (AKR1B7 and AKR1B8) and aldehyde dehydrogenases (ALDH1L2, ALDH5A1, and ALDH7A1) was detected in IKARS1 cells compared with 1970C3 cells. Exposure to reactive aldehydes (3-deoxyglucosone, methylglyoxal, and 4-hydroxynonenal) significantly up-regulated the mRNA expression of AKR1B7 and AKR1B8 in IKARS1 cells, but not in 1970C3 cells. Because no significant differences in viability between these two cell lines after exposure to these aldehydes were observed, it can be assumed that the aldehyde detoxification is taken over by AKR1B7 and AKR1B8 in the absence of AR. © 2017 International Society for Neurochemistry.

  15. Catalytic properties of thermophilic lactate dehydrogenase and halophilic malate dehydrogenase at high temperature and low water activity.

    PubMed

    Hecht, K; Wrba, A; Jaenicke, R

    1989-07-15

    Thermophilic lactate dehydrogenases from Thermotoga maritima and Bacillus stearothermophilus are stable up to temperature limits close to the optimum growth temperature of their parent organisms. Their catalytic properties are anomalous in that Km shows a drastic increase with increasing temperature. At low temperatures, the effect levels off. Extreme halophilic malate dehydrogenase from Halobacterium marismortui exhibits a similar anomaly. Increasing salt concentration (NaCl) leads to an optimum curve for Km, oxaloacctate while Km, NADH remains constant. Previous claims that the activity of halophilic malate dehydrogenase shows a maximum at 1.25 M NaCl are caused by limiting substrate concentration; at substrate saturation, specific activity of halophilic malate dehydrogenase reaches a constant value at ionic strengths I greater than or equal to 1 M. Non-halophilic (mitochondrial) malate dehydrogenase shows Km characteristics similar to those observed for the halophilic enzyme. The drastic decrease in specific activity of the mitochondrial enzyme at elevated salt concentrations is caused by the salt-induced increase in rigidity of the enzyme, rather than gross structural changes.

  16. Ebselen: Mechanisms of Glutamate Dehydrogenase and Glutaminase Enzyme Inhibition.

    PubMed

    Yu, Yan; Jin, Yanhong; Zhou, Jie; Ruan, Haoqiang; Zhao, Han; Lu, Shiying; Zhang, Yue; Li, Di; Ji, Xiaoyun; Ruan, Benfang Helen

    2017-12-15

    Ebselen modulates target proteins through redox reactions with selenocysteine/cysteine residues, or through binding to the zinc finger domains. However, a recent contradiction in ebselen inhibition of kidney type glutaminase (KGA) stimulated our interest in investigating its inhibition mechanism with glutamate dehydrogenase (GDH), KGA, thioredoxin reductase (TrxR), and glutathione S-transferase. Fluorescein- or biotin-labeled ebselen derivatives were synthesized for mechanistic analyses. Biomolecular interaction analyses showed that only GDH, KGA, and TrxR proteins can bind to the ebselen derivative, and the binding to GDH and KGA could be competed off by glutamine or glutamate. From the gel shift assays, the fluorescein-labeled ebselen derivative could co-migrate with hexameric GDH and monomeric/dimeric TrxR in a dose-dependent manner; it also co-migrated with KGA but disrupted the tetrameric form of the KGA enzyme at a high compound concentration. Further proteomic analysis demonstrated that the ebselen derivative could cross-link with proteins through a specific cysteine at the active site of GDH and TrxR proteins, but for KGA protein, the binding site is at the N-terminal appendix domain outside of the catalytic domain, which might explain why ebselen is not a potent KGA enzyme inhibitor in functional assays. In conclusion, ebselen could inhibit enzyme activity by binding to the catalytic domain or disruption of the protein complex. In addition, ebselen is a relatively potent selective GDH inhibitor that might provide potential therapeutic opportunities for hyperinsulinism-hyperammonemia syndrome patients who have the mutational loss of GTP inhibition.

  17. Aldehyde-forming fatty acyl-CoA reductase from cyanobacteria: expression, purification and characterization of the recombinant enzyme.

    PubMed

    Lin, Fengming; Das, Debasis; Lin, Xiaoxia N; Marsh, E Neil G

    2013-10-01

    Long-chain acyl-CoA reductases (ACRs) catalyze a key step in the biosynthesis of hydrocarbon waxes. As such they are attractive as components in engineered metabolic pathways for 'drop in' biofuels. Most ACR enzymes are integral membrane proteins, but a cytosolic ACR was recently discovered in cyanobacteria. The ACR from Synechococcus elongatus was overexpressed in Escherichia coli, purified and characterized. The enzyme was specific for NADPH and catalyzed the reduction of fatty acyl-CoA esters to the corresponding aldehydes, rather than alcohols. Stearoyl-CoA was the most effective substrate, being reduced more rapidly than either longer or shorter chain acyl-CoAs. ACR required divalent metal ions, e.g. Mg(2+), for activity and was stimulated ~ 10-fold by K(+). The enzyme was inactivated by iodoacetamide and was acylated on incubation with stearoyl-CoA, suggesting that reduction occurs through an enzyme-thioester intermediate. Consistent with this, steady state kinetic analysis indicates that the enzyme operates by a 'ping-pong' mechanism with kcat = 0.36 ± 0.023 min(-1), K(m)(stearoyl-CoA) = 31.9 ± 4.2 μM and K(m)(NADPH) = 35.6 ± 4.9 μM. The slow turnover number measured for ACR poses a challenge for its use in biofuel applications where highly efficient enzymes are needed. © 2013 FEBS.

  18. Association between aldehyde dehydrogenase 2 polymorphisms and the incidence of diabetic retinopathy among Japanese subjects with type 2 diabetes mellitus.

    PubMed

    Morita, Kazunori; Saruwatari, Junji; Miyagawa, Haruna; Uchiyashiki, Yoshihiro; Oniki, Kentaro; Sakata, Misaki; Kajiwara, Ayami; Yoshida, Akira; Jinnouchi, Hideaki; Nakagawa, Kazuko

    2013-09-13

    Mitochondrial aldehyde dehydrogenase 2 (ALDH2) detoxifies reactive aldehydes in the micro- and macrovasculature. These substrates, including methylglyoxal and 4-hydroxynonenal formed from glucose and lipids, cause protein carbonylation and mitochondrial dysfunction, forming advanced glycation end products (AGEs). The present study aimed to confirm the association between the inactive ALDH2*2 allele and diabetic retinopathy (DR). A retrospective longitudinal analysis was conducted, among 234 Japanese patients with type 2 diabetes mellitus (DM) (156 males and 78 females) who had no DR signs at baseline and were treated for more than half a year. The ALDH2*1/*2 alleles were determined using a real-time TaqMan allelic discrimination assay. Multivariate-adjusted hazard ratios (HRs) and 95% confidential intervals (CIs) for the cumulative incidence of the development of DR were examined using a Cox proportional hazard model, taking drinking habits and the serum γ-glutamyltransferase (GGT) level into consideration. The frequency of the ALDH2*2 allele was 22.3%. Fifty-two subjects cumulatively developed DR during the follow-up period of 5.5 ± 2.5 years. The ALDH2*2 allele carriers had a significantly higher incidence of DR than the non-carriers (HR: 1.92; P = 0.02). The incidence of DR was significantly higher in the drinkers with the ALDH2*2 allele than in those with the ALDH2*1/*1 genotype (HR: 2.61; P = 0.03), while the incidence of DR in the non-drinkers did not differ significantly between the ALDH2 genotype groups (P > 0.05). The incidence of DR was significantly higher in the ALDH2*2 allele carriers with a high GGT level than in the non-carriers with a high or low GGT level (HR: 2.45; P = 0.03; and HR: 2.63; P = 0.03, respectively). To the best of our knowledge, this is the first report of a significant association between the ALDH2*2 allele and the incidence of DR. These findings provide additional evidence that ALDH2 protects both microvasculature and

  19. Aldehyde dehydrogenase-2 genotypes and HLA haplotypes in Japanese patients with esophageal cancer.

    PubMed

    Watanabe, Seishiro; Sasahara, Katsuyuki; Kinekawa, Fumihiko; Uchida, Naohito; Masaki, Tsutomu; Kurokohchi, Kazutaka; Murota, Masayuki; Touge, Tetsuo; Kawauchi, Kazuyoshi; Oda, Syuji; Kuriyama, Shigeki

    2002-01-01

    The aim of this study was to examine how aldehyde dehydrogenase-2 (ALDH2) genotypes and human leukocyte antigen (HLA) haplotypes contribute to the risk for esophageal cancer. We examined ALDH2 genotypes and HLA haplotypes in 29 Japanese patients with esophageal cancer. The ratio of patients who experienced current or former intense vasodilatation upon consuming alcohol (flushing type) was much higher in individuals with the inactive form of ALDH2 encoded by the ALDH2(2)/2(2) or ALDH2(1)/2(2) genotype than in those with the active form of ALDH2 encoded by the ALDH2(1)/2(1) genotype. The ratio of inactive ALDH2 was significantly higher in patients with esophageal cancer than in control normal subjects, suggesting that alcoholics with inactive ALDH2 were susceptible to esophageal cancer. HLA haplotypes A24, A26, B54, B61 and DR9 were prevalent in patients with esophageal cancer (82.8, 24.1, 34.5, 37.9 and 44.8%, respectively). HLA haplotype of A24 and inactive ALDH2 were simultaneously found in 58.6% of patients with esophageal cancer. Furthermore, we found other primary malignancies in 6 of 29 (20.7%) patients with esophageal cancer, and 4 of these 6 patients had both the inactive form of ALDH2 and the HLA A24 haplotype. The present study showed the high prevalence of the inactive form of ALDH2 and HLA haplotypes A24, A26, B54, B61 and DR9 in Japanese patients with esophageal cancer. Therefore, the examination of genotypes of ALDH2 loci and HLA haplotypes may allow the early detection of esophageal cancer in the Japanese population.

  20. Activation of human liver 3 alpha-hydroxysteroid dehydrogenase by sulphobromophthalein.

    PubMed Central

    Matsuura, K; Tamada, Y; Deyashiki, Y; Miyabe, Y; Nakanishi, M; Ohya, I; Hara, A

    1996-01-01

    Human liver contains at least two isoenzymes (DD2 and DD4) of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase. The NADP(H)-linked oxidoreductase activities of DD4 were activated more than 4-fold by sulphobromophthalein at concentrations above 20 microM and under physiological pH conditions. Sulphobromophthalein did not stimulate the activities of DD2 and human liver aldehyde reductase, which are functionally and/or structurally related to DD4. No stimulatory effect on the activity of DD4 was observed with other organic anions such as Indocyanine Green, haematin and Rose Bengal. The binding of sulphobromophthalein to DD4 was instantaneous and reversible, and was detected by fluorescence and ultrafiltration assays. The activation by sulphobromophthalein decreased the activation energy in the dehydrogenation reaction for the enzyme, and increased both kcat, and Km values for the coenzymes and substrates. Kinetic analyses with respect to concentrations of NADP+ and (S)-(+)-indan-1-ol indicated that sulphobromophthalein was a non-essential activator of mixed type showing a dissociation constant of 2.6 microM. Thus, the human 3 alpha-hydroxysteroid dehydrogenase isoenzyme has a binding site specific to sulphobromophthalein, and the hepatic metabolism mediated by this isoenzyme may be influenced when this drug is administered. PMID:8546681

  1. Metabolism of 2-phenylethylamine and phenylacetaldehyde by precision-cut guinea pig fresh liver slices.

    PubMed

    Panoutsopoulos, Georgios I; Kouretas, Demetrios; Gounaris, Elias G; Beedham, Christine

    2004-01-01

    2-Phenylethylamine is an endogenous constituent of human brain and is implicated in cerebral transmission. It is also found in certain foodstuffs and may cause toxic side-effects in susceptible individuals. Metabolism of 2-phenylethylamine to phenylacetaldehyde is catalyzed by monoamine oxidase and the oxidation of the reactive aldehyde to its acid derivative is catalyzed mainly by aldehyde dehydrogenase and perhaps aldehyde oxidase, with xanthine oxidase having minimal transformation. The present investigation examines the metabolism of 2-phenylethylamine to phenylacetaldehyde in liver slices and compares the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase activity in the oxidation of phenylacetaldehyde with precision-cut fresh liver slices in the presence/absence of specific inhibitors of each enzyme. In liver slices, phenylacetaldehyde was rapidly converted to phenylacetic acid. Phenylacetic acid was the main metabolite of 2-phenylethylamine, via the intermediate phenylacetaldehyde. Phenylacetic acid formation was completely inhibited by disulfiram (specific inhibitor of aldehyde dehydrogenase), whereas isovanillin (specific inhibitor of aldehyde oxidase) inhibited acid formation to a lesser extent and allopurinol (specific inhibitor of xanthine oxidase) had little or no effect. Therefore, in liver slices, phenylacetaldehyde is rapidly oxidized by aldehyde dehydrogenase and aldehyde oxidase with little or no contribution from xanthine oxidase.

  2. Sequence of the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Nicotiana plumbaginifolia and phylogenetic origin of the gene family.

    PubMed

    Habenicht, A; Quesada, A; Cerff, R

    1997-10-01

    A cDNA-library has been constructed from Nicotiana plumbaginifolia seedlings, and the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GapN, EC 1.2.1.9) was isolated by plaque hybridization using the cDNA from pea as a heterologous probe. The cDNA comprises the entire GapN coding region. A putative polyadenylation signal is identified. Phylogenetic analysis based on the deduced amino acid sequences revealed that the GapN gene family represents a separate ancient branch within the aldehyde dehydrogenase superfamily. It can be shown that the GapN gene family and other distinct branches of the superfamily have its phylogenetic origin before the separation of primary life-forms. This further demonstrates that already very early in evolution, a broad diversification of the aldehyde dehydrogenases led to the formation of the superfamily.

  3. Hangover symptoms in Asian Americans with variations in the aldehyde dehydrogenase (ALDH2) gene.

    PubMed

    Wall, T L; Horn, S M; Johnson, M L; Smith, T L; Carr, L G

    2000-01-01

    Hangovers are not experienced by all people and whether they contribute to the development of alcoholism is unclear. One population that might provide some insight into the role of hangover in the etiology of alcohol use disorders is that of individuals of Asian heritage. Certain Asians have lower rates of alcohol use and alcoholism, findings associated with a mutation in the aldehyde dehydrogenase (ALDH2) gene. Asians with ALDH2*2 alleles drink less and are less likely to be alcoholic than Asians without this mutation. Following alcohol ingestion, they exhibit more intense reactions to alcohol and generate higher levels of the metabolite acetaldehyde. This study evaluated hangover symptoms in Asian Americans with variations in the ALDH2 gene. Men and women of Chinese, Japanese and Korean heritage (N = 140) were asked about their drinking history and a blood sample was collected for genotyping at the ALDH2 locus. Subjects used a Likert-type scale to estimate their severity of hangover and completed a 13-item hangover scale assessing the frequency of hangover symptoms during the previous 6 months. With abstainers (n = 17) excluded and with the effects of gender and recent drinking history controlled, ALDH2 genotype accounted for a significant amount of additional variability in the estimated severity of hangover score with a similar, but nonsignificant, trend for a five-item subscale score derived from the hangover scale. These results suggest that Asian Americans with ALDH2*2 alleles may experience more severe hangovers that may contribute, in part, to protection against the development of excessive or problematic drinking in this population.

  4. Characterization of aldehyde dehydrogenase 1 high ovarian cancer cells: Towards targeted stem cell therapy.

    PubMed

    Sharrow, Allison C; Perkins, Brandy; Collector, Michael I; Yu, Wayne; Simons, Brian W; Jones, Richard J

    2016-08-01

    The cancer stem cell (CSC) paradigm hypothesizes that successful clinical eradication of CSCs may lead to durable remission for patients with ovarian cancer. Despite mounting evidence in support of ovarian CSCs, their phenotype and clinical relevance remain unclear. We and others have found high aldehyde dehydrogenase 1 (ALDH(high)) expression in a variety of normal and malignant stem cells, and sought to better characterize ALDH(high) cells in ovarian cancer. We compared ALDH(high) to ALDH(low) cells in two ovarian cancer models representing distinct subtypes: FNAR-C1 cells, derived from a spontaneous rat endometrioid carcinoma, and the human SKOV3 cell line (described as both serous and clear cell subtypes). We assessed these populations for stem cell features then analyzed expression by microarray and qPCR. ALDH(high) cells displayed CSC properties, including: smaller size, quiescence, regenerating the phenotypic diversity of the cell lines in vitro, lack of contact inhibition, nonadherent growth, multi-drug resistance, and in vivo tumorigenicity. Microarray and qPCR analysis of the expression of markers reported by others to enrich for ovarian CSCs revealed that ALDH(high) cells of both models showed downregulation of CD24, but inconsistent expression of CD44, KIT and CD133. However, the following druggable targets were consistently expressed in the ALDH(high) cells from both models: mTOR signaling, her-2/neu, CD47 and FGF18/FGFR3. Based on functional characterization, ALDH(high) ovarian cancer cells represent an ovarian CSC population. Differential gene expression identified druggable targets that have the potential for therapeutic efficacy against ovarian CSCs from multiple subtypes. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Four distinct types of E.C. 1.2.1.30 enzymes can catalyze the reduction of carboxylic acids to aldehydes.

    PubMed

    Stolterfoht, Holly; Schwendenwein, Daniel; Sensen, Christoph W; Rudroff, Florian; Winkler, Margit

    2017-09-10

    Increasing demand for chemicals from renewable resources calls for the development of new biotechnological methods for the reduction of oxidized bio-based compounds. Enzymatic carboxylate reduction is highly selective, both in terms of chemo- and product selectivity, but not many carboxylate reductase enzymes (CARs) have been identified on the sequence level to date. Thus far, their phylogeny is unexplored and very little is known about their structure-function-relationship. CARs minimally contain an adenylation domain, a phosphopantetheinylation domain and a reductase domain. We have recently identified new enzymes of fungal origin, using similarity searches against genomic sequences from organisms in which aldehydes were detected upon incubation with carboxylic acids. Analysis of sequences with known CAR functionality and CAR enzymes recently identified in our laboratory suggests that the three-domain architecture mentioned above is modular. The construction of a distance tree with a subsequent 1000-replicate bootstrap analysis showed that the CAR sequences included in our study fall into four distinct subgroups (one of bacterial origin and three of fungal origin, respectively), each with a bootstrap value of 100%. The multiple sequence alignment of all experimentally confirmed CAR protein sequences revealed fingerprint sequences of residues which are likely to be involved in substrate and co-substrate binding and one of the three catalytic substeps, respectively. The fingerprint sequences broaden our understanding of the amino acids that might be essential for the reduction of organic acids to the corresponding aldehydes in CAR proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Measurement of crude-cell-extract glycerol dehydratase activity in recombinant Escherichia coli using coupled-enzyme reactions.

    PubMed

    Sankaranarayanan, Mugesh; Seol, Eunhee; Kim, Yeonhee; Chauhan, Ashish Singh; Park, Sunghoon

    2017-03-01

    Glycerol dehydratase (GDHt), which converts glycerol to 3-hydroxypropionaldehyde, is essential to the production of 1,3-propanediol (1,3-PDO) or 3-hydroxypropionic acid (3-HP). A reliable GDHt activity assay in crude-cell extract was developed. In the assay, GDHt converted 1,2-propanediol (1,2-PDO) to propionaldehyde, which was further converted to 1-propionic acid by aldehyde dehydrogenase (KGSADH) or to 1-propanol by yeast-alcohol dehydrogenase (yADH), while the NADH concentration change was monitored spectrophotometrically. Cells should be disintegrated by Bead Beater/French Press, not by chemical methods (BugBuster ® /B-PER™), because the reagents significantly inactivated GDHt and coupling enzymes. Furthermore, in the assay mixture, a much higher activity of KGSADH (>200-fold) or yADH (>400-fold) than that of GDHt should have been maintained. Under optimal conditions, both KGSADH and yADH showed practically the same activity. The coupled-enzyme assay method established here should prove to be applicable to recombinant strains developed for the production of 3-HP and/or 1,3-PDO from glycerol.

  7. Biochemical and structural characterization of Cryptosporidium parvum Lactate dehydrogenase.

    PubMed

    Cook, William J; Senkovich, Olga; Hernandez, Agustin; Speed, Haley; Chattopadhyay, Debasish

    2015-03-01

    The protozoan parasite Cryptosporidium parvum causes waterborne diseases worldwide. There is no effective therapy for C. parvum infection. The parasite depends mainly on glycolysis for energy production. Lactate dehydrogenase is a major regulator of glycolysis. This paper describes the biochemical characterization of C. parvum lactate dehydrogenase and high resolution crystal structures of the apo-enzyme and four ternary complexes. The ternary complexes capture the enzyme bound to NAD/NADH or its 3-acetylpyridine analog in the cofactor binding pocket, while the substrate binding site is occupied by one of the following ligands: lactate, pyruvate or oxamate. The results reveal distinctive features of the parasitic enzyme. For example, C. parvum lactate dehydrogenase prefers the acetylpyridine analog of NADH as a cofactor. Moreover, it is slightly less sensitive to gossypol inhibition compared with mammalian lactate dehydrogenases and not inhibited by excess pyruvate. The active site loop and the antigenic loop in C. parvum lactate dehydrogenase are considerably different from those in the human counterpart. Structural features and enzymatic properties of C. parvum lactate dehydrogenase are similar to enzymes from related parasites. Structural comparison with malate dehydrogenase supports a common ancestry for the two genes. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Intracellular NADPH Levels Affect the Oligomeric State of the Glucose 6-Phosphate Dehydrogenase

    PubMed Central

    Tramonti, Angela; Lanini, Claudio; Cialfi, Samantha; De Biase, Daniela; Falcone, Claudio

    2012-01-01

    In the yeast Kluyveromyces lactis, glucose 6-phosphate dehydrogenase (G6PDH) is detected as two differently migrating forms on native polyacrylamide gels. The pivotal metabolic role of G6PDH in K. lactis led us to investigate the mechanism controlling the two activities in respiratory and fermentative mutant strains. An extensive analysis of these mutants showed that the NAD+(H)/NADP+(H)-dependent cytosolic alcohol (ADH) and aldehyde (ALD) dehydrogenase balance affects the expression of the G6PDH activity pattern. Under fermentative/ethanol growth conditions, the concomitant activation of ADH and ALD activities led to cytosolic accumulation of NADPH, triggering an alteration in the oligomeric state of the G6PDH caused by displacement/release of the structural NADP+ bound to each subunit of the enzyme. The new oligomeric G6PDH form with faster-migrating properties increases as a consequence of intracellular redox unbalance/NADPH accumulation, which inhibits G6PDH activity in vivo. The appearance of a new G6PDH-specific activity band, following incubation of Saccharomyces cerevisiae and human cellular extracts with NADP+, also suggests that a regulatory mechanism of this activity through NADPH accumulation is highly conserved among eukaryotes. PMID:23064253

  9. Existence of a novel enzyme, pyrroloquinoline quinone-dependent polyvinyl alcohol dehydrogenase, in a bacterial symbiont, Pseudomonas sp. strain VM15C.

    PubMed Central

    Shimao, M; Ninomiya, K; Kuno, O; Kato, N; Sakazawa, C

    1986-01-01

    A novel enzyme, pyrroloquinoline quinone (PQQ)-dependent polyvinyl alcohol (PVA) dehydrogenase, was found in and partially purified from the membrane fraction of a PVA-degrading symbiont, Pseudomonas sp. strain VM15C. The enzyme required PQQ for PVA dehydrogenation with phenazine methosulfate, phenazine ethosulfate, and 2,6-dichlorophenolindophenol as electron acceptors and did not show PVA oxidase activity leading to H2O2 formation. The enzyme was active toward low-molecular-weight secondary alcohols rather than primary alcohols. A membrane-bound PVA oxidase was also present in cells of VM15C. Although the purified oxidase showed a substrate specificity similar to that of PQQ-dependent PVA dehydrogenase and about threefold-higher PVA-dehydrogenating activity with phenazine methosulfate or phenazine ethosulfate than PVA oxidase activity with H2O2 formation, it was shown that the enzyme does not contain PQQ as the coenzyme, and PQQ did not affect its activity. Incubation of the membrane fraction of cells with PVA caused a reduction in the cytochrome(s) of the fraction. Images PMID:3513704

  10. Biotransformation enzymes in the rodent nasal mucosa: the value of a histochemical approach.

    PubMed Central

    Bogdanffy, M S

    1990-01-01

    An increasing number of chemicals have been identified as being toxic to the nasal mucosa of rats. While many chemicals exert their effects only after inhalation exposure, others are toxic following systemic administration, suggesting that factors other than direct deposition on the nasal mucosa may be important in mechanisms of nasal toxicity. The mucosal lining of the nasal cavity consists of a heterogeneous population of ciliated and nonciliated cells, secretory cells, sensory cells, and glandular and other cell types. For chemicals that are metabolized in the nasal mucosa, the balance between metabolic activation and detoxication within a cell type may be a key factor in determining whether that cell type will be a target for toxicity. Recent research in the area of xenobiotic metabolism in nasal mucosa has demonstrated the presence of many enzymes previously described in other tissues. In particular, carboxylesterase, aldehyde dehydrogenase, cytochromes P-450, epoxide hydrolase, and glutathione S-transferases have been localized by histochemical techniques. The distribution of these enzymes appears to be cell-type-specific and the presence of the enzyme may predispose particular cell types to enhanced susceptibility or resistance to chemical-induced injury. This paper reviews the distribution of these enzymes within the nasal mucosa in the context of their contribution to xenobiotic metabolism. The localization of the enzymes by histochemical techniques has provided important information on the potential mechanism of action of esters, aldehydes, and cytochrome P-450 substrates known to injure the nasal mucosa. Images PLATE 1. PLATE 2. PLATE 3. PMID:2200661

  11. 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

  12. Identification and functional evaluation of the reductases and dehydrogenases from Saccharomyces cerevisiae involved in vanillin resistance.

    PubMed

    Wang, Xinning; Liang, Zhenzhen; Hou, Jin; Bao, Xiaoming; Shen, Yu

    2016-04-01

    Vanillin, a type of phenolic released during the pre-treatment of lignocellulosic materials, is toxic to microorganisms and therefore its presence inhibits the fermentation. The vanillin can be reduced to vanillyl alcohol, which is much less toxic, by the ethanol producer Saccharomyces cerevisiae. The reducing capacity of S. cerevisiae and its vanillin resistance are strongly correlated. However, the specific enzymes and their contribution to the vanillin reduction are not extensively studied. In our previous work, an evolved vanillin-resistant strain showed an increased vanillin reduction capacity compared with its parent strain. The transcriptome analysis suggested the reductases and dehydrogenases of this vanillin resistant strain were up-regulated. Using this as a starting point, 11 significantly regulated reductases and dehydrogenases were selected in the present work for further study. The roles of these reductases and dehydrogenases in the vanillin tolerance and detoxification abilities of S. cerevisiae are described. Among the candidate genes, the overexpression of the alcohol dehydrogenase gene ADH6, acetaldehyde dehydrogenase gene ALD6, glucose-6-phosphate 1-dehydrogenase gene ZWF1, NADH-dependent aldehyde reductase gene YNL134C, and aldo-keto reductase gene YJR096W increased 177, 25, 6, 15, and 18 % of the strain μmax in the medium containing 1 g L(-1) vanillin. The in vitro detected vanillin reductase activities of strain overexpressing ADH6, YNL134C and YJR096W were notably higher than control. The vanillin specific reduction rate increased by 8 times in ADH6 overexpressed strain but not in YNL134C and YJR096W overexpressed strain. This suggested that the enzymes encoded by YNL134C and YJR096W might prefer other substrate and/or could not show their effects on vanillin on the high background of Adh6p in vivo. Overexpressing ALD6 and ZWF1 mainly increased the [NADPH]/[NADP(+)] and [GSH]/[GSSG] ratios but not the vanillin reductase activities. Their

  13. Mechanisms of aldehyde-induced adenosinetriphosphatase activities of kinases.

    PubMed

    Rendina, A R; Cleland, W W

    1984-10-23

    Aldehyde analogues of the normal alcohol substrates induce ATPase activities by glycerokinase (D-glyceraldehyde), fructose-6-phosphate kinase (2,5-anhydromannose 6-phosphate), fructokinase (2,5-anhydromannose or 2,5-anhydrotalose), hexokinase (D-gluco-hexodialdose), choline kinase (betaine aldehyde), and pyruvate kinase (glyoxylate). Since purified deuterated aldehydes give V and V/K isotope effects near 1.0 for glycerokinase, fructokinase with 2,5-anhydro[1-2H]talose, hexokinase, choline kinase, and pyruvate kinase, the hydrates of these almost fully hydrated aldehydes are the activators of the ATPase reactions. Fructose-6-phosphate kinase and fructokinase with 2,5-anhydro[1-2H]mannose show V/K deuterium isotope effects of 1.10 and 1.22, respectively, suggesting either that both hydrate and free aldehyde may be activators (predicted values are 1.37 if only the free aldehyde activates the ATPase) or, more likely, that the phosphorylated hydrate breaks down in a rate-limiting step on the enzyme while MgADP is still present and the back-reaction to yield free hydrate in solution is still possible. 18O was transferred from the aldehyde hydrate to phosphate during the ATPase reactions of glycerokinase, fructose-6-phosphate kinase, fructokinase, and hexokinase but not with choline kinase or pyruvate kinase. Thus, direct phosphorylation of the hydrates by the first four enzymes gives the phosphate adduct of the aldehyde, which decomposes nonenzymatically, while with choline kinase and pyruvate kinase the hydrates induce transfer to water (metal-bound hydroxide or water with pyruvate kinase on the basis of pH profiles). Observation of a lag in the release of phosphate from the glycerokinase ATPase reaction at 15 degrees C supports the existence of a phosphorylated hydrate intermediate with a rate constant for breakdown of 0.035-0.043 s-1 at this temperature. Kinases that phosphorylate creatine, 3-phosphoglycerate, and acetate did not exhibit ATPase activities in the

  14. The effect of peroxynitrite decomposition catalyst MnTBAP on aldehyde dehydrogenase-2 nitration by organic nitrates: role in nitrate tolerance.

    PubMed

    Mollace, Vincenzo; Muscoli, Carolina; Dagostino, Concetta; Giancotti, Luigino Antonio; Gliozzi, Micaela; Sacco, Iolanda; Visalli, Valeria; Gratteri, Santo; Palma, Ernesto; Malara, Natalia; Musolino, Vincenzo; Carresi, Cristina; Muscoli, Saverio; Vitale, Cristiana; Salvemini, Daniela; Romeo, Francesco

    2014-11-01

    Bioconversion of glyceryl trinitrate (GTN) into nitric oxide (NO) by aldehyde dehydrogenase-2 (ALDH-2) is a crucial mechanism which drives vasodilatory and antiplatelet effect of organic nitrates in vitro and in vivo. Oxidative stress generated by overproduction of free radical species, mostly superoxide anions and NO-derived peroxynitrite, has been suggested to play a pivotal role in the development of nitrate tolerance, though the mechanism still remains unclear. Here we studied the free radical-dependent impairment of ALDH-2 in platelets as well as vascular tissues undergoing organic nitrate ester tolerance and potential benefit when using the selective peroxynitrite decomposition catalyst Mn(III) tetrakis (4-Benzoic acid) porphyrin (MnTBAP). Washed human platelets were made tolerant to nitrates via incubation with GTN for 4h. This was expressed by attenuation of platelet aggregation induced by thrombin (40U/mL), an effect accompanied by GTN-related induction of cGMP levels in platelets undergoing thrombin-induced aggregation. Both effects were associated to attenuated GTN-induced nitrite formation in platelets supernatants and to prominent nitration of ALDH-2, the GTN to NO metabolizing enzyme, suggesting that GTN tolerance was associated to reduced NO formation via impairment of ALDH-2. These effects were all antagonized by co-incubation of platelets with MnTBAP, which restored GTN-induced responses in tolerant platelets. Comparable effect was found under in in vivo settings. Indeed, MnTBAP (10mg/kg, i.p.) significantly restored the hypotensive effect of bolus injection of GTN in rats made tolerants to organic nitrates via chronic administration of isosorbide-5-mononitrate (IS-5-MN), thus confirming the role of peroxynitrite overproduction in the development of tolerance to vascular responses induced by organic nitrates. In conclusion, oxidative stress subsequent to prolonged use of organic nitrates, which occurs via nitration of ALDH-2, represents a key event

  15. 21 CFR 862.1670 - Sorbitol dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Sorbitol dehydrogenase test system. 862.1670... Systems § 862.1670 Sorbitol dehydrogenase test system. (a) Identification. A sorbitol dehydrogenase test system is a device intended to measure the activity of the enzyme sorbitol dehydrogenase in serum...

  16. Methanol-Water Aqueous-Phase Reforming with the Assistance of Dehydrogenases at Near-Room Temperature.

    PubMed

    Shen, Yangbin; Zhan, Yulu; Li, Shuping; Ning, Fandi; Du, Ying; Huang, Yunjie; He, Ting; Zhou, Xiaochun

    2018-03-09

    As an excellent hydrogen-storage medium, methanol has many advantages, such as high hydrogen content (12.6 wt %), low cost, and availability from biomass or photocatalysis. However, conventional methanol-water reforming usually proceeds at high temperatures. In this research, we successfully designed a new effective strategy to generate hydrogen from methanol at near-room temperature. The strategy involved two main processes: CH 3 OH→HCOOH→H 2 and NADH→HCOOH→H 2 . The first process (CH 3 OH→HCOOH→H 2 ) was performed by an alcohol dehydrogenase (ADH), an aldehyde dehydrogenase (ALDH), and an Ir catalyst. The second procedure (NADH→HCOOH→H 2 ) was performed by formate dehydrogenase (FDH) and the Ir catalyst. The Ir catalyst used was a previously reported polymer complex catalyst [Cp*IrCl 2 (ppy); Cp*=pentamethylcyclopentadienyl, ppy=polypyrrole] with high catalytic activity for the decomposition of formic acid at room temperature and is compatible with enzymes, coenzymes, and poisoning chemicals. Our results revealed that the optimum hydrogen generation rate could reach up to 17.8 μmol h -1  g cat -1 under weak basic conditions at 30 °C. This will have high impact on hydrogen storage, production, and applications and should also provide new inspiration for hydrogen generation from methanol. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. 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 Central

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

    2014-01-01

    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

  18. 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. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  19. NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2

    USDA-ARS?s Scientific Manuscript database

    Aldehyde dehydrogenase 2 (ALDH2) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions influence enzyme activity in part by increasing NADH binding affinity. Traditional fluorescence measurements monitor the blue shift of the NADH fluorescence spectrum to study ...

  20. [Hepatic allopurinol oxidizing enzyme in mice].

    PubMed

    Huh, K; Iwata, H; Yamamoto, I

    1975-03-01

    The relationship between allopurinol oxidizing enzyme and aldehyde oxidase was investaged in mice. The oxidation of both N-methylnicotinamide and allopurinol appears to be catalized by a single enzyme, aldehyde oxidase (aldehyde-oxygen oxidoreductase EC, 1.2.3.1.). This conclusion is based on the following evidence; The postnatal changes of allopurinol and N-methylnicotinamide oxidizing activities were similar during growth and the levels of both activities increased in a parallel fashion upon the attainment of sexual maturity. The rates of loss of the activities of both enzymes by heat denaturation as well as dexamethasone administration were similar. The inhibitors of allopurinol oxidizing enzyme also suppressed N-methylnicotinamide oxidation. Competition of N-methylnicotineamide and allopurinol for oxidation was demonstrated. The rate of increase of the activities in both enzymes was almost parallel during each step of the purification from mouse liver supernatant. It was ascertained that xanthine oxidase in the enzyme preparation does not influence allopurinol oxidation.

  1. The first mammalian aldehyde oxidase crystal structure: insights into substrate specificity.

    PubMed

    Coelho, Catarina; Mahro, Martin; Trincão, José; Carvalho, Alexandra T P; Ramos, Maria João; Terao, Mineko; Garattini, Enrico; Leimkühler, Silke; Romão, Maria João

    2012-11-23

    Aldehyde oxidases have pharmacological relevance, and AOX3 is the major drug-metabolizing enzyme in rodents. The crystal structure of mouse AOX3 with kinetics and molecular docking studies provides insights into its enzymatic characteristics. Differences in substrate and inhibitor specificities can be rationalized by comparing the AOX3 and xanthine oxidase structures. The first aldehyde oxidase structure represents a major advance for drug design and mechanistic studies. 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.

  2. Polymorphisms of alcohol dehydrogenase-1B and aldehyde dehydrogenase-2 and the blood and salivary ethanol and acetaldehyde concentrations of Japanese alcoholic men.

    PubMed

    Yokoyama, Akira; Tsutsumi, Eri; Imazeki, Hiromi; Suwa, Yoshihide; Nakamura, Chizu; Yokoyama, Tetsuji

    2010-07-01

    The effects of genetic polymorphism of aldehyde dehydrogenase-2 (ALDH2) on alcohol metabolism are striking in nonalcoholics, and the effects of genetic polymorphism of alcohol dehydrogenase-1B (ADH1B) are modest at most, whereas genetic polymorphisms of both strongly affect the susceptibility to alcoholism and upper aerodigestive tract (UADT) cancer of drinkers. We evaluated associations between ADH1B/ADH1C/ALDH2 genotypes and the blood and salivary ethanol and acetaldehyde levels of 168 Japanese alcoholic men who came to our hospital for the first time in the morning and had been drinking until the day before. The ethanol levels in their blood and saliva were similar, but the acetaldehyde levels in their saliva were much higher than in their blood, probably because of acetaldehyde production by oral bacteria. Blood and salivary ethanol and acetaldehyde levels were both significantly higher in the subjects with the less active ADH1B*1/*1 genotype than in the ADH1B*2 carriers, but none of the levels differed according to ALDH2 genotype. Significant linkage disequilibrium was detected between the ADH1B and ADH1C genotypes, but ADH1C genotype did not affect the blood or salivary ethanol or acetaldehyde levels. High blood acetaldehyde levels were found even in the active ALDH2*1/*1 alcoholics, which were comparable with the levels of the inactive heterozygous ALDH2*1/*2 alcoholics with less active ADH1B*1/*1. The slope of the increase in blood acetaldehyde level as the blood ethanol level increased was significantly steeper in alcoholics with inactive heterozygous ALDH2*1/*2 plus ADH1B*2 allele than with any other genotype combinations, but the slopes of the increase in salivary acetaldehyde level as the salivary ethanol level increased did not differ between the groups of subjects with any combinations of ALDH2 and ADH1B genotypes. The ADH1B/ALDH2 genotype affected the blood and salivary ethanol and acetaldehyde levels of nonabstinent alcoholics in a different manner

  3. 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

  4. Recent advances in biotechnological applications of alcohol dehydrogenases.

    PubMed

    Zheng, Yu-Guo; Yin, Huan-Huan; Yu, Dao-Fu; Chen, Xiang; Tang, Xiao-Ling; Zhang, Xiao-Jian; Xue, Ya-Ping; Wang, Ya-Jun; Liu, Zhi-Qiang

    2017-02-01

    Alcohol dehydrogenases (ADHs), which belong to the oxidoreductase superfamily, catalyze the interconversion between alcohols and aldehydes or ketones with high stereoselectivity under mild conditions. ADHs are widely employed as biocatalysts for the dynamic kinetic resolution of racemic substrates and for the preparation of enantiomerically pure chemicals. This review provides an overview of biotechnological applications for ADHs in the production of chiral pharmaceuticals and fine chemicals.

  5. Inhibition of Aldehyde Dehydrogenase-Activity Expands Multipotent Myeloid Progenitor Cells with Vascular Regenerative Function.

    PubMed

    Cooper, Tyler T; Sherman, Stephen E; Kuljanin, Miljan; Bell, Gillian I; Lajoie, Gilles A; Hess, David A

    2018-05-01

    Blood-derived progenitor cell transplantation holds potential for the treatment of severe vascular diseases. Human umbilical cord blood (UCB)-derived hematopoietic progenitor cells purified using high aldehyde dehydrogenase (ALDH hi ) activity demonstrate pro-angiogenic functions following intramuscular (i.m.) transplantation into immunodeficient mice with hind-limb ischemia. Unfortunately, UCB ALDH hi cells are rare and prolonged ex vivo expansion leads to loss of high ALDH-activity and diminished vascular regenerative function. ALDH-activity generates retinoic acid, a potent driver of hematopoietic differentiation, creating a paradoxical challenge to expand UCB ALDH hi cells while limiting differentiation and retaining pro-angiogenic functions. We investigated whether inhibition of ALDH-activity during ex vivo expansion of UCB ALDH hi cells would prevent differentiation and expand progeny that retained pro-angiogenic functions after transplantation into non-obese diabetic/severe combined immunodeficient mice with femoral artery ligation-induced unilateral hind-limb ischemia. Human UCB ALDH hi cells were cultured under serum-free conditions for 9 days, with or without the reversible ALDH-inhibitor, diethylaminobenzaldehyde (DEAB). Although total cell numbers were increased >70-fold, the frequency of cells that retained ALDH hi /CD34+ phenotype was significantly diminished under basal conditions. In contrast, DEAB-inhibition increased total ALDH hi /CD34+ cell number by ≥10-fold, reduced differentiation marker (CD38) expression, and enhanced the retention of multipotent colony-forming cells in vitro. Proteomic analysis revealed that DEAB-treated cells upregulated anti-apoptotic protein expression and diminished production of proteins implicated with megakaryocyte differentiation. The i.m. transplantation of DEAB-treated cells into mice with hind-limb ischemia stimulated endothelial cell proliferation and augmented recovery of hind-limb perfusion. DEAB

  6. RNA-sequencing quantification of hepatic ontogeny of phase-I enzymes in mice.

    PubMed

    Peng, Lai; Cui, Julia Y; Yoo, Byunggil; Gunewardena, Sumedha S; Lu, Hong; Klaassen, Curtis D; Zhong, Xiao-Bo

    2013-12-01

    Phase-I drug metabolizing enzymes catalyze reactions of hydrolysis, reduction, and oxidation of drugs and play a critical role in drug metabolism. However, the functions of most phase-I enzymes are not mature at birth, which markedly affects drug metabolism in newborns. Therefore, characterization of the expression profiles of phase-I enzymes and the underlying regulatory mechanisms during liver maturation is needed for better estimation of using drugs in pediatric patients. The mouse is an animal model widely used for studying the mechanisms in the regulation of developmental expression of phase-I genes. Therefore, we applied RNA sequencing to provide a "true quantification" of the mRNA expression of phase-I genes in the mouse liver during development. Liver samples of male C57BL/6 mice at 12 different ages from prenatal to adulthood were used for defining the ontogenic mRNA profiles of phase-I families, including hydrolysis: carboxylesterase (Ces), paraoxonase (Pon), and epoxide hydrolase (Ephx); reduction: aldo-keto reductase (Akr), quinone oxidoreductase (Nqo), and dihydropyrimidine dehydrogenase (Dpyd); and oxidation: alcohol dehydrogenase (Adh), aldehyde dehydrogenase (Aldh), flavin monooxygenases (Fmo), molybdenum hydroxylase (Aox and Xdh), cytochrome P450 (P450), and cytochrome P450 oxidoreductase (Por). Two rapidly increasing stages of total phase-I gene expression after birth reflect functional transition of the liver during development. Diverse expression patterns were identified, and some large gene families contained the mRNA of genes that are enriched at different stages of development. Our study reveals the mRNA abundance of phase-I genes in the mouse liver during development and provides a valuable foundation for mechanistic studies in the future.

  7. Effect of Aldehyde Dehydrogenase 2 Gene Polymorphism on Hemodynamics After Nitroglycerin Intervention in Northern Chinese Han Population

    PubMed Central

    Xia, Jia-Qi; Song, Jie; Zhang, Yi; An, Ni-Na; Ding, Lei; Zhang, Zheng

    2015-01-01

    Background: Nitroglycerin (NTG) is one of the few immediate treatments for acute angina. Aldehyde dehydrogenase 2 (ALDH2) is a key enzyme in the human body that facilitates the biological metabolism of NTG. The biological mechanism of NTG serves an important function in NTG efficacy. Some reports still contradict the results that the correlation between ALDH2 gene polymorphisms and NTG and its clinical efficacy is different. However, data on NTG measurement by pain relief are subjective. This study aimed to investigate the influence of ALDH2 gene polymorphism on intervention with sublingual NTG using noninvasive hemodynamic parameters of cardiac output (CO) and systemic vascular resistance (SVR) in Northern Chinese Han population. Methods: This study selected 559 patients from the Affiliated Hospital of Qingdao University. A total of 203 patients presented with coronary heart disease (CHD) and 356 had non-CHD (NCHD) cases. All patient ALDH2 genotypes (G504A) were detected and divided into two types: Wild (GG) and mutant (GA/AA). Among the CHD group, 103 were wild-type cases, and 100 were mutant-type cases. Moreover, 196 cases were wild-type, and 160 cases were mutant type among the NCHD volunteers. A noninvasive hemodynamic detector was used to monitor the CO and the SVR at the 0, 5, and 15 minute time points after medication with 0.5 mg sublingual NTG. Two CO and SVR indicators were used for a comparative analysis of all case genotypes. Results: Both CO and SVR indicators significantly differed between the wild and mutant genotypes at various time points after intervention with sublingual NTG at 5 and 15 minutes in the NCHD (F = 16.460, 15.003, P = 0.000, 0.000) and CHD groups (F = 194.482, 60.582, P = 0.000, 0.000). All CO values in the wild-type case of both NCHD and CHD groups increased, whereas those in the mutant type decreased. The CO and ΔCO differences were statistically significant (P < 0.05; P < 0.05). The SVR and ΔSVR changed between the wild- and

  8. Enantiocomplementary Yarrowia lipolytica Oxidoreductases: Alcohol Dehydrogenase 2 and Short Chain Dehydrogenase/Reductase

    PubMed Central

    Napora-Wijata, Kamila; Strohmeier, Gernot A.; Sonavane, Manoj N.; Avi, Manuela; Robins, Karen; Winkler, Margit

    2013-01-01

    Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases. PMID:24970175

  9. Enantiocomplementary Yarrowia lipolytica Oxidoreductases: Alcohol Dehydrogenase 2 and Short Chain Dehydrogenase/Reductase.

    PubMed

    Napora-Wijata, Kamila; Strohmeier, Gernot A; Sonavane, Manoj N; Avi, Manuela; Robins, Karen; Winkler, Margit

    2013-08-12

    Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases.

  10. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Biochemical and structural characterization of recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius highly enantioselective on diaryl diketone benzil.

    PubMed

    Pennacchio, Angela; Sannino, Vincenzo; Sorrentino, Giosuè; Rossi, Mosè; Raia, Carlo A; Esposito, Luciana

    2013-05-01

    The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh2 gene was heterologously overexpressed in Escherichia coli, and the resulting protein (SaADH2) was purified to homogeneity and both biochemically and structurally characterized. The crystal structure of the SaADH2 NADH-bound form reveals that the enzyme is a tetramer consisting of identical 27,024-Da subunits, each composed of 255 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 80 °C and a 30-min half-inactivation temperature of ∼88 °C. It also shows good tolerance to common organic solvents and a strict requirement for NAD(H) as the coenzyme. SaADH2 displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and α-ketoesters, but is poorly active on aliphatic, cyclic and aromatic alcohols, showing no activity on aldehydes. Interestingly, the enzyme catalyses the asymmetric reduction of benzil to (R)-benzoin with both excellent conversion (98 %) and optical purity (98 %) by way of an efficient in situ NADH-recycling system involving a second thermophilic ADH. The crystal structure of the binary complex SaADH2-NADH, determined at 1.75 Å resolution, reveals details of the active site providing hints on the structural basis of the enzyme enantioselectivity.

  12. Alcohol Selectivity in a Synthetic Thermophilic n-Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes

    PubMed Central

    Loder, Andrew J.; Zeldes, Benjamin M.; Garrison, G. Dale; Lipscomb, Gina L.; Adams, Michael W. W.

    2015-01-01

    n-Butanol is generated as a natural product of metabolism by several microorganisms, but almost all grow at mesophilic temperatures. A synthetic pathway for n-butanol production from acetyl coenzyme A (acetyl-CoA) that functioned at 70°C was assembled in vitro from enzymes recruited from thermophilic bacteria to inform efforts for engineering butanol production into thermophilic hosts. Recombinant versions of eight thermophilic enzymes (β-ketothiolase [Thl], 3-hydroxybutyryl-CoA dehydrogenase [Hbd], and 3-hydroxybutyryl-CoA dehydratase [Crt] from Caldanaerobacter subterraneus subsp. tengcongensis; trans-2-enoyl-CoA reductase [Ter] from Spirochaeta thermophila; bifunctional acetaldehyde dehydrogenase/alcohol dehydrogenase [AdhE] from Clostridium thermocellum; and AdhE, aldehyde dehydrogenase [Bad], and butanol dehydrogenase [Bdh] from Thermoanaerobacter sp. strain X514) were utilized to examine three possible pathways for n-butanol. These pathways differed in the two steps required to convert butyryl-CoA to n-butanol: Thl-Hbd-Crt-Ter-AdhE (C. thermocellum), Thl-Hbd-Crt-Ter-AdhE (Thermoanaerobacter X514), and Thl-Hbd-Crt-Ter-Bad-Bdh. n-Butanol was produced at 70°C, but with different amounts of ethanol as a coproduct, because of the broad substrate specificities of AdhE, Bad, and Bdh. A reaction kinetics model, validated via comparison to in vitro experiments, was used to determine relative enzyme ratios needed to maximize n-butanol production. By using large relative amounts of Thl and Hbd and small amounts of Bad and Bdh, >70% conversion to n-butanol was observed in vitro, but with a 60% decrease in the predicted pathway flux. With more-selective hypothetical versions of Bad and Bdh, >70% conversion to n-butanol is predicted, with a 19% increase in pathway flux. Thus, more-selective thermophilic versions of Bad, Bdh, and AdhE are needed to fully exploit biocatalytic n-butanol production at elevated temperatures. PMID:26253677

  13. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Systems § 862.1440 Lactate dehydrogenase test system. (a) Identification. A lactate dehydrogenase test system is a device intended to measure the activity of the enzyme lactate dehydrogenase in serum. Lactate... hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial infarction...

  14. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Systems § 862.1420 Isocitric dehydrogenase test system. (a) Identification. An isocitric dehydrogenase test system is a device intended to measure the activity of the enzyme isocitric dehydrogenase in serum... disease such as viral hepatitis, cirrhosis, or acute inflammation of the biliary tract; pulmonary disease...

  15. Purification and characterization of a novel recombinant highly enantioselective short-chain NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus.

    PubMed

    Pennacchio, Angela; Pucci, Biagio; Secundo, Francesco; La Cara, Francesco; Rossi, Mosè; Raia, Carlo A

    2008-07-01

    The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenase/reductase (SDR) superfamily was identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The T. thermophilus ADH gene (adh(Tt)) was heterologously overexpressed in Escherichia coli, and the protein (ADH(Tt)) was purified to homogeneity and characterized. ADH(Tt) is a tetrameric enzyme consisting of identical 26,961-Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to approximately 73 degrees C and a 30-min half-inactivation temperature of approximately 90 degrees C, as well as good tolerance to common organic solvents. ADH(Tt) has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and alpha-keto esters, and poor activity on aromatic alcohols and aldehydes. This thermophilic enzyme catalyzes the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, alpha-tetralone, and alpha-methyl and alpha-ethyl benzoylformates to (S)-(-)-1-phenylethanol (>99% enantiomeric excess [ee]), (R)-alpha-(trifluoromethyl)benzyl alcohol (93% ee), (S)-alpha-tetralol (>99% ee), methyl (R)-(-)-mandelate (92% ee), and ethyl (R)-(-)-mandelate (95% ee), respectively, by way of an efficient in situ NADH-recycling system involving 2-propanol and a second thermophilic ADH. This study further supports the critical role of the D37 residue in discriminating NAD(H) from NADP(H) in members of the SDR superfamily.

  16. Synthesis of robalzotan, ebalzotan, and rotigotine precursors via the stereoselective multienzymatic cascade reduction of α,β-unsaturated aldehydes.

    PubMed

    Brenna, Elisabetta; Gatti, Francesco G; Malpezzi, Luciana; Monti, Daniela; Parmeggiani, Fabio; Sacchetti, Alessandro

    2013-05-17

    A stereoselective synthesis of bicyclic primary or secondary amines, based on tetralin or chroman structural moieties, is reported. These amines are precursors of important active pharmaceutical ingredients such as rotigotine (Neupro), robalzotan, and ebalzotan. The key step is based on a multienzymatic reduction of an α,β-unsaturated aldehyde or ketone to give the saturated primary or secondary alcohol, in a high yield and with a high ee. The catalytic system consists of the combination of an ene-reductase (ER; i.e., OYE2 or OYE3 belonging to the Old Yellow Enzyme family) with an alcohol dehydrogenase (ADH), applying the in situ substrate feeding product removal technology. By this system the formation of the allylic alcohol side product and the racemization of the chirally unstable α-substituted aldehyde intermediate are minimized. The primary alcohols were elaborated via a Curtius rearrangement. The combination of OYE2 with a Prelog or an anti-Prelog ADH allowed the preparation of the secondary alcohols with ee > 99% and de > 87%. The absolute configuration of the primary amines was unambiguously assigned by comparison with authentic samples. The stereochemistry of secondary alcohols was assigned by X-ray crystal structure and NMR analysis of Mosher esters.

  17. Purification and characterization of vanillin dehydrogenases from alkaliphile Micrococcus sp. TA1 and neutrophile Burkholderia cepacia TM1.

    PubMed

    Mitsui, Ryoji; Hirota, Mizuho; Tsuno, Takuo; Tanaka, Mitsuo

    2010-02-01

    Vanillin dehydrogenases (VDHs) were purified and characterized from two bacterial strains that have different pH dependencies for growth. The alkaliphile Micrococcus sp. TA1, isolated from an alkaline spa, can grow on several aromatic compounds such as ferulic acid, vanillin, vanillic acid, and protocatechuic acid under alkaline conditions. The neutrophile Burkholderia cepacia TM1, which was isolated previously, also grew on the above-mentioned compounds because they functioned as the sole carbon source under neutral conditions. Purified VDHs showed activities toward some aromatic aldehydes. These enzymes have the same subunit molecular mass of about 57 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but differed in some of their observed properties. Native molecular masses also differed between the purified enzymes. These were 250 kDa for the enzyme from alkaliphilic strain TA1 and 110 kDa for that from neutrophilic strain TM1, as determined by gel filtration. The enzyme from strain TA1 required NADP(+) as a coenzyme for its activity, but that from strain TM1 required NAD(+). These results are important because this is the first report of an alkaliphilic bacterium consuming lignin monomers.

  18. Metabolism of 2-phenylethylamine to phenylacetic acid, via the intermediate phenylacetaldehyde, by freshly prepared and cryopreserved guinea pig liver slices.

    PubMed

    Panoutsopoulos, Georgios I

    2004-01-01

    2-Phenylethylamine is an endogenous amine, which acts as a neuromodulator of dopaminergic responses. Exogenous 2-phenylethylamine is found in certain foodstuffs and may cause toxic side-effects in susceptible individuals. The present investigation examined the metabolism of 2-phenylethylamine to phenylacetic acid, via phenylacetaldehyde, in freshly prepared and cryopreserved liver slices. Additionally, it compared the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase by using specific inhibitors for each oxidizing enzyme. In freshly prepared and cryopreserved liver slices, phenylacetic acid was the main metabolite of 2-phenylethalamine. In freshly prepared liver slices, phenylacetic acid was completely inhibited by disulfiram (inhibitor of aldehyde dehydrogenase), whereas isovanillin (inhibitor of aldehyde oxidase) inhibited acid formation to a lesser extent and allopurinol (inhibitor of xanthine oxidase) had no effect. In cryopreserved liver slices, isovanillin inhibited phenylacetic acid by 85%, whereas disulfiram inhibited acid formation to a lesser extent and allopurinol had no effect. In liver slices, 2-phenylethylamine is rapidly oxidized to phenylacetic acid, via phenylacetaldehyde, by aldehyde dehydrogenase and aldehyde oxidase with no contribution from xanthine oxidase.

  19. 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. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Effect of Lipid Peroxidation Products on the Activity of Human Retinol Dehydrogenase 12 (RDH12) and Retinoid Metabolism

    PubMed Central

    Lee, Seung-Ah; Belyaeva, Olga V.; Kedishvili, Natalia Y.

    2008-01-01

    SUMMARY Mutations in human Retinol Dehydrogenase 12 (RDH12) are known to cause photoreceptor cell death but the physiological function of RDH12 in photoreceptors remains poorly understood. In vitro, RDH12 recognizes both retinoids and medium-chain aldehydes as substrates. Our previous study suggested that RDH12 protects cells against toxic levels of retinaldehyde and retinoic acid [Lee et al., J. Biol. Chem. 282 (2007) 35621–35628]. Here, we investigated whether RDH12 can also protect cells against highly reactive medium-chain aldehydes. Analysis of cell survival demonstrated that RDH12 was protective against nonanal but not against 4-hydroxynonenal. At high concentrations, nonanal inhibited the activity of RDH12 towards retinaldehyde, suggesting that nonanal was metabolized by RDH12. 4-Hydroxynonenal did not inhibit the RDH12 retinaldehyde reductase activity, but it strongly inhibited the activities of lecithin:retinol acyl transferase and aldehyde dehydrogenase, resulting in decreased levels of retinyl esters and retinoic acid and accumulation of unesterified retinol. Thus, the results of this study showed that RDH12 is more effective in protection against retinaldehyde than against medium-chain aldehydes, and that medium-chain aldehydes, especially 4-hydroxynonenal, severely disrupt cellular retinoid homeostasis. Together, these findings provide a new insight into the effects of lipid peroxidation products and the impact of oxidative stress on retinoid metabolism. PMID:18396173

  1. Reconstitution of the Escherichia coli pyruvate dehydrogenase complex.

    PubMed Central

    Reed, L J; Pettit, F H; Eley, M H; Hamilton, L; Collins, J H; Oliver, R M

    1975-01-01

    The binding of pyruvate dehydrogenase and dihydrolipoyl dehydrogenase (flavoprotein) to dihydrolipoyl transacetylase, the core enzyme of the E. coli pyruvate dehydrogenase complex [EC 1.2.4.1:pyruvate:lipoate oxidoreductase (decaryboxylating and acceptor-acetylating)], has been studied using sedimentation equilibrium analysis and radioactive enzymes in conjunction with gel filtration chromatography. The results show that the transacetylase, which consists of 24 apparently identical polypeptide chains organized into a cube-like structure, has the potential to bind 24 pyruvate dehydrogenase dimers in the absence of flavoprotein and 24 flavoprotein dimers in the absence of pyruvate dehydrogenase. The results of reconstitution experiments, utilizing binding and activity measurements, indicate that the transacetylase can accommodate a total of only about 12 pyruvate dehydrogenase dimers and six flavoprotein dimers and that this stoichiometry, which is the same as that of the native pyruvate dehydrogenase complex, produces maximum activity. It appears that steric hindrance between the relatively bulky pyruvate dehydrogenase and flavoprotein molecules prevents the transacetylase from binding 24 molecules of each ligand. A structural model for the native and reconstituted pyruvate dehydrogenase complexes is proposed in which the 12 pyruvate dehydrogenase dimers are distributed symmetrically on the 12 edges of the transacetylase cube and the six flavoprotein dimers are distributed in the six faces of the cube. Images PMID:1103138

  2. Aldehyde dehydrogenase, Ald4p, is a major component of mitochondrial fluorescent inclusion bodies in the yeast Saccharomyces cerevisiae

    PubMed Central

    Misonou, Yoshiko; Kikuchi, Maiko; Sato, Hiroshi; Inai, Tomomi; Kuroiwa, Tsuneyoshi; Tanaka, Kenji; Miyakawa, Isamu

    2014-01-01

    ABSTRACT When Saccharomyces cerevisiae strain 3626 was cultured to the stationary phase in a medium that contained glucose, needle-like structures that emitted autofluorescence were observed in almost all cells by fluorescence microscopy under UV excitation. The needle-like structures completely overlapped with the profile of straight elongated mitochondria. Therefore, these structures were designated as mitochondrial fluorescent inclusion bodies (MFIBs). The MFIB-enriched mitochondrial fractions were successfully isolated and 2D-gel electrophoresis revealed that a protein of 54 kDa was only highly concentrated in the fractions. Determination of the N-terminal amino acid sequence of the 54-kDa protein identified it as a mitochondrial aldehyde dehydrogenase, Ald4p. Immunofluorescence microscopy showed that anti-Ald4p antibody specifically stained MFIBs. Freeze-substitution electron microscopy demonstrated that cells that retained MFIBs had electron-dense filamentous structures with a diameter of 10 nm in straight elongated mitochondria. Immunoelectron microscopy showed that Ald4p was localized to the electron-dense filamentous structures in mitochondria. These results together showed that a major component of MFIBs is Ald4p. In addition, we demonstrate that MFIBs are common features that appear in mitochondria of many species of yeast. PMID:24771619

  3. Identification and characterization of aldehyde oxidases (AOXs) in the cotton bollworm

    NASA Astrophysics Data System (ADS)

    Xu, Wei; Liao, Yalin

    2017-12-01

    Aldehyde oxidases (AOXs) are a family of metabolic enzymes that oxidize aldehydes into carboxylic acids; therefore, they play critical roles in detoxification and degradation of chemicals. By using transcriptomic and genomic approaches, we successfully identified six putative AOX genes (HarmAOX1-6) from cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). In silico expression profile, reverse transcription (RT)-PCR, and quantitative PCR (qPCR) analyses showed that HarmAOX1 is highly expressed in adult antennae, tarsi, and larval mouthparts, so they may play an important role in degrading plant-derived compounds. HarmAOX2 is highly and specifically expressed in adult antennae, suggesting a candidate pheromone-degrading enzyme (PDE) to inactivate the sex pheromone components (Z)-11-hexadecenal and (Z)-9-hexadecenal. RNA sequencing data further demonstrated that a number of host plants they feed on could significantly upregulate the expression levels of HarmAOX1 in larvae. This study improves our understanding of insect aldehyde oxidases and insect-plant interactions.

  4. 21 CFR 862.1670 - Sorbitol dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Systems § 862.1670 Sorbitol dehydrogenase test system. (a) Identification. A sorbitol dehydrogenase test system is a device intended to measure the activity of the enzyme sorbitol dehydrogenase in serum... cirrhosis or acute hepatitis. (b) Classification. Class I (general controls). The device is exempt from the...

  5. Cloning, functional expression and characterization of a bifunctional 3-hydroxybutanal dehydrogenase /reductase involved in acetone metabolism by Desulfococcus biacutus.

    PubMed

    Frey, Jasmin; Rusche, Hendrik; Schink, Bernhard; Schleheck, David

    2016-11-25

    The strictly anaerobic, sulfate-reducing bacterium Desulfococcus biacutus can utilize acetone as sole carbon and energy source for growth. Whereas in aerobic and nitrate-reducing bacteria acetone is activated by carboxylation with CO 2 to acetoacetate, D. biacutus involves CO as a cosubstrate for acetone activation through a different, so far unknown pathway. Proteomic studies indicated that, among others, a predicted medium-chain dehydrogenase/reductase (MDR) superfamily, zinc-dependent alcohol dehydrogenase (locus tag DebiaDRAFT_04514) is specifically and highly produced during growth with acetone. The MDR gene DebiaDRAFT_04514 was cloned and overexpressed in E. coli. The purified recombinant protein required zinc as cofactor, and accepted NADH/NAD + but not NADPH/NADP + as electron donor/acceptor. The pH optimum was at pH 8, and the temperature optimum at 45 °C. Highest specific activities were observed for reduction of C 3 - C 5 -aldehydes with NADH, such as propanal to propanol (380 ± 15 mU mg -1 protein), butanal to butanol (300 ± 24 mU mg -1 ), and 3-hydroxybutanal to 1,3-butanediol (248 ± 60 mU mg -1 ), however, the enzyme also oxidized 3-hydroxybutanal with NAD + to acetoacetaldehyde (83 ± 18 mU mg -1 ). The enzyme might play a key role in acetone degradation by D. biacutus, for example as a bifunctional 3-hydroxybutanal dehydrogenase/reductase. Its recombinant production may represent an important step in the elucidation of the complete degradation pathway.

  6. 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.

  7. Structural and kinetic studies of a novel nerol dehydrogenase from Persicaria minor, a nerol-specific enzyme for citral biosynthesis.

    PubMed

    Tan, Cheng Seng; Hassan, Maizom; Mohamed Hussein, Zeti Azura; Ismail, Ismanizan; Ho, Kok Lian; Ng, Chyan Leong; Zainal, Zamri

    2018-02-01

    Geraniol degradation pathway has long been elucidated in microorganisms through bioconversion studies, yet weakly characterised in plants; enzyme with specific nerol-oxidising activity has not been reported. A novel cDNA encodes nerol dehydrogenase (PmNeDH) was isolated from Persicaria minor. The recombinant PmNeDH (rPmNeDH) is a homodimeric enzyme that belongs to MDR (medium-chain dehydrogenases/reductases) superfamily that catalyses the first oxidative step of geraniol degradation pathway in citral biosynthesis. Kinetic analysis revealed that rPmNeDH has a high specificity for allylic primary alcohols with backbone ≤10 carbons. rPmNeDH has ∼3 fold higher affinity towards nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) than its trans-isomer, geraniol. To our knowledge, this is the first alcohol dehydrogenase with higher preference towards nerol, suggesting that nerol can be effective substrate for citral biosynthesis in P. minor. The rPmNeDH crystal structure (1.54 Å) showed high similarity with enzyme structures from MDR superfamily. Structure guided mutation was conducted to describe the relationships between substrate specificity and residue substitutions in the active site. Kinetics analyses of wild-type rPmNeDH and several active site mutants demonstrated that the substrate specificity of rPmNeDH can be altered by changing any selected active site residues (Asp 280 , Leu 294 and Ala 303 ). Interestingly, the L294F, A303F and A303G mutants were able to revamp the substrate preference towards geraniol. Furthermore, mutant that exhibited a broader substrate range was also obtained. This study demonstrates that P. minor may have evolved to contain enzyme that optimally recognise cis-configured nerol as substrate. rPmNeDH structure provides new insights into the substrate specificity and active site plasticity in MDR superfamily. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  8. Widespread Non-Hematopoietic Tissue Distribution by Transplanted Human Progenitor Cells with High Aldehyde Dehydrogenase Activity

    PubMed Central

    Hess, David A.; Craft, Timothy P.; Wirthlin, Louisa; Hohm, Sarah; Zhou, Ping; Eades, William C.; Creer, Michael H.; Sands, Mark S.; Nolta, Jan A.

    2011-01-01

    Transplanted adult progenitor cells distribute to peripheral organs and can promote endogenous cellular repair in damaged tissues. However, development of cell-based regenerative therapies has been hindered by the lack of pre-clinical models to efficiently assess multiple organ distribution and difficulty defining human cells with regenerative function. After transplantation into beta-glucuronidase (GUSB)-deficient NOD/SCID/MPSVII mice, we characterized the distribution of lineage depleted human umbilical cord blood-derived cells purified by selection using high aldehyde dehydrogenase activity (ALDH) with CD133 co-expression. ALDHhi or ALDHhiCD133+ cells produced robust hematopoietic reconstitution, and variable levels of tissue distribution in multiple organs. GUSB+ donor cells that co-expressed human (HLA-A,B,C) and hematopoietic (CD45+) cell surface markers were the primary cell phenotype found adjacent to the vascular beds of several tissues, including islet and ductal regions of mouse pancreata. In contrast, variable phenotypes were detected in the chimeric liver, with HLA+/CD45+ cells demonstrating robust GUSB expression adjacent to blood vessels, and CD45−/HLA− cells with diluted GUSB expression predominant in the liver parenchyma. However, true non-hematopoietic human (HLA+/CD45−) cells were rarely detected in other peripheral tissues, suggesting that these GUSB+/HLA−/CD45− cells in the liver were a result of downregulated human surface marker expression in vivo, not widespread seeding of non-hematopoietic cells. However, relying solely on continued expression of cell surface markers, as employed in traditional xenotransplantation models, may underestimate true tissue distribution. ALDH-expressing progenitor cells demonstrated widespread and tissue-specific distribution of variable cellular phenotypes, indicating that these adult progenitor cells should be explored in transplantation models of tissue damage. PMID:18055447

  9. A search for microorganisms producing medium-chain alkanes from aldehydes.

    PubMed

    Ito, Masakazu; Kambe, Hiromi; Kishino, Shigenobu; Muramatsu, Masayoshi; Ogawa, Jun

    2018-01-01

    Microorganisms with medium-chain alkane-producing activity are promising for the bio-production of drop-in fuel. In this study, we screened for microorganisms producing tridecane from tetradecanal. The activity of aldehyde decarbonylation was found in a wide range of microbes. In particular, the genus Klebsiella in the Enterobacteriaceae family was found to have a high ability to produce alkanes from aldehydes via enzyme catalyzed reaction. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  10. Coniferyl aldehyde 5-hydroxylation and methylation direct syringyl lignin biosynthesis in angiosperms

    PubMed Central

    Osakabe, Keishi; Tsao, Cheng Chung; Li, Laigeng; Popko, Jacqueline L.; Umezawa, Toshiaki; Carraway, Daniel T.; Smeltzer, Richard H.; Joshi, Chandrashekhar P.; Chiang, Vincent L.

    1999-01-01

    A central question in lignin biosynthesis is how guaiacyl intermediates are hydroxylated and methylated to the syringyl monolignol in angiosperms. To address this question, we cloned cDNAs encoding a cytochrome P450 monooxygenase (LsM88) and a caffeate O-methyltransferase (COMT) from sweetgum (Liquidambar styraciflua) xylem. Mass spectrometry-based functional analysis of LsM88 in yeast identified it as coniferyl aldehyde 5-hydroxylase (CAld5H). COMT expressed in Escherichia coli methylated 5-hydroxyconiferyl aldehyde to sinapyl aldehyde. Together, CAld5H and COMT converted coniferyl aldehyde to sinapyl aldehyde, suggesting a CAld5H/COMT-mediated pathway from guaiacyl to syringyl monolignol biosynthesis via coniferyl aldehyde that contrasts with the generally accepted route to sinapate via ferulate. Although the CAld5H/COMT enzyme system can mediate the biosynthesis of syringyl monolignol intermediates through either route, kcat/Km of CAld5H for coniferyl aldehyde was ≈140 times greater than that for ferulate. More significantly, when coniferyl aldehyde and ferulate were present together, coniferyl aldehyde was a noncompetitive inhibitor (Ki = 0.59 μM) of ferulate 5-hydroxylation, thereby eliminating the entire reaction sequence from ferulate to sinapate. In contrast, ferulate had no effect on coniferyl aldehyde 5-hydroxylation. 5-Hydroxylation also could not be detected for feruloyl-CoA or coniferyl alcohol. Therefore, in the presence of coniferyl aldehyde, ferulate 5-hydroxylation does not occur, and the syringyl monolignol can be synthesized only from coniferyl aldehyde. Endogenous coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes were detected, consistent with in vivo operation of the CAld5H/COMT pathway from coniferyl to sinapyl aldehydes via 5-hydroxyconiferyl aldehyde for syringyl monolignol biosynthesis. PMID:10430877

  11. Synthesis and accumulation of aromatic aldehydes in an engineered strain of Escherichia coli.

    PubMed

    Kunjapur, Aditya M; Tarasova, Yekaterina; Prather, Kristala L J

    2014-08-20

    Aromatic aldehydes are useful in numerous applications, especially as flavors, fragrances, and pharmaceutical precursors. However, microbial synthesis of aldehydes is hindered by rapid, endogenous, and redundant conversion of aldehydes to their corresponding alcohols. We report the construction of an Escherichia coli K-12 MG1655 strain with reduced aromatic aldehyde reduction (RARE) that serves as a platform for aromatic aldehyde biosynthesis. Six genes with reported activity on the model substrate benzaldehyde were rationally targeted for deletion: three genes that encode aldo-keto reductases and three genes that encode alcohol dehydrogenases. Upon expression of a recombinant carboxylic acid reductase in the RARE strain and addition of benzoate during growth, benzaldehyde remained in the culture after 24 h, with less than 12% conversion of benzaldehyde to benzyl alcohol. Although individual overexpression results demonstrated that all six genes could contribute to benzaldehyde reduction in vivo, additional experiments featuring subset deletion strains revealed that two of the gene deletions were dispensable under the conditions tested. The engineered strain was next investigated for the production of vanillin from vanillate and succeeded in preventing formation of the byproduct vanillyl alcohol. A pathway for the biosynthesis of vanillin directly from glucose was introduced and resulted in a 55-fold improvement in vanillin titer when using the RARE strain versus the wild-type strain. Finally, synthesis of the chiral pharmaceutical intermediate L-phenylacetylcarbinol (L-PAC) was demonstrated from benzaldehyde and glucose upon expression of a recombinant mutant pyruvate decarboxylase in the RARE strain. Beyond allowing accumulation of aromatic aldehydes as end products in E. coli, the RARE strain expands the classes of chemicals that can be produced microbially via aldehyde intermediates.

  12. Novel fungal FAD glucose dehydrogenase derived from Aspergillus niger for glucose enzyme sensor strips.

    PubMed

    Sode, Koji; Loew, Noya; Ohnishi, Yosuke; Tsuruta, Hayato; Mori, Kazushige; Kojima, Katsuhiro; Tsugawa, Wakako; LaBelle, Jeffrey T; Klonoff, David C

    2017-01-15

    In this study, a novel fungus FAD dependent glucose dehydrogenase, derived from Aspergillus niger (AnGDH), was characterized. This enzyme's potential for the use as the enzyme for blood glucose monitor enzyme sensor strips was evaluated, especially by investigating the effect of the presence of xylose during glucose measurements. The substrate specificity of AnGDH towards glucose was investigated, and only xylose was found as a competing substrate. The specific catalytic efficiency for xylose compared to glucose was 1.8%. The specific activity of AnGDH for xylose at 5mM concentration compared to glucose was 3.5%. No other sugars were used as substrate by this enzyme. The superior substrate specificity of AnGDH was also demonstrated in the performance of enzyme sensor strips. The impact of spiking xylose in a sample with physiological glucose concentrations on the sensor signals was investigated, and it was found that enzyme sensor strips using AnGDH were not affected at all by 5mM (75mg/dL) xylose. This is the first report of an enzyme sensor strip using a fungus derived FADGDH, which did not show any positive bias at a therapeutic level xylose concentration on the signal for a glucose sample. This clearly indicates the superiority of AnGDH over other conventionally used fungi derived FADGDHs in the application for SMBG sensor strips. The negligible activity of AnGDH towards xylose was also explained on the basis of a 3D structural model, which was compared to the 3D structures of A. flavus derived FADGDH and of two glucose oxidases. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Biotechnological Production of Methyl-Branched Aldehydes.

    PubMed

    Fraatz, Marco Alexander; Goldmann, Michael; Geissler, Torsten; Gross, Egon; Backes, Michael; Hilmer, Jens-Michael; Ley, Jakob; Rost, Johanna; Francke, Alexander; Zorn, Holger

    2018-03-14

    A number of methyl-branched aldehydes impart interesting flavor impressions, and especially 12-methyltridecanal is a highly sought after flavoring compound for savory foods. Its smell is reminiscent of cooked meat and tallow. For the biotechnological production of 12-methyltridecanal, the literature was screened for fungi forming iso-fatty acids. Suitable organisms were identified and successfully grown in submerged cultures. The culture medium was optimized to increase the yields of branched fatty acids. A recombinant carboxylic acid reductase was used to reduce 12-methyltridecanoic acid to 12-methyltridecanal. The efficiency of whole-cell catalysis was compared to that of the purified enzyme preparation. After lipase-catalyzed hydrolysis of the fungal lipid extracts, the released fatty acids were converted to the corresponding aldehydes, including 12-methyltridecanal and 12-methyltetradecanal.

  14. Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

    PubMed Central

    Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2012-01-01

    Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor. PMID:23203056

  15. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of lactate...

  16. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of lactate...

  17. Isolation and characterization of full-length putative alcohol dehydrogenase genes from polygonum minus

    NASA Astrophysics Data System (ADS)

    Hamid, Nur Athirah Abd; Ismail, Ismanizan

    2013-11-01

    Polygonum minus, locally named as Kesum is an aromatic herb which is high in secondary metabolite content. Alcohol dehydrogenase is an important enzyme that catalyzes the reversible oxidation of alcohol and aldehyde with the presence of NAD(P)(H) as co-factor. The main focus of this research is to identify the gene of ADH. The total RNA was extracted from leaves of P. minus which was treated with 150 μM Jasmonic acid. Full-length cDNA sequence of ADH was isolated via rapid amplification cDNA end (RACE). Subsequently, in silico analysis was conducted on the full-length cDNA sequence and PCR was done on genomic DNA to determine the exon and intron organization. Two sequences of ADH, designated as PmADH1 and PmADH2 were successfully isolated. Both sequences have ORF of 801 bp which encode 266 aa residues. Nucleotide sequence comparison of PmADH1 and PmADH2 indicated that both sequences are highly similar at the ORF region but divergent in the 3' untranslated regions (UTR). The amino acid is differ at the 107 residue; PmADH1 contains Gly (G) residue while PmADH2 contains Cys (C) residue. The intron-exon organization pattern of both sequences are also same, with 3 introns and 4 exons. Based on in silico analysis, both sequences contain "classical" short chain alcohol dehydrogenases/reductases ((c) SDRs) conserved domain. The results suggest that both sequences are the members of short chain alcohol dehydrogenase family.

  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. Analysis of nucleoside-binding proteins by ligand-specific elution from dye resin: application to Mycobacterium tuberculosis aldehyde dehydrogenases.

    PubMed

    Kim, Chang-Yub; Webster, Cecelia; Roberts, Justin K M; Moon, Jin Ho; Alipio Lyon, Emily Z; Kim, Heungbok; Yu, Minmin; Hung, Li-Wei; Terwilliger, Thomas C

    2009-12-01

    We show that Cibacron Blue F3GA dye resin chromatography can be used to identify ligands that specifically interact with proteins from Mycobacterium tuberculosis, and that the identification of these ligands can facilitate structure determination by enhancing the quality of crystals. Four native Mtb proteins of the aldehyde dehydrogenase (ALDH) family were previously shown to be specifically eluted from a Cibacron Blue F3GA dye resin with nucleosides. In this study we characterized the nucleoside-binding specificity of one of these ALDH isozymes (recombinant Mtb Rv0223c) and compared these biochemical results with co-crystallization experiments with different Rv0223c-nucleoside pairings. We found that the strongly interacting ligands (NAD and NADH) aided formation of high-quality crystals, permitting solution of the first Mtb ALDH (Rv0223c) structure. Other nucleoside ligands (AMP, FAD, adenosine, GTP and NADP) exhibited weaker binding to Rv0223c, and produced co-crystals diffracting to lower resolution. Difference electron density maps based on crystals of Rv0223c with various nucleoside ligands show most share the binding site where the natural ligand NAD binds. From the high degree of similarity of sequence and structure compared to human mitochondrial ALDH-2 (BLAST Z-score = 53.5 and RMSD = 1.5 A), Rv0223c appears to belong to the ALDH-2 class. An altered oligomerization domain in the Rv0223c structure seems to keep this protein as monomer whereas native human ALDH-2 is a multimer.

  20. Three-dimensional structure of holo 3 alpha,20 beta-hydroxysteroid dehydrogenase: a member of a short-chain dehydrogenase family.

    PubMed Central

    Ghosh, D; Weeks, C M; Grochulski, P; Duax, W L; Erman, M; Rimsay, R L; Orr, J C

    1991-01-01

    The x-ray structure of a short-chain dehydrogenase, the bacterial holo 3 alpha,20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53), is described at 2.6 A resolution. This enzyme is active as a tetramer and crystallizes with four identical subunits in the asymmetric unit. It has the alpha/beta fold characteristic of the dinucleotide binding region. The fold of the rest of the subunit, the quaternary structure, and the nature of the cofactor-enzyme interactions are, however, significantly different from those observed in the long-chain dehydrogenases. The architecture of the postulated active site is consistent with the observed stereospecificity of the enzyme and the fact that the tetramer is the active form. There is only one cofactor and one substrate-binding site per subunit; the specificity for both 3 alpha- and 20 beta-ends of the steroid results from the binding of the steroid in two orientations near the same cofactor at the same catalytic site. Images PMID:1946424

  1. Crystal structure of homoisocitrate dehydrogenase from Schizosaccharomyces pombe

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bulfer, Stacie L.; Hendershot, Jenna M.; Trievel, Raymond C.

    Lysine biosynthesis in fungi, euglena, and certain archaebacteria occurs through the {alpha}-aminoadipate pathway. Enzymes in the first steps of this pathway have been proposed as potential targets for the development of antifungal therapies, as they are absent in animals but are conserved in several pathogenic fungi species, including Candida, Cryptococcus, and Aspergillus. One potential antifungal target in the {alpha}-aminoadipate pathway is the third enzyme in the pathway, homoisocitrate dehydrogenase (HICDH), which catalyzes the divalent metal-dependent conversion of homoisocitrate to 2-oxoadipate (2-OA) using nicotinamide adenine dinucleotide (NAD{sup +}) as a cofactor. HICDH belogns to a family of {beta}-hydroxyacid oxidative decarboxylases thatmore » includes malate dehydrogenase, tartrate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase (ICDH), and 3-isopropylmalte dehydrogenase (IPMDH). ICDH and IPMDH are well-characterized enzymes that catalyze the decarboxylation of isocitrate to yield 2-oxoglutarate (2-OG) in the citric acid cycle and the conversion of 3-isopropylmalate to 2-oxoisovalerate in the leucine biosynthetic pathway, respectively. Recent structural and biochemical studies of HICDH reveal that this enzyme shares sequence, structural, and mechanistic homology with ICDH and IPMDH. To date, the only published structures of HICDH are from the archaebacteria Thermus thermophilus (TtHICDH). Fungal HICDHs diverge from TtHICDH in several aspects, including their thermal stability, oligomerization state, and substrate specificity, thus warranting further characterization. To gain insights into these differences, they determined crystal structures of a fungal Schizosaccharomyces pombe HICDH (SpHICDH) as an apoenzyme and as a binary complex with additive tripeptide glycyl-glycyl-glycine (GGG) to 1.55 {angstrom} and 1.85 {angstrom} resolution, respectively. Finally, a comparison of the SpHICDH and TtHICDH structures reveal

  2. Genetic polymorphisms of alcohol and aldehyde dehydrogenases and glutathione S-transferase M1 and drinking, smoking, and diet in Japanese men with esophageal squamous cell carcinoma.

    PubMed

    Yokoyama, Akira; Kato, Hoichi; Yokoyama, Tetsuji; Tsujinaka, Toshimasa; Muto, Manabu; Omori, Tai; Haneda, Tatsumasa; Kumagai, Yoshiya; Igaki, Hiroyasu; Yokoyama, Masako; Watanabe, Hiroshi; Fukuda, Haruhiko; Yoshimizu, Haruko

    2002-11-01

    The genetic polymorphisms of aldehyde dehydrogenase-2 (ALDH2), alcohol dehydrogenase-2 (ADH2), ADH3, and glutathione S-transferase M1 (GSTM1) influence the metabolism of alcohol and other carcinogens. The ALDH2*1/2*2 genotype, which encodes inactive ALDH2, and ADH2*1/2*1, which encodes the low-activity form of ADH2, enhance the risk for esophageal cancer in East Asian alcoholics. This case-control study of whether the enzyme-related vulnerability for esophageal cancer can be extended to a general population involved 234 Japanese men with esophageal squamous cell carcinoma and 634 cancer-free Japanese men who received annual health checkups. The GSTM1 genotype was not associated with the risk for this cancer. Light drinkers (1-8.9 units/week) with ALDH2*1/2*2 had an esophageal cancer risk 5.82 times that of light drinkers with ALDH2*1/2*1 (reference category), and their risk was similar to that of moderate drinkers (9-17.9 units/week) with ALDH2*1/2*1 (odds ratio = 5.58). The risk for moderate drinkers with ALDH2*1/2*2 (OR = 55.84) exceeded that for heavy drinkers (18+ units/week) with ALDH2*1/2*1 (OR = 10.38). Similar increased risks were observed for those with ADH2*1/2*1. A multiple logistic model including ALDH2, ADH2, and ADH3 genotypes showed that the ADH3 genotype does not significantly affect the risk for esophageal cancer. For individuals with both ALDH2*1/2*2 and ADH2*1/2*1, the risk of esophageal cancer was enhanced in a multiplicative fashion (OR = 30.12), whereas for those with either ALDH2*1/2*2 or ADH2*1/2*1 alone the ORs were 7.36 and 4.11. In comparison with the estimated population-attributable risks for preference for strong alcoholic beverages (30.7%), smoking (53.6%) and for lower intake of green and yellow vegetables (25.7%) and fruit (37.6%), an extraordinarily high proportion of the excessive risk for esophageal cancer in the Japanese males can be attributed to drinking (90.9%), particularly drinking by persons with inactive heterozygous ALDH

  3. Cloning, expression, purification and preliminary crystallographic analysis of the short-chain dehydrogenase enzymes WbmF, WbmG and WbmH from Bordetella bronchiseptica

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Harmer, Nicholas J., E-mail: nic@cryst.bioc.cam.ac.uk; King, Jerry D.; Department of Veterinary Medicine, Cambridge CB3 0ES

    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, whichmore » 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.« less

  4. Purification, Characterization, and Potential Bacterial Wax Production Role of an NADPH-Dependent Fatty Aldehyde Reductase from Marinobacter aquaeolei VT8▿ †

    PubMed Central

    Wahlen, Bradley D.; Oswald, Whitney S.; Seefeldt, Lance C.; Barney, Brett M.

    2009-01-01

    Wax esters, ester-linked fatty acids and long-chain alcohols, are important energy storage compounds in select bacteria. The synthesis of wax esters from fatty acids is proposed to require the action of a four-enzyme pathway. An essential step in the pathway is the reduction of a fatty aldehyde to the corresponding fatty alcohol, although the enzyme responsible for catalyzing this reaction has yet to be identified in bacteria. We report here the purification and characterization of an enzyme from the wax ester-accumulating bacterium Marinobacter aquaeolei VT8, which is a proposed fatty aldehyde reductase in this pathway. The enzyme, a 57-kDa monomer, was expressed in Escherichia coli as a fusion protein with the maltose binding protein on the N terminus and was purified to near homogeneity by using amylose affinity chromatography. The purified enzyme was found to reduce a number of long-chain aldehydes to the corresponding alcohols coupled to the oxidation of NADPH. The highest specific activity was observed for the reduction of decanal (85 nmol decanal reduced/min/mg). Short-chain and aromatic aldehydes were not substrates. The enzyme showed no detectable catalysis of the reverse reaction, the oxidation of decanol by NADP+. The mechanism of the enzyme was probed with several site-specific chemical probes. The possible uses of this enzyme in the production of wax esters are discussed. PMID:19270127

  5. Regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+ ions within toluene-permeabilized rat heart mitochondria. Interactions with regulation by adenine nucleotides and NADH/NAD+ ratios.

    PubMed Central

    Rutter, G A; Denton, R M

    1988-01-01

    1. Toluene-permeabilized rat heart mitochondria have been used to study the regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+, adenine and nicotinamide nucleotides, and to compare the properties of the enzymes in situ, with those in mitochondrial extracts. 2. Although K0.5 values (concn. giving half-maximal effect) for Ca2+ of 2-oxoglutarate dehydrogenase were around 1 microM under all conditions, corresponding values for NAD+-linked isocitrate dehydrogenase were in the range 5-43 microM. 3. For both enzymes, K0.5 values for Ca2+ observed in the presence of ATP were 3-10-fold higher than those in the presence of ADP, with values increasing over the ADP/ATP range 0.0-1.0. 4. 2-Oxoglutarate dehydrogenase was less sensitive to inhibition by NADH when assayed in permeabilized mitochondria than in mitochondrial extracts. Similarly, the Km of NAD+-linked isocitrate dehydrogenase for threo-Ds-isocitrate was lower in permeabilized mitochondria than in extracts under all the conditions investigated. 5. It is concluded that in the intact heart Ca2+ activation of NAD+-linked isocitrate dehydrogenase may not necessarily occur in parallel with that of the other mitochondrial Ca2+-sensitive enzymes, 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase system. PMID:3421900

  6. Deletion Mapping of zwf, the Gene for a Constitutive Enzyme, Glucose 6-Phosphate Dehydrogenase in ESCHERICHIA COLI

    PubMed Central

    Fraenkel, D. G.; Banerjee, Santimoy

    1972-01-01

    Genes for three enzymes of intermediary sugar metabolism in E. coli, zwf (glucose 6-phosphate dehydrogenase, constitutive), edd (gluconate 6-phosphate dehydrase, inducible), and eda (2-keto-3-deoxygluconate 6-phosphate aldolase, differently inducible) are closely linked on the E. coli genetic map, the overall gene order being man... old... eda. edd. zwf... cheB... uvrC... his. One class of apparent revertants of an eda mutant strain contains a secondary mutation in edd, and some of these mutations are deletions extending into zwf. We have used a series of spontaneous edd-zwf deletions to map a series of point mutants in zwf and thus report the first fine structure map of a gene for a constitutive enzyme (zwf). PMID:4560065

  7. Central carbon metabolism in marine bacteria examined with a simplified assay for dehydrogenases.

    PubMed

    Wen, Weiwei; Wang, Shizhen; Zhou, Xiaofen; Fang, Baishan

    2013-06-01

    A simplified assay platform was developed to measure the activities of the key oxidoreductases in central carbon metabolism of various marine bacteria. Based on microplate assay, the platform was low-cost and simplified by unifying the reaction conditions of enzymes including temperature, buffers, and ionic strength. The central carbon metabolism of 16 marine bacteria, involving Pseudomonas, Exiguobacterium, Marinobacter, Citreicella, and Novosphingobium were studied. Six key oxidoreductases of central carbon metabolism, glucose-6-phosphate dehydrogenase, pyruvate dehydrogenase, 2-ketoglutarate dehydrogenase, malate dehydrogenase, malic enzyme, and isocitrate dehydrogenase were investigated by testing their activities in the pathway. High activity of malate dehydrogenase was found in Citreicella marina, and the specific activity achieved 22 U/mg in cell crude extract. The results also suggested that there was a considerable variability on key enzymes' activities of central carbon metabolism in some strains which have close evolutionary relationship while they adapted to the requirements of the niche they (try to) occupy.

  8. Alcohol Selectivity in a Synthetic Thermophilic n-Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes.

    PubMed

    Loder, Andrew J; Zeldes, Benjamin M; Garrison, G Dale; Lipscomb, Gina L; Adams, Michael W W; Kelly, Robert M

    2015-10-01

    n-Butanol is generated as a natural product of metabolism by several microorganisms, but almost all grow at mesophilic temperatures. A synthetic pathway for n-butanol production from acetyl coenzyme A (acetyl-CoA) that functioned at 70°C was assembled in vitro from enzymes recruited from thermophilic bacteria to inform efforts for engineering butanol production into thermophilic hosts. Recombinant versions of eight thermophilic enzymes (β-ketothiolase [Thl], 3-hydroxybutyryl-CoA dehydrogenase [Hbd], and 3-hydroxybutyryl-CoA dehydratase [Crt] from Caldanaerobacter subterraneus subsp. tengcongensis; trans-2-enoyl-CoA reductase [Ter] from Spirochaeta thermophila; bifunctional acetaldehyde dehydrogenase/alcohol dehydrogenase [AdhE] from Clostridium thermocellum; and AdhE, aldehyde dehydrogenase [Bad], and butanol dehydrogenase [Bdh] from Thermoanaerobacter sp. strain X514) were utilized to examine three possible pathways for n-butanol. These pathways differed in the two steps required to convert butyryl-CoA to n-butanol: Thl-Hbd-Crt-Ter-AdhE (C. thermocellum), Thl-Hbd-Crt-Ter-AdhE (Thermoanaerobacter X514), and Thl-Hbd-Crt-Ter-Bad-Bdh. n-Butanol was produced at 70°C, but with different amounts of ethanol as a coproduct, because of the broad substrate specificities of AdhE, Bad, and Bdh. A reaction kinetics model, validated via comparison to in vitro experiments, was used to determine relative enzyme ratios needed to maximize n-butanol production. By using large relative amounts of Thl and Hbd and small amounts of Bad and Bdh, >70% conversion to n-butanol was observed in vitro, but with a 60% decrease in the predicted pathway flux. With more-selective hypothetical versions of Bad and Bdh, >70% conversion to n-butanol is predicted, with a 19% increase in pathway flux. Thus, more-selective thermophilic versions of Bad, Bdh, and AdhE are needed to fully exploit biocatalytic n-butanol production at elevated temperatures. Copyright © 2015, American Society for

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 identificationmore » 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.« less

  10. 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...

  11. Glucose-6-phosphate dehydrogenase enzyme stability in filter paper dried blood spots.

    PubMed

    Flores, Sharon R; Hall, Elizabeth M; De Jesús, Víctor R

    2017-10-01

    Prior to initial distribution of Glucose-6-phosphate dehydrogenase (G6PD) proficiency testing (PT) materials, we evaluated G6PD enzyme stability in dried blood spots (DBS) under various temperature and humidity environments to develop storage and usage guidelines for our new materials. We prepared fresh G6PD-normal DBS materials and conducted stability evaluations of daily use and short and long-term storage under various temperature and humidity environments. G6PD DBS PT materials retained 92% of initial activity after 30days of use at 4°C. Materials stored at -20°C and 4°C with desiccant for 30days retained 95% and 90% of initial activity, respectively. When stored for one year at -20°C or six months at 4°C specimens retained >90% of initial activity. Specimens stored at 37°C with desiccant lost 10% activity in three days. At the end of 30days, specimens stored under 'Extreme'-humidity >50% without desiccant- conditions at 37°C assayed below the NSQAP cut off for G6PD. Humidity exacerbated loss of enzyme activity with increasing temperature and time duration. Data suggest that G6PD PT materials can be stored at 4°C and used for up to one month and can be stored at -20°C for one year and yield >90% enzyme activity. Exposure to warm temperatures, especially with elevated humidity, should be avoided. Desiccant should always be used to mitigate humidity effects. Published by Elsevier Inc.

  12. Activity of select dehydrogenases with sepharose-immobilized N(6)-carboxymethyl-NAD.

    PubMed

    Beauchamp, Justin; Vieille, Claire

    2015-01-01

    N(6)-carboxymethyl-NAD (N(6)-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N(6)-CM-NAD onto sepharose beads and showed that Thermotoga maritima glycerol dehydrogenase could use the immobilized cofactor with cofactor recycling. We now show that Saccharomyces cerevisiae alcohol dehydrogenase, rabbit muscle L-lactate dehydrogenase (type XI), bovine liver L-glutamic dehydrogenase (type III), Leuconostoc mesenteroides glucose-6-phosphate dehydro-genase, and Thermotoga maritima mannitol dehydrogenase are active with soluble N(6)-CM-NAD. The products of all enzymes but 6-phospho-D-glucono-1,5-lactone were formed when sepharose-immobilized N(6)-CM-NAD was recycled by T. maritima glycerol dehydrogenase, indicating that N(6)-immobilized NAD is suitable for use by a variety of different dehydrogenases. Observations of the enzyme active sites suggest that steric hindrance plays a greater role in limiting or allowing activity with the modified cofactor than do polarity and charge of the residues surrounding the N(6)-amine group on NAD.

  13. Cloning and sequencing of the cDNA species for mammalian dimeric dihydrodiol dehydrogenases.

    PubMed Central

    Arimitsu, E; Aoki, S; Ishikura, S; Nakanishi, K; Matsuura, K; Hara, A

    1999-01-01

    Cynomolgus and Japanese monkey kidneys, dog and pig livers and rabbit lens contain dimeric dihydrodiol dehydrogenase (EC 1.3.1.20) associated with high carbonyl reductase activity. Here we have isolated cDNA species for the dimeric enzymes by reverse transcriptase-PCR from human intestine in addition to the above five animal tissues. The amino acid sequences deduced from the monkey, pig and dog cDNA species perfectly matched the partial sequences of peptides digested from the respective enzymes of these animal tissues, and active recombinant proteins were expressed in a bacterial system from the monkey and human cDNA species. Northern blot analysis revealed the existence of a single 1.3 kb mRNA species for the enzyme in these animal tissues. The human enzyme shared 94%, 85%, 84% and 82% amino acid identity with the enzymes of the two monkey strains (their sequences were identical), the dog, the pig and the rabbit respectively. The sequences of the primate enzymes consisted of 335 amino acid residues and lacked one amino acid compared with the other animal enzymes. In contrast with previous reports that other types of dihydrodiol dehydrogenase, carbonyl reductases and enzymes with either activity belong to the aldo-keto reductase family or the short-chain dehydrogenase/reductase family, dimeric dihydrodiol dehydrogenase showed no sequence similarity with the members of the two protein families. The dimeric enzyme aligned with low degrees of identity (14-25%) with several prokaryotic proteins, in which 47 residues are strictly or highly conserved. Thus dimeric dihydrodiol dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and is suggested to constitute a novel protein family with the prokaryotic proteins. PMID:10477285

  14. Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells

    PubMed Central

    Hess, David A.; Wirthlin, Louisa; Craft, Timothy P.; Herrbrich, Phillip E.; Hohm, Sarah A.; Lahey, Ryan; Eades, William C.; Creer, Michael H.; Nolta, Jan A.

    2006-01-01

    The development of novel cell-based therapies requires understanding of distinct human hematopoietic stem and progenitor cell populations. We recently isolated reconstituting hematopoietic stem cells (HSCs) by lineage depletion and purification based on high aldehyde dehydrogenase activity (ALDHhiLin- cells). Here, we further dissected the ALDHhi-Lin- population by selection for CD133, a surface molecule expressed on progenitors from hematopoietic, endothelial, and neural lineages. ALDHhiCD133+Lin- cells were primarily CD34+, but also included CD34-CD38-CD133+ cells, a phenotype previously associated with repopulating function. Both ALDHhiCD133-Lin- and ALDHhiCD133+Lin- cells demonstrated distinct clonogenic progenitor function in vitro, whereas only the ALDHhiCD133+Lin- population seeded the murine bone marrow 48 hours after transplantation. Significant human cell repopulation was observed only in NOD/SCID and NOD/SCID β2M-null mice that received transplants of ALDHhiCD133+Lin- cells. Limiting dilution analysis demonstrated a 10-fold increase in the frequency of NOD/SCID repopulating cells compared with CD133+Lin- cells, suggesting that high ALDH activity further purified cells with repopulating function. Transplanted ALDHhiCD133+Lin- cells also maintained primitive hematopoietic phenotypes (CD34+CD38-) and demonstrated enhanced repopulating function in recipients of serial, secondary transplants. Cell selection based on ALDH activity and CD133 expression provides a novel purification of HSCs with long-term repopulating function and may be considered an alternative to CD34 cell selection for stem cell therapies. PMID:16269619

  15. 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.

  16. Cyclohexanecarboxyl-Coenzyme A (CoA) and Cyclohex-1-ene-1-Carboxyl-CoA Dehydrogenases, Two Enzymes Involved in the Fermentation of Benzoate and Crotonate in Syntrophus aciditrophicus

    PubMed Central

    Kung, Johannes W.; Seifert, Jana; von Bergen, Martin

    2013-01-01

    The strictly anaerobic Syntrophus aciditrophicus is a fermenting deltaproteobacterium that is able to degrade benzoate or crotonate in the presence and in the absence of a hydrogen-consuming partner. During growth in pure culture, both substrates are dismutated to acetate and cyclohexane carboxylate. In this work, the unknown enzymes involved in the late steps of cyclohexane carboxylate formation were studied. Using enzyme assays monitoring the oxidative direction, a cyclohex-1-ene-1-carboxyl-CoA (Ch1CoA)-forming cyclohexanecarboxyl-CoA (ChCoA) dehydrogenase was purified and characterized from S. aciditrophicus and after heterologous expression of its gene in Escherichia coli. In addition, a cyclohexa-1,5-diene-1-carboxyl-CoA (Ch1,5CoA)-forming Ch1CoA dehydrogenase was characterized after purification of the heterologously expressed gene. Both enzymes had a native molecular mass of 150 kDa and were composed of a single, 40- to 45-kDa subunit; both contained flavin adenine dinucleotide (FAD) as a cofactor. While the ChCoA dehydrogenase was competitively inhibited by Ch1CoA in the oxidative direction, Ch1CoA dehydrogenase further converted the product Ch1,5CoA to benzoyl-CoA. The results obtained suggest that Ch1,5CoA is a common intermediate in benzoate and crotonate fermentation that serves as an electron-accepting substrate for the two consecutively operating acyl-CoA dehydrogenases characterized in this work. In the case of benzoate fermentation, Ch1,5CoA is formed by a class II benzoyl-CoA reductase; in the case of crotonate fermentation, Ch1,5CoA is formed by reversing the reactions of the benzoyl-CoA degradation pathway that are also employed during the oxidative (degradative) branch of benzoate fermentation. PMID:23667239

  17. Activation of liver alcohol dehydrogenase by glycosylation.

    PubMed Central

    Tsai, C S; White, J H

    1983-01-01

    D-Fructose and D-glucose activate alcohol dehydrogenase from horse liver to oxidize ethanol. One mol of D-[U-14C]fructose or D-[U-14C]glucose is covalently incorporated per mol of the maximally activated enzyme. Amino acid and N-terminal analyses of the 14C-labelled glycopeptide isolated from a proteolytic digest of the [14C]glycosylated enzyme implicate lysine-315 as the site of the glycosylation. 13C-n.m.r.-spectroscopic studies indicate that D-[13C]glucose is covalently linked in N-glucosidic and Amadori-rearranged structures in the [13C]glucosylated alcohol dehydrogenase. Experimental results are consistent with the formation of the N-glycosylic linkage between glycose and lysine-315 of liver alcohol dehydrogenase in the initial step that results in an enhanced catalytic efficiency to oxidize ethanol. PMID:6342612

  18. Oxidative bioactivation of abacavir in subcellular fractions of human antigen presenting cells.

    PubMed

    Bell, Catherine C; Santoyo Castelazo, Anahi; Yang, Emma L; Maggs, James L; Jenkins, Rosalind E; Tugwood, Jonathan; O'Neill, Paul M; Naisbitt, Dean J; Park, B Kevin

    2013-07-15

    Human exposure to abacavir, a primary alcohol antiretroviral, is associated with the development of immunological drug reactions in individuals carrying the HLA risk allele B*57:01. Interaction of abacavir with antigen presenting cells results in cell activation through an Hsp70-mediated Toll-like receptor pathway and the provision of T-cell antigenic determinants. Abacavir's electrophilic aldehyde metabolites are potential precursors of neoantigens. Herein, we have used mass spectrometry to study the oxidative metabolism of abacavir in EBV-transformed human B-cells. RNA and protein were isolated from the cells and subjected to transcriptomic and mass spectrometric analyses to identify the redox enzymes expressed. Low levels of isomeric abacavir carboxylic acids were detected in subcellular fractions of EBV-transformed human B-cells incubated with abacavir. Metabolite formation was time-dependent but was not reduced by an inhibitor of Class I alcohol dehydrogenases. Relatively high levels of mRNA were detected for several redox enzymes, including alcohol dehydrogenase 5 (Class III), aldehyde dehydrogenases (ALDH3A2, ALDH6A1, and ALDH9A1), CYP1B1, CYP2R1, CYP7B1, and hydroxysteroid dehydrogenase 10. Over 2600 proteins were identified by mass spectrometry. More than 1000 of these proteins exhibited catalytic activity, and 80 were oxido-reductases. This is the first proteomic inventory of enzymes in antigen presenting cells. However, neither of the hepatic alcohol dehydrogenases of Class I which metabolize abacavir in vitro was expressed at the protein level. Nevertheless the metabolic production of abacavir carboxylic acids by B-cell fractions implies abacavir-treated immune cells might be exposed to the drug's protein-reactive aldehyde metabolites in vivo.

  19. 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

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

    2015-01-01

    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. 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. 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). 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-metabolizing ADH1B was associated with lower serum LDL

  20. Dysfunction of mitochondria and oxidative stress in the pathogenesis of Alzheimer's disease: on defects in the cytochrome c oxidase complex and aldehyde detoxification.

    PubMed

    Ohta, Shigeo; Ohsawa, Ikuroh

    2006-07-01

    The mitochondrion is an organelle that plays a central role in energy production. It, at the same time, generates reactive oxygen species as by-products. Large-scale epidemiological case-control studies suggest the involvements of dihydrolipoamide succinyltransferase (DLST) of the mitochondrial Krebs cycle and mitochondrial aldehyde dehydrogenase-2 (ALDH2) in Alzheimer's disease (AD). The DLST gene has two gene-products, one of which, a novel gene product MIRTD, mediates the molecular assembly of the cytochrome c oxidase complex whose defect has been a candidate of the causes of AD. Since levels of MIRTD mRNA in the brains of AD patients were significantly low, a decrease in MIRTD could affect energy production. ALDH2, a matrix enzyme, was found to act as a protector against oxidative stress through oxidizing toxic aldehydes, such as 4-hydroxy-2-nonenal, that are spontaneously produced from lipid peroxides. Hence, a decrease in ALDH2 activity is proposed to contribute to AD. Indeed, transgenic mice with low activity of ALDH2 exhibited an age-dependent neurodegeneration accompanying memory loss. Since amyloid beta peptide has been recently shown to be present in neuronal mitochondria to decline energy production and enhance ROS production, it has become possible to link AD more closely with roles of mitochondria in the pathogenesis.

  1. Oxidative damage in keratinocytes exposed to cigarette smoke and aldehydes.

    PubMed

    Avezov, Katia; Reznick, Abraham Z; Aizenbud, Dror

    2014-06-01

    Cigarette smoke (CS) is a significant environmental source of human exposure to chemically active saturated (acetaldehyde) and α,β-unsaturated aldehydes (acrolein) inducing protein carbonylation and dysfunction. The exposure of oral tissues to environmental hazards is immense, especially in smokers. The objectives of the current study were to examine the effect of aldehydes originating from CS on intracellular proteins of oral keratinocytes and to observe the antioxidant response in these cells. Intracellular protein carbonyl modification under CS, acrolein and acetaldehyde exposure in the HaCaT keratinocyte cell line, representing oral keratinocytes was examined by Western blot. Possible intracellular enzymatic dysfunction under the above conditions was examined by lactate dehydrogenase (LDH) activity assay. Oxidative stress response was investigated, by DCF (2,7-dichlorodihydrofluorescein) assay and GSH (glutathione) oxidation. Intracellular protein carbonyls increased 5.2 times after CS exposure and 2.7 times after exposure to 1 μmol of acrolein. DCF assay revealed an increase of fluorescence intensity 3.2 and 3.1 times after CS and acrolein exposure, respectively. CS caused a 72.5% decrease in intracellular GSH levels compared to controls. Activity of intracellular LDH was preserved. α,β-Unsaturated aldehydes from CS are capable of intracellular protein carbonylation and have a role in intracellular oxidative stress elevation in keratinocytes, probably due to the reduction in GSH levels. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Genetic polymorphisms of alcohol dehydrogense-1B and aldehyde dehydrogenase-2, alcohol flushing, mean corpuscular volume, and aerodigestive tract neoplasia in Japanese drinkers.

    PubMed

    Yokoyama, Akira; Mizukami, Takeshi; Yokoyama, Tetsuji

    2015-01-01

    Genetic polymorphisms of alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) modulate exposure levels to ethanol/acetaldehyde. Endoscopic screening of 6,014 Japanese alcoholics yielded high detection rates of esophageal squamous cell carcinoma (SCC; 4.1%) and head and neck SCC (1.0%). The risks of upper aerodigestive tract SCC/dysplasia, especially of multiple SCC/dysplasia, were increased in a multiplicative fashion by the presence of a combination of slow-metabolizing ADH1B*1/*1 and inactive heterozygous ALDH2*1/*2 because of prolonged exposure to higher concentrations of ethanol/acetaldehyde. A questionnaire asking about current and past facial flushing after drinking a glass (≈180 mL) of beer is a reliable tool for detecting the presence of inactive ALDH2. We invented a health-risk appraisal (HRA) model including the flushing questionnaire and drinking, smoking, and dietary habits. Esophageal SCC was detected at a high rate by endoscopic mass-screening in high HRA score persons. A total of 5.0% of 4,879 alcoholics had a history of (4.0%) or newly diagnosed (1.0%) gastric cancer. Their high frequency of a history of gastric cancer is partly explained by gastrectomy being a risk factor for alcoholism because of altered ethanol metabolism, e.g., by blood ethanol level overshooting. The combination of H. pylori-associated atrophic gastritis and ALDH2*1/*2 showed the greatest risk of gastric cancer in alcoholics. High detection rates of advanced colorectal adenoma/carcinoma were found in alcoholics, 15.7% of 744 immunochemical fecal occult blood test (IFOBT)-negative alcoholics and 31.5% of the 393 IFOBT-positive alcoholics. Macrocytosis with an MCV≥106 fl increased the risk of neoplasia in the entire aerodigestive tract of alcoholics, suggesting that poor nutrition as well as ethanol/acetaldehyde exposure plays an important role in neoplasia.

  3. Functional analysis of aldehyde oxidase using expressed chimeric enzyme between monkey and rat.

    PubMed

    Itoh, Kunio; Asakawa, Tasuku; Hoshino, Kouichi; Adachi, Mayuko; Fukiya, Kensuke; Watanabe, Nobuaki; Tanaka, Yorihisa

    2009-01-01

    Aldehyde oxidase (AO) is a homodimer with a subunit molecular mass of approximately 150 kDa. Each subunit consists of about 20 kDa 2Fe-2S cluster domain storing reducing equivalents, about 40 kDa flavine adenine dinucleotide (FAD) domain and about 85 kDa molybdenum cofactor (MoCo) domain containing a substrate binding site. In order to clarify the properties of each domain, especially substrate binding domain, chimeric cDNAs were constructed by mutual exchange of 2Fe-2S/FAD and MoCo domains between monkey and rat. Chimeric monkey/rat AO was referred to one with monkey type 2Fe-2S/FAD domains and a rat type MoCo domain. Rat/monkey AO was vice versa. AO-catalyzed 2-oxidation activities of (S)-RS-8359 were measured using the expressed enzyme in Escherichia coli. Substrate inhibition was seen in rat AO and chimeric monkey/rat AO, but not in monkey AO and chimeric rat/monkey AO, suggesting that the phenomenon might be dependent on the natures of MoCo domain of rat. A biphasic Eadie-Hofstee profile was observed in monkey AO and chimeric rat/monkey AO, but not rat AO and chimeric monkey/rat AO, indicating that the biphasic profile might be related to the properties of MoCo domain of monkey. Two-fold greater V(max) values were observed in monkey AO than in chimeric rat/monkey AO, and in chimeric monkey/rat AO than in rat AO, suggesting that monkey has the more effective electron transfer system than rat. Thus, the use of chimeric enzymes revealed that 2Fe-2S/FAD and MoCo domains affect the velocity and the quantitative profiles of AO-catalyzed (S)-RS-8359 2-oxidation, respectively.

  4. Maternal aldehyde elimination during pregnancy preserves the fetal genome.

    PubMed

    Oberbeck, Nina; Langevin, Frédéric; King, Gareth; de Wind, Niels; Crossan, Gerry P; Patel, Ketan J

    2014-09-18

    Maternal metabolism provides essential nutrients to enable embryonic development. However, both mother and embryo produce reactive metabolites that can damage DNA. Here we discover how the embryo is protected from these genotoxins. Pregnant mice lacking Aldh2, a key enzyme that detoxifies reactive aldehydes, cannot support the development of embryos lacking the Fanconi anemia DNA repair pathway gene Fanca. Remarkably, transferring Aldh2(-/-)Fanca(-/-) embryos into wild-type mothers suppresses developmental defects and rescues embryonic lethality. These rescued neonates have severely depleted hematopoietic stem and progenitor cells, indicating that despite intact maternal aldehyde catabolism, fetal Aldh2 is essential for hematopoiesis. Hence, maternal and fetal aldehyde detoxification protects the developing embryo from DNA damage. Failure of this genome preservation mechanism might explain why birth defects and bone marrow failure occur in Fanconi anemia, and may have implications for fetal well-being in the many women in Southeast Asia that are genetically deficient in ALDH2. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Maternal Aldehyde Elimination during Pregnancy Preserves the Fetal Genome

    PubMed Central

    Oberbeck, Nina; Langevin, Frédéric; King, Gareth; de Wind, Niels; Crossan, Gerry P.; Patel, Ketan J.

    2014-01-01

    Summary Maternal metabolism provides essential nutrients to enable embryonic development. However, both mother and embryo produce reactive metabolites that can damage DNA. Here we discover how the embryo is protected from these genotoxins. Pregnant mice lacking Aldh2, a key enzyme that detoxifies reactive aldehydes, cannot support the development of embryos lacking the Fanconi anemia DNA repair pathway gene Fanca. Remarkably, transferring Aldh2−/−Fanca−/− embryos into wild-type mothers suppresses developmental defects and rescues embryonic lethality. These rescued neonates have severely depleted hematopoietic stem and progenitor cells, indicating that despite intact maternal aldehyde catabolism, fetal Aldh2 is essential for hematopoiesis. Hence, maternal and fetal aldehyde detoxification protects the developing embryo from DNA damage. Failure of this genome preservation mechanism might explain why birth defects and bone marrow failure occur in Fanconi anemia, and may have implications for fetal well-being in the many women in Southeast Asia that are genetically deficient in ALDH2. PMID:25155611

  6. Two-carbon homologation of aldehydes and ketones to α,β-unsaturated aldehydes.

    PubMed

    Petroski, Richard J; Vermillion, Karl; Cossé, Allard A

    2011-06-17

    Phosphonate reagents were developed for the two-carbon homologation of aldehydes or ketones to unbranched- or methyl-branched α,β-unsaturated aldehydes. The phosphonate reagents, diethyl methylformyl-2-phosphonate dimethylhydrazone and diethyl ethylformyl-2-phosphonate dimethylhydrazone, contained a protected aldehyde group instead of the usual ester group. A homologation cycle entailed condensation of the reagent with the starting aldehyde, followed by removal of the dimethylhydrazone protective group with a biphasic mixture of 1 M HCl and petroleum ether. This robust two-step process worked with a variety of aldehydes and ketones. Overall isolated yields of unsaturated aldehyde products ranged from 71% to 86% after the condensation and deprotection steps.

  7. Evolutionary genetics of the Drosophila alcohol dehydrogenase gene-enzyme system.

    PubMed

    Heinstra, P W

    1993-01-01

    Evolutionary genetics embodies a broad research area that ranges from the DNA level to studies of genetic aspects in populations. In all cases the purpose is to determine the impact of genetic variation on evolutionary change. The broad range of evolutionary genetics requires the involvement of a diverse group of researchers: molecular biologists, (population) geneticists, biochemists, physiologists, ecologists, ethologists and theorists, each of which has its own insights and interests. For example, biochemists are often not concerned with the physiological function of a protein (with respect to pH, substrates, temperature, etc.), while ecologists, in turn, are often not interested in the biochemical-physiological aspects underlying the traits they study. This review deals with several evolutionary aspects of the Drosophila alcohol dehydrogenase gene-enzyme system, and includes my own personal viewpoints. I have tried to condense and integrate the current knowledge in this field as it has developed since the comprehensive review by van Delden (1982). Details on specific issues may be gained from Sofer and Martin (1987), Sullivan, Atkinson and Starmer (1990); Chambers (1988, 1991); Geer, Miller and Heinstra (1991); and Winberg and McKinley-McKee (1992).

  8. Expression and kinetic properties of a recombinant 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isoenzyme of human liver.

    PubMed

    Deyashiki, Y; Tamada, Y; Miyabe, Y; Nakanishi, M; Matsuura, K; Hara, A

    1995-08-01

    Human liver cytosol contains multiple forms of 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase with hydroxysteroid dehydrogenase activity, and multiple cDNAs for the enzymes have been cloned from human liver cDNA libraries. To understand the relationship of the multiple enzyme froms to the genes, a cDNA, which has been reported to code for an isoenzyme of human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase, was expressed in Escherichia coli. The recombinant enzyme showed structural and functional properties almost identical to those of the isoenzyme purified from human liver. In addition, the recombinant isoenzyme efficiently reduced 5 alpha-dihydrotestosterone and 5 beta-dihydrocortisone, the known substrates of human liver 3 alpha-hydroxysteroid dehydrogenase and chlordecone reductase previously purified, which suggests that these human liver enzymes are identical. Furthermore, the steady-state kinetic data for NADP(+)-linked (S)-1-indanol oxidation by the recombinant isoenzyme were consistent with a sequential ordered mechanism in which NADP+ binds first. Phenolphthalein inhibited this isoenzyme much more potently than it did the other human liver dihydrodiol dehydrogenases, and was a competitive inhibitor (Ki = 20 nM) that bound to the enzyme-NADP+ complex.

  9. Radiation-induced enzyme efflux from rat heart: sedentary animals. [Gamma radiation, lactate dehydrogenase, creative kinase, glutamate oxaloacetate transaminase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    MacWilliam, L.D.; Bhakthan, N.M.G.

    1976-01-01

    Serum levels of lactate dehydrogenase, creatine kinase, and glutamate oxaloacetate transaminase show initial elevations within 12 hr of exposure to 2,000 rads of ..gamma..-radiation to the thoracic region of rats. Significant decreases in heart muscle homogenate levels of these enzymes parallel initial elevations in the serum and may suggest that enhanced leakage of enzymes is a consequence of radiation injury to heart muscle. Insignificant alterations in mitochondrial glutamate oxaloacetate transaminase levels after exposure indicate that in vivo injury to the mitochondria from therapeutic levels of ..gamma..-radiation is questionable. The results support the contention that ionizing radiation instigates alterations in themore » dynamic permeability of membranes, allowing leakage of biologically active material out of the injured cell.« less

  10. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nikolau, Basil J; Wurtele, Eve S; Oliver, David J

    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 ofmore » 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.« less

  11. Thermostable Alcohol Dehydrogenase from Thermococcus kodakarensis KOD1 for Enantioselective Bioconversion of Aromatic Secondary Alcohols

    PubMed Central

    Wu, Xi; Zhang, Chong; Orita, Izumi; Imanaka, Tadayuki

    2013-01-01

    A novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkADH). The gene, tk0845, which encodes an aldo-keto reductase, was heterologously expressed in Escherichia coli. The enzyme was found to be a monomer with a molecular mass of 31 kDa. It was highly thermostable with an optimal temperature of 90°C and a half-life of 4.5 h at 95°C. The apparent Km values for the cofactors NAD(P)+ and NADPH were similar within a range of 66 to 127 μM. TkADH preferred secondary alcohols and accepted various ketones and aldehydes as substrates. Interestingly, the enzyme could oxidize 1-phenylethanol and its derivatives having substituents at the meta and para positions with high enantioselectivity, yielding the corresponding (R)-alcohols with optical purities of greater than 99.8% enantiomeric excess (ee). TkADH could also reduce 2,2,2-trifluoroacetophenone to (R)-2,2,2-trifluoro-1-phenylethanol with high enantioselectivity (>99.6% ee). Furthermore, the enzyme showed high resistance to organic solvents and was particularly highly active in the presence of H2O–20% 2-propanol and H2O–50% n-hexane or n-octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols. PMID:23354700

  12. HIBCH mutations can cause Leigh-like disease with combined deficiency of multiple mitochondrial respiratory chain enzymes and pyruvate dehydrogenase.

    PubMed

    Ferdinandusse, Sacha; Waterham, Hans R; Heales, Simon J R; Brown, Garry K; Hargreaves, Iain P; Taanman, Jan-Willem; Gunny, Roxana; Abulhoul, Lara; Wanders, Ronald J A; Clayton, Peter T; Leonard, James V; Rahman, Shamima

    2013-12-04

    Deficiency of 3-hydroxy-isobutyryl-CoA hydrolase (HIBCH) caused by HIBCH mutations is a rare cerebral organic aciduria caused by disturbance of valine catabolism. Multiple mitochondrial respiratory chain (RC) enzyme deficiencies can arise from a number of mechanisms, including defective maintenance or expression of mitochondrial DNA. Impaired biosynthesis of iron-sulphur clusters and lipoic acid can lead to pyruvate dehydrogenase complex (PDHc) deficiency in addition to multiple RC deficiencies, known as the multiple mitochondrial dysfunctions syndrome. Two brothers born to distantly related Pakistani parents presenting in early infancy with a progressive neurodegenerative disorder, associated with basal ganglia changes on brain magnetic resonance imaging, were investigated for suspected Leigh-like mitochondrial disease. The index case had deficiencies of multiple RC enzymes and PDHc in skeletal muscle and fibroblasts respectively, but these were normal in his younger brother. The observation of persistently elevated hydroxy-C4-carnitine levels in the younger brother led to suspicion of HIBCH deficiency, which was investigated by biochemical assay in cultured skin fibroblasts and molecular genetic analysis. Specific spectrophotometric enzyme assay revealed HIBCH activity to be below detectable limits in cultured skin fibroblasts from both brothers. Direct Sanger sequence analysis demonstrated a novel homozygous pathogenic missense mutation c.950G dehydrogenase deficiency.

  13. Enzymatic transformation of hydrocarbons by methanotrophic organisms

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Patel, R.N.; Hou, C.T.

    Soluble methane monooxygenase from a facultative methane-utilizing organism, Methylobacterium sp. CRL-26 or R6, catalyzed the NAD(P)H-dependent epoxidation/hydroxylation of a variety of hydrocarbons, including terminal alkenes, internal alkenes, substituted alkenes, branch-chain alkenes, alkanes (C1-C8), substituted alkanes, branch-chain alkanes, carbon monoxide, ether, cyclic and aromatic compounds. The NAD -linked dehydrogenases such as formate dehydrogenase or secondary alcohol dehydrogenase in the presence of formate or secondary alcohol, respectively, regenerated NAD/NADH required for the methane monooxygenase in a coupled enzymes reactions. Oxidation of secondary alcohols to the corresponding methylketones in methanotrophs is catalyzed by an NAD -dependent, zinc-containing, secondary alcohol hydrogenase. Primary alcohols weremore » oxidized to the corresponding aldehydes by a phenazine methosulfate-dependent, pyrollo quinoline quinone (methoxatin or PQQ) containing, methanol dehydrogenase. Oxidation of aldehydes (C1 to C10) to the corresponding carboxylic acids is catalyzed by a heme-containing aldehyde dehydrogenase. Methanotrophs have been considered potentially useful for single cell protein (SCP), amino acids, and biopolymer production at the expense of growth on cheap and readily available C1 compounds. 80 references, 1 figure, 6 tables.« less

  14. Subcellular localization of celery mannitol dehydrogenase. A cytosolic metabolic enzyme in nuclei.

    PubMed Central

    Yamamoto, Y T; Zamski, E; Williamson, J D; Conkling, M A; Pharr, D M

    1997-01-01

    Mannitol dehydrogenase (MTD) is the first enzyme in mannitol catabolism in celery (Apium graveolens L. var dulce [Mill] Pers. cv Florida 638). Mannitol is an important photoassimilate, as well as providing plants with resistance to salt and osmotic stress. Previous work has shown that expression of the celery Mtd gene is regulated by many factors, such as hexose sugars, salt and osmotic stress, and salicylic acid. Furthermore, MTD is present in cells of sink organs, phloem cells, and mannitol-grown suspension cultures. Immunogold localization and biochemical analyses presented here demonstrate that celery MTD is localized in the cytosol and nuclei. Although the cellular density of MTD varies among different cell types, densities of nuclear and cytosolic MTD in a given cell are approximately equal. Biochemical analyses of nuclear extracts from mannitol-grown cultured cells confirmed that the nuclear-localized MTD is enzymatically active. The function(s) of nuclear-localized MTD is unknown. PMID:9414553

  15. Formation of Nitric Oxide by Aldehyde Dehydrogenase-2 Is Necessary and Sufficient for Vascular Bioactivation of Nitroglycerin.

    PubMed

    Opelt, Marissa; Eroglu, Emrah; Waldeck-Weiermair, Markus; Russwurm, Michael; Koesling, Doris; Malli, Roland; Graier, Wolfgang F; Fassett, John T; Schrammel, Astrid; Mayer, Bernd

    2016-11-11

    Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN), resulting in activation of soluble guanylate cyclase (sGC) and cGMP-mediated vasodilation. We have previously shown that a minor reaction of ALDH2-catalyzed GTN bioconversion, accounting for about 5% of the main clearance-based turnover yielding inorganic nitrite, results in direct NO formation and concluded that this minor pathway could provide the link between vascular GTN metabolism and activation of sGC. However, lack of detectable NO at therapeutically relevant GTN concentrations (≤1 μm) in vascular tissue called into question the biological significance of NO formation by purified ALDH2. We addressed this issue and used a novel, highly sensitive genetically encoded fluorescent NO probe (geNOp) to visualize intracellular NO formation at low GTN concentrations (≤1 μm) in cultured vascular smooth muscle cells (VSMC) expressing an ALDH2 mutant that reduces GTN to NO but lacks clearance-based GTN denitration activity. NO formation was compared with GTN-induced activation of sGC. The addition of 1 μm GTN to VSMC expressing either wild-type or C301S/C303S ALDH2 resulted in pronounced intracellular NO elevation, with maximal concentrations of 7 and 17 nm, respectively. Formation of GTN-derived NO correlated well with activation of purified sGC in VSMC lysates and cGMP accumulation in intact porcine aortic endothelial cells infected with wild-type or mutant ALDH2. Formation of NO and cGMP accumulation were inhibited by ALDH inhibitors chloral hydrate and daidzin. The present study demonstrates that ALDH2-catalyzed NO formation is necessary and sufficient for GTN bioactivation in VSMC. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Aldehyde dehydrogenase 1 (ALDH1) expression is an independent prognostic factor in triple negative breast cancer (TNBC).

    PubMed

    Ma, Fei; Li, Huihui; Li, Yiqun; Ding, Xiaoyan; Wang, Haijuan; Fan, Ying; Lin, Chen; Qian, Haili; Xu, Binghe

    2017-04-01

    Triple negative breast cancer (TNBC) is a subset of breast cancer that is highly aggressive and has a poor prognosis. Meanwhile, cancer stem cells (CSCs) are also characterized by a strong tumorigenic potential, which might be partly responsible for the aggressive behavior of TNBC. We previously showed that CSCs are enriched in TNBC cell lines and tissues. Further experiments in animal models revealed higher tumorigenicity of CSCs sorted from TNBC cell lines. In this study, we aimed to determine the clinical relationship between CSCs and TNBC by exploring the expression of aldehyde dehydrogenase 1 (ALDH1), which is a putative marker of breast CSCs, in TNBC tissues.ALDH1 levels in paraffin-embedded tumor tissues from 158 TNBC patients were evaluated by immunohistochemistry staining using an ALDH1A1 primary antibody. Staining evaluation was performed independently by two pathologists, and the expression level of ALDH1 was evaluated in terms of the percentage and intensity of positive cells. The association of immunohistochemistry staining of ALDH1 expression with clinical parameters was also analyzed.ALDH1 expression in tumor cells was observed in 88 out of 158 cases (55.7%). Analysis of clinicopathological parameters showed that the immunohistochemistry staining of ALDH1 was significantly correlated with tumor size (P = 0.02) and stage (P = 0.04). Survival analysis in patients with ALDH1 expression demonstrated shorter relapse-free survival (RFS) and overall survival (OS) times (P = 0.01; P = 0.001). Moreover, Cox multivariate analysis revealed that ALDH1 expression was an independent prognostic indicator of RFS and OS (P = 0.04; P = 0.04).Immunohistochemistry staining of ALDH1 in tumor cells is an independent prognostic indicator of RFS and OS in TNBC patients.

  17. Analysis of nucleotide diphosphate sugar dehydrogenases reveals family and group-specific relationships.

    PubMed

    Freas, Nicholas; Newton, Peter; Perozich, John

    2016-01-01

    UDP-glucose dehydrogenase (UDPGDH), UDP-N-acetyl-mannosamine dehydrogenase (UDPNAMDH) and GDP-mannose dehydrogenase (GDPMDH) belong to a family of NAD (+)-linked 4-electron-transfering oxidoreductases called nucleotide diphosphate sugar dehydrogenases (NDP-SDHs). UDPGDH is an enzyme responsible for converting UDP-d-glucose to UDP-d-glucuronic acid, a product that has different roles depending on the organism in which it is found. UDPNAMDH and GDPMDH convert UDP-N-acetyl-mannosamine to UDP-N-acetyl-mannosaminuronic acid and GDP-mannose to GDP-mannuronic acid, respectively, by a similar mechanism to UDPGDH. Their products are used as essential building blocks for the exopolysaccharides found in organisms like Pseudomonas aeruginosa and Staphylococcus aureus. Few studies have investigated the relationships between these enzymes. This study reveals the relationships between the three enzymes by analysing 229 amino acid sequences. Eighteen invariant and several other highly conserved residues were identified, each serving critical roles in maintaining enzyme structure, coenzyme binding or catalytic function. Also, 10 conserved motifs that included most of the conserved residues were identified and their roles proposed. A phylogenetic tree demonstrated relationships between each group and verified group assignment. Finally, group entropy analysis identified novel conservations unique to each NDP-SDH group, including residue positions critical to NDP-sugar substrate interaction, enzyme structure and intersubunit contact. These positions may serve as targets for future research. UDP-glucose dehydrogenase (UDPGDH, EC 1.1.1.22).

  18. Comparison of the effects of Ca2+, adenine nucleotides and pH on the kinetic properties of mitochondrial NAD(+)-isocitrate dehydrogenase and oxoglutarate dehydrogenase from the yeast Saccharomyces cerevisiae and rat heart.

    PubMed Central

    Nichols, B J; Rigoulet, M; Denton, R M

    1994-01-01

    The regulatory properties of NAD(+)-isocitrate dehydrogenase and oxoglutarate dehydrogenase in extracts of yeast and rat heart mitochondria were studied under identical conditions. Yeast NAD(+)-isocitrate dehydrogenase exhibits a low K0.5 for isocitrate and is activated by AMP and ADP, but is insensitive to ATP and Ca2+. In contrast, the rat heart NAD(+)-isocitrate dehydrogenase was insensitive to AMP, but was activated by ADP and by Ca2+ in the presence of ADP or ATP. Both yeast and rat heart oxoglutarate dehydrogenase were stimulated by ADP, but only the heart enzyme was activated by Ca2+. All the enzymes studied were activated by decreases in pH, but to differing extents. The effects of Ca2+, adenine nucleotides and pH were through K0.5 for isocitrate or 2-oxoglutarate. These observations are discussed with reference to the deduced amino acid sequences of the constituent subunits of the enzymes, where they are available. PMID:7980405

  19. Increased IMP dehydrogenase gene expression in solid tumor tissues and tumor cell lines

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Collart, F.R.; Chubb, C.B.; Mirkin, B.L.

    1992-07-10

    IMP dehydrogenase, a regulatory enzyme of guanine nucleotide biosynthesis, may play a role in cell proliferation and malignancy. To assess this possibility, we examined IMP dehydrogenase expression in a series of human solid tumor tissues and tumor cell lines in comparison with their normal counterparts. Increased IMP dehydrogenase gene expression was observed in brain tumors relative to normal brain tissue and in sarcoma cells relative to normal fibroblasts. Similarly, in several B- and T-lymphoid leukemia cell lines, elevated levels of IMP dehydrogenase mRNA and cellular enzyme were observed in comparison with the levels in peripheral blood lymphocytes. These results aremore » consistent with an association between increased IMP dehydrogenase expression and either enhanced cell proliferation or malignant transformation.« less

  20. 21 CFR 862.1565 - 6-Phosphogluconate dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Test Systems § 862.1565 6-Phosphogluconate dehydrogenase test system. (a) Identification. A 6-phosphogluconate dehydrogenase test system is a device intended to measure the activity of the enzyme 6... are used in the diagnosis and treatment of certain liver diseases (such as hepatitis) and anemias. (b...

  1. 21 CFR 862.1565 - 6-Phosphogluconate dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Test Systems § 862.1565 6-Phosphogluconate dehydrogenase test system. (a) Identification. A 6-phosphogluconate dehydrogenase test system is a device intended to measure the activity of the enzyme 6... are used in the diagnosis and treatment of certain liver diseases (such as hepatitis) and anemias. (b...

  2. Characterization of an Arxula adeninivorans alcohol dehydrogenase involved in the metabolism of ethanol and 1-butanol.

    PubMed

    Kasprzak, Jakub; Rauter, Marion; Riechen, Jan; Worch, Sebastian; Baronian, Kim; Bode, Rüdiger; Schauer, Frieder; Kunze, Gotthard

    2016-05-01

    In this study, alcohol dehydrogenase 1 from Arxula adeninivorans (Aadh1p) was identified and characterized. Aadh1p showed activity with short and medium chain length primary alcohols in the forward reaction and their aldehydes in the reverse reaction. Aadh1p has 64% identity with Saccharomyces cerevisiae Adh1p, is localized in the cytoplasm and uses NAD(+) as cofactor. Gene expression analysis showed a low level increase in AADH1 gene expression with ethanol, pyruvate or xylose as the carbon source. Deletion of the AADH1 gene affects growth of the cells with 1-butanol, ethanol and glucose as the carbon source, and a strain which overexpressed the AADH1 gene metabolized 1-butanol more rapidly. An ADH activity assay indicated that Aadh1p is a major enzyme for the synthesis of ethanol and the degradation of 1-butanol in A. adeninivorans. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. 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.

  4. ENZYME ACTIVITIES DURING THE ASEXUAL CYCLE OF NEUROSPORA CRASSA

    PubMed Central

    Stine, G. J.

    1968-01-01

    Three enzymes, (a) nicotinamide adenine diphosphate-dependent glutamic dehydrogenase (NAD enzyme), (b) nictoinamide adenine triphosphate-dependent glutamic dehydrogenase (NADP enzyme), and (c) nicotinamide-adenine dinucleotidase (NADase), were measured in separate extracts of Neurospora crassa grown in Vogel's medium N and medium N + glutamate. Specific activities and total units per culture of each enzyme were determined at nine separate intervals phased throughout the asexual cycle. The separate dehydrogenases were lowest in the conidia, increased slowly during germination, and increased rapidly during logarithmic mycelial growth. The amounts of these enzymes present during germination were small when compared with those found later during the production of the conidiophores. The NAD enzyme may be necessary for pregermination synthesis. The NADP-enzyme synthesis was associated with the appearance of the germ tube. Although higher levels of the dehydrogenases in the conidiophores resulted in more enzyme being found in the differentiated conidia, the rate of germination was uneffected. The greatest activity for the NADase enzyme was associated with the conidia, early phases of germination, and later production of new conidia. NADase decreased significantly with the onset of logarithmic growth, remained low during the differentiation of conidiophores, and increased considerably as the conidiophores aged. PMID:4384627

  5. Mathematical modelling of metabolic pathways affected by an enzyme deficiency. Energy and redox metabolism of glucose-6-phosphate-dehydrogenase-deficient erythrocytes.

    PubMed

    Schuster, R; Jacobasch, G; Holzhütter, H G

    1989-07-01

    The effects of various forms of glucose-6-phosphate dehydrogenase deficiency on erythrocyte metabolism have been studied on the basis of a complex mathematical model which comprises the main pathways of this cell: glycolysis, pentose pathway, reactions of the glutathione and adenine nucleotide metabolism. The calculated flux rates through the oxidative pentose pathway with and without methylene blue are in good accord with experimental results. The degree of deficiency as predicted by the model on the basis of calculated upper oxidative load boundaries, as well as of maximal methylene blue stimulation, correlates with the individual clinical manifestation of the metabolic disease. Therefore, the model allows one to judge the degree of metabolic disorder in the presence of glucose-6-phosphate dehydrogenase enzymopathies if the kinetic properties of the defect enzyme are known. Experimentally accessible parameters for an assessment of the oxidative load capacity of cells in vivo are proposed. It is pointed out that the threshold of tolerance as to energetic load is drastically reduced in the case of severe glucose-6-phosphate dehydrogenase deficiency.

  6. Reversible inactivation of CO dehydrogenase with thiol compounds

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kreß, Oliver; Gnida, Manuel; Pelzmann, Astrid M.

    2014-05-09

    Highlights: • Rather large thiols (e.g. coenzyme A) can reach the active site of CO dehydrogenase. • CO- and H{sub 2}-oxidizing activity of CO dehydrogenase is inhibited by thiols. • Inhibition by thiols was reversed by CO or upon lowering the thiol concentration. • Thiols coordinate the Cu ion in the [CuSMo(=O)OH] active site as a third ligand. - Abstract: Carbon monoxide dehydrogenase (CO dehydrogenase) from Oligotropha carboxidovorans is a structurally characterized member of the molybdenum hydroxylase enzyme family. It catalyzes the oxidation of CO (CO + H{sub 2}O → CO{sub 2} + 2e{sup −} + 2H{sup +}) which proceedsmore » at a unique [CuSMo(=O)OH] metal cluster. Because of changing activities of CO dehydrogenase, particularly in subcellular fractions, we speculated whether the enzyme would be subject to regulation by thiols (RSH). Here we establish inhibition of CO dehydrogenase by thiols and report the corresponding K{sub i}-values (mM): L-cysteine (5.2), D-cysteine (9.7), N-acetyl-L-cysteine (8.2), D,L-homocysteine (25.8), L-cysteine–glycine (2.0), dithiothreitol (4.1), coenzyme A (8.3), and 2-mercaptoethanol (9.3). Inhibition of the enzyme was reversed by CO or upon lowering the thiol concentration. Electron paramagnetic resonance spectroscopy (EPR) and X-ray absorption spectroscopy (XAS) of thiol-inhibited CO dehydrogenase revealed a bimetallic site in which the RSH coordinates to the Cu-ion as a third ligand ([Mo{sup VI}(=O)OH{sub (2)}SCu{sup I}(SR)S-Cys]) leaving the redox state of the Cu(I) and the Mo(VI) unchanged. Collectively, our findings establish a regulation of CO dehydrogenase activity by thiols in vitro. They also corroborate the hypothesis that CO interacts with the Cu-ion first. The result that thiol compounds much larger than CO can freely travel through the substrate channel leading to the bimetallic cluster challenges previous concepts involving chaperone function and is of importance for an understanding how the sulfuration

  7. Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase.

    PubMed

    Bomati, Erin K; Noel, Joseph P

    2005-05-01

    We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities.

  8. Cellular distribution, purification and electrophoretic properties of malate dehydrogenase in Trichuris ovis and inhibition by benzimidazoles and pyrimidine derivatives.

    PubMed

    Sanchez-Moreno, M; Ortega, J E; Valero, A

    1989-12-01

    High levels of malate dehydrogenase were found in Trichuris ovis. Two molecular forms of the enzyme, of different cellular location and electrophoretic pattern, were isolated and purified. The activity of soluble malate dehydrogenase was greater than that of mitochondrial malate dehydrogenase. Both forms also displayed different electrophoretic profiles in comparison with purified extracts from goat (Capra hircus) liver. Substrate concentration directly affected enzyme activity. Host and parasite malate dehydrogenase activity were both inhibited by a series of benzimidazoles and pyrimidine-derived compounds, some of which markedly reduced parasite enzyme activity, but not host enzyme activity. Percentage inhibition by some pyrimidine derivatives was greater than that produced by benzimidazoles.

  9. Substrate specificity and inhibitor sensitivity of rabbit 20α-hydroxysteroid dehydrogenase.

    PubMed

    Endo, Satoshi; Arai, Yuki; Hara, Akira; Kitade, Yukio; Bunai, Yasuo; El-Kabbani, Ossama; Matsunaga, Toshiyuki

    2013-01-01

    In this study, we examined the substrate specificity and inhibitor sensitivity of rabbit 20α-hydroxysteroid dehydrogenase (AKR1C5), which plays a role in the termination of pregnancy by progesterone inactivation. AKR1C5 moderately reduced the 3-keto group of only 5α-dihydrosteroids with 17β- or 20α/β-hydroxy group among 3-ketosteroids. In contrast, the enzyme reversibly and efficiently catalyzed the reduction of various 17- and 20-ketosteroids, including estrogen precursors (dehydroepiandrosterone, estrone and 5α-androstan-3β-ol-17-one) and tocolytic 5β-pregnane-3,20-dione. In addition to the progesterone inactivation, the formation of estrogens and metabolism of the tocolytic steroid by AKR1C5 may be related to its role in rabbit parturition. AKR1C5 also reduced various non-steroidal carbonyl compounds, including isatin, an antagonist of the C-type natriuretic peptide receptor, and 4-oxo-2-nonenal, suggesting its roles in controlling the bioactive isatin and detoxification of cytotoxic aldehydes. AKR1C5 was potently and competitively inhibited by flavonoids such as kaempferol and quercetin, suggesting that its activity is affected by ingested flavonoids.

  10. 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. Copyright © 2016 John Wiley & Sons, Ltd.

  11. Energetic aspects of the light activation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase.

    PubMed

    Miginiac-Maslow, M; Jacquot, J P; Droux, M

    1985-09-01

    The light energy requirements for photoactivation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were studied in a reconstituted chloroplast system. This system comprised isolated pea thylakoids, ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) thioredoxinm and f (Tdm, Tdf) and the photoactivatable enzyme. Light-saturation curves of the photoactivation process were established with once washed thylakoids which did not require the addition of Td for light activation. They exhibited a plateau at 10 W·m(-2) under nitrogen and 50 W·m(-2) under air, while NADP photoreduction was saturated at 240 W·m(-2). Cyclic and pseudocyclic phosphorylations saturated at identical levels as enzyme photoactivations. All these observations suggested that the shift of the light saturation plateau towards higher values under air was due to competing oxygen-dependent reactions. With twice washed thylakoids, which required Td for enzyme light-activation, photophosphorylation was stimulated under N2 by the addition of the components of the photoactivation system. Its rate increased with increasing Td concentrations, just as did the enzyme photoactivation rate, while varying the target enzyme concentration had only a weak effect. Considering that Td concentrations were in a large excess over target enzyme concentrations, it may be assumed that the observed ATP synthesis was essentially dependent on the rate of Td reduction.Under air, Fd-dependent pseudo-cyclic photophosphorylation was not stimulated by the addition of the other enzyme photoactivation components, suggesting that an important site of action of O2 was located at the level of Fd.

  12. Ultra-high-pressure processing improves proteolysis and release of bioactive peptides with activation activities on alcohol metabolic enzymes in vitro from mushroom foot protein.

    PubMed

    Zhao, Rui-Jie; Huo, Chun-Yan; Qian, Yang; Ren, Di-Feng; Lu, Jun

    2017-09-15

    This study was to find an effective process to extract bioactive peptides from mushroom foot and determine their effects on activation of alcohol metabolic enzymes in vitro. The optimum extraction assisted by ultra-high-pressure processing of mushroom foot peptides was obtained with a pressure of 400MPa and a processing time of 10min. After ultrafiltration, peptides with molecular weight of 0-3kDa had the highest activity to activate alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) by 70.79% and 71.35%, respectively. Following dextran gel chromatography, two peaks (p-I and p-II) appeared and the activation activities on ADH and ALDH of p-I were 72.00% and 73.43%, both higher than p-II. Nine peptides were found in p-I as determined by LC-MS/MS, and two of them (IPLH and IPIVLL) were synthesized. IPLH activated ADH and ALDH by 42.7% and 29.2% respectively, which were higher than IPIVLL. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Evaluation of aldehyde dehydrogenase 1 and transcription factors in both primary breast cancer and axillary lymph node metastases as a prognostic factor.

    PubMed

    Ito, Maiko; Shien, Tadahiko; Omori, Masako; Mizoo, Taeko; Iwamoto, Takayuki; Nogami, Tomohiro; Motoki, Takayuki; Taira, Naruto; Doihara, Hiroyoshi; Miyoshi, Shinichiro

    2016-05-01

    Aldehyde dehydrogenase 1 (ALDH1) is a marker of breast cancer stem cells, and the expression of ALDH1 may be a prognostic factor of poor clinical outcome. The epithelial-mesenchymal transition may produce cells with stem-cell-like properties promoted by transcription factors. We investigated the expression of ALDH1 and transcription factors in both primary and metastatic lesions, and prognostic value of them in breast cancer patients with axillary lymph node metastasis (ALNM). Forty-seven breast cancer patients with ALNM who underwent surgery at Okayama University Hospital from 2002 to 2008 were enrolled. We retrospectively evaluated the levels of ALDH1 and transcription factors, such as Snail, Slug and Twist, in both primary and metastatic lesions by immunohistochemistry. In primary lesions, the positive rate of ALDH1, Snail, Slug and Twist was 19, 49, 40 and 26%, respectively. In lymph nodes, that of ALDH1, Snail, Slug and Twist was 21, 32, 13 and 23%, respectively. The expression of ALDH1 or transcription factors alone was not significantly associated with a poor prognosis. However, co-expression of ALDH1 and Slug in primary lesions was associated with a shorter DFS (P = 0.009). The evaluation of the co-expression of ALDH1 and transcription factors in primary lesions may be useful in prognosis of node-positive breast cancers.

  14. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.

    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 ofmore » 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.« less

  15. Inducible NAD(H)-linked methylglyoxal oxidoreductase regulates cellular methylglyoxal and pyruvate through enhanced activities of alcohol dehydrogenase and methylglyoxal-oxidizing enzymes in glutathione-depleted Candida albicans.

    PubMed

    Kwak, Min-Kyu; Ku, MyungHee; Kang, Sa-Ouk

    2018-01-01

    High methylglyoxal content disrupts cell physiology, but mammals have scavengers to prevent glycolytic and mitochondrial dysfunctions. In yeast, methylglyoxal accumulation triggers methylglyoxal-oxidizing alcohol dehydrogenase (Adh1) activity. While methylglyoxal reductases and glyoxalases have been well studied in prokaryotes and eukaryotes, experimental evidence for methylglyoxal dehydrogenase (Mgd) and other catalytic activities of this enzyme affecting glycolysis and the tricarboxylic acid cycle is lacking. A glycine-rich cytoplasmic Mgd protein, designated as Mgd1/Grp2, was isolated from glutathione-depleted Candida albicans. The effects of Mgd1/Grp2 activities on metabolic pathophysiology were investigated using knockout and overexpression mutants. We measured glutathione-(in)dependent metabolite contents and metabolic effects, including viability, oxygen consumption, ADH1 transcripts, and glutathione reductase and α-ketoglutarate dehydrogenase activities in the mutants. Based on the findings, methylglyoxal-oxidizing proteins were monitored to determine effects of MGD1/GRP2 disruption on methylglyoxal-scavenging traits during glutathione deprivation. Methylglyoxal-oxidizing NAD(H)-linked Mgd1/Grp2 was found solely in glutathione auxotrophs, and it catalyzed the reduction of both methylglyoxal and pyruvate. MGD1/GRP2 disruptants showed growth defects, cell-cycle arrest, and methylglyoxal and pyruvate accumulation with mitochondrial impairment, regardless of ADH1 compensation. Other methylglyoxal-oxidizing enzymes were identified as key glycolytic enzymes with enhanced activity and transcription in MGD1/GRP2 disruptants, irrespective of glutathione content. Failure of methylglyoxal and pyruvate dissimilation by Mgd1/Grp2 deficiency leads to poor glutathione-dependent redox regulation despite compensation by Adh1. This is the first report that multifunctional Mgd activities contribute to scavenging methylglyoxal and pyruvate to maintain metabolic homeostasis

  16. Functional characterization of SlscADH1, a fruit-ripening-associated short-chain alcohol dehydrogenase of tomato.

    PubMed

    Moummou, Hanane; Tonfack, Libert Brice; Chervin, Christian; Benichou, Mohamed; Youmbi, Emmanuel; Ginies, Christian; Latché, Alain; Pech, Jean-Claude; van der Rest, Benoît

    2012-10-15

    A tomato short-chain dehydrogenase-reductase (SlscADH1) is preferentially expressed in fruit with a maximum expression at the breaker stage while expression in roots, stems, leaves and flowers is very weak. It represents a potential candidate for the formation of aroma volatiles by interconverting alcohols and aldehydes. The SlscADH1 recombinant protein produced in Escherichia coli exhibited dehydrogenase-reductase activity towards several volatile compounds present in tomato flavour with a strong preference for the NAD/NADH co-factors. The strongest activity was observed for the reduction of hexanal (K(m)=0.175mM) and phenylacetaldehyde (K(m)=0.375mM) in the presence of NADH. The oxidation process of hexanol and 1-phenylethanol was much less efficient (K(m)s of 2.9 and 23.0mM, respectively), indicating that the enzyme preferentially acts as a reductase. However activity was observed only for hexanal, phenylacetaldehyde, (E)-2-hexenal and acetaldehyde and the corresponding alcohols. No activity could be detected for other aroma volatiles important for tomato flavour, such as methyl-butanol/methyl-butanal, 5-methyl-6-hepten-2-one/5-methyl-6-hepten-2-ol, citronellal/citronellol, neral/nerol, geraniol. In order to assess the function of the SlscADH1 gene, transgenic plants have been generated using the technique of RNA interference (RNAi). Constitutive down-regulation using the 35S promoter resulted in the generation of dwarf plants, indicating that the SlscADH1 gene, although weakly expressed in vegetative tissues, had a function in regulating plant development. Fruit-specific down-regulation using the 2A11 promoter had no morphogenetic effect and did not alter the aldehyde/alcohol balance of the volatiles compounds produced by the fruit. Nevertheless, SlscADH1-inhibited fruit unexpectedly accumulated higher concentrations of C5 and C6 volatile compounds of the lipoxygenase pathway, possibly as an indirect effect of the suppression of SlscADH1 on the catabolism of

  17. Structural and Kinetic Basis for Substrate Selectivity in Populus tremuloides Sinapyl Alcohol Dehydrogenase

    PubMed Central

    Bomati, Erin K.; Noel, Joseph P.

    2005-01-01

    We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities. PMID:15829607

  18. Aldehyde dehydrogenase inhibition combined with phenformin treatment reversed NSCLC through ATP depletion.

    PubMed

    Kang, Joon Hee; Lee, Seon-Hyeong; Lee, Jae-Seon; Nam, Boas; Seong, Tae Wha; Son, Jaekyoung; Jang, Hyonchol; Hong, Kyeong Man; Lee, Cheolju; Kim, Soo-Youl

    2016-08-02

    Among ALDH isoforms, ALDH1L1 in the folate pathway showed highly increased expression in non-small-cell lung cancer cells (NSCLC). Based on the basic mechanism of ALDH converting aldehyde to carboxylic acid with by-product NADH, we suggested that ALDH1L1 may contribute to ATP production using NADH through oxidative phosphorylation. ALDH1L1 knockdown reduced ATP production by up to 60% concomitantly with decrease of NADH in NSCLC. ALDH inhibitor, gossypol, also reduced ATP production in a dose dependent manner together with decrease of NADH level in NSCLC. A combination treatment of gossypol with phenformin, mitochondrial complex I inhibitor, synergized ATP depletion, which efficiently induced cell death. Pre-clinical xenograft model using human NSCLC demonstrated a remarkable therapeutic response to the combined treatment of gossypol and phenformin.

  19. Optimized dosing schedule based on circadian dynamics of mouse breast cancer stem cells improves the anti-tumor effects of aldehyde dehydrogenase.

    PubMed

    Matsunaga, Naoya; Ogino, Takashi; Hara, Yukinori; Tanaka, Takahiro; Koyanagi, Satoru; Ohdo, Shigehiro

    2018-05-07

    Although malignant phenotypes of triple-negative breast cancer (TNBC) are subject to circadian alterations, the role of cancer stem cells (CSC) in defining this circadian change remains unclear. CSC are often characterized by high aldehyde dehydrogenase (ALDH) activity, which is associated with the malignancy of cancer cells and used for identification and isolation of CSC. Here we show that the popultation of ALDH-positive cells in a mouse 4T1 breast tumor model exhibits pronounced circadian alterations. Alterations in the number of ALDH-positive cells was generated by time-dependent increases and decreases in the expression of Aldh3a1. Importantly, circadian clock genes were rhythmically expressed in ALDH-negative cells, but not in ALDH-positive cells. Circadian expression of Aldh3a1 in ALDH-positive cells was dependent on the time-dependent release of Wingless-type mmtv integration site family 10a (WNT10a) from ALDH-negative cells. Furthermore, anti-tumor and anti-metastatic effects of ALDH inhibitor N,N-diethylaminobenzaldehyde were enhanced by administration at the time of day when ALDH activity was increased in 4T1 tumor cells. Our findings reveal a new role for the circadian clock within the tumor microenvironment in regulating the circadian dynamics of CSC. These results should enable the development of novel therapeutic strategies for treatment of TNBC with ALDH inhibitors. Copyright ©2018, American Association for Cancer Research.

  20. Candidate enzymes for saffron crocin biosynthesis are localized in multiple cellular compartments.

    PubMed

    Demurtas, Olivia Costantina; Frusciante, Sarah; Ferrante, Paola; Diretto, Gianfranco; Azad, Noraddin Hosseinpour; Pietrella, Marco; Aprea, Giuseppe; Taddei, Anna Rita; Romano, Elena; Mi, Jianing; Al-Babili, Salim; Frigerio, Lorenzo; Giuliano, Giovanni

    2018-05-29

    Saffron is composed of the dried stigmas of Crocus sativus and is the most expensive spice on Earth. Its red color is due to the apocarotenoid glycosides, crocins, which accumulate in the vacuole and reach up to 10% of the stigma dry weight. We have previously characterized the first dedicated enzyme in crocin biosynthesis, CsCCD2, which cleaves zeaxanthin to yield crocetin dialdehyde. In this work, we identified six putative aldehyde dehydrogenase (ALDH) transcripts expressed in saffron stigmas. When expressed in E. coli, only one of corresponding proteins (CsALDH3I1), was able to convert crocetin dialdehyde into the crocin precursor, crocetin. CsALDH3I1 carries a C-terminal hydrophobic domain, similar to that of a Neurospora membrane-associated apocarotenoid dehydrogenase, YLO-1. We also characterized a UDP-glycosyltransferase enzyme, CsUGT74AD1, able to convert crocetin to crocins 1 and 2'. In vitro assays showed high specificity of CsALDH3I1 for crocetin dialdehyde and long chain apocarotenals, and of CsUGT74AD1 for crocetin. Upon extract fractionation, the CsCCD2, CsALDH3I1 and CsUGT74AD1 enzymes partitioned in the insoluble fraction, suggesting that they are associated to membranes or to large insoluble complexes. Immunogold labeling of saffron stigmas and confocal microscopy of fusions to Green Fluorescent Protein expressed in N. benthamiana leaves revealed that CsCCD2 localizes to plastids, CsALDH3I1 to the endoplasmic reticulum (ER) and CsUGT74AD1 to the cytoplasm, in association with cytoskeletal-like structures. Based on our and on literature data, we propose that the ER and cytoplasm function as "transit centers" for metabolites whose biosynthesis starts in the plastid and are accumulated in the vacuole. {copyright, serif} 2018 American Society of Plant Biologists. All rights reserved.

  1. The substrate oxidation mechanism of pyranose 2-oxidase and other related enzymes in the glucose-methanol-choline superfamily.

    PubMed

    Wongnate, Thanyaporn; Chaiyen, Pimchai

    2013-07-01

    Enzymes in the glucose-methanol-choline (GMC) oxidoreductase superfamily catalyze the oxidation of an alcohol moiety to the corresponding aldehyde. In this review, the current understanding of the sugar oxidation mechanism in the reaction of pyranose 2-oxidase (P2O) is highlighted and compared with that of other enzymes in the GMC family for which structural and mechanistic information is available, including glucose oxidase, choline oxidase, cholesterol oxidase, cellobiose dehydrogenase, aryl-alcohol oxidase, and pyridoxine 4-oxidase. Other enzymes in the family that have been newly discovered or for which less information is available are also discussed. A large primary kinetic isotope effect was observed for the flavin reduction when 2-d-D-glucose was used as a substrate, but no solvent kinetic isotope effect was detected for the flavin reduction step. The reaction of P2O is consistent with a hydride transfer mechanism in which there is stepwise formation of d-glucose alkoxide prior to the hydride transfer. Site-directed mutagenesis of P2O and pH-dependence studies indicated that His548 is a catalytic base that facilitates the deprotonation of C2-OH in D-glucose. This finding agrees with the current mechanistic model for aryl-alcohol oxidase, glucose oxidase, cellobiose dehydrogenase, methanol oxidase, and pyridoxine 4-oxidase, but is different from that of cholesterol oxidase and choline oxidase. Although all of the GMC enzymes share similar structural folding and use the hydride transfer mechanism for flavin reduction, they appear to have subtle differences in the fine-tuned details of how they catalyze substrate oxidation. © 2013 The Authors Journal compilation © 2013 FEBS.

  2. Substrate specificity and subcellular localization of the aldehyde-alcohol redox-coupling reaction in carp cones.

    PubMed

    Sato, Shinya; Fukagawa, Takashi; Tachibanaki, Shuji; Yamano, Yumiko; Wada, Akimori; Kawamura, Satoru

    2013-12-20

    Our previous study suggested the presence of a novel cone-specific redox reaction that generates 11-cis-retinal from 11-cis-retinol in the carp retina. This reaction is unique in that 1) both 11-cis-retinol and all-trans-retinal were required to produce 11-cis-retinal; 2) together with 11-cis-retinal, all-trans-retinol was produced at a 1:1 ratio; and 3) the addition of enzyme cofactors such as NADP(H) was not necessary. This reaction is probably part of the reactions in a cone-specific retinoid cycle required for cone visual pigment regeneration with the use of 11-cis-retinol supplied from Müller cells. In this study, using purified carp cone membrane preparations, we first confirmed that the reaction is a redox-coupling reaction between retinals and retinols. We further examined the substrate specificity, reaction mechanism, and subcellular localization of this reaction. Oxidation was specific for 11-cis-retinol and 9-cis-retinol. In contrast, reduction showed low specificity: many aldehydes, including all-trans-, 9-cis-, 11-cis-, and 13-cis-retinals and even benzaldehyde, supported the reaction. On the basis of kinetic studies of this reaction (aldehyde-alcohol redox-coupling reaction), we found that formation of a ternary complex of a retinol, an aldehyde, and a postulated enzyme seemed to be necessary, which suggested the presence of both the retinol- and aldehyde-binding sites in this enzyme. A subcellular fractionation study showed that the activity is present almost exclusively in the cone inner segment. These results suggest the presence of an effective production mechanism of 11-cis-retinal in the cone inner segment to regenerate visual pigment.

  3. Purification and Characterization of a Novel NAD(P)+-Farnesol Dehydrogenase from Polygonum minus Leaves.

    PubMed

    Ahmad-Sohdi, Nor-Ain-Shahajar; Seman-Kamarulzaman, Ahmad-Faris; Mohamed-Hussein, Zeti-Azura; Hassan, Maizom

    2015-01-01

    Juvenile hormones have attracted attention as safe and selective targets for the design and development of environmentally friendly and biorational insecticides. In the juvenile hormone III biosynthetic pathway, the enzyme farnesol dehydrogenase catalyzes the oxidation of farnesol to farnesal. In this study, farnesol dehydrogenase was extracted from Polygonum minus leaves and purified 204-fold to apparent homogeneity by ion-exchange chromatography using DEAE-Toyopearl, SP-Toyopearl, and Super-Q Toyopearl, followed by three successive purifications by gel filtration chromatography on a TSK-gel GS3000SW. The enzyme is a heterodimer comprised of subunits with molecular masses of 65 kDa and 70 kDa. The optimum temperature and pH were 35°C and pH 9.5, respectively. Activity was inhibited by sulfhydryl reagents, metal-chelating agents and heavy metal ions. The enzyme utilized both NAD+ and NADP+ as coenzymes with Km values of 0.74 mM and 40 mM, respectively. Trans, trans-farnesol was the preferred substrate for the P. minus farnesol dehydrogenase. Geometrical isomers of trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol were also oxidized by the enzyme with lower activity. The Km values for trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol appeared to be 0.17 mM, 0.33 mM and 0.42 mM, respectively. The amino acid sequences of 4 tryptic peptides of the enzyme were analyzed by MALDI-TOF/TOF-MS spectrometry, and showed no significant similarity to those of previously reported farnesol dehydrogenases. These results suggest that the purified enzyme is a novel NAD(P)+-dependent farnesol dehydrogenase. The purification and characterization established in the current study will serve as a basis to provide new information for recombinant production of the enzyme. Therefore, recombinant farnesol dehydrogenase may provide a useful molecular tool in manipulating juvenile hormone biosynthesis to generate transgenic plants for pest control.

  4. Purification and Characterization of a Novel NAD(P)+-Farnesol Dehydrogenase from Polygonum minus Leaves

    PubMed Central

    Seman-Kamarulzaman, Ahmad-Faris; Mohamed-Hussein, Zeti-Azura

    2015-01-01

    Juvenile hormones have attracted attention as safe and selective targets for the design and development of environmentally friendly and biorational insecticides. In the juvenile hormone III biosynthetic pathway, the enzyme farnesol dehydrogenase catalyzes the oxidation of farnesol to farnesal. In this study, farnesol dehydrogenase was extracted from Polygonum minus leaves and purified 204-fold to apparent homogeneity by ion-exchange chromatography using DEAE-Toyopearl, SP-Toyopearl, and Super-Q Toyopearl, followed by three successive purifications by gel filtration chromatography on a TSK-gel GS3000SW. The enzyme is a heterodimer comprised of subunits with molecular masses of 65 kDa and 70 kDa. The optimum temperature and pH were 35°C and pH 9.5, respectively. Activity was inhibited by sulfhydryl reagents, metal-chelating agents and heavy metal ions. The enzyme utilized both NAD+ and NADP+ as coenzymes with K m values of 0.74 mM and 40 mM, respectively. Trans, trans-farnesol was the preferred substrate for the P. minus farnesol dehydrogenase. Geometrical isomers of trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol were also oxidized by the enzyme with lower activity. The K m values for trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol appeared to be 0.17 mM, 0.33 mM and 0.42 mM, respectively. The amino acid sequences of 4 tryptic peptides of the enzyme were analyzed by MALDI-TOF/TOF-MS spectrometry, and showed no significant similarity to those of previously reported farnesol dehydrogenases. These results suggest that the purified enzyme is a novel NAD(P)+-dependent farnesol dehydrogenase. The purification and characterization established in the current study will serve as a basis to provide new information for recombinant production of the enzyme. Therefore, recombinant farnesol dehydrogenase may provide a useful molecular tool in manipulating juvenile hormone biosynthesis to generate transgenic plants for pest control. PMID:26600471

  5. Xenobiotic metabolizing enzyme activities in cells used for testing skin sensitization in vitro.

    PubMed

    Fabian, E; Vogel, D; Blatz, V; Ramirez, T; Kolle, S; Eltze, T; van Ravenzwaay, B; Oesch, F; Landsiedel, R

    2013-09-01

    For ethical and regulatory reasons, in vitro tests for scoring potential toxicities of cosmetics are essential. A test strategy for investigating potential skin sensitization using two human keratinocytic and two human dendritic cell lines has been developed (Mehling et al. Arch Toxicol 86:1273–1295, 2012). Since prohaptens may be metabolically activated in the skin, information on xenobiotic metabolizing enzyme (XME) activities in these cell lines is of high interest. In this study, XME activity assays, monitoring metabolite or cofactor, showed the following: all three passages of keratinocytic (KeratinoSens® and LuSens) and dendritic (U937 und THP-1) cells displayed N-acetyltransferase 1 (NAT1) activities (about 6–60 nmol/min/mg S9-protein for acetylation of para-aminobenzoic acid). This is relevant since reactive species of many cosmetics are metabolically controlled by cutaneous NAT1. Esterase activities of about 1–4 nmol fluorescein diacetate/min/mg S9-protein were observed in all passages of investigated keratinocytic and about 1 nmol fluorescein diacetate/min/mg S9-protein in dendritic cell lines. This is also of practical relevance since many esters and amides are detoxified and others activated by cutaneous esterases. In both keratinocytic cell lines, activities of aldehyde dehydrogenase (ALDH) were observed (5–17 nmol product/min/mg cytosolic protein). ALDH is relevant for the detoxication of reactive aldehydes. Activities of several other XME were below detection, namely the investigated cytochrome P450-dependent alkylresorufin O-dealkylases 7-ethylresorufin O-deethylase, 7-benzylresorufin O-debenzylase and 7-pentylresorufin O-depentylase (while NADPH cytochrome c reductase activities were much above the limit of quantification), the flavin-containing monooxygenase, the alcohol dehydrogenase as well as the UDP glucuronosyl transferase activities.

  6. The multifunctional isopropyl alcohol dehydrogenase of Phytomonas sp. could be the result of a horizontal gene transfer from a bacterium to the trypanosomatid lineage.

    PubMed

    Molinas, Sara M; Altabe, Silvia G; Opperdoes, Fred R; Rider, Mark H; Michels, Paul A M; Uttaro, Antonio D

    2003-09-19

    Isopropyl alcohol dehydrogenase (iPDH) is a dimeric mitochondrial alcohol dehydrogenase (ADH), so far detected within the Trypanosomatidae only in the genus Phytomonas. The cloning, sequencing, and heterologous expression of the two gene alleles of the enzyme revealed that it is a zinc-dependent medium-chain ADH. Both polypeptides have 361 amino acids. A mitochondrial targeting sequence was identified. The mature proteins each have 348 amino acids and a calculated molecular mass of 37 kDa. They differ only in one amino acid, which can explain the three isoenzymes and their respective isoelectric points previously found. A phylogenetic analysis locates iPDH within a cluster with fermentative ADHs from bacteria, sharing 74% similarity and 60% identity with Ralstonia eutropha ADH. The characterization of the two bacterially expressed Phytomonas enzymes and the comparison of their kinetic properties with those of the wild-type iPDH and of the R. eutropha ADH strongly support the idea of a horizontal gene transfer event from a bacterium to a trypanosomatid to explain the origin of the iPDH in Phytomonas. Phytomonas iPDH and R. eutropha ADH are able to use a wide range of substrates with similar Km values such as primary and secondary alcohols, diols, and aldehydes, as well as ketones such as acetone, diacetyl, and acetoin. We speculate that, as for R. eutropha ADH, Phytomonas iPDH acts as a safety valve for the release of excess reducing power.

  7. Structure and Function of the Unusual Tungsten Enzymes Acetylene Hydratase and Class II Benzoyl-Coenzyme A Reductase.

    PubMed

    Boll, Matthias; Einsle, Oliver; Ermler, Ulrich; Kroneck, Peter M H; Ullmann, G Matthias

    2016-01-01

    In biology, tungsten (W) is exclusively found in microbial enzymes bound to a bis-pyranopterin cofactor (bis-WPT). Previously known W enzymes catalyze redox oxo/hydroxyl transfer reactions by directly coordinating their substrates or products to the metal. They comprise the W-containing formate/formylmethanofuran dehydrogenases belonging to the dimethyl sulfoxide reductase (DMSOR) family and the aldehyde:ferredoxin oxidoreductase (AOR) families, which form a separate enzyme family within the Mo/W enzymes. In the last decade, initial insights into the structure and function of two unprecedented W enzymes were obtained: the acetaldehyde forming acetylene hydratase (ACH) belongs to the DMSOR and the class II benzoyl-coenzyme A (CoA) reductase (BCR) to the AOR family. The latter catalyzes the reductive dearomatization of benzoyl-CoA to a cyclic diene. Both are key enzymes in the degradation of acetylene (ACH) or aromatic compounds (BCR) in strictly anaerobic bacteria. They are unusual in either catalyzing a nonredox reaction (ACH) or a redox reaction without coordinating the substrate or product to the metal (BCR). In organic chemical synthesis, analogous reactions require totally nonphysiological conditions depending on Hg2+ (acetylene hydration) or alkali metals (benzene ring reduction). The structural insights obtained pave the way for biological or biomimetic approaches to basic reactions in organic chemistry. © 2016 S. Karger AG, Basel.

  8. Aldehyde dehydrogenase inhibition combined with phenformin treatment reversed NSCLC through ATP depletion

    PubMed Central

    Lee, Jae-Seon; Nam, Boas; Seong, Tae Wha; Son, Jaekyoung; Jang, Hyonchol; Hong, Kyeong Man; Lee, Cheolju; Kim, Soo-Youl

    2016-01-01

    Among ALDH isoforms, ALDH1L1 in the folate pathway showed highly increased expression in non-small-cell lung cancer cells (NSCLC). Based on the basic mechanism of ALDH converting aldehyde to carboxylic acid with by-product NADH, we suggested that ALDH1L1 may contribute to ATP production using NADH through oxidative phosphorylation. ALDH1L1 knockdown reduced ATP production by up to 60% concomitantly with decrease of NADH in NSCLC. ALDH inhibitor, gossypol, also reduced ATP production in a dose dependent manner together with decrease of NADH level in NSCLC. A combination treatment of gossypol with phenformin, mitochondrial complex I inhibitor, synergized ATP depletion, which efficiently induced cell death. Pre-clinical xenograft model using human NSCLC demonstrated a remarkable therapeutic response to the combined treatment of gossypol and phenformin. PMID:27384481

  9. Some enzymes of carbohydrate metabolism in Mesocestoides corti and Heterakis spumosa.

    PubMed

    Dubinský, P; Ruscinová, B; Hetmanski, S L; Arme, C; Turceková, L; Rybos, M

    1991-09-01

    The activities of selected enzymes of carbohydrate metabolism were measured in tetrathyridia of Mesocestoides corti and in adult females and males of Heterakis spumosa. When the species were compared, only lactate dehydrogenase and phosphoenolpyruvate carboxykinase activities were considerably higher in M. corti. Activities of other enzymes were higher in H. spumosa, with malate dehydrogenase activity being considerably so. In H. spumosa, enzyme activity was higher, and succinate dehydrogenase markedly so in males, when compared with females. Tetrathyridia aged 170 and 210 days show relatively stable malate and lactate dehydrogenase activities, and mice of ICR and BALB/c strains are suitable for the maintenance of tetrathyridia.

  10. 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.

  11. Regulation of 11 beta-hydroxysteroid dehydrogenase enzymes in the rat kidney by estradiol.

    PubMed

    Gomez-Sanchez, Elise P; Ganjam, Venkataseshu; Chen, Yuan Jian; Liu, Ying; Zhou, Ming Yi; Toroslu, Cigdem; Romero, Damian G; Hughson, Michael D; de Rodriguez, Angela; Gomez-Sanchez, Celso E

    2003-08-01

    The 11beta-hydroxysteroid dehydrogenase (11betaHSD) type 1 (11betaHSD1) enzyme is an NADP+-dependent oxidoreductase, usually reductase, of major glucocorticoids. The NAD+-dependent type 2 (11betaHSD2) enzyme is an oxidase that inactivates cortisol and corticosterone, conferring extrinsic specificity of the mineralocorticoid receptor for aldosterone. We reported that addition of a reducing agent to renal homogenates results in the monomerization of 11betaHSD2 dimers and a significant increase in NAD+-dependent corticosterone conversion. Estrogenic effects on expression, dimerization, and activity of the kidney 11betaHSD1 and -2 enzymes are described herein. Renal 11betaHSD1 mRNA and protein expressions were decreased to very low levels by estradiol (E2) treatment of both intact and castrated male rats; testosterone had no effect. NADP+-dependent enzymatic activity of renal homogenates from E2-treated rats measured under nonreducing conditions was less than that of homogenates from intact animals. Addition of 10 mM DTT to aliquots from these same homogenates abrogated the difference in NADP+-dependent activity between E2-treated and control rats. In contrast, 11betaHSD2 mRNA and protein expressions were significantly increased by E2 treatment. There was a marked increase in the number of juxtamedullary proximal tubules stained by the antibody against 11betaHSD2 after the administration of E2. Notwithstanding, neither the total corticosterone and 11-dehydrocorticosterone excreted in the urine nor their ratio differed between E2- and vehicle-treated rats. NAD+-dependent enzymatic activity in the absence or presence of a reducing agent demonstrated that the increase in 11betaHSD2 protein was not associated with an increase in in vitro activity unless the dimers were reduced to monomers.

  12. Cloning, expression, and sequence analysis of the Bacillus methanolicus C1 methanol dehydrogenase gene.

    PubMed Central

    de Vries, G E; Arfman, N; Terpstra, P; Dijkhuizen, L

    1992-01-01

    The gene (mdh) coding for methanol dehydrogenase (MDH) of thermotolerant, methylotroph Bacillus methanolicus C1 has been cloned and sequenced. The deduced amino acid sequence of the mdh gene exhibited similarity to those of five other alcohol dehydrogenase (type III) enzymes, which are distinct from the long-chain zinc-containing (type I) or short-chain zinc-lacking (type II) enzymes. Highly efficient expression of the mdh gene in Escherichia coli was probably driven from its own promoter sequence. After purification of MDH from E. coli, the kinetic and biochemical properties of the enzyme were investigated. The physiological effect of MDH synthesis in E. coli and the role of conserved sequence patterns in type III alcohol dehydrogenases have been analyzed and are discussed. Images PMID:1644761

  13. Enzymes of Glucose Catabolism in a Member of the Psittacosis Group

    PubMed Central

    Moulder, James W.; Grisso, Dorothy L.; Brubaker, Robert R.

    1965-01-01

    Moulder, James W. (University of Chicago, Chicago, Ill.), Dorothy L. Grisso, and Robert R. Brubaker. Enzymes of glucose catabolism in a member of the psittacosis group. J. Bacteriol. 89:810–812. 1965.—Extracts of preparations of the agent of meningopneumonitis made from infected chick-embryo allantoic fluid contained three enzymes of the pentose pathway of glucose degradation: glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and phosphoglucose isomerase. Vertical starch-gel electrophoresis showed that the two dehydrogenases were qualitatively different from the corresponding enzymes of the host. Enzymes of the Embden-Meyerhof and Entner-Doudoroff pathways were not found. Images PMID:14273665

  14. Substrate specificity of sheep liver sorbitol dehydrogenase.

    PubMed Central

    Lindstad, R I; Köll, P; McKinley-McKee, J S

    1998-01-01

    The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo> D-ribo > L-xylo > D-lyxo approximately L-arabino > D-arabino > L-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH > -CH2NH2 > -CH2OCH3 approximately -CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of

  15. Structural Basis for "Flip-Flop" Action of Human Pyruvate Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Ciszak, Ewa; Korotchkina, Lioubov; Dominiak, Paulina; Sidhu, Sukhdeep; Patel, Mulchand

    2003-01-01

    The derivative of vitamin B1, thiamin pyrophosphate is a cofactor of pyruvate dehydrogenase, a component enzyme of the mitochondrial pyruvate dehydrogenase multienzyme complex that plays a major role in directing energy metabolism in the cell. This cofactor is used to cleave the C(sup alpha)-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. In alpha(sub 2)beta(sub 2)-tetrameric human pyruvate dehydrogenase, there are two cofactor binding sites, each of them being a center of independently conducted, although highly coordinated enzymatic reactions. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites can now be understood based on the recently determined crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95A resolution. The structure of pyruvate dehydrogenase was determined using a combination of MAD phasing and molecular replacement followed by rounds of torsion-angles molecular-dynamics simulated-annealing refinement. The final pyruvate dehydrogenase structure included coordinates for all protein amino acids two cofactor molecules, two magnesium and two potassium ions, and 742 water molecules. The structure was refined to R = 0.202 and R(sub free) = 0.244. Our structural analysis of the enzyme folding and domain assembly identified a simple mechanism of this protein motion required for the conduct of catalytic action.

  16. The effects of iron deficiency on rat liver enzymes.

    PubMed Central

    Bailey-Wood, R.; Blayney, L. M.; Muir, J. R.; Jacobs, A.

    1975-01-01

    The effect of iron deficiency on a number or iron containing enzymes in rat liver has been examined. In addition, 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase have been assayed. Of the mitochondrial electron transport reactions only succinate-cytochrome C reductase activity was decreased in iron deficient animals. Microsomal reductase enzymes associated with the NADPH-oxidase system were also markedly decreased although cytochrome P450 concentrations were unaffected. Both 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase were reduced in young iron deficient rats but the former had returned to control levels at the age of 14 weeks. PMID:172099

  17. Inhibition of Wnt/beta-catenin signaling downregulates expression of aldehyde dehydrogenase isoform 3A1 (ALDH3A1) to reduce resistance against temozolomide in glioblastoma in vitro.

    PubMed

    Suwala, Abigail Kora; Koch, Katharina; Rios, Dayana Herrera; Aretz, Philippe; Uhlmann, Constanze; Ogorek, Isabella; Felsberg, Jörg; Reifenberger, Guido; Köhrer, Karl; Deenen, René; Steiger, Hans-Jakob; Kahlert, Ulf D; Maciaczyk, Jaroslaw

    2018-04-27

    Glioblastoma is the most aggressive type of glioma. The Wingless (Wnt) signaling pathway has been shown to promote stem cell properties and resistance to radio- and chemotherapy in glioblastoma. Here, we demonstrate that pharmacological Wnt pathway inhibition using the porcupine inhibitor LGK974 acts synergistically with temozolomide (TMZ), the chemotherapeutic drug currently used as standard treatment for glioblastoma, to suppress in vitro growth of glioma cells. Synergistic growth inhibition was independent of the O 6 -alkylguanine DNA alkyltransferase ( MGMT ) promoter methylation status. Transcriptomic analysis revealed that expression of aldehyde dehydrogenase 3A1 ( ALDH3A1 ) was significantly down-regulated when cells were treated with LGK974 and TMZ. Suppressing ALDH3A1 expression increased the efficacy of TMZ and reduced clonogenic potential accompanied by decreased expression of stem cell markers CD133, Nestin and Sox2. Taken together, our study suggests that previous observations concerning Wnt signaling blockade to reduce chemoresistance in glioblastoma is at least in part mediated by inhibition of ALDH3A1.

  18. Protein-protein interactions and substrate channeling in orthologous and chimeric aldolase-dehydrogenase complexes.

    PubMed

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

    2012-03-06

    Bacterial aldolase-dehydrogenase complexes catalyze the last steps in the meta cleavage pathway of aromatic hydrocarbon degradation. The aldolase (TTHB246) and dehydrogenase (TTHB247) from Thermus thermophilus were separately expressed and purified from recombinant Escherichia coli. The aldolase forms a dimer, while the dehydrogenase is a monomer; these enzymes can form a stable tetrameric complex in vitro, consisting of two aldolase and two dehydrogenase subunits. Upon complex formation, the K(m) value of 4-hydroxy-2-oxopentanoate, the substrate of TTHB246, is decreased 4-fold while the K(m) of acetaldehyde, the substrate of TTHB247, is increased 3-fold. The k(cat) values of each enzyme were reduced by ~2-fold when they were in a complex. The half-life of TTHB247 at 50 °C increased by ~4-fold when it was in a complex with TTHB246. The acetaldehyde product from TTHB246 could be efficiently channelled directly to TTHB247, but the channeling efficiency for the larger propionaldehyde was ~40% lower. A single A324G substitution in TTHB246 increased the channeling efficiency of propionaldehyde to a value comparable to that of acetaldehyde. Stable and catalytically competent chimeric complexes could be formed between the T. thermophilus enzymes and the orthologous aldolase (BphI) and dehydrogenase (BphJ) from the biphenyl degradation pathway of Burkholderia xenovorans LB400. However, channeling efficiencies for acetaldehyde in these chimeric complexes were ~10%. Structural and sequence analysis suggests that interacting residues in the interface of the aldolase-dehydrogenase complex are highly conserved among homologues, but coevolution of partner enzymes is required to fine-tune this interaction to allow for efficient substrate channeling.

  19. Cloning and expression studies of the Dunaliella salina UDP-glucose dehydrogenase cDNA.

    PubMed

    Qinghua, He; Dairong, Qiao; Qinglian, Zhang; Shunji, He; Yin, Li; Linhan, Bai; Zhirong, Yang; Yi, Cao

    2005-06-01

    The enzyme UDP-glucose dehydrogenase (EC 1.1.1.22) converts UDP-glucose to UDP-glucuronate. Plant UDP-glucose dehydrogenase (UGDH) is an important enzyme in the formation of hemicellulose and pectin, the components of primary cell walls. A cDNA, named DsUGDH, (GeneBank accession number: AY795899) corresponding to UGDH was cloned by RT-PCR approach from Dunaliella salina. The cDNA is 1941-bp long and has an open reading frame encoded a protein of 483 amino acids with a calculated molecular weight of 53 kDa. The derived amino acids sequence shows high homology with reported plants UGDHs, and has highly conserved amino acids motifs believed to be NAD binding site and catalytic site. Although UDP-glucose dehydrogenase is a comparatively well characterized enzyme, the cloning and characterization of the green alga Dunaliella salina UDP-glucose dehydrogenase gene is very important to understand the salt tolerance mechanism of Dunaliella salina. Northern analyses indicate that NaCl can induce the expression the DsUGDH.

  20. Pyruvate decarboxylase and alcohol dehydrogenase overexpression in Escherichia coli resulted in high ethanol production and rewired metabolic enzyme networks.

    PubMed

    Yang, Mingfeng; Li, Xuefeng; Bu, Chunya; Wang, Hui; Shi, Guanglu; Yang, Xiushan; Hu, Yong; Wang, Xiaoqin

    2014-11-01

    Pyruvate decarboxylase and alcohol dehydrogenase are efficient enzymes for ethanol production in Zymomonas mobilis. These two enzymes were over-expressed in Escherichia coli, a promising candidate for industrial ethanol production, resulting in high ethanol production in the engineered E. coli. To investigate the intracellular changes to the enzyme overexpression for homoethanol production, 2-DE and LC-MS/MS were performed. More than 1,000 protein spots were reproducibly detected in the gel by image analysis. Compared to the wild-type, 99 protein spots showed significant changes in abundance in the recombinant E. coli, in which 46 were down-regulated and 53 were up-regulated. Most proteins related to tricarboxylic acid cycle, glycerol metabolism and other energy metabolism were up-regulated, whereas proteins involved in glycolysis and glyoxylate pathway were down-regulated, indicating the rewired metabolism in the engineered E. coli. As glycolysis is the main pathway for ethanol production, and it was inhibited significantly in engineered E. coli, further efforts should be directed at minimizing the repression of glycolysis to optimize metabolism network for higher yields of ethanol production.

  1. Amine oxidation by d-arginine dehydrogenase in Pseudomonas aeruginosa.

    PubMed

    Ouedraogo, Daniel; Ball, Jacob; Iyer, Archana; Reis, Renata A G; Vodovoz, Maria; Gadda, Giovanni

    2017-10-15

    d-Arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) is a flavin-dependent oxidoreductase, which is part of a novel two-enzyme racemization system that functions to convert d-arginine to l-arginine. PaDADH contains a noncovalently linked FAD that shows the highest activity with d-arginine. The enzyme exhibits broad substrate specificity towards d-amino acids, particularly with cationic and hydrophobic d-amino acids. Biochemical studies have established the structure and the mechanistic properties of the enzyme. The enzyme is a true dehydrogenase because it displays no reactivity towards molecular oxygen. As established through solvent and multiple kinetic isotope studies, PaDADH catalyzes an asynchronous CH and NH bond cleavage via a hydride transfer mechanism. Steady-state kinetic studies with d-arginine and d-histidine are consistent with the enzyme following a ping-pong bi-bi mechanism. As shown by a combination of crystallography, kinetic and computational data, the shape and flexibility of loop L1 in the active site of PaDADH are important for substrate capture and broad substrate specificity. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Advanced development of immobilized enzyme reactors

    NASA Technical Reports Server (NTRS)

    Jolly, Clifford D.; Schussel, Leonard J.; Carter, Layne

    1991-01-01

    Fixed-bed reactors have been used at NASA-Marshall to purify wastewater generated by an end-use equipment facility, on the basis of a combination of multifiltration unibeds and enzyme unibeds. The enzyme beds were found to effectively remove such targeted organics as urea, alcohols, and aldehydes, down to levels lying below detection limits. The enzyme beds were also found to remove organic contaminants not specifically targeted.

  3. Substitutions of S101 decrease proton and hydride transfers in the oxidation of betaine aldehyde by choline oxidase.

    PubMed

    Gadda, Giovanni; Yuan, Hongling

    2017-11-15

    Choline oxidase oxidizes choline to glycine betaine, with two flavin-mediated reactions to convert the alcohol substrate to the carbon acid product. Proton abstraction from choline or hydrated betaine aldehyde in the wild-type enzyme occurs in the mixing time of the stopped-flow spectrophotometer, thereby precluding a mechanistic investigation. Mutagenesis of S101 rendered the proton transfer reaction amenable to study. Here, we have investigated the aldehyde oxidation reaction catalyzed by the mutant enzymes using steady-state and rapid kinetics with betaine aldehyde. Stopped-flow traces for the reductive half-reaction of the S101T/V/C variants were biphasic, corresponding to the reactions of proton abstraction and hydride transfer. In contrast, the S101A enzyme yielded monophasic traces like wild-type choline oxidase. The rate constants for proton transfer in the S101T/C/V variants decreased logarithmically with increasing hydrophobicity of residue 101, indicating a behavior different from that seen previously with choline for which no correlation was determined. The rate constants for hydride transfer also showed a logarithmic decrease with increasing hydrophobicity at position 101, which was similar to previous results with choline as a substrate for the enzyme. Thus, the hydrophilic character of S101 is necessary not only for efficient hydride transfer but also for the proton abstraction reaction. Copyright © 2017. Published by Elsevier Inc.

  4. Determination of hydride transfer stereospecificity of NADH-dependent alcohol-aldehyde/ketone oxidoreductase from Sulfolobus solfataricus.

    PubMed

    Trincone, A; Lama, L; Rella, R; D'Auria, S; Raia, C A; Nicolaus, B

    1990-10-18

    This paper describes the determination of stereospecificity of hydride transfer reaction of an alcohol dehydrogenase isolated from the archaebacterium Sulfolobus solfataricus. The 1H-NMR and EI-MS data indicate that the enzyme transfers the pro-R hydrogen from coenzyme to substrate and is therefore an A-specific dehydrogenase.

  5. A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD(+) regeneration.

    PubMed

    Wang, Jizhong; Yang, Chengli; Chen, Xing; Bao, Bingxin; Zhang, Xuan; Li, Dali; Du, Xingfan; Shi, Ruofu; Yang, Junfang; Zhu, Ronghui

    2016-08-01

    To find an efficient and cheap system for NAD(+) regeneration A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K m value for NADH of 10 μM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD(+) regeneration was established. The system is attractive in that the O2 consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD(+) was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C CONCLUSION: The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD(+) regeneration.

  6. Inhibition of human cytochrome P450 2E1 and 2A6 by aldehydes: structure and activity relationships.

    PubMed

    Kandagatla, Suneel K; Mack, Todd; Simpson, Sean; Sollenberger, Jill; Helton, Eric; Raner, Gregory M

    2014-08-05

    The purpose of this study was to probe active site structure and dynamics of human cytochrome P4502E1 and P4502A6 using a series of related short chain fatty aldehydes. Binding efficiency of the aldehydes was monitored via their ability to inhibit the binding and activation of the probe substrates p-nitrophenol (2E1) and coumarin (2A6). Oxidation of the aldehydes was observed in reactions with individually expressed 2E1, but not 2A6, suggesting alternate binding modes. For saturated aldehydes the optimum chain length for inhibition of 2E1 was 9 carbons (KI=7.8 ± 0.3 μM), whereas for 2A6 heptanal was most potent (KI=15.8 ± 1.1 μM). A double bond in the 2-position of the aldehyde significantly decreased the observed KI relative to the corresponding saturated compound in most cases. A clear difference in the effect of the double bond was observed between the two isoforms. With 2E1, the double bond appeared to remove steric constraints on aldehyde binding with KI values for the 5-12 carbon compounds ranging between 2.6 ± 0.1 μM and 12.8 ± 0.5 μM, whereas steric effects remained the dominant factor in the binding of the unsaturated aldehydes to 2A6 (observed KI values between 7.0 ± 0.5 μM and >1000 μM). The aldehyde function was essential for effective inhibition, as the corresponding carboxylic acids had very little effect on enzyme activity over the same range of concentrations, and branching at the 3-position of the aldehydes increased the corresponding KI value in all cases examined. The results suggest that a conjugated π-system may be a key structural determinant in the binding of these compounds to both enzymes, and may also be an important feature for the expansion of the active site volume in 2E1. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  7. Inhibition of human Cytochrome P450 2E1 and 2A6 by aldehydes: Structure and activity relationships

    PubMed Central

    Kandagatla, Suneel K.; Mack, Todd; Simpson, Sean; Sollenberger, Jill; Helton, Eric; Raner, Gregory M.

    2014-01-01

    The purpose of this study was to probe active site structure and dynamics of human cytochrome P4502E1 and P4502A6 using a series of related short chain fatty aldehydes. Binding efficiency of the aldehydes was monitored via their ability to inhibit the binding and activation of the probe substrates p-nitrophenol (2E1) and coumarin (2A6). Oxidation of the aldehydes was observed in reactions with individually expressed 2E1, but not 2A6, suggesting alternate binding modes. For saturated aldehydes the optimum chain length for inhibition of 2E1 was 9 carbons (KI=7.8 ±0.3 μM), whereas for 2A6 heptanal was most potent (KI=15.8 ±1.1 μM). A double bond in the 2-position of the aldehyde significantly decreased the observed KI relative to the corresponding saturated compound in most cases. A clear difference in the effect of the double bond was observed between the two isoforms. With 2E1, the double bond appeared to remove steric constraints on aldehyde binding with KI values for the 5–12 carbon compounds ranging between 2.6 ± 0.1 μM and 12.8± 0.5 μM, whereas steric effects remained the dominant factor in the binding of the unsaturated aldehydes to 2A6 (observed KI values between 7.0± 0.5 μM and >1000 μM). The aldehyde function was essential for effective inhibition, as the corresponding carboxylic acids had very little effect on enzyme activity over the same range of concentrations, and branching at the 3-position of the aldehydes increased the corresponding KI value in all cases examined. The results suggest that a conjugated π-system may be a key structural determinant in the binding of these compounds to both enzymes, and may also be an important feature for the expansion of the active site volume in 2E1. PMID:24924949

  8. Aldehyde dehydrogenase 2 protects human umbilical vein endothelial cells against oxidative damage and increases endothelial nitric oxide production to reverse nitroglycerin tolerance.

    PubMed

    Hu, X Y; Fang, Q; Ma, D; Jiang, L; Yang, Y; Sun, J; Yang, C; Wang, J S

    2016-06-10

    Medical nitroglycerin (glyceryl trinitrate, GTN) use is limited principally by tolerance typified by a decrease in nitric oxide (NO) produced by biotransformation. Such tolerance may lead to endothelial dysfunction by inducing oxidative stress. In vivo studies have demonstrated that aldehyde dehydrogenase 2 (ALDH2) plays important roles in GTN biotransformation and tolerance. Thus, modification of ALDH2 expression represents a potentially effective strategy to prevent and reverse GTN tolerance and endothelial dysfunction. In this study, a eukaryotic expression vector containing the ALDH2 gene was introduced into human umbilical vein endothelial cells (HUVECs) by liposome-mediated transfection. An indirect immunofluorescence assay showed that ALDH2 expression increased 24 h after transfection. Moreover, real-time polymerase chain reaction and western blotting revealed significantly higher ALDH2 mRNA and protein expression in the gene-transfected group than in the two control groups. GTN tolerance was induced by treating HUVECs with 10 mM GTN for 16 h + 10 min, which significantly decreased NO levels in control cells, but not in those transfected with ALDH2. Overexpression of ALDH2 increased cell survival against GTN-induced cytotoxicity and conferred protection from oxidative damage resulting from nitrate tolerance, accompanied by decreased production of intracellular reactive oxygen species and reduced expression of heme oxygenase 1. Furthermore, ALDH2 overexpression promoted Akt phosphorylation under GTN tolerance conditions. ALDH2 gene transfection can reverse and prevent tolerance to GTN through its bioactivation and protect against oxidative damage, preventing the development of endothelial dysfunction.

  9. Malate dehydrogenase of the cytosol. A kinetic investigation of the reaction mechanism and a comparison with lactate dehydrogenase.

    PubMed Central

    Lodola, A; Shore, J D; Parker, D M; Holbrook, J

    1978-01-01

    1. The mechanisms of the reduction of oxaloacetate and of 3-fluoro-oxaloacetate by NADH catalysed by cytoplasmic pig heart malate dehydrogenase (MDH) were investigated. 2. One mol of dimeric enzyme produces 1.7+/-0.4 mol of enzyme-bound NADH when mixed with saturating NAD+ and L-malate at a rate much higher than the subsequent turnover at pH 7.5. 3. Transient measurements of protein and nucleotide fluorescence show that the steady-state complex in the forward direction is MDH-NADH and in the reverse direction MDH-NADH-oxaloacetate. 4. The rate of dissociation of MDH-NADH was measured and is the same as Vmax. in the forward direction at pH 7.5. Both NADH-binding sites are kinetically equivalent. The rate of dissociation varies with pH, as does the equilibrium binding constant for NADH. 5. 3-Fluoro-oxaloacetate is composed of three forms (F1, F2 and S) of which F1 and F2 are immediately substrates for the enzyme. The third form, S, is not a substrate, but when the F forms are used up form S slowly and non-enzymically equilibrates to yield the active substrate forms. S is 2,2-dihydroxy-3-fluorosuccinate. 6. The steady-state compound during the reduction of form F1 is an enzyme form that does not contain NADH, probably MDH-NAD+-fluoromalate. The steady-state compound for form F2 is an enzyme form containing NADH, probably MDH-NADH-fluoro-oxaloacetate. 7. The rate-limiting reaction in the reduction of form F2 shows a deuterium isotope rate ratio of 4 when NADH is replaced by its deuterium analogue, and the rate-limiting reaction is concluded to be hydride transfer. 8. A novel titration was used to show that dimeric cytoplasmic malate dehydrogenase contains two sites that can rapidly reduce the F1 form of 3-fluoro-oxaloacetate. The enzyme shows 'all-of-the-sites' behaviour. 9. Partial mechanisms are proposed to explain the enzyme-catalysed transformations of the natural and the fluoro substrates. These mechanisms are similar to the mechanism of pig heart lactate

  10. Effects of biogenic aldehydes and aldehyde dehydrogenase inhibitors on rat brain tryptophan hydroxylase activity in vitro.

    PubMed

    Nilsson, G E; Tottmar, O

    1987-04-21

    The effect of indole-3-acetaldehyde, 5-hydroxyindole-3-acetaldehyde, disulfiram, diethyldithiocarbamate, coprine, and 1-amino-cyclopropanol on tryptophan hydroxylase activity was studied in vitro using high performance liquid chromatography with electro-chemical detection. With the analytical method developed, 5-hydroxytryptophan, serotonin, and 5-hydroxyindole-3-acetic acid could be measured simultaneously. Indole-3-acetaldehyde (12-1200 microM) was found to cause a 6-33% inhibition of the enzyme. Dependent upon the nature of the sulfhydryl- or reducing-agent (dithiotreitol, glutathione, or ascorbate) present in the incubates, the degree of inhibition by disulfiram varied, probably due to the formation of various mixed disulfides. Also the presence of diethyldithiocarbamate (160-1600 microM) was found to inhibit tryptophan hydroxylase (28-91%), while 5-hydroxyindole-3-acetaldehyde, coprine, or 1-aminocyclopropanol appeared to have no effect on the enzyme activity.

  11. Cloning, expression, and characterization of bacterial L-arabinose 1-dehydrogenase involved in an alternative pathway of L-arabinose metabolism.

    PubMed

    Watanabe, Seiya; Kodaki, Tsutomu; Kodak, Tsutomu; Makino, Keisuke

    2006-02-03

    Azospirillum brasiliense converts L-arabinose to alpha-ketoglutarate via five hypothetical enzymatic steps. We purified and characterized L-arabinose 1-dehydrogenase (EC 1.1.1.46), catalyzing the conversion of L-arabinose to L-arabino-gamma-lactone as an enzyme responsible for the first step of this alternative pathway of L-arabinose metabolism. The purified enzyme preferred NADP+ to NAD+ as a coenzyme. Kinetic analysis revealed that the enzyme had high catalytic efficiency for both L-arabinose and D-galactose. The gene encoding L-arabinose 1-dehydrogenase was cloned using a partial peptide sequence of the purified enzyme and was overexpressed in Escherichia coli as a fully active enzyme. The enzyme consists of 308 amino acids and has a calculated molecular mass of 33,663.92 Da. The deduced amino acid sequence had some similarity to glucose-fructose oxidoreductase, D-xylose 1-dehydrogenase, and D-galactose 1-dehydrogenase. Site-directed mutagenesis revealed that the enzyme possesses unique catalytic amino acid residues. Northern blot analysis showed that this gene was induced by L-arabinose but not by D-galactose. Furthermore, a disruptant of the L-arabinose 1-dehydrogenase gene did not grow on L-arabinose but grew on D-galactose at the same growth rate as the wild-type strain. There was a partial gene for L-arabinose transport in the flanking region of the L-arabinose 1-dehydrogenase gene. These results indicated that the enzyme is involved in the metabolism of L-arabinose but not D-galactose. This is the first identification of a gene involved in an alternative pathway of L-arabinose metabolism in bacterium.

  12. Characterization of the membrane-bound succinic dehydrogenase of Micrococcus lysodeikticus.

    PubMed

    Pollock, J J; Linder, R; Salton, M R

    1971-07-01

    The occurrence of succinic dehydrogenase [succinic:(acceptor) oxidoreductase, EC 1.3.99.1] in membrane fractions of Micrococcus lysodeikticus was investigated. The enzyme could be purified 10-fold, by deoxycholate treatment. Butanol extraction of membranes yielded an active fraction, nonsedimentable at 130,000 x g for 2 hr and altered in its phospholipid content relative to membranes. The activity of the enzyme in particulate preparations was decreased in the presence of competitive inhibitors and by compounds known to react with iron, sulfhydryl groups, and flavine. In this respect, the bacterial succinic dehydrogenase is similar to the enzyme derived from yeast and mammalian sources. In certain membrane fractions, Ca(2+) and Mg(2+) exhibited inhibitory effects whereas Triton X-100 caused activation. The enzyme could also be activated by substrate. In the phenazine reductase assay, incomplete reduction of electron acceptor was observed upon addition of divalent cations and iron binding agents.

  13. Inhibition of several enzymes by gold compounds. II. beta-Glucuronidase, acid phosphatase and L-malate dehydrogenase by sodium thiomalatoraurate (I), sodium thiosulfatoaurate (I) and thioglucosoaurate (I).

    PubMed

    Lee, M T; Ahmed, T; Haddad, R; Friedman, M E

    1989-01-01

    Bovine liver beta-D-glucuronide glucuronohydrolase, EC 3.2.1.32), wheat germ acid phosphatase (orthophosphoric monoesterphosphohydrolase, EC 3.1.3.2) and bovine liver L-malate dehydrogenase (L-malate: NAD oxidoreductase, EC 1.1.1.37) were inhibited by a series of gold (I) complexes that have been used as anti-inflammatory drugs. Both sodium thiosulfatoaurate (I) (Na AuTs) and sodium thiomalatoraurate (NaAuTM) effectively inhibited all three enzymes, while thioglucosoaurate (I) (AuTG) only inhibited L-malate dehydrogenase. The equilibrium constants (K1) ranged from nearly 4000 microM for the NaAuTM-beta-glucuronidase interaction to 24 microM for the NaAuTS-beta-glucuronidase interaction. The rate of covalent bond formation (kp) ranged from 0.00032 min-1 for NaAuTM-beta-glucuronidase formation to 1.7 min-1 for AuTG-L-malate dehydrogenase formation. The equilibrium data shows that the gold (I) drugs bind by several orders lower than the gold (III) compounds, suggesting a significantly stronger interaction between the more highly charged gold ion and the enzyme. Yet the rate of covalent bond formation depends as much on the structure of the active site as upon the lability of the gold-ligand bond. It was also observed that the more effective the gold inhibition the more toxic the compound.

  14. Diaphorase Coupling Protocols for Red-Shifting Dehydrogenase Assays

    PubMed Central

    Davis, Mindy I.; Shen, Min; Simeonov, Anton

    2016-01-01

    Abstract Dehydrogenases are an important target for the development of cancer therapeutics. Dehydrogenases either produce or consume NAD(P)H, which is fluorescent but at a wavelength where many compounds found in chemical libraries are also fluorescent. By coupling dehydrogenases to diaphorase, which utilizes NAD(P)H to produce the fluorescent molecule resorufin from resazurin, the assay can be red-shifted into a spectral region that reduces interference from compound libraries. Dehydrogenases that produce NAD(P)H, such as isocitrate dehydrogenase 1 (IDH1), can be read in kinetic mode. Dehydrogenases that consume NAD(P)H, such as mutant IDH1 R132H, can be read in endpoint mode. Here, we report protocols for robust and miniaturized 1,536-well assays for WT IDH1 and IDH1 R132H coupled to diaphorase, and the counterassays used to further detect compound interference with the coupling reagents. This coupling technique is applicable to dehydrogenases that either produce or consume NAD(P)H, and the examples provided here can act as guidelines for the development of high-throughput screens against this enzyme class. PMID:27078681

  15. Structure and Mechanism of ArnA: Conformational Change Implies Ordered Dehydrogenase Mechanism in Key Enzyme for Polymyxin Resistance

    PubMed Central

    Gatzeva-Topalova, Petia Z.; May, Andrew P.; Sousa, Marcelo C.

    2010-01-01

    Summary The modification of lipid A with 4-amino-4-deoxy-L-arabinose (Ara4N) allows gram-negative bacteria to resist the antimicrobial activity of cationic antimicrobial peptides and antibiotics such as polymyxin. ArnA is the first enzyme specific to the lipid A-Ara4N pathway. It contains two functionally and physically separable domains: a dehydrogenase domain (ArnA_DH) catalyzing the NAD+-dependent oxidative decarboxylation of UDP-Glucuronic acid (UDP-GlcA), and a transformylase domain that formylates UDP-Ara4N. Here, we describe the crystal structure of the full-length bifunctional ArnA with UDP-GlcA and ATP bound to the dehydrogenase domain. Binding of UDP-GlcA triggers a 17 Å conformational change in ArnA_DH that opens the NAD+ binding site while trapping UDP-GlcA. We propose an ordered mechanism of substrate binding and product release. Mutation of residues R619 and S433 demonstrates their importance in catalysis and suggests that R619 functions as a general acid in catalysis. The proposed mechanism for ArnA_DH has important implications for the design of selective inhibitors. PMID:15939024

  16. An efficient ribitol-specific dehydrogenase from Enterobacter aerogenes.

    PubMed

    Singh, Ranjitha; Singh, Raushan; Kim, In-Won; Sigdel, Sujan; Kalia, Vipin C; Kang, Yun Chan; Lee, Jung-Kul

    2015-05-01

    An NAD(+)-dependent ribitol dehydrogenase from Enterobacter aerogenes KCTC 2190 (EaRDH) was cloned and successfully expressed in Escherichia coli. The complete 729-bp gene was amplified, cloned, expressed, and subsequently purified in an active soluble form using nickel affinity chromatography. The enzyme had an optimal pH and temperature of 11.0 and 45°C, respectively. Among various polyols, EaRDH exhibited activity only toward ribitol, with Km, Vmax, and kcat/Km values of 10.3mM, 185Umg(-1), and 30.9s(-1)mM(-1), respectively. The enzyme showed strong preference for NAD(+) and displayed no detectable activity with NADP(+). Homology modeling and sequence analysis of EaRDH, along with its biochemical properties, confirmed that EaRDH belongs to the family of NAD(+)-dependent ribitol dehydrogenases, a member of short-chain dehydrogenase/reductase (SCOR) family. EaRDH showed the highest activity and unique substrate specificity among all known RDHs. Homology modeling and docking analysis shed light on the molecular basis of its unusually high activity and substrate specificity. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. 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.

  18. 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

  19. 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

  20. Substrate specificity and catalytic efficiency of aldo-keto reductases with phospholipid aldehydes

    PubMed Central

    Spite, Matthew; Baba, Shahid P.; Ahmed, Yonis; Barski, Oleg A.; Nijhawan, Kanchan; Petrash, J. Mark; Bhatnagar, Aruni; Srivastava, Sanjay

    2007-01-01

    Phospholipid oxidation generates several bioactive aldehydes that remain esterified to the glycerol backbone (‘core’ aldehydes). These aldehydes induce endothelial cells to produce monocyte chemotactic factors and enhance monocyte–endothelium adhesion. They also serve as ligands of scavenger receptors for the uptake of oxidized lipoproteins or apoptotic cells. The biochemical pathways involved in phospholipid aldehyde metabolism, however, remain largely unknown. In the present study, we have examined the efficacy of the three mammalian AKR (aldo-keto reductase) families in catalysing the reduction of phospholipid aldehydes. The model phospholipid aldehyde POVPC [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine] was efficiently reduced by members of the AKR1, but not by the AKR6 or the ARK7 family. In the AKR1 family, POVPC reductase activity was limited to AKR1A and B. No significant activity was observed with AKR1C enzymes. Among the active proteins, human AR (aldose reductase) (AKR1B1) showed the highest catalytic activity. The catalytic efficiency of human small intestinal AR (AKR1B10) was comparable with the murine AKR1B proteins 1B3 and 1B8. Among the murine proteins AKR1A4 and AKR1B7 showed appreciably lower catalytic activity as compared with 1B3 and 1B8. The human AKRs, 1B1 and 1B10, and the murine proteins, 1B3 and 1B8, also reduced C-7 and C-9 sn-2 aldehydes as well as POVPE [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphoethanolamine]. AKR1A4, B1, B7 and B8 catalysed the reduction of aldehydes generated in oxidized C16:0-20:4 phosphatidylcholine with acyl, plasmenyl or alkyl linkage at the sn-1 position or C16:0-20:4 phosphatidylglycerol or phosphatidic acid. AKR1B1 displayed the highest activity with phosphatidic acids; AKR1A4 was more efficient with long-chain aldehydes such as 5-hydroxy-8-oxo-6-octenoyl derivatives, whereas AKR1B8 preferred phosphatidylglycerol. These results suggest that proteins of the AKR1A and B families are

  1. Genetic polymorphisms of alcohol dehydrogenase-1B and aldehyde dehydrogenase-2 and liver cirrhosis, chronic calcific pancreatitis, diabetes mellitus, and hypertension among Japanese alcoholic men.

    PubMed

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

    2013-08-01

    The presence of the less-active form of alcohol dehydrogenase-1B encoded by ADH1B*1/*1 (vs. *2 allele) and active form of aldehyde dehydrogenase-2 (ALDH2) encoded by ALDH2*1/*1 (vs. *2 allele) increases the risk of alcoholism in East Asians. The subjects in this cross-sectional survey were 1,902 Japanese alcoholic men (≥40 years) who underwent ADH1B/ALDH2 genotyping. Age-adjusted daily alcohol consumption did not differ according to the ADH1B/ALDH2 genotypes. The age-adjusted odds ratios (AORs; 95% confidence interval) for liver cirrhosis (LC; n = 359, 1.58 [1.19 to 2.09]), chronic calcific pancreatitis (CP; n = 80, 2.24 [1.20 to 4.20]), and diabetes mellitus (DM; n = 383, 1.51 [1.15 to 1.99]) were higher in the ADH1B*2 allele carriers than in the ADH1B*1/*1 carriers. The AORs for LC (1.43 [1.01 to 2.02]), CP (1.68 [0.80 to 3.53]), DM (1.63 [1.15 to 2.30]), and hypertension (HT; n = 495, 1.52 [1.11 to 2.07]) were higher in the ALDH2*1/*1 carriers than in the ALDH2*1/*2 carriers. The ADH1B*2-associated AOR for LC was 2.08 (1.46 to 2.94) among those aged 40 to 59 years, but 0.89 (0.56 to 1.43) among those aged 60 years or over, and the interaction between ADH1B genotype and age on the LC risk was significant (p = 0.009). When the group with non-LC and no/mild fibrosis was used as controls, the ADH1B*2-associated AORs increased according to the severity of their liver disease: 1.67 (1.32 to 2.11) for the group with non-LC and serum type IV collagen values ≥200 ng/ml, 1.81 (1.24 to 2.63) for the group of Child-Pugh class A LC, and 3.17 (1.98 to 5.07) for the group with Child-Pugh class B/C LC. Anti-hepatitis C virus (HCV) antibody was positive in 103 patients, and the groups with a high anti-HCV antibody titer and either the ADH1B*2/*2 genotype or the ALDH2*1/*1 genotype had the highest AORs (8.83 and 4.90, respectively). The population attributable fraction (PAF) due to the ADH1B*2 allele was 29% for LC, 47% for CP, and 27% for DM, and the PAF due to the ALDH2

  2. Regulation of retinoic acid synthetic enzymes by WT1 and HDAC inhibitors in 293 cells.

    PubMed

    Li, Yifan; Wang, Lei; Ai, Weipeng; He, Nianhui; Zhang, Lin; Du, Jihui; Wang, Yong; Mao, Xingjian; Ren, Junqi; Xu, Dan; Zhou, Bei; Li, Rong; Mai, Liwen

    2017-09-01

    All-trans retinoic acid (atRA), which is mainly generated endogenously via two steps of oxidation from vitamin A (retinol), plays an indispensible role in the development of the kidney and many other organs. Enzymes that catalyze the oxidation of retinol to generate atRA, including aldehyde dehydrogenase 1 family (ALDH1)A1, ALDH1A2 and ALDH1A3, exhibit complex expression patterns at different stages of renal development. However, molecular triggers that control these differential expression levels are poorly understood. In this study, we provide in vitro evidence to demonstrate that Wilms' tumor 1 (WT1) negatively regulates the expression of the atRA synthetic enzymes, ALDH1A1, ALDH1A2 and ALDH1A3, in the 293 cell line, leading to significant blockage of atRA production. Furthermore, we demonstrate that the suppression of ALDH1A1 by WT1 can be markedly attenuated by histone deacetylase inhibitors (HDACis). Taken together, we provide evidence to indicate that WT1 and HDACs are strong regulators of endogenous retinoic acid synthetic enzymes in 293 cells, indicating that they may be involved in the regulation of atRA synthesis.

  3. Physiological Regulation of Isocitrate Dehydrogenase and the Role of 2-Oxoglutarate in Prochlorococcus sp. Strain PCC 9511

    PubMed Central

    Diez, Jesús; Gómez-Baena, Guadalupe; Rangel-Zúñiga, Oriol Alberto; García-Fernández, José Manuel

    2014-01-01

    The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42) catalyzes the oxidative decarboxylation of isocitrate, to produce 2-oxoglutarate. The incompleteness of the tricarboxylic acids cycle in marine cyanobacteria confers a special importance to isocitrate dehydrogenase in the C/N balance, since 2-oxoglutarate can only be metabolized through the glutamine synthetase/glutamate synthase pathway. The physiological regulation of isocitrate dehydrogenase was studied in cultures of Prochlorococcus sp. strain PCC 9511, by measuring enzyme activity and concentration using the NADPH production assay and Western blotting, respectively. The enzyme activity showed little changes under nitrogen or phosphorus starvation, or upon addition of the inhibitors DCMU, DBMIB and MSX. Azaserine, an inhibitor of glutamate synthase, induced clear increases in the isocitrate dehydrogenase activity and icd gene expression after 24 h, and also in the 2-oxoglutarate concentration. Iron starvation had the most significant effect, inducing a complete loss of isocitrate dehydrogenase activity, possibly mediated by a process of oxidative inactivation, while its concentration was unaffected. Our results suggest that isocitrate dehydrogenase responds to changes in the intracellular concentration of 2-oxoglutarate and to the redox status of the cells in Prochlorococcus. PMID:25061751

  4. Characterization of the Membrane-Bound Succinic Dehydrogenase of Micrococcus lysodeikticus

    PubMed Central

    Pollock, Jerry J.; Linder, Regina; Salton, Milton R. J.

    1971-01-01

    The occurrence of succinic dehydrogenase [succinic:(acceptor) oxidoreductase, EC 1.3.99.1] in membrane fractions of Micrococcus lysodeikticus was investigated. The enzyme could be purified 10-fold, by deoxycholate treatment. Butanol extraction of membranes yielded an active fraction, nonsedimentable at 130,000 × g for 2 hr and altered in its phospholipid content relative to membranes. The activity of the enzyme in particulate preparations was decreased in the presence of competitive inhibitors and by compounds known to react with iron, sulfhydryl groups, and flavine. In this respect, the bacterial succinic dehydrogenase is similar to the enzyme derived from yeast and mammalian sources. In certain membrane fractions, Ca2+ and Mg2+ exhibited inhibitory effects whereas Triton X-100 caused activation. The enzyme could also be activated by substrate. In the phenazine reductase assay, incomplete reduction of electron acceptor was observed upon addition of divalent cations and iron binding agents. Images PMID:4327510

  5. 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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    McIntire, W.S.; Wemmer, D.E.; Chistoserdov, A.

    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 spectralmore » 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.« less

  7. Insight into the stereospecificity of short-chain thermus thermophilus alcohol dehydrogenase showing pro-S hydride transfer and prelog enantioselectivity.

    PubMed

    Pennacchio, Angela; Giordano, Assunta; Esposito, Luciana; Langella, Emma; Rossi, Mosè; Raia, Carlo A

    2010-04-01

    The stereochemistry of the hydride transfer in reactions catalyzed by NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus HB27 was determined by means of (1)H-NMR spectroscopy. The enzyme transfers the pro-S hydrogen of [4R-(2)H]NADH and exhibits Prelog specificity. Enzyme-substrate docking calculations provided structural details about the enantioselectivity of this thermophilic enzyme. These results give additional insights into the diverse active site architectures of the largely versatile short-chain dehydrogenase superfamily enzymes. A feasible protocol for the synthesis of [4R-(2)H]NADH with high yield was also set up by enzymatic oxidation of 2-propanol-d(8) catalyzed by Bacillus stearothermophilus alcohol dehydrogenase.

  8. Retinoic acid biosynthesis catalyzed by retinal dehydrogenases relies on a rate-limiting conformational transition associated with substrate recognition

    PubMed Central

    Bchini, Raphaël; Vasiliou, Vasilis; Branlant, Guy; Talfournier, François; Rahuel-Clermont, Sophie

    2012-01-01

    Retinoic acid (RA), a metabolite of vitamin A, exerts pleiotropic effects throughout life in vertebrate organisms. Thus, RA action must be tightly regulated through the coordinated action of biosynthetic and degradating enzymes. The last step of retinoic acid biosynthesis is irreversibly catalyzed by the NAD-dependent retinal dehydrogenases (RALDH), which are members of the aldehyde dehydrogenase (ALDH) superfamily. Low intracellular retinal concentrations imply efficient substrate molecular recognition to ensure high affinity and specificity of RALDHs for retinal. This study addresses the molecular basis of retinal recognition in human ALDH1A1 (or RALDH1) and rat ALDH1A2 (or RALDH2), through the comparison of the catalytic behavior of retinal analogs and use of the fluorescence properties of retinol. We show that, in contrast to long chain unsaturated substrates, the rate-limiting step of retinal oxidation by RALDHs is associated with acylation. Use of the fluorescence resonance energy transfer upon retinol interaction with RALDHs provides evidence that retinal recognition occurs in two steps: binding into the substrate access channel, and a slower structural reorganization with a rate constant of the same magnitude as the kcat for retinal oxidation: 0.18 vs. 0.07 s−1 and 0.25 vs. 0.1 s−1 for ALDH1A1 and ALDH1A2, respectively. This suggests that the conformational transition of the RALDH-retinal complex significantly contributes to the rate-limiting step that controls the kinetics of retinal oxidation, as a prerequisite for the formation of a catalytically competent Michaelis complex. This conclusion is consistent with the general notion that structural flexibility within the active site of ALDH enzymes has been shown to be an integral component of catalysis. PMID:23220587

  9. Two-carbon homologation of aldehydes and ketones to a,ß-unsaturated aldehydes

    USDA-ARS?s Scientific Manuscript database

    Phosphonate reagents were developed for the two-carbon homologation of aldehydes or ketones to unbranched- or methyl-branched a,ß-unsaturated aldehydes. The phosphonate reagents, diethyl methylformyl-2-phosphonate dimethylhydrazone and diethyl ethylformyl-2-phosphonate dimethylhydrazone, contained a...

  10. The activity of the artemisinic aldehyde Δ11(13) reductase promoter is important for artemisinin yield in different chemotypes of Artemisia annua L.

    PubMed

    Yang, Ke; Monfared, Sajad Rashidi; Monafared, Rashidi Sajad; Wang, Hongzhen; Lundgren, Anneli; Brodelius, Peter E

    2015-07-01

    The artemisinic aldehyde double bond reductase (DBR2) plays an important role in the biosynthesis of the antimalarial artemisinin in Artemisia annua. Artemisinic aldehyde is reduced into dihydroartemisinic aldehyde by DBR2. Artemisinic aldehyde can also be oxidized by amorpha-4,11-diene 12-hydroxylase and/or aldehyde dehydrogenase 1 to artemisinic acid, a precursor of arteannuin B. In order to better understand the effects of DBR2 expression on the flow of artemisinic aldehyde into either artemisinin or arteannuin B, we determined the content of dihydroartemisinic aldehyde, artemisinin, artemisinic acid and arteannuin B content of A. annua varieties sorted into two chemotypes. The high artemisinin producers (HAPs), which includes the '2/39', 'Chongqing' and 'Anamed' varieties, produce more artemisinin than arteannuin B; the low artemisinin producers (LAPs), which include the 'Meise', 'Iran#8', 'Iran#14', 'Iran#24' and 'Iran#47' varieties, produce more arteannuin B than artemisinin. Quantitative PCR showed that the relative expression of DBR2 was significantly higher in the HAP varieties. We cloned and sequenced the promoter of the DBR2 gene from varieties of both the LAP and the HAP groups. There were deletions/insertions in the region just upstream of the ATG start codon in the LAP varities, which might be the reason for the different promoter activities of the HAP and LAP varieties. The relevance of promoter variation, DBR2 expression levels and artemisinin biosynthesis capabilities are discussed and a selection method for HAP varieties with a DNA marker is suggested. Furthermore, putative cis-acting regulatory elements differ between the HAP and LAP varieties.

  11. Human class II (pi) alcohol dehydrogenase has a redox-specific function in norepinephrine metabolism.

    PubMed Central

    Mårdh, G; Dingley, A L; Auld, D S; Vallee, B L

    1986-01-01

    Studies of the function of human alcohol dehydrogenase (ADH) have revealed substrates that are virtually unique for class II ADH (pi ADH). It catalyzes the formation of the intermediary glycols of norepinephrine metabolism, 3,4-dihydroxyphenylglycol and 4-hydroxy-3-methoxyphenylglycol, from the corresponding aldehydes 3,4-dihydroxymandelaldehyde and 4-hydroxy-3-methoxymandelaldehyde with Km values of 55 and 120 microM and kcat/Km ratios of 14,000 and 17,000 mM-1 X min-1; these are from 60- to 210-fold higher than those obtained with class I ADH isozymes. The catalytic preference of class II ADH also extends to benzaldehydes. The kcat/Km values for the reduction of benzaldehyde, 3,4-dihydroxybenzaldehyde and 4-hydroxy-3-methoxybenzaldehyde by pi ADH are from 9- to 29-fold higher than those for a class I isozyme, beta 1 gamma 2 ADH. Furthermore, the norepinephrine aldehydes are potent inhibitors of alcohol (ethanol) oxidation by pi ADH. The high catalytic activity of pi ADH-catalyzed reduction of the aldehydes in combination with a possible regulatory function of the aldehydes in the oxidative direction leads to essentially "unidirectional" catalysis by pi ADH. These features and the presence of pi ADH in human liver imply a physiological role for pi ADH in the degradation of circulating epinephrine and norepinephrine. PMID:3466164

  12. UDP-glucose Dehydrogenase Polymorphisms from Patients with Congenital Heart Valve Defects Disrupt Enzyme Stability and Quaternary Assembly*

    PubMed Central

    Hyde, Annastasia S.; Farmer, Erin L.; Easley, Katherine E.; van Lammeren, Kristy; Christoffels, Vincent M.; Barycki, Joseph J.; Bakkers, Jeroen; Simpson, Melanie A.

    2012-01-01

    Cardiac valve defects are a common congenital heart malformation and a significant clinical problem. Defining molecular factors in cardiac valve development has facilitated identification of underlying causes of valve malformation. Gene disruption in zebrafish revealed a critical role for UDP-glucose dehydrogenase (UGDH) in valve development, so this gene was screened for polymorphisms in a patient population suffering from cardiac valve defects. Two genetic substitutions were identified and predicted to encode missense mutations of arginine 141 to cysteine and glutamate 416 to aspartate, respectively. Using a zebrafish model of defective heart valve formation caused by morpholino oligonucleotide knockdown of UGDH, transcripts encoding the UGDH R141C or E416D mutant enzymes were unable to restore cardiac valve formation and could only partially rescue cardiac edema. Characterization of the mutant recombinant enzymes purified from Escherichia coli revealed modest alterations in the enzymatic activity of the mutants and a significant reduction in the half-life of enzyme activity at 37 °C. This reduction in activity could be propagated to the wild-type enzyme in a 1:1 mixed reaction. Furthermore, the quaternary structure of both mutants, normally hexameric, was destabilized to favor the dimeric species, and the intrinsic thermal stability of the R141C mutant was highly compromised. The results are consistent with the reduced function of both missense mutations significantly reducing the ability of UGDH to provide precursors for cardiac cushion formation, which is essential to subsequent valve formation. The identification of these polymorphisms in patient populations will help identify families genetically at risk for valve defects. PMID:22815472

  13. Carboxylic acid reductase enzymes (CARs).

    PubMed

    Winkler, Margit

    2018-04-01

    Carboxylate reductases (CARs) are emerging as valuable catalysts for the selective one-step reduction of carboxylic acids to their corresponding aldehydes. The substrate scope of CARs is exceptionally broad and offers potential for their application in diverse synthetic processes. Two major fields of application are the preparation of aldehydes as end products for the flavor and fragrance sector and the integration of CARs in cascade reactions with aldehydes as the key intermediates. The latest applications of CARs are dominated by in vivo cascades and chemo-enzymatic reaction sequences. The challenge to fully exploit product selectivity is discussed. Recent developments in the characterization of CARs are summarized, with a focus on aspects related to the domain architecture and protein sequences of CAR enzymes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Characterization of human DHRS6, an orphan short chain dehydrogenase/reductase enzyme: a novel, cytosolic type 2 R-beta-hydroxybutyrate dehydrogenase.

    PubMed

    Guo, Kunde; Lukacik, Petra; Papagrigoriou, Evangelos; Meier, Marc; Lee, Wen Hwa; Adamski, Jerzy; Oppermann, Udo

    2006-04-14

    Human DHRS6 is a previously uncharacterized member of the short chain dehydrogenases/reductase family and displays significant homologies to bacterial hydroxybutyrate dehydrogenases. Substrate screening reveals sole NAD(+)-dependent conversion of (R)-hydroxybutyrate to acetoacetate with K(m) values of about 10 mm, consistent with plasma levels of circulating ketone bodies in situations of starvation or ketoacidosis. The structure of human DHRS6 was determined at a resolution of 1.8 A in complex with NAD(H) and reveals a tetrameric organization with a short chain dehydrogenases/reductase-typical folding pattern. A highly conserved triad of Arg residues ("triple R" motif consisting of Arg(144), Arg(188), and Arg(205)) was found to bind a sulfate molecule at the active site. Docking analysis of R-beta-hydroxybutyrate into the active site reveals an experimentally consistent model of substrate carboxylate binding and catalytically competent orientation. GFP reporter gene analysis reveals a cytosolic localization upon transfection into mammalian cells. These data establish DHRS6 as a novel, cytosolic type 2 (R)-hydroxybutyrate dehydrogenase, distinct from its well characterized mitochondrial type 1 counterpart. The properties determined for DHRS6 suggest a possible physiological role in cytosolic ketone body utilization, either as a secondary system for energy supply in starvation or to generate precursors for lipid and sterol synthesis.

  15. Prospects for robust biocatalysis: engineering of novel specificity in a halophilic amino acid dehydrogenase.

    PubMed

    Munawar, Nayla; Engel, Paul C

    2013-01-01

    Heat- and solvent-tolerant enzymes from halophiles, potentially important industrially, offer a robust framework for protein engineering, but few solved halophilic structures exist to guide this. Homology modelling has guided mutations in glutamate dehydrogenase (GDH) from Halobacterium salinarum to emulate conversion of a mesophilic GDH to a methionine dehydrogenase. Replacement of K89, A163 and S367 by leucine, glycine and alanine converted halophilic GDH into a dehydrogenase accepting L-methionine, L-norleucine and L-norvaline as substrates. Over-expression in the halophilic expression host Haloferax volcanii and three-step purification gave ~98 % pure protein exhibiting maximum activity at pH 10. This enzyme also showed enhanced thermostability and organic solvent tolerance even at 70 °C, offering a biocatalyst resistant to harsh industrial environments. To our knowledge, this is the first reported amino acid specificity change engineered in a halophilic enzyme, encouraging use of mesophilic models to guide engineering of novel halophilic biocatalysts for industrial application. Calibrated gel filtration experiments show that both the mutant and the wild-type enzyme are stable hexamers.

  16. Single nucleotide polymorphism detection in aldehyde dehydrogenase 2 (ALDH2) gene using bacterial magnetic particles based on dissociation curve analysis.

    PubMed

    Maruyama, Kohei; Takeyama, Haruko; Nemoto, Etsuo; Tanaka, Tsuyoshi; Yoda, Kiyoshi; Matsunaga, Tadashi

    2004-09-20

    Single nucleotide polymorphism (SNP) detection for aldehyde dehydrogenase 2 (ALDH2) gene based on DNA thermal dissociation curve analysis was successfully demonstrated using an automated system with bacterial magnetic particles (BMPs) by developing a new method for avoiding light scattering caused by nanometer-size particles when using commercially available fluorescent dyes such as FITC, Cy3, and Cy5 as labeling chromophores. Biotin-labeled PCR products in ALDH2, two allele-specific probes (Cy3-labeled detection probe for ALDH2*1 and Cy5-labeled detection probe for ALDH2*2), streptavidin-immobilized BMPs (SA-BMPs) were simultaneously mixed. The mixture was denatured at 70 degrees C for 3 min, cooled slowly to 25 degrees C, and incubated for 10 min, allowing the DNA duplex to form between Cy3- or Cy5-labeled detection probes and biotin-labeled PCR products on SA-BMPs. Then duplex DNA-BMP complex was heated to 58 degrees C, a temperature determined by dissociation curve analysis and a dissociated single-base mismatched detection probe was removed at the same temperature under precise control. Furthermore, fluorescence signal from the detection probe was liberated into the supernatant from completely matched duplex DNA-BMP complex by heating to 80 degrees C and measured. In the homozygote target DNA (ALDH2*1/*1 and ALDH2*2/*2), the fluorescence signals from single-base mismatched were decreased to background level, indicating that mismatched hybridization was efficiently removed by the washing process. In the heterozygote target DNA (ALDH2*1/*2), each fluorescence signals was at a similar level. Therefore, three genotypes of SNP in ALDH2 gene were detected using the automated detection system with BMPs. Copyright 2004 Wiley Periodicals, Inc.

  17. Role of quinate dehydrogenase in quinic acid metabolism in conifers

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Osipov, V.I.; Shein, I.V.

    1986-08-10

    Quinate dehydrogenase was isolated from young needles of the Siberian larch and partially purified by ammonium sulfate fractionation. It was found that in conifers, in contrast to other plants, quinate dehydrogenase is active both with NAD and with NADP. The values of K/sub m/ for quinate and NADP were 1.8 and 0.18 mM. The enzyme exhibits maximum activity at pH 9.0. It was assumed that NADP-dependent quinate dehydrogenase is responsible for quinic acid synthesis. The special features of the organization and regulation of the initial stages of the shikimate pathway in conifers are discussed.

  18. DIFFERENTIATING THE TOXICITY OF CARCINOGENIC ALDEHYDES FROM NONCARCINOGENIC ALDEHYDES IN THE RAT NOSE USING CDNA ARRAYS

    EPA Science Inventory

    Differentiating the Toxicity of Carcinogenic Aldehydes from Noncarcinogenic Aldehydes in the Rat Nose Using cDNA Arrays.

    Formaldehyde is a widely used aldehyde in many industrial settings, the tanning process, household products, and is a contaminant in cigarette smoke. H...

  19. Identification and Molecular Characterization of Genes Coding Pharmaceutically Important Enzymes from Halo-Thermo Tolerant Bacillus

    PubMed Central

    Safary, Azam; Moniri, Rezvan; Hamzeh-Mivehroud, Maryam; Dastmalchi, Siavoush

    2016-01-01

    Purpose: Robust pharmaceutical and industrial enzymes from extremophile microorganisms are main source of enzymes with tremendous stability under harsh conditions which make them potential tools for commercial and biotechnological applications. Methods: The genome of a Gram-positive halo-thermotolerant Bacillus sp. SL1, new isolate from Saline Lake, was investigated for the presence of genes coding for potentially pharmaceutical enzymes. We determined gene sequences for the enzymes laccase (CotA), l-asparaginase (ansA3, ansA1), glutamate-specific endopeptidase (blaSE), l-arabinose isomerase (araA2), endo-1,4-β mannosidase (gmuG), glutaminase (glsA), pectate lyase (pelA), cellulase (bglC1), aldehyde dehydrogenase (ycbD) and allantoinases (pucH) in the genome of Bacillus sp. SL1. Results: Based on the DNA sequence alignment results, six of the studied enzymes of Bacillus sp. SL-1 showed 100% similarity at the nucleotide level to the same genes of B. licheniformis 14580 demonstrating extensive organizational relationship between these two strains. Despite high similarities between the B. licheniformis and Bacillus sp. SL-1 genomes, there are minor differences in the sequences of some enzyme. Approximately 30% of the enzyme sequences revealed more than 99% identity with some variations in nucleotides leading to amino acid substitution in protein sequences. Conclusion: Molecular characterization of this new isolate provides useful information regarding evolutionary relationship between B. subtilis and B. licheniformis species. Since, the most industrial processes are often performed in harsh conditions, enzymes from such halo-thermotolerant bacteria may provide economically and industrially appealing biocatalysts to be used under specific physicochemical situations in medical, pharmaceutical, chemical and other industries. PMID:28101462

  20. Identification and Molecular Characterization of Genes Coding Pharmaceutically Important Enzymes from Halo-Thermo Tolerant Bacillus.

    PubMed

    Safary, Azam; Moniri, Rezvan; Hamzeh-Mivehroud, Maryam; Dastmalchi, Siavoush

    2016-12-01

    Purpose: Robust pharmaceutical and industrial enzymes from extremophile microorganisms are main source of enzymes with tremendous stability under harsh conditions which make them potential tools for commercial and biotechnological applications. Methods: The genome of a Gram-positive halo-thermotolerant Bacillus sp. SL1, new isolate from Saline Lake, was investigated for the presence of genes coding for potentially pharmaceutical enzymes. We determined gene sequences for the enzymes laccase (CotA), l-asparaginase (ansA3, ansA1), glutamate-specific endopeptidase (blaSE), l-arabinose isomerase (araA2), endo-1,4-β mannosidase (gmuG), glutaminase (glsA), pectate lyase (pelA), cellulase (bglC1), aldehyde dehydrogenase (ycbD) and allantoinases (pucH) in the genome of Bacillus sp. SL1. Results: Based on the DNA sequence alignment results, six of the studied enzymes of Bacillus sp. SL-1 showed 100% similarity at the nucleotide level to the same genes of B. licheniformis 14580 demonstrating extensive organizational relationship between these two strains. Despite high similarities between the B. licheniformis and Bacillus sp. SL-1 genomes, there are minor differences in the sequences of some enzyme. Approximately 30% of the enzyme sequences revealed more than 99% identity with some variations in nucleotides leading to amino acid substitution in protein sequences. Conclusion: Molecular characterization of this new isolate provides useful information regarding evolutionary relationship between B. subtilis and B. licheniformis species. Since, the most industrial processes are often performed in harsh conditions, enzymes from such halo-thermotolerant bacteria may provide economically and industrially appealing biocatalysts to be used under specific physicochemical situations in medical, pharmaceutical, chemical and other industries.

  1. Effect of feeding and of DDT on the activity of hepatic glucose 6- phosphate dehydrogenase in two salmonids

    USGS Publications Warehouse

    Buhler, Donald R.; Benville, P.

    1969-01-01

    The specific activity of liver glucose 6-phosphate dehydrogenase in yearling rainbow trout remained unchanged when the fish were starved for periods as long as 8 weeks and when starved animals were fed diets of various compositions. Injection of insulin concurrently with refeeding also failed to alter the specific activity of the enzyme in trout. The absence of a dietary or insulin influence on the teleost enzyme system is to be contrasted with studies in mammals in which the activity of hepatic glucose 6-P dehydrogenase was markedly stimulated after refeeding starved animals or injection of insulin.Ingestion of the pesticide DDT by juvenile coho salmon or adult rainbow trout also had no effect on the specific activity of liver glucose 6-P dehydrogenase and DDT failed to inhibit the rainbow trout enzyme in vitro. These results also differ considerably from those found in higher animals.These results suggest that the glucose 6-P dehydrogenase enzyme in teleosts may be under a different type of regulatory control from that found in mammals.

  2. Glucose-6-phosphate dehydrogenase Buenos Aires: a novel de novo missense mutation associated with severe enzyme deficiency.

    PubMed

    Minucci, Angelo; Concolino, Paola; Vendittelli, Francesca; Giardina, Bruno; Zuppi, Cecilia; Capoluongo, Ettore

    2008-06-01

    : Glucose 6-phosphate dehydrogenase (G6PD) catalyzes the first committed steps in the pentose phosphate pathway: the generation of NADPH by this enzyme is essential for protection against oxidative stress. The human enzyme is in a dimer<-->tetramer equilibrium and its stability depends on NADP(+) concentration. Herein, we report a case of a symptomatic baby affected by severe deficiency of G6PD activity due to a novel de novo genetic mutation (g1465C>T) in the thirteenth exon of its gene. : Clinical, biochemical and genetic evaluations of the affected baby and his mother were performed. : We found the g1465C>T novel mutation, in the thirteenth exon of G6PD gene (named "G6PD Buenos Aires variant"). This g1465C>T mutation produce a P489S substitution at protein level. The P489S mutation was absent in his mother, suggesting that G6PD Buenos Aires resulted from a de novo mutation. : The absence of mosaicism in the baby's DNA (from saliva and blood samples) suggests that a de novo mutation event may occur in the very early stages in embryogenesis or in the mother's germ cell lines.

  3. First general methods toward aldehyde enolphosphates.

    PubMed

    Barthes, Nicolas; Grison, Claude

    2012-02-01

    We herein report two innovative methods toward aldehyde enolphosphates and the first saccharidic aldehyde enolphosphates. Aldehyde enolphosphate function is worthwhile to be considered as a good phosphoenolpyruvate analogue. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Differential levels of metabolites and enzymes related to aroma formation in aromatic indica rice varieties: comparison with non-aromatic varieties.

    PubMed

    Ghosh, Puja; Roychoudhury, Aryadeep

    2018-01-01

    Accounting for aroma production in different aromatic indica rice varieties based on variations in the levels of concerned metabolites and enzymes is poorly explored. The present investigation was, therefore, focused on unraveling the differential levels of metabolites and activities of enzymes related to aroma formation in eleven indigenous aromatic rice varieties, as compared with four non-aromatic varieties. The levels of metabolites such as proline (Pro) and Δ 1 -pyrroline-5-carboxylate (P5C), and the activity of related enzymes such as proline dehydrogenase (PDH), Δ 1 -pyrroline-5-carboxylate synthetase (P5CS), and ornithine aminotransferase (OAT) were comparatively higher in the aromatic varieties, with Kalonunia and Tulaipanji registering the highest Pro, Kalonunia the highest P5C content, Gobindobhog with the highest PDH activity, Gobindobhog and Tulaipanji with the highest P5CS, and Pusa Basmati-1 with the highest OAT activity. The levels of putrescine (Put) and γ-aminobutyric acid (GABA) were comparatively lower in aromatic varieties, with concomitant higher diamine oxidase (DAO) activity, especially in the varieties Gobindobhog and Tulaipanji. The betaine-aldehyde dehydrogenase 2 (BADH2) enzyme activity was remarkably lesser in aromatic varieties, especially Radhunipagal and Gobindobhog. Though the metabolites such as glycine-betaine and higher polyamines such as spermidine and spermine showed no specific trend with respect to their quantitative level in either aromatic or non-aromatic varieties, they were notably lower in the aromatic varieties such as Gobindobhog, Kalonunia, and Tulaipanji, indicating a possibility of their involvement in aroma formation. Therefore, the levels of metabolites such as Pro, P5C and methylglyoxal (MG), and the activity of enzymes such as PDH, P5CS, OAT, and DAO were comparatively higher in the aromatic rice varieties than the non-aromatic ones, whereas the levels of Put, GABA, and BADH2 were lower. Overall, the present

  5. Structural characterization of the thermostable Bradyrhizobium japonicumD-sorbitol dehydrogenase.

    PubMed

    Fredslund, Folmer; Otten, Harm; Gemperlein, Sabrina; Poulsen, Jens Christian N; Carius, Yvonne; Kohring, Gert Wieland; Lo Leggio, Leila

    2016-11-01

    Bradyrhizobium japonicum sorbitol dehydrogenase is NADH-dependent and is active at elevated temperatures. The best substrate is D-glucitol (a synonym for D-sorbitol), although L-glucitol is also accepted, giving it particular potential in industrial applications. Crystallization led to a hexagonal crystal form, with crystals diffracting to 2.9 Å resolution. In attempts to phase the data, a molecular-replacement solution based upon PDB entry 4nbu (33% identical in sequence to the target) was found. The solution contained one molecule in the asymmetric unit, but a tetramer similar to that found in other short-chain dehydrogenases, including the search model, could be reconstructed by applying crystallographic symmetry operations. The active site contains electron density consistent with D-glucitol and phosphate, but there was not clear evidence for the binding of NADH. In a search for the features that determine the thermostability of the enzyme, the T m for the orthologue from Rhodobacter sphaeroides, for which the structure was already known, was also determined, and this enzyme proved to be considerably less thermostable. A continuous β-sheet is formed between two monomers in the tetramer of the B. japonicum enzyme, a feature not generally shared by short-chain dehydrogenases, and which may contribute to thermostability, as may an increased Pro/Gly ratio.

  6. A dual substrate kinetic model for cytochrome P450BM3-F87G catalysis: simultaneous binding of long chain aldehydes and 4-fluorophenol.

    PubMed

    Ledford, Chelsea; McMahon, Monica; Whitesell, Ashley; Khan, Ghalib; Kandagatla, Suneel K; Hurst, Dow P; Reggio, Patricia H; Raner, Gregory M

    2017-02-01

    To develop a model for binding and catalysis associated with the stimulation of 4-fluorophenol (4-FP) oxidation in the presence of long chain aldehydes by the enzymatic catalyst, cytochrome P450 BM3 -F87G. A variation of the Michaeli-Menten kinetic model was employed to describe interactions at the active site of the enzyme, along with computer aided modeling approaches. In addition to the hydroquinone product arising from de-fluorination of 4-FP, a second product (p-fluorocatechol) was also observed and, like the hydroquinone, its rate of formation increased in the presence of the aldehyde. When only aldehyde was present with the enzyme, BM3-F87G catalyzed its oxidation to the corresponding carboxylic acid; however, this activity was inhibited when 4-FP was added to the reaction. A 3D computer model of the active site containing both aldehyde and 4-FP was generated, guided by these kinetic observations. Finally, partitioning between the two phenolic products was examined with an emphasis on the conditions directing the initial epoxidation at either the 2,3- or 3,4-positions on the substrate. Temperature, reaction time, substrate concentration, and the structure of the aldehyde had no substantial effect on the overall product ratios, however the NADPH coupling efficiency decreased when unsaturated aldehydes were included, or when the temperature of the reaction was reduced. The unsaturated aldehyde, trans-2-decenal, stimulates BM3-F87G catalyzed oxidation of 4-fluorophenol through a cooperative active site binding mode that doesn't influence product distributions or coupling efficiencies, while 4-fluorophenol acts as a competitive inhibitor of aldehyde oxidation.

  7. Roles of the C-terminal domains of human dihydrodiol dehydrogenase isoforms in the binding of substrates and modulators: probing with chimaeric enzymes.

    PubMed Central

    Matsuura, K; Hara, A; Deyashiki, Y; Iwasa, H; Kume, T; Ishikura, S; Shiraishi, H; Katagiri, Y

    1998-01-01

    Human liver dihydrodiol dehydrogenase (DD; EC 1.3.1.20) exists in isoforms (DD1, DD2 and DD4) composed of 323 amino acids. DD1 and DD2 share 98% amino acid sequence identity, but show lower identities (approx. 83%) with DD4, in which a marked difference is seen in the C-terminal ten amino acids. DD4 exhibits unique catalytic properties, such as the ability to oxidize both (R)- and (S)-alicyclic alcohols equally, high dehydrogenase activity for bile acids, potent inhibition by steroidal anti-inflammatory drugs and activation by sulphobromophthalein and clofibric acid derivatives. In this study, we have prepared chimaeric enzymes, in which we exchanged the C-terminal 39 residues between the two enzymes. Compared with DD1, CDD1-4 (DD1 with the C-terminal sequence of DD4) had increased kcat/Km values for 3alpha-hydroxy-5beta-androstanes and bile acids of 3-9-fold and decreased values for the other substrates by 5-100-fold. It also became highly sensitive to DD4 inhibitors such as phenolphthalein and hexoestrol. Another chimaeric enzyme, CDD4-1 (DD4 with the C-terminal sequence of DD1), showed the same (S)-stereospecificity for the alicyclic alcohols as DD1, had decreased kcat/Km values for bile acids with 7beta- or 12alpha-hydroxy groups by more than 120-fold and was resistant to inhibition by betamethasone. In addition, the activation effects of sulphobromophthalein and bezafibrate decreased or disappeared for CDD4-1. The recombinant DD4 with the His314-->Pro (the corresponding residue of DD1) mutation showed intermediate changes in the properties between those of wild-type DD4 and CDD4-1. The results indicate that the binding of substrates, inhibitors and activators to the enzymes is controlled by residues in their C-terminal domains; multiple residues co-ordinately act as determinants for substrate specificity and inhibitor sensitivity. PMID:9820821

  8. Aldehyde Detection in Electronic Cigarette Aerosols

    PubMed Central

    2017-01-01

    Acetaldehyde, acrolein, and formaldehyde are the principal toxic aldehydes present in cigarette smoke and contribute to the risk of cardiovascular disease and noncancerous pulmonary disease. The rapid growth of the use of electronic cigarettes (e-cigarettes) has raised concerns over emissions of these harmful aldehydes. This work determines emissions of these aldehydes in both free and bound (aldehyde–hemiacetal) forms and other carbonyls from the use of e-cigarettes. A novel silicon microreactor with a coating phase of 4-(2-aminooxyethyl)-morpholin-4-ium chloride (AMAH) was used to trap carbonyl compounds in the aerosols of e-cigarettes via oximation reactions. AMAH–aldehyde adducts were measured using gas chromatography–mass spectrometry. 1H nuclear magnetic resonance spectroscopy was used to analyze hemiacetals in the aerosols. These aldehydes were detected in the aerosols of all e-cigarettes. Newer-generation e-cigarette devices generated more aldehydes than the first-generation e-cigarettes because of higher battery power output. Formaldehyde–hemiacetal was detected in the aerosols generated from some e-liquids using the newer e-cigarette devices at a battery power output of 11.7 W and above. The emission of these aldehydes from all e-cigarettes, especially higher levels of aldehydes from the newer-generation e-cigarette devices, indicates the risk of using e-cigarettes. PMID:28393137

  9. Response surface methodology as an approach to determine the optimal activities of xylose reductase and xylitol dehydrogenase enzymes from Candida Mogii.

    PubMed

    Mayerhoff, Zea D V L; Roberto, Inês C; Franco, Telma T

    2006-05-01

    A central composite experimental design leading to a set of 16 experiments with different combinations of pH and temperature was performed to attain the optimal activities of xylose reductase (XR) and xylitol dehydrogenase (XDH) enzymes from Candida mogii cell extract. Under optimized conditions (pH 6.5 and 38 degrees C), the XR and XDH activities were found to be 0.48 U/ml and 0.22 U/ml, respectively, resulting in an XR to XDH ratio of 2.2. Stability, cofactor specificity and kinetic parameters of the enzyme XR were also evaluated. XR activity remained stable for 3 h under 4 and 38 degrees C and for 4 months of storage at -18 degrees C. Studies on cofactor specificity showed that only NADPH-dependent XR was obtained under the cultivation conditions employed. The XR present in C. mogii extracts showed a superior Km value for xylose when compared with other yeast strains. Besides, this parameter was not modified after enzyme extraction by aqueous two-phase system.

  10. 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

  11. Alcohol, Aldehydes, Adducts and Airways.

    PubMed

    Sapkota, Muna; Wyatt, Todd A

    2015-11-05

    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.

  12. Formate Dehydrogenase from Clostridium acidiurici

    PubMed Central

    Kearny, James J.; Sagers, Richard D.

    1972-01-01

    Partial purification of formate dehydrogenase from Clostridium acidiurici has been accomplished, and some properties of the enzyme have been determined. The molecular weight of the protein is at least 200,000 daltons. The enzyme showed marked instability to freezing and thawing and was inhibited strongly by oxygen and by light. Such inhibition was not reversed by incubation in the presence of thiol compounds. Cyanide inhibited the enzyme 90% at 0.1 mm concentrations, but ethylenediaminetetraacetate produced only slight inhibition at concentrations as high as 50 mm. The purified enzyme showed no ferredoxin activity in the Clostridium pasteurianum clastic system during pyruvate oxidation. Crude preparations of the enzyme could be coupled through ferredoxin to the reduction of nicotinamide adenine dinucleotide during formate oxidation, but the purified enzyme could not catalyze the reduction of pyridine nucleotides by formate in the presence of ferredoxin. Formate oxidation with the purified enzyme was readily coupled to benzyl viologen reduction, in which case ferredoxin was not required. An exchange between formate and bicarbonate was catalyzed by both crude and purified preparations of the enzyme, but the net synthesis of formate from CO2 was not accomplished. PMID:4333376

  13. Adrenal 11-beta hydroxysteroid dehydrogenase activity in response to stress.

    PubMed

    Zallocchi, Marisa; Matković, Laura; Damasco, María C

    2004-06-01

    This work studied the effect of stresses produced by simulated gavage or gavage with 200 mmol/L HCl two hours before adrenal extraction, on the activities of the 11beta-hydroxysteroid dehydrogenase 1 and 11beta-hydroxysteroid dehydrogenase 2 isoforms present in the rat adrenal gland. These activities were determined on immediately prepared adrenal microsomes following incubations with 3H-corticosterone and NAD+ or NADP+. 11-dehydrocorticosterone was measured as an end-product by TLC, and controls were adrenal microsomes from rats kept under basal (unstressed) conditions. 11beta-hydroxysteroid dehydrogenase 1 activity, but not 11beta-hydroxysteroid dehydrogenase 2 activity, was increased under both stress-conditions. Homeostatically, the stimulation of 11beta-hydroxysteroid dehydrogenase 1 activity would increase the supply of glucocorticoids. These, in turn, would activate the enzyme phenylethanolamine N-methyl transferase, thereby improving the synthesis of epinephrine as part of the stress-response.

  14. The crystallogenesis of a human estradiol dehydrogenase-substrate complex

    NASA Astrophysics Data System (ADS)

    Zhu, Dao-Wei; Azzi, Arezki; Rehse, Peter; Lin, Sheng-Xiang

    1996-10-01

    Human 17β-hydroxysteroid dehydrogenase type 1 is an important steroidogenic enzyme catalyzing the synthesis of the most active estrogen: estradiol. The enzyme is formed by two identical subunits (34.5 kDa). In this paper, we report the preparation of a stoichiometric 17β-HSD1-estradiol complex sample at a much higher concentration than the solubility of the free substrate, using a gradual concentration of the enzyme-substrate mixture starting at low concentration. The complex is successfully crystallized by vapor diffusion at pH 7.5 with polyethyleneglycol 4000 as the precipitating agent. The space group is C2 with a = 123.56 Å, b = 45.21 Å, c = 61.30 Å and β = 99.06°. There is one monomer in the asymmetric unit and two molecules of the enzyme in a unit cell. A diffraction data set to 2.5 Å has been collected to 86% completeness on native crystals. The high quality of the electronic density map of estradiol supports the full occupancy of the binding site, thus confirming the efficiency of the complex preparation. This method will also be useful in crystallizing other steroid-dehydrogenase complexes.

  15. YNL134C from Saccharomyces cerevisiae encodes a novel protein with aldehyde reductase activity for detoxification of furfural derived from lignocellulosic biomass.

    PubMed

    Zhao, Xianxian; Tang, Juan; Wang, Xu; Yang, Ruoheng; Zhang, Xiaoping; Gu, Yunfu; Li, Xi; Ma, Menggen

    2015-05-01

    Furfural and 5-hydroxymethylfurfural (HMF) are the two main aldehyde compounds derived from pentoses and hexoses, respectively, during lignocellulosic biomass pretreatment. These two compounds inhibit microbial growth and interfere with subsequent alcohol fermentation. Saccharomyces cerevisiae has the in situ ability to detoxify furfural and HMF to the less toxic 2-furanmethanol (FM) and furan-2,5-dimethanol (FDM), respectively. Herein, we report that an uncharacterized gene, YNL134C, was highly up-regulated under furfural or HMF stress and Yap1p and Msn2/4p transcription factors likely controlled its up-regulated expression. Enzyme activity assays showed that YNL134C is an NADH-dependent aldehyde reductase, which plays a role in detoxification of furfural to FM. However, no NADH- or NADPH-dependent enzyme activity was observed for detoxification of HMF to FDM. This enzyme did not catalyse the reverse reaction of FM to furfural or FDM to HMF. Further studies showed that YNL134C is a broad-substrate aldehyde reductase, which can reduce multiple aldehydes to their corresponding alcohols. Although YNL134C is grouped into the quinone oxidoreductase family, no quinone reductase activity was observed using 1,2-naphthoquinone or 9,10-phenanthrenequinone as a substrate, and phylogenetic analysis indicates that it is genetically distant to quinone reductases. Proteins similar to YNL134C in sequence from S. cerevisiae and other microorganisms were phylogenetically analysed. Copyright © 2015 John Wiley & Sons, Ltd.

  16. Optical characterization of glutamate dehydrogenase monolayers chemisorbed on SiO2

    NASA Astrophysics Data System (ADS)

    Pompa, P. P.; Blasi, L.; Longo, L.; Cingolani, R.; Ciccarella, G.; Vasapollo, G.; Rinaldi, R.; Rizzello, A.; Storelli, C.; Maffia, M.

    2003-04-01

    This paper describes the formation of glutamate dehydrogenase monolayers on silicon dioxide, and their characterization by means of physical techniques, i.e., fluorescence spectroscopy and Fourier-transform infrared spectroscopy. Detailed investigations of the intrinsic stability of native proteins in solution were carried out to elucidate the occurrence of conformational changes induced by the immobilization procedure. The enzyme monolayers were deposited on SiO2 after preexposing silicon surfaces to 3-aminopropyltriethoxysilane and reacting the silylated surfaces with glutaric dialdehyde. The optical characterization demonstrates that the immobilization does not interfere with the fold pattern of the native enzyme. In addition, fluorescence spectroscopy, thermal denaturation, and quenching studies performed on the enzyme in solution well describe the folding and unfolding properties of glutamate dehydrogenase. The photophysical studies reported here are relevant for nanobioelectronics applications requiring protein immobilization on a chip.

  17. Regulation of aflatoxin biosynthesis: effect of glucose on activities of various glycolytic enzymes.

    PubMed Central

    Buchanan, R L; Lewis, D F

    1984-01-01

    Catabolism of carbohydrates has been implicated in the regulation of aflatoxin synthesis. To characterize this effect further, the activities of various enzymes associated with glucose catabolism were determined in Aspergillus parasiticus organisms that were initially cultured in peptone-mineral salts medium and then transferred to glucose-mineral salts and peptone-mineral salts media. After an initial increase in activity, the levels of glucose 6-phosphate dehydrogenase, mannitol dehydrogenase, and malate dehydrogenase were lowered in the presence of glucose. Phosphofructokinase activity was greater in the peptone-grown mycelium, but fructose diphosphatase was largely unaffected by carbon source. Likewise, carbon source had relatively little effect on the activities of pyruvate kinase, malic enzyme, isocitrate-NADP dehydrogenase, and isocitrate-NAD dehydrogenase. The results suggest that glucose may, in part, regulate aflatoxin synthesis via a carbon catabolite repression of NADPH-generating and tricarboxylic acid cycle enzymes. PMID:6091545

  18. Kinetic mechanism of Escherichia coli isocitrate dehydrogenase and its inhibition by glyoxylate and oxaloacetate.

    PubMed Central

    Nimmo, H G

    1986-01-01

    The inhibition of Escherichia coli isocitrate dehydrogenase by glyoxylate and oxaloacetate was examined. The shapes of the progress curves in the presence of the inhibitors depended on the order of addition of the assay components. When isocitrate dehydrogenase or NADP+ was added last, the rate slowly decreased until a new, inhibited, steady state was obtained. When isocitrate was added last, the initial rate was almost zero, but the rate increased slowly until the same steady-state value was obtained. Glyoxylate and oxaloacetate gave competitive inhibition against isocitrate and uncompetitive inhibition against NADP+. Product-inhibition studies showed that isocitrate dehydrogenase obeys a compulsory-order mechanism, with coenzyme binding first. Glyoxylate and oxaloacetate bind to and dissociate from isocitrate dehydrogenase slowly. These observations can account for the shapes of the progress curves observed in the presence of the inhibitors. Condensation of glyoxylate and oxaloacetate produced an extremely potent inhibitor of isocitrate dehydrogenase. Analysis of the reaction by h.p.l.c. showed that this correlated with the formation of oxalomalate. This compound decomposed spontaneously in assay mixtures, giving 4-hydroxy-2-oxoglutarate, which was a much less potent inhibitor of the enzyme. Oxalomalate inhibited isocitrate dehydrogenase competitively with respect to isocitrate and was a very poor substrate for the enzyme. The data suggest that the inhibition of isocitrate dehydrogenase by glyoxylate and oxaloacetate is not physiologically significant. PMID:3521584

  19. Galectin-9 Produced by Intestinal Epithelial Cells Enhances Aldehyde Dehydrogenase Activity in Dendritic Cells in a PI3K- and p38-Dependent Manner.

    PubMed

    de Kivit, Sander; Kostadinova, Atanaska I; Kerperien, JoAnn; Ayechu Muruzabal, Veronica; Morgan, Mary E; Knippels, Leon M J; Kraneveld, Aletta D; Garssen, Johan; Willemsen, Linette E M

    2017-01-01

    Intestinal epithelial cells (IEC) drive regulatory T cell (Treg) responses by promoting the differentiation of aldehyde dehydrogenase (ALDH)-expressing CD103+ dendritic cells (DC). Apical stimulation of TLR9 by CpG DNA on IEC supports galectin-9 expression by IEC, which is promoted by short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides (GF). While galectin-9 can induce the maturation of monocyte-derived DC (moDC), the contribution of galectin-9 on the induction of ALDH activity in DC is not known. To this end, DC were stimulated with galectin-9, and ALDH activity and the expression of CD103 were assessed. ALDH activity was increased by moDC exposed to galectin-9, while the expression of CD103 remained unaltered. Galectin-9 secreted by IEC apically exposed to CpG DNA and GF enhanced ALDH activity, but not CD103 expression by moDC, which was abrogated upon galectin-9 neutralization. Similar observations were found in murine GM-CSF-cultured bone marrow-derived DC (BMDC). Using Flt3L-cultured BMDC and ex vivo murine splenic DC, it was observed that galectin-9 only enhanced ALDH activity in the presence of GM-CSF in CD103- cells. The induction of ALDH activity in BMDC was dependent on p38 and PI3K signaling. These data indicate a novel role for galectin-9 in modulating innate immunity by inducing ALDH activity in DC. © 2017 S. Karger AG, Basel.

  20. AMP-Dependent Kinase and Autophagic Flux are Involved in Aldehyde Dehydrogenase 2-Offered Protection against Cardiac Toxicity of Ethanol

    PubMed Central

    Ge, Wei; Guo, Rui; Ren, Jun

    2011-01-01

    Mitochondrial aldehyde dehydrogenase-2 (ALDH2) alleviates ethanol toxicity although the precise mechanism is unclear. This study was designed to evaluate the effect of ALDH2 on ethanol-induced myocardial damage with a focus on autophagy. Wild-type FVB and transgenic mice overexpressing ALDH2 were challenged with ethanol (3 g/kg/d, i.p.) for 3 days and cardiac mechanical function was assessed using the echocardiographic and IonOptix systems. Western blot analysis was used to evaluate essential autophagy markers, Akt and AMPK and their downstream signaling mTOR. Ethanol challenge altered cardiac geometry and function evidenced by enlarged ventricular end systolic and diastolic diameters, decreased cell shortening and intracellular Ca2+ rise, prolonged relengthening and intracellular Ca2+ decay, as well as reduced SERCA Ca2+ uptake, the effects of which were mitigated by ALDH2. Ethanol challenge facilitated myocardial autophagy as evidenced by enhanced expression of Beclin, ATG7 and LC3B II, as well as mTOR dephosphorylation, which was alleviated by ALDH2. Ethanol challenge-induced cardiac defect and apoptosis were reversed by the ALDH-2 agonist Alda-1, the autophagy inhibitor 3-MA, and the AMPK inhibitor compound C whereas the autophagy inducer rapamycin and the AMPK activator AICAR mimicked or exacerbated ethanol-induced cell injury. Ethanol promoted or suppressed phosphorylation of AMPK and Akt, respectively, in FVB but not ALDH2 murine hearts. Moreover, AICAR nullified Alda-1-induced protection against ethanol-triggered autophagic and functional changes. Ethanol increased GFP-LC3 puncta in H9c2 cells, the effect of which was ablated by Alda-1 and 3-MA. Lysosomal inhibition using bafilomycin A1, E64D and pepstatin A obliterated Alda-1- but not ethanol-induced responses in GFP-LC3 puncta. Our results suggested that ALDH2 protects against ethanol toxicity through altered Akt and AMPK signaling and regulation of autophagic flux. PMID:21871561

  1. 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. Copyright 1999 Academic Press.

  2. Isolation, sequence, and characterization of the Cercospora nicotianae phytoene dehydrogenase gene.

    PubMed Central

    Ehrenshaft, M; Daub, M E

    1994-01-01

    We have cloned and sequenced the Cercospora nicotianae gene for the carotenoid biosynthetic enzyme phytoene dehydrogenase. Analysis of the derived amino acid sequence revealed it has greater than 50% identity with its counterpart in Neurospora crassa and approximately 30% identity with prokaryotic phytoene dehydrogenases and is related, but more distantly, to phytoene dehydrogenases from plants and cyanobacteria. Our analysis confirms that phytoene dehydrogenase proteins fall into two groups: those from plants and cyanobacteria and those from eukaryotic and noncyanobacter prokaryotic microbes. Southern analysis indicated that the C. nicotianae phytoene dehydrogenase gene is present in a single copy. Extraction of beta-carotene, the sole carotenoid accumulated by C. nicotianae, showed that both light- and dark-grown cultures synthesize carotenoids, but higher levels accumulate in the light. Northern (RNA) analysis of poly(A)+ RNA, however, showed no differential accumulation of phytoene dehydrogenase mRNA between light- and dark-grown fungal cultures. Images PMID:8085820

  3. Digitalis metabolism and human liver alcohol dehydrogenase.

    PubMed Central

    Frey, W A; Vallee, B L

    1980-01-01

    Human liver alcohol dehydrogenase (alcohol: NAD" oxidoreductase, EC 1.1.1.1) catalyzes the oxidation of the 3 beta-OH group of digitoxigenin, digoxigenin, and gitoxigenin to their 3-keto derivatives, which have been characterized by high performance liquid chromatography and mass spectrometry. These studies have identified human liver alcohol dehydrogenase as the unknown NAD(H)-dependent liver enzyme specific for the free hydroxyl group at C3 of the cardiac genins; this hydroxyl is the critical site of the genins' enzymatic oxidation and concomitant pharmacological inactivation in humans. Several kinetic approaches have demonstrated that ethanol and the pharmacologically active components of the digitalis glycosides are oxidized with closely similar kcat/Km values at the same site on human liver alcohol dehydrogenase, for which they compete. Human liver alcohol dehydrogenase thereby becomes an important biochemical link in the metabolism, pharmacology, and toxicology of ethanol and these glycosides, structurally unrelated agents that are both used widely. Both the competition of ethanol with these cardiac sterols and the narrow margin of safety in the therapeutic use of digitalis derivatives would seem to place at increased risk those individuals who receive digitalis and simultaneously consume large amounts of ethanol or whose alcohol dehydrogenase function is impaired. PMID:6987673

  4. Glutathione-related enzymes and the eye.

    PubMed

    Ganea, Elena; Harding, John J

    2006-01-01

    Glutathione and the related enzymes belong to the defence system protecting the eye against chemical and oxidative stress. This review focuses on GSH and two key enzymes, glutathione reductase and glucose-6-phosphate dehydrogenase in lens, cornea, and retina. Lens contains a high concentration of reduced glutathione, which maintains the thiol groups in the reduced form. These contribute to lens complete transparency as well as to the transparent and refractive properties of the mammalian cornea, which are essential for proper image formation on the retina. In cornea, gluthatione also plays an important role in maintaining normal hydration level, and in protecting cellular membrane integrity. In retina, glutathione is distributed in the different types of retinal cells. Intracellular enzyme, glutathione reductase, involved in reducing the oxidized glutathione has been found at highest activity in human and primate lenses, as compared to other species. Besides the enzymes directly involved in maintaining the normal redox status of the cell, glucose-6-phosphate dehydrogenase which catalyzes the first reaction of the pentose phosphate pathway, plays a key role in protection of the eye against reactive oxygen species. Cornea has a high activity of the pentose phosphate pathway and glucose-6-phosphate dehydrogenase activity. Glycation, the non-enzymic reaction between a free amino group in proteins and a reducing sugar, slowly inactivates gluthathione-related and other enzymes. In addition, glutathione can be also glycated. The presence of glutathione, and of the related enzymes has been also reported in other parts of the eye, such as ciliary body and trabecular meshwork, suggesting that the same enzyme systems are present in all tissues of the eye to generate NADPH and to maintain gluthatione in the reduced form. Changes of glutathione and related enzymes activity in lens, cornea, retina and other eye tissues, occur with ageing, cataract, diabetes, irradiation and

  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. Cloning and mRNA Expression of NADH Dehydrogenase during Ochlerotatus taeniorhynchus Development and Pesticide Response

    USDA-ARS?s Scientific Manuscript database

    NADH dehydrogenase, the largest of the respiratory complexes, is the first enzyme of the mitochondrial electron transport chain. We have cloned and sequenced cDNA of NADH dehydrogenase gene from Ochlerotatus (Ochlerotatus) taeniorhynchus (Wiedemann) adult (GeneBank Accession number: FJ458415). The ...

  7. Novel chiral tool, (R)-2-octanol dehydrogenase, from Pichia finlandica: purification, gene cloning, and application for optically active α-haloalcohols.

    PubMed

    Yamamoto, Hiroaki; Kudoh, Masatake

    2013-09-01

    A novel enantioselective alcohol dehydrogenase, (R)-2-octanol dehydrogenase (PfODH), was discovered among methylotrophic microorganisms. The enzyme was purified from Pichia finlandica and characterized. The molecular mass of the enzyme was estimated to be 83,000 and 30,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The enzyme was an NAD(+)-dependent secondary alcohol dehydrogenase and showed a strict enantioselectivity, very broad substrate specificity, and high tolerance to SH reagents. A gene-encoding PfODH was cloned and sequenced. The gene consisted of 765 nucleotides, coding polypeptides of 254 amino acids. The gene was singly expressed and coexpressed together with a formate dehydrogenase as an NADH regenerator in an Escherichia coli. Ethyl (S)-4-chloro-3-hydroxybutanoate and (S)-2-chloro-1-phenylethanol were synthesized using a whole-cell biocatalyst in more than 99 % optical purity.

  8. Effect of cigarette smoke on salivary proteins and enzyme activities.

    PubMed

    Nagler, R; Lischinsky, S; Diamond, E; Drigues, N; Klein, I; Reznick, A Z

    2000-07-15

    Exposure of human plasma in vitro to gas-phase cigarette smoke (CS) causes a marked modification of plasma proteins as measured by protein carbonyl assay. Aldehydes present in CS may cause this elevation of protein carbonyls by reacting with sulfhydryl groups of proteins. Saliva is the first body fluid to confront the inhaled CS. Thus, in vitro exposure of saliva to nine "puffs" of CS also showed a distinct increase in protein carbonyls. Ascorbate and desferrioxamine mesylate had little effect on protein carbonyl formation, while GSH and N-acetylcysteine considerably inhibited the accumulation of protein carbonyls due to CS exposure. Following the exposure to CS, the activities of several salivary enzymes-amylase, lactic dehydrogenase (LDH), and acid phosphatase-were found to be significantly reduced (34, 57, and 77%, respectively). However, CS had no effect on the activities of aspartate aminotransferase and alkaline phosphatase. Addition of 1 mM of GSH and N-acetylcysteine considerably protected LDH and amylase activities, suggesting that sulfhydryl groups are affected in LDH and amylase. On the other hand, addition of 1 mM ascorbate caused a further loss of LDH and amylase activities, which could be partially prevented by the addition of desferrioxamine mesylate, implicating metal-catalyzed oxidation processes. Finally, loss of acid phosphatase activity was completely unaffected by any of the above antioxidants. It is concluded that the loss of salivary enzyme activities may be due to various agents in the CS that affect the enzyme activities via different mechanisms. Copyright 2000 Academic Press.

  9. Application of a new chemiluminescence method for the determination of glucose-6-phosphate dehydrogenase activity in healthy and enzyme-deficient individuals.

    PubMed

    Gumuslu, Saadet; Yucel, Gultekin; Sarikcioglu, Sureyya Bilmen; Serteser, Mustafa

    2005-01-01

    A chemiluminescence (CL) technique, which determines the glucose-6-phosphate dehydrogenase (G-6-PD) activities in healthy, heterozygous, and completely enzyme-deficient individuals was applied. CL intensities were detected for 4 h at 15-min intervals in each sample with or without addition of G-6-PD substrates into the reaction mixture. The results revealed an inverse correlation to the reference UV method (Zinkham method; r=-0.80). Furthermore, the CL assay was able to detect G-6-PD activities as low as 0.2 IU/gHb, which was not possible by the UV method. In conclusion, we believe that this method offers a new diagnostic tool for the detection of G-6-PD activities in enzyme-deficient individuals and, because of its increased sensitivity, makes it amenable for determining the effects of different pharmaceutical agents on G-6-PD activity in tissue or cell cultures.

  10. Expression pattern, ethanol-metabolizing activities, and cellular localization of alcohol and aldehyde dehydrogenases in human large bowel: association of the functional polymorphisms of ADH and ALDH genes with hemorrhoids and colorectal cancer.

    PubMed

    Chiang, Chien-Ping; Jao, Shu-Wen; Lee, Shiao-Pieng; Chen, Pei-Chi; Chung, Chia-Chi; Lee, Shou-Lun; Nieh, Shin; Yin, Shih-Jiun

    2012-02-01

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are principal enzymes responsible for metabolism of ethanol. Functional polymorphisms of ADH1B, ADH1C, and ALDH2 genes occur among racial populations. The goal of this study was to systematically determine the functional expressions and cellular localization of ADHs and ALDHs in human rectal mucosa, the lesions of adenocarcinoma and hemorrhoid, and the genetic association of allelic variations of ADH and ALDH with large bowel disorders. Twenty-one surgical specimens of rectal adenocarcinoma and the adjacent normal mucosa, including 16 paired tissues of rectal tumor, normal mucosae of rectum and sigmoid colon from the same individuals, and 18 surgical mixed hemorrhoid specimens and leukocyte DNA samples from 103 colorectal cancer patients, 67 hemorrhoid patients, and 545 control subjects recruited in previous study, were investigated. The isozyme/allozyme expression patterns of ADH and ALDH were identified by isoelectric focusing and the activities were assayed spectrophotometrically. The protein contents of ADH/ALDH isozymes were determined by immunoblotting using the corresponding purified class-specific antibodies; the cellular activity and protein localizations were detected by immunohistochemistry and histochemistry, respectively. Genotypes of ADH1B, ADH1C, and ALDH2 were determined by polymerase chain reaction-restriction fragment length polymorphisms. At 33mM ethanol, pH 7.5, the activity of ADH1C*1/1 phenotypes exhibited 87% higher than that of the ADH1C*1/*2 phenotypes in normal rectal mucosa. The activity of ALDH2-active phenotypes of rectal mucosa was 33% greater than ALDH2-inactive phenotypes at 200μM acetaldehyde. The protein contents in normal rectal mucosa were in the following order: ADH1>ALDH2>ADH3≈ALDH1A1, whereas those of ADH2, ADH4, and ALDH3A1 were fairly low. Both activity and content of ADH1 were significantly decreased in rectal tumors, whereas the ALDH activity remained

  11. Homogeneous bioluminescence competitive binding assay for folate based on a coupled glucose-6-phosphate dehydrogenase--bacterial luciferase enzyme system.

    PubMed

    Huang, W; Feltus, A; Witkowski, A; Daunert, S

    1996-05-01

    A homogeneous bioluminescence competitive binding assay for folate was developed by using a coupled enzyme system of glucose-6-phosphate dehydrogenase (G6PDH) and bacterial luciferase. A highly substituted G6PDH-folate conjugate was prepared by employing an N-hydroxysuccinimide/carbodiimide method. Folate binding protein inhibits the activity of the conjugate. In the presence of folate, there is a competition between folate and the G6PDH-folate conjugate for the binding site of the folate binding protein, and the activity of the conjugate is recovered. Thus, the concentration of folate can be related to the activity of the G6PDH-folate conjugate, which is directly related to the bioluminescence produced by the coupled enzyme reaction. Using this assay, dose-response curves with a detection limit of 2.5 x 10(-8) M folate were obtained, which is an improvement of an order of magnitude with respect to an assay that monitors G6PDH activity spectrophotometrically. The assay was validated using vitamin tablets and a cell culture medium.

  12. 9-Hydroxyprostaglandin dehydrogenase activity in the adult rat kidney. Regional distribution and sub-fractionation.

    PubMed

    Asciak, C P; Domazet, Z

    1975-02-20

    1. Catabolism of prostaglandin F2alpha in the adult rat kidney takes place by the following sequence of enzymatic steps: (1) 15-hydroxyprostaglandin dehydrogenase; (2) prostaglandin delta13-reductase; and (3) 9-hydroxyprostaglandin dehydrogenase. 2. 9-Hydroxyprostaglandin dehydrogenase activity was highest in the cortex with lesser amounts in the medulla and negligible activity detected in the papilla. A similar distribution was observed for 15-hydroxyprostaglandin dehydrogenase and prostaglandin delta13-reductase. 3. Most of the 9-hydroxyprostaglandin dehydrogenase activity in the homogenate was found in the high-speed supernatant as also observed for 15-hydroxyprostaglandin dehydrogenase and prostaglandin delta13-reductase. 4. These observations indicate that the rat kidney contains an abundance of prostaglandin-catabolising enzymes which favour formation of metabolites of the E-type.

  13. Tungsten and Molybdenum Regulation of Formate Dehydrogenase Expression in Desulfovibrio vulgaris Hildenborough ▿

    PubMed Central

    da Silva, Sofia M.; Pimentel, Catarina; Valente, Filipa M. A.; Rodrigues-Pousada, Claudina; Pereira, Inês A. C.

    2011-01-01

    Formate is an important energy substrate for sulfate-reducing bacteria in natural environments, and both molybdenum- and tungsten-containing formate dehydrogenases have been reported in these organisms. In this work, we studied the effect of both metals on the levels of the three formate dehydrogenases encoded in the genome of Desulfovibrio vulgaris Hildenborough, with lactate, formate, or hydrogen as electron donors. Using Western blot analysis, quantitative real-time PCR, activity-stained gels, and protein purification, we show that a metal-dependent regulatory mechanism is present, resulting in the dimeric FdhAB protein being the main enzyme present in cells grown in the presence of tungsten and the trimeric FdhABC3 protein being the main enzyme in cells grown in the presence of molybdenum. The putatively membrane-associated formate dehydrogenase is detected only at low levels after growth with tungsten. Purification of the three enzymes and metal analysis shows that FdhABC3 specifically incorporates Mo, whereas FdhAB can incorporate both metals. The FdhAB enzyme has a much higher catalytic efficiency than the other two. Since sulfate reducers are likely to experience high sulfide concentrations that may result in low Mo bioavailability, the ability to use W is likely to constitute a selective advantage. PMID:21498650

  14. Electrophilic aldehyde products of lipid peroxidation selectively adduct to heat shock protein 90 and arylsulfatase A in stallion spermatozoa.

    PubMed

    Hall, Sally E; Aitken, R John; Nixon, Brett; Smith, Nathan D; Gibb, Zamira

    2017-01-01

    Oxidative stress is a major determinant of mammalian sperm function stimulating lipid peroxidation cascades that culminate in the generation of potentially cytotoxic aldehydes. The aim of this study was to assess the impact of such aldehydes on the functionality of stallion spermatozoa. The impact of exposure to exogenous acrolein (ACR) and 4-hydroxynonenal (4HNE) was manifested in a highly significant dose- and time-dependent increase in mitochondrial reactive oxygen species (ROS), total cellular ROS, a decrease in sperm motility, and a time-dependent increase in lipid peroxidation. Notably, low doses of ACR and 4HNE also caused a significant decrease in zona binding. In contrast, exogenous malondialdehyde, a commonly used marker of oxidative stress, had little impact on the various sperm parameters assessed. In accounting for the negative physiological impact of ACR and 4HNE, it was noted that both aldehydes readily adducted to sperm proteins located predominantly within the head, proximal centriole, and tail. The detoxifying activity of mitochondrial aldehyde dehydrogenase 2 appeared responsible for a lack of adduction in the midpiece; however, this activity was overwhelmed by 24 h of electrophilic aldehyde exposure. Sequencing of the dominant proteins targeted for ACR and 4HNE covalent modification identified heat shock protein 90 alpha (cytosolic) class A member 1 and arylsulfatase A, respectively. These collective findings may prove useful in the identification of diagnostic biomarkers of stallion fertility and resolving the mechanistic basis of sperm dysfunction in this species. © The Authors 2016. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please journals.permissions@oup.com.

  15. Human dehydrogenase/reductase (SDR family) member 11 is a novel type of 17β-hydroxysteroid dehydrogenase.

    PubMed

    Endo, Satoshi; Miyagi, Namiki; Matsunaga, Toshiyuki; Hara, Akira; Ikari, Akira

    2016-03-25

    We report characterization of a member of the short-chain dehydrogenase/reductase superfamily encoded in a human gene, DHRS11. The recombinant protein (DHRS11) efficiently catalyzed the conversion of the 17-keto group of estrone, 4- and 5-androstenes and 5α-androstanes into their 17β-hydroxyl metabolites with NADPH as a coenzyme. In contrast, it exhibited reductive 3β-hydroxysteroid dehydrogenase activity toward 5β-androstanes, 5β-pregnanes, 4-pregnenes and bile acids. Additionally, DHRS11 reduced α-dicarbonyls (such as diacetyl and methylglyoxal) and alicyclic ketones (such as 1-indanone and loxoprofen). The enzyme activity was inhibited in a mixed-type manner by flavonoids, and competitively by carbenoxolone, glycyrrhetinic acid, zearalenone, curcumin and flufenamic acid. The expression of DHRS11 mRNA was observed widely in human tissues, most abundantly in testis, small intestine, colon, kidney and cancer cell lines. Thus, DHRS11 represents a novel type of 17β-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Pyruvate dehydrogenase complex and lactate dehydrogenase are targets for therapy of acute liver failure.

    PubMed

    Ferriero, Rosa; Nusco, Edoardo; De Cegli, Rossella; Carissimo, Annamaria; Manco, Giuseppe; Brunetti-Pierri, Nicola

    2018-03-24

    Acute liver failure is a rapidly progressive deterioration of hepatic function resulting in high mortality and morbidity. Metabolic enzymes can translocate to the nucleus to regulate histone acetylation and gene expression. Levels and activities of pyruvate dehydrogenase complex (PDHC) and lactate dehydrogenase (LDH) were evaluated in nuclear fractions of livers of mice exposed to various hepatotoxins including CD95-antibody, α-amanitin, and acetaminophen. Whole-genome gene expression profiling by RNA-seq was performed in livers of mice with acute liver failure and analyzed by gene ontology enrichment analysis. Cell viability was evaluated in cell lines knocked-down for PDHA1 or LDH-A and in cells incubated with the LDH inhibitor galloflavin after treatment with CD95-antibody. We evaluated whether the histone acetyltransferase inhibitor garcinol or galloflavin could reduce liver damage in mice with acute liver failure. Levels and activities of PDHC and LDH were increased in nuclear fractions of livers of mice with acute liver failure. The increase of nuclear PDHC and LDH was associated with increased concentrations of acetyl-CoA and lactate in nuclear fractions, and histone H3 hyper-acetylation. Gene expression in livers of mice with acute liver failure suggested that increased histone H3 acetylation induces the expression of genes related to damage response. Reduced histone acetylation by the histone acetyltransferase inhibitor garcinol decreased liver damage and improved survival in mice with acute liver failure. Knock-down of PDHC or LDH improved viability in cells exposed to a pro-apoptotic stimulus. Treatment with the LDH inhibitor galloflavin that was also found to inhibit PDHC, reduced hepatic necrosis, apoptosis, and expression of pro-inflammatory cytokines in mice with acute liver failure. Mice treated with galloflavin also showed a dose-response increase in survival. PDHC and LDH translocate to the nucleus, leading to increased nuclear concentrations of

  17. Crystal structure of a chimaeric bacterial glutamate dehydrogenase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Oliveira, Tânia; Sharkey, Michael A.; Engel, Paul C.

    2016-05-23

    Glutamate dehydrogenases (EC 1.4.1.2–4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate using NAD(P) +as a cofactor. The bacterial enzymes are hexameric, arranged with 32 symmetry, and each polypeptide consists of an N-terminal substrate-binding segment (domain I) followed by a C-terminal cofactor-binding segment (domain II). The catalytic reaction takes place in the cleft formed at the junction of the two domains. Distinct signature sequences in the nucleotide-binding domain have been linked to the binding of NAD +versusNADP +, but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies,more » as shown by the apo structure of glutamate dehydrogenase fromClostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia colienzyme has been determined in the apo state. The enzyme is fully functional and reveals possible determinants of interdomain flexibility at a hinge region following the pivot helix. The enzyme retains the preference for NADP +cofactor from the parentE. colidomain II, although there are subtle differences in catalytic activity.« less

  18. DNA-Templated Polymerization of Side-Chain-Functionalized Peptide Nucleic Acid Aldehydes

    PubMed Central

    Kleiner, Ralph E.; Brudno, Yevgeny; Birnbaum, Michael E.; Liu, David R.

    2009-01-01

    The DNA-templated polymerization of synthetic building blocks provides a potential route to the laboratory evolution of sequence-defined polymers with structures and properties not necessarily limited to those of natural biopolymers. We previously reported the efficient and sequence-specific DNA-templated polymerization of peptide nucleic acid (PNA) aldehydes. Here, we report the enzyme-free, DNA-templated polymerization of side-chain-functionalized PNA tetramer and pentamer aldehydes. We observed that the polymerization of tetramer and pentamer PNA building blocks with a single lysine-based side chain at various positions in the building block could proceed efficiently and sequence-specifically. In addition, DNA-templated polymerization also proceeded efficiently and in a sequence-specific manner with pentamer PNA aldehydes containing two or three lysine side chains in a single building block to generate more densely functionalized polymers. To further our understanding of side-chain compatibility and expand the capabilities of this system, we also examined the polymerization efficiencies of 20 pentamer building blocks each containing one of five different side-chain groups and four different side-chain regio- and stereochemistries. Polymerization reactions were efficient for all five different side-chain groups and for three of the four combinations of side-chain regio- and stereochemistries. Differences in the efficiency and initial rate of polymerization correlate with the apparent melting temperature of each building block, which is dependent on side-chain regio- and stereochemistry, but relatively insensitive to side-chain structure among the substrates tested. Our findings represent a significant step towards the evolution of sequence-defined synthetic polymers and also demonstrate that enzyme-free nucleic acid-templated polymerization can occur efficiently using substrates with a wide range of side-chain structures, functionalization positions within each

  19. Mitochondrial Aldehyde Dehydrogenase 2 Regulates Revascularization in Chronic Ischemia: Potential Impact on the Development of Coronary Collateral Circulation.

    PubMed

    Liu, Xiangwei; Sun, Xiaolei; Liao, Hua; Dong, Zhen; Zhao, Jingjing; Zhu, Hong; Wang, Peng; Shen, Li; Xu, Lei; Ma, Xin; Shen, Cheng; Fan, Fan; Wang, Cong; Hu, Kai; Zou, Yunzeng; Ge, Junbo; Ren, Jun; Sun, Aijun

    2015-10-01

    Revascularization is an essential process to compensate for cardiac underperfusion and, therefore, preserves cardiac function in the face of chronic ischemic injury. Recent evidence suggested a vital role of aldehyde dehydrogenase 2 (ALDH2) in cardiac protection after ischemia. This study was designed to determine whether ALDH2 regulates chronic ischemia-induced angiogenesis and to explore the underlying mechanism involved. Moreover, the clinical impact of the ALDH2 mutant allele on the development of coronary collateral circulation (CCC) was evaluated. Mice limb ischemia was performed. Compared with wild-type, ALDH2 deletion significantly reduced perfusion recovery, small artery and capillary density, and increased muscle atrophy in this ischemic model. In vitro, ALDH2-knockdown reduced proliferation, migration and hypoxia triggered endothelial tube formation of endothelial cells, the effects of which were restored by ALDH2 transfection. Further examination revealed that ALDH2 regulated angiogenesis possibly through hypoxia-inducible factor-1α/vascular endothelial growth factor pathways. To further discern the role of ALDH2 deficiency in the function of bone marrow stem/progenitor cells, cross bone marrow transplantation was performed between wild-type and ALDH2-knockout mice. However, there was no significant improvement for wild-type bone marrow transplantation into knockout mice. ALDH2 genotyping was screened in 139 patients with chronic total occlusion recruited to Zhongshan Hospital (2011.10-2014.4). Patients with poor CCC (Rentrop 0-1; n=51) exhibited a higher frequency of the AA genotype than those with enriched CCC (Rentrop 2-3; n=88; 11.76% versus 1.14%; P=0 0.01). However, the AA group displayed less enriched CCC frequency in Logistic regression model when compared with the GG group (odds ratio=0.08; 95% confidence interval, 0.009-0.701; P=0 0.026). Furthermore, serum vascular endothelial growth factor level tended to be lower in patients with ALDH2

  20. Estrogen Modification of Human Glutamate Dehydrogenases Is Linked to Enzyme Activation State*

    PubMed Central

    Borompokas, Nikolas; Papachatzaki, Maria-Martha; Kanavouras, Konstantinos; Mastorodemos, Vasileios; Zaganas, Ioannis; Spanaki, Cleanthe; Plaitakis, Andreas

    2010-01-01

    Mammalian glutamate dehydrogenase (GDH) is a housekeeping enzyme central to the metabolism of glutamate. Its activity is potently inhibited by GTP (IC50 = 0.1–0.3 μm) and thought to be controlled by the need of the cell in ATP. Estrogens are also known to inhibit mammalian GDH, but at relatively high concentrations. Because, in addition to this housekeeping human (h) GDH1, humans have acquired via a duplication event an hGDH2 isoform expressed in human cortical astrocytes, we tested here the interaction of estrogens with the two human isoenzymes. The results showed that, under base-line conditions, diethylstilbestrol potently inhibited hGDH2 (IC50 = 0.08 ± 0.01 μm) and with ∼18-fold lower affinity hGDH1 (IC50 = 1.67 ± 0.06 μm; p < 0.001). Similarly, 17β-estradiol showed a ∼18-fold higher affinity for hGDH2 (IC50 = 1.53 ± 0.24 μm) than for hGDH1 (IC50 = 26.94 ± 1.07 μm; p < 0.001). Also, estriol and progesterone were more potent inhibitors of hGDH2 than hGDH1. Structure/function analyses revealed that the evolutionary R443S substitution, which confers low basal activity, was largely responsible for sensitivity of hGDH2 to estrogens. Inhibition of both human GDHs by estrogens was inversely related to their state of activation induced by ADP, with the slope of this correlation being steeper for hGDH2 than for hGDH1. Also, the study of hGDH1 and hGDH2 mutants displaying different states of activation revealed that the affinity of estrogen for these enzymes correlated inversely (R = 0.99; p = 0.0001) with basal catalytic activity. Because astrocytes are known to synthesize estrogens, these hormones, by interacting potently with hGDH2 in its closed state, may contribute to regulation of glutamate metabolism in brain. PMID:20628048

  1. The X-Ray Crystal Structure of Escherichia coli Succinic Semialdehyde Dehydrogenase; Structural Insights into NADP+/Enzyme Interactions

    PubMed Central

    Langendorf, Christopher G.; Key, Trevor L. G.; Fenalti, Gustavo; Kan, Wan-Ting; Buckle, Ashley M.; Caradoc-Davies, Tom; Tuck, Kellie L.; Law, Ruby H. P.; Whisstock, James C.

    2010-01-01

    Background In mammals succinic semialdehyde dehydrogenase (SSADH) plays an essential role in the metabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) to succinic acid (SA). Deficiency of SSADH in humans results in elevated levels of GABA and γ-Hydroxybutyric acid (GHB), which leads to psychomotor retardation, muscular hypotonia, non-progressive ataxia and seizures. In Escherichia coli, two genetically distinct forms of SSADHs had been described that are essential for preventing accumulation of toxic levels of succinic semialdehyde (SSA) in cells. Methodology/Principal Findings Here we structurally characterise SSADH encoded by the E coli gabD gene by X-ray crystallographic studies and compare these data with the structure of human SSADH. In the E. coli SSADH structure, electron density for the complete NADP+ cofactor in the binding sites is clearly evident; these data in particular revealing how the nicotinamide ring of the cofactor is positioned in each active site. Conclusions/Significance Our structural data suggest that a deletion of three amino acids in E. coli SSADH permits this enzyme to use NADP+, whereas in contrast the human enzyme utilises NAD+. Furthermore, the structure of E. coli SSADH gives additional insight into human mutations that result in disease. PMID:20174634

  2. Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase

    PubMed Central

    Lloyd, Julie C.; Raines, Christine A.

    2011-01-01

    In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein–protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the ‘non-regulatory’ A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed. PMID:21498632

  3. Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase.

    PubMed

    Howard, Thomas P; Lloyd, Julie C; Raines, Christine A

    2011-07-01

    In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein-protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the 'non-regulatory' A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed.

  4. Changes in cinnamyl alcohol dehydrogenase activities from sugarcane cultivars inoculated with Sporisorium scitamineum sporidia.

    PubMed

    Santiago, Rocío; Alarcón, Borja; de Armas, Roberto; Vicente, Carlos; Legaz, María Estrella

    2012-06-01

    This study describes a method for determining cinnamyl alcohol dehydrogenase activity in sugarcane stems using reverse phase (RP) high-performance liquid chromatography to elucidate their possible lignin origin. Activity is assayed using the reverse mode, the oxidation of hydroxycinnamyl alcohols into hydroxycinnamyl aldehydes. Appearance of the reaction products, coniferaldehyde and sinapaldehyde is determined by measuring absorbance at 340 and 345 nm, respectively. Disappearance of substrates, coniferyl alcohol and sinapyl alcohol is measured at 263 and 273 nm, respectively. Isocratic elution with acetonitrile:acetic acid through an RP Mediterranea sea C18 column is performed. As case examples, we have examined two different cultivars of sugarcane; My 5514 is resistant to smut, whereas B 42231 is susceptible to the pathogen. Inoculation of sugarcane stems elicits lignification and produces significant increases of coniferyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD). Production of lignin increases about 29% in the resistant cultivar and only 13% in the susceptible cultivar after inoculation compared to uninoculated plants. Our results show that the resistance of My 5514 to smut is likely derived, at least in part, to a marked increase of lignin concentration by the activation of CAD and SAD. Copyright © Physiologia Plantarum 2012.

  5. Characterization of human short chain dehydrogenase/reductase SDR16C family members related to retinol dehydrogenase 10.

    PubMed

    Adams, Mark K; Lee, Seung-Ah; Belyaeva, Olga V; Wu, Lizhi; Kedishvili, Natalia Y

    2017-10-01

    All-trans-retinoic acid (RA) is a bioactive derivative of vitamin A that serves as an activating ligand for nuclear transcription factors, retinoic acid receptors. RA biosynthesis is initiated by the enzymes that oxidize retinol to retinaldehyde. It is well established that retinol dehydrogenase 10 (RDH10, SDR16C4), which belongs to the 16C family of the short chain dehydrogenase/reductase (SDR) superfamily of proteins, is the major enzyme responsible for the oxidation of retinol to retinaldehyde for RA biosynthesis during embryogenesis. However, several lines of evidence point towards the existence of additional retinol dehydrogenases that contribute to RA biosynthesis in vivo. In close proximity to RDH10 gene on human chromosome 8 are located two genes that are phylogenetically related to RDH10. The predicted protein products of these genes, retinol dehydrogenase epidermal 2 (RDHE2, SDR16C5) and retinol dehydrogenase epidermal 2-similar (RDHE2S, SDR16C6), share 59% and 56% sequence similarity with RDH10, respectively. Previously, we showed that the single ortholog of the human RDHE2 and RDHE2S in frogs, Xenopus laevis rdhe2, oxidizes retinol to retinaldehyde and is essential for frog embryonic development. In this study, we explored the potential of each of the two human proteins to contribute to RA biosynthesis. The results of this study demonstrate that human RDHE2 exhibits a relatively low but reproducible activity when expressed in either HepG2 or HEK293 cells. Expression of the native RDHE2 is downregulated in the presence of elevated levels of RA. On the other hand, the protein encoded by the human RDHE2S gene is unstable when expressed in HEK293 cells. RDHE2S protein produced in Sf9 cells is stable but has no detectable catalytic activity towards retinol. We conclude that the human RDHE2S does not contribute to RA biosynthesis, whereas the low-activity RA-sensitive human RDHE2 may have a role in adjusting the cellular levels of RA in accord with

  6. Direct catalytic asymmetric alpha-amination of aldehydes.

    PubMed

    List, Benjamin

    2002-05-22

    The first direct catalytic asymmetric alpha-amination of aldehydes is described herein. alpha-Unbranched aldehydes react in this novel proline-catalyzed reaction with dialkyl azodicarboxylates to give alpha-amino aldehydes in excellent yields and enantioselectivities.

  7. The Pivotal Role of Aldehyde Toxicity in Autism Spectrum Disorder: The Therapeutic Potential of Micronutrient Supplementation

    PubMed Central

    Jurnak, Frances

    2015-01-01

    Autism spectrum disorder (ASD) is characterized by social and communication impairments as well as by restricted, repetitive patterns of behavior and interests. Genomic studies have not revealed dominant genetic errors common to all forms of ASD. So ASD is assumed to be a complex disorder due to mutations in hundreds of common variants. Other theories argue that spontaneous DNA mutations and/or environmental factors contribute to as much as 50% of ASD. In reviewing potential genetic linkages between autism and alcoholism, it became apparent that all theories of ASD are consistent with aldehyde toxicity, in which endogenous and exogenous aldehydes accumulate as a consequence of mutations in key enzymes. Aldehyde toxicity is characterized by cell-localized, micronutrient deficiencies in sulfur-containing antioxidants, thiamine (B1), pyridoxine (B6), folate, Zn2+, possibly Mg2+, and retinoic acid, causing oxidative stress and a cascade of metabolic disturbances. Aldehydes also react with selective cytosolic and membrane proteins in the cell of origin; then some types migrate to damage neighboring cells. Reactive aldehydes also form adducts with DNA, selectively mutating bases and inducing strand breakage. This article reviews the relevant genomic, biochemical, and nutritional literature, which supports the central hypothesis that most ASD symptoms are consistent with symptoms of aldehyde toxicity. The hypothesis represents a paradigm shift in thinking and has profound implications for clinical detection, treatment, and even prevention of ASD. Insight is offered as to which neurologically afflicted children might successfully be treated with micronutrients and which children are unlikely to be helped. The aldehyde toxicity hypothesis likely applies to other neurological disorders. PMID:27330305

  8. Acetylene hydratase: a non-redox enzyme with tungsten and iron-sulfur centers at the active site.

    PubMed

    Kroneck, Peter M H

    2016-03-01

    In living systems, tungsten is exclusively found in microbial enzymes coordinated by the pyranopterin cofactor, with additional metal coordination provided by oxygen and/or sulfur, and/or selenium atoms in diverse arrangements. Prominent examples are formate dehydrogenase, formylmethanofuran dehydrogenase, and aldehyde oxidoreductase all of which catalyze redox reactions. The bacterial enzyme acetylene hydratase (AH) stands out of its class as it catalyzes the conversion of acetylene to acetaldehyde, clearly a non-redox reaction and a reaction distinct from the reduction of acetylene to ethylene by nitrogenase. AH harbors two pyranopterins bound to W, and a [4Fe-4S] cluster. W is coordinated by four dithiolene sulfur atoms, one cysteine sulfur, and one oxygen ligand. AH activity requires a strong reductant suggesting W(IV) as the active oxidation state. Two different types of reaction pathways have been proposed. The 1.26 Å structure reveals a water molecule coordinated to W which could gain a partially positive net charge by the adjacent protonated Asp-13, enabling a direct attack of C2H2. To access the W-Asp site, a substrate channel was evolved distant from where it is found in other members of the DMSOR family. Computational studies of this second shell mechanism led to unrealistically high energy barriers, and alternative pathways were proposed where C2H2 binds directly to W. The architecture of the catalytic cavity, the specificity for C2H2 and the results from site-directed mutagenesis do not support this first shell mechanism. More investigations including structural information on the binding of C2H2 are needed to present a conclusive answer.

  9. The Role and Regulation of the 11 Beta-Hydroxysteroid Dehydrogenase Enzyme System in Patients with Inflammatory Bowel Disease.

    PubMed

    Hussey, M; Holleran, G; Smith, S; Sherlock, Mark; McNamara, D

    2017-12-01

    Glucocorticoids are known to modulate a number of immunological responses including counteracting inflammation. Within tissues expressing the glucocorticoid and mineralocorticoid receptors including the colon, glucocorticoid metabolism is regulated by the isoenzymes of 11ß-hydroxysteroid dehydrogenase (11β-HSD). 11β-HSD1 acts as an oxidoreductase converting inactive cortisone into active cortisol, while 11β-HSD2 acts as a dehydrogenase converting active cortisol to inactive cortisone. Hexose-6 phosphate dehydrogenase (H6PDH) is a key regulator of 11β-HSD1 activity via its generation of NADPH. Variations in the 11β-HSD enzyme system in relation to levels of expression and regulation may have a role in IBD. The aim of this study was to investigate possible abnormalities of 11β-HSD enzyme system in the colon of patients with IBD. By using quantitative real-time PCR, we investigated the transcription levels of 11β-HSD1 and 2 in colonic tissue from IBD patients and healthy controls undergoing a colonoscopy for disease assessment. Disease activity was recorded using clinical (Mayo Score/Harvey-Bradshaw Index), Biochemical (C-reactive protein), histological, and endoscopic parameters. In addition, transcription levels of H6PDH and the glucocorticoid receptor alpha (GR-α) as well as key pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, Rela (subunit for NF Kappa B)) were later examined among this group, and results were correlated with 11β-HSD2 gene expression. Results and patient demographics were expressed as a mean (and SD), and differences between IBD patients and control groups were analyzed using a Student's t test or Mann-Whitney U test as appropriate, with a p value of ≤0.05 considered significant. Results were controlled for disease activity as outlined above. Results have demonstrated a significant downregulation in 11β-HSD2 expression in IBD patients compared with controls (13.8 ± 17.1 au vs. 318.4 ± 521.1 au, p = 0.01), whereas levels of

  10. Myoglobin-Catalyzed Olefination of Aldehydes.

    PubMed

    Tyagi, Vikas; Fasan, Rudi

    2016-02-12

    The olefination of aldehydes constitutes a most valuable and widely adopted strategy for constructing carbon-carbon double bonds in organic chemistry. While various synthetic methods have been made available for this purpose, no biocatalysts are known to mediate this transformation. Reported herein is that engineered myoglobin variants can catalyze the olefination of aldehydes in the presence of α-diazoesters with high catalytic efficiency (up to 4,900 turnovers) and excellent E diastereoselectivity (92-99.9 % de). This transformation could be applied to the olefination of a variety of substituted benzaldehydes and heteroaromatic aldehydes, also in combination with different alkyl α-diazoacetate reagents. This work provides a first example of biocatalytic aldehyde olefination and extends the spectrum of synthetically valuable chemical transformations accessible using metalloprotein-based catalysts. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Differential accumulation of proteins in oil palms affected by fatal yellowing disease

    PubMed Central

    do Nascimento, Sidney Vasconcelos; Magalhães, Marcelo Murad; Cunha, Roberto Lisboa; Costa, Paulo Henrique de Oliveira; Alves, Ronnie Cley de Oliveira; de Oliveira, Guilherme Corrêa

    2018-01-01

    There is still no consensus on the true origin of fatal yellowing, one of the most important diseases affecting oil palm (Elaeis guineensis Jacq.) plantations. This study involved two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D-UPLC-MSE) analyses to identify changes in protein profiles of oil palms affected by FY disease. Oil palm roots were sampled from two growing areas. Differential accumulation of proteins was assessed by comparing plants with and without symptoms and between plants at different stages of FY development. Most of the proteins identified with differential accumulation were those related to stress response and energy metabolism. The latter proteins include the enzymes alcohol dehydrogenase and aldehyde dehydrogenase, related to alcohol fermentation, which were identified in plants with and without symptoms. The presence of these enzymes suggests an anaerobic condition before or during FY. Transketolase, isoflavone reductase, cinnamyl alcohol dehydrogenase, caffeic acid 3-O-methyltransferase, S-adenosylmethionine synthase, aldehyde dehydrogenase and ferritin, among others, were identified as potential marker proteins and could be used to guide selection of FY-tolerant oil palm genotypes or to understand the source of this anomaly. When comparing different stages of FY, we observed high accumulation of alcohol dehydrogenase and other abiotic stress related-proteins at all disease stages. On the other hand, biological stress-related proteins were more accumulated at later stages of the disease. These results suggest that changes in abiotic factors can trigger FY development, creating conditions for the establishment of opportunistic pathogens. PMID:29621343

  12. Deodorants: an experimental provocation study with cinnamic aldehyde.

    PubMed

    Bruze, Magnus; Johansen, J D; Andersen, K E; Frosch, P; Lepoittevin, J-P; Rastogi, S; Wakelin, S; White, I; Menné, T

    2003-02-01

    Axillary dermatitis is common and overrepresented in individuals with contact allergy to fragrances. Many individuals suspect their deodorants to be the incriminating products. Our aim was to investigate the significance of cinnamic aldehyde in deodorants for the development of axillary dermatitis when used by individuals with and without contact allergy to cinnamic aldehyde. Patch tests with deodorants and ethanol solutions with cinnamic aldehyde, and repeated open application tests with roll-on deodorants without and with cinnamic aldehyde at different concentrations, were performed in 37 patients with dermatitis, 20 without and 17 with contact allergy to cinnamic aldehyde. A repeated open application test with positive findings was noted only in patients hypersensitive to cinnamic aldehyde (P <.001) and only in the axilla to which the deodorants containing cinnamic aldehyde had been applied (P <.001). Deodorants containing cinnamic aldehyde in the concentration range 0.01% to 0.32%, used twice daily on healthy skin, can elicit axillary dermatitis within a few weeks.

  13. In vivo Post-Cardiac Arrest Myocardial Dysfunction is Supported by CaMKII-Mediated Calcium Long-Term Potentiation and Mitigated by Alda-1, an Agonist of Aldehyde Dehydrogenase Type 2

    PubMed Central

    Downey, Peter; Zalewski, Adrian; Rubio, Gabriel R.; Liu, Jing; Homburger, Julian R.; Grunwald, Zachary; Qi, Wei; Bollensdorff, Christian; Thanaporn, Porama; Ali, Ayyaz; Riemer, Kirk; Kohl, Peter; Mochly-Rosen, Daria; Gerstenfeld, Edward; Large, Stephen; Ali, Ziad; Ashley, Euan

    2016-01-01

    Background Survival after sudden cardiac arrest is limited by post-arrest myocardial dysfunction but understanding of this phenomenon is constrained by lack of data from a physiological model of disease. In this study, we established an in vivo model of cardiac arrest and resuscitation, characterized the biology of the associated myocardial dysfunction, and tested novel therapeutic strategies. Methods We developed rodent models of in vivo post-arrest myocardial dysfunction using extra-corporeal membrane oxygenation (ECMO) resuscitation followed by invasive hemodynamics measurement. In post-arrest isolated cardiomyocytes, we assessed mechanical load and Ca2+ induced Ca2+ release (CICR) simultaneously using the micro-carbon-fiber technique and observed reduced function and myofilament calcium sensitivity. We used a novel-designed fiber optic catheter imaging system, and a genetically encoded calcium sensor GCaMP6f, to image CICR in vivo. Results We found potentiation of CICR in isolated cells from this ECMO model and also in cells isolated from an ischemia-reperfusion Langendorff model perfused with oxygenated blood from an arrested animal, but not when reperfused in saline. We established that CICR potentiation begins in vivo. The augmented CICR observed post-arrest was mediated by the activation of Ca2+/calmodulin kinase II (CaMKII). Increased phosphorylation of CaMKII, phospholamban and ryanodine receptor 2 (RyR2) was detected in the post-arrest period. Exogenous adrenergic activation in vivo recapitulated Ca2+ potentiation but was associated with lesser CaMKII activation. Since oxidative stress and aldehydic adduct formation were high post arrest, we tested a small molecule activator of aldehyde dehydrogenase type 2, Alda-1, which reduced oxidative stress, restored calcium and CaMKII homeostasis, and improved cardiac function and post-arrest outcome in vivo. Conclusions Cardiac arrest and reperfusion lead to CaMKII activation and calcium long-term potentiation

  14. Dissimilar Deficiency of Glucose-6-Phosphate Dehydrogenase (G-6-PD) among the AFARS and the Somalis of Djibouti

    DTIC Science & Technology

    1991-01-01

    DEFICIENCY OF GLUCOSE - 6 - PHOSPHATE DEHYDROGENASE (G- 6 ...the prevalence of deficient activity of the enzyme glucose - 6 - phosphate dehydrogenase (G- 6 -PD) among - Ces difficiences enzymatiques sant plus particu...Screening for glucose - 6 - 3 - CaosBy W.H. - Hematologic diseases. In : I lunter’s Tropical phosphate dehydrogenase (G- 6 -PD) deficiency by a simple

  15. Antisense RNA Downregulation of Coenzyme A Transferase Combined with Alcohol-Aldehyde Dehydrogenase Overexpression Leads to Predominantly Alcohologenic Clostridium acetobutylicum Fermentations

    PubMed Central

    Tummala, Seshu B.; Junne, Stefan G.; Papoutsakis, Eleftherios T.

    2003-01-01

    Plasmid pAADB1 for the overexpression of the alcohol-aldehyde dehydrogenase (aad) gene and downregulation of the coenzyme A transferase (CoAT) using antisense RNA (asRNA) against ctfB (the second CoAT gene on the polycistronic aad-ctfA-ctfB message) was used in order to increase the butanol/acetone ratio of Clostridium acetobutylicum ATCC 824 fermentations. Acetone and butanol levels were drastically reduced in 824(pCTFB1AS) (expresses only an asRNA against ctfB) compared to 824(pSOS95del) (plasmid control). Compared to strain 824(pCTFB1AS), 824(pAADB1) fermentations exhibited two profound differences. First, butanol levels were ca. 2.8-fold higher in 824(pAADB1) and restored back to plasmid control levels, thus supporting the hypothesis that asRNA downregulation of ctfB leads to degradation of the whole aad-ctfA-ctfB transcript. Second, ethanol titers in 824(pAADB1) were ca. 23-fold higher and the highest (ca. 200 mM) ever reported in C. acetobutylicum. Western blot analysis confirmed that CoAT was downregulated in 824(pAADB1) at nearly the same levels as in strain 824(pCTFB1AS). Butyrate depletion in 824(pAADB1) fermentations suggested that butyryl-CoA was limiting butanol production in 824(pAADB1). This was confirmed by exogenously adding butyric acid to 824(pAADB1) fermentations to increase the butanol/ethanol ratio. DNA microarray analysis showed that aad overexpression profoundly affects the large-scale transcriptional program of the cells. Several classes of genes were differentially expressed [strain 824(pAADB1) versus strain 824(pCTFB1AS)], including genes of the stress response, sporulation, and chemotaxis. The expression patterns of the CoAT genes (ctfA and ctfB) and aad were consistent with the overexpression of aad and asRNA downregulation of ctfB. PMID:12775702

  16. Antisense RNA downregulation of coenzyme A transferase combined with alcohol-aldehyde dehydrogenase overexpression leads to predominantly alcohologenic Clostridium acetobutylicum fermentations.

    PubMed

    Tummala, Seshu B; Junne, Stefan G; Papoutsakis, Eleftherios T

    2003-06-01

    Plasmid pAADB1 for the overexpression of the alcohol-aldehyde dehydrogenase (aad) gene and downregulation of the coenzyme A transferase (CoAT) using antisense RNA (asRNA) against ctfB (the second CoAT gene on the polycistronic aad-ctfA-ctfB message) was used in order to increase the butanol/acetone ratio of Clostridium acetobutylicum ATCC 824 fermentations. Acetone and butanol levels were drastically reduced in 824(pCTFB1AS) (expresses only an asRNA against ctfB) compared to 824(pSOS95del) (plasmid control). Compared to strain 824(pCTFB1AS), 824(pAADB1) fermentations exhibited two profound differences. First, butanol levels were ca. 2.8-fold higher in 824(pAADB1) and restored back to plasmid control levels, thus supporting the hypothesis that asRNA downregulation of ctfB leads to degradation of the whole aad-ctfA-ctfB transcript. Second, ethanol titers in 824(pAADB1) were ca. 23-fold higher and the highest (ca. 200 mM) ever reported in C. acetobutylicum. Western blot analysis confirmed that CoAT was downregulated in 824(pAADB1) at nearly the same levels as in strain 824(pCTFB1AS). Butyrate depletion in 824(pAADB1) fermentations suggested that butyryl-CoA was limiting butanol production in 824(pAADB1). This was confirmed by exogenously adding butyric acid to 824(pAADB1) fermentations to increase the butanol/ethanol ratio. DNA microarray analysis showed that aad overexpression profoundly affects the large-scale transcriptional program of the cells. Several classes of genes were differentially expressed [strain 824(pAADB1) versus strain 824(pCTFB1AS)], including genes of the stress response, sporulation, and chemotaxis. The expression patterns of the CoAT genes (ctfA and ctfB) and aad were consistent with the overexpression of aad and asRNA downregulation of ctfB.

  17. Effects of cannabis and tobacco on the enzymes of alcohol metabolism in the rat.

    PubMed

    Marselos, M; Vasiliou, V; Malamas, M; Alikaridis, F; Kefalas, T

    1991-01-01

    The effects of cannabis and tobacco on the enzymes of ethanol metabolism were studied in the Wistar rat. The activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AlDH) were measured in the liver and the brain, after treatment with an extract of cannabis resin, with an extract of tobacco leaves, or with nicotine. A condensate of cannabis resin extract was collected in a smoking machine, using a tobacco cigarette as the vehicle. Unsmoked or smoked cannabis extracts were dissolved in olive oil and were given i.p. (twice daily, for 7 days). In both cases, a similar dose level was used in terms of starting material (raw cannabis resin), estimated at about 100 mg/kg body weight. Control animals were treated either with olive oil, or with the same amount of smoked condensate obtained from a reference cigarette. Nicotine was dissolved in olive oil and it was given i.p. (10 micrograms/kg, twice daily for 7 days). An extract of unsmoked tobacco was dissolved in olive oil and was given with the same schedule, at a dose which was estimated to correspond to about 10 micrograms nicotine/kg b.w. All groups of animals received an additional i.p. injection on day 8, one hour before sacrifice. Our results showed that unsmoked cannabis inhibited the hepatic activities of the microchondrial AlDH (low-Km and high-Km), the hepatic low-Km cytosolic AlDH (p less than 0.001), and the low-Km mitochondrial AlDH of the brain (p less than 0.001). Administration of smoked cannabis to the animals inhibited the hepatic mitochondrial low-Km AlDH (p less than 0.001), but it did not influence the brain enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Subcellular distribution of delta 5-3 beta-hydroxy steroid dehydrogenase in the granulosa cells of the domestic fowl (Gallus domesticus).

    PubMed Central

    Armstrong, D G

    1979-01-01

    1. The distribution of 3 beta-hydroxy steroid dehydrogenase was examined in the subcellular fractions of granulosa cells collected from the ovary of the domestic fowl. 2. 3 beta-hydroxy steroid dehydrogenase activity was observed in the mitochondrial (4000g for 20min) and microsomal (105 000g for 120min) fractions. 3. Approximately three times more 3 beta-hydroxy steroid dehydrogenase activity was associated with the cytochrome oxidase activity (a mitochondrial marker enzyme) in anteovulatory-follicle granulosa cells than with that of the postovulatory follicle. 4. Comparison of the latent properties of mitochondrial 3 beta-hydroxy steroid dehydrogenase with those of cytochrome oxidase and isocitrate dehydrogenase indicated that 3 beta-hydroxy steroid dehydrogenase is located extramitochondrially. 5. This apparent distribution of 3 beta-hydroxy steroid dehydrogenase is explained on the basis that the mitochondrial activity is either an artefact caused by a redistribution in the subcellular location of the enzyme, occurring during homogenization, or by the existence of a functionally heterogeneous endoplasmic reticulum that yields particles of widely differing sedimentation properties. PMID:518548

  19. 13-cis-retinoic acid competitively inhibits 3 alpha-hydroxysteroid oxidation by retinol dehydrogenase RoDH-4: a mechanism for its anti-androgenic effects in sebaceous glands?

    PubMed

    Karlsson, Teresa; Vahlquist, Anders; Kedishvili, Natalia; Törmä, Hans

    2003-03-28

    Retinol dehydrogenase-4 (RoDH-4) converts retinol and 13-cis-retinol to corresponding aldehydes in human liver and skin in the presence of NAD(+). RoDH-4 also converts 3 alpha-androstanediol and androsterone into dihydrotestosterone and androstanedione, which may stimulate sebum secretion. This oxidative 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) activity of RoDH-4 is competitively inhibited by retinol and 13-cis-retinol. Here, we further examine the substrate specificity of RoDH-4 and the inhibition of its 3 alpha-HSD activity by retinoids. Recombinant RoDH-4 oxidized 3,4-didehydroretinol-a major form of vitamin A in the skin-to its corresponding aldehyde. 13-cis-retinoic acid (isotretinoin), 3,4-didehydroretinoic acid, and 3,4-didehydroretinol, but not all-trans-retinoic acid or the synthetic retinoids acitretin and adapalene, were potent competitive inhibitors of the oxidative 3 alpha-HSD activity of RoDH-4, i.e., reduced the formation of dihydrotestosterone and androstandione in vitro. Extrapolated to the in vivo situation, this effect might explain the unique sebosuppressive effect of isotretinoin when treating acne.

  20. [Characterization of D-lactate dehydrogenase isozymes from a D-lactic acid producing bacterium Sporolactobacillus inulinus].

    PubMed

    Zhang, Danru; Zheng, Lu; Wu, Bin; He, Bingfang

    2016-11-04

    Sporolactobacillus inulinus, a typical homofermentative lactic acid bacterium, is an efficient D-lactic acid producer. Various environment factors affect the productivity of S. inulinus. Glucokinase, phosphofructokinase, pyruvate kinase and lactic dehydrogenase are the key enzymes of D-lactic acid production from glucose by S. inulinus. The characteristics of these enzymes are important in controlling and regulating the fermentation process. According to the genome bioinformatics analysis of S. inulinus CASD, three putative D-lactate dehydrogenases were identified, among which the bifunctional protein had been reported. In this study, we provided insights into the characteristics of the other two D-lactate dehydrogenase isozymes. S. inulinus Y2-8 genome was used as the template to amplify D-lactate dehydrogenase gene (dldh) and D-isomer specific 2-hydroxyacid dehydrogenase gene (dhdh). The two recombinant strains E-pET-28a/dldh and E-pET-28a/dhdh were constructed for enzyme expression. Both recombinants DLDH and DHDH could convert pyruvic acid into D-lactic acid. Enzymes expressed by recombinant strains were purified by Ni-NTA chromatography. The apparent molecular mass of DLDH was approximately 37 kDa by SDS-PAGE analysis, and DLDH showed a high affinity to pyruvate with the Km value of (0.58±0.04) mmol/L. The optimal reaction temperature and pH for DLDH was 35℃ and 6.5, respectively. The apparent molecular mass of DHDH was approximately 39 kDa, and the Km of DHDH toward pyruvate was (1.70±0.08) mmol/L. The optimum catalysis temperature and pH of DHDH were 30℃ and 7.5, respectively. According to the Km and optimal reaction pH, DLDH was suggested as the main catalyst in formation D-lactic acid from pyruvate during the fermentation. The enzymatic properties would contribute to the regulation of the fermentation of S. inulinus.

  1. Cofactor specificity switch in Shikimate dehydrogenase by rational design and consensus engineering.

    PubMed

    García-Guevara, Fernando; Bravo, Iris; Martínez-Anaya, Claudia; Segovia, Lorenzo

    2017-08-01

    Consensus engineering has been used to design more stable variants using the most frequent amino acid at each site of a multiple sequence alignment; sometimes consensus engineering modifies function, but efforts have mainly been focused on studying stability. Here we constructed a consensus Rossmann domain for the Shikimate dehydrogenase enzyme; separately we decided to switch the cofactor specificity through rational design in the Escherichia coli Shikimate dehydrogenase enzyme and then analyzed the effect of consensus mutations on top of our design. We found that consensus mutations closest to the 2' adenine moiety increased the activity in our design. Consensus engineering has been shown to result in more stable proteins and our findings suggest it could also be used as a complementary tool for increasing or modifying enzyme activity during design. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  2. Light-regulation of enzyme activity in anacystis nidulans (Richt.).

    PubMed

    Duggan, J X; Anderson, L E

    1975-01-01

    The effect of light on the levels of activity of six enzymes which are light-modulated in higher plants was examined in the photosynthetic procaryot Anacystis nidulans. Ribulose-5-phosphate kinase (EC 2.7.1.19) was found to be light-activated in vivo and dithiothreitol-activated in vitro while glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was light-inactivated and dithiothreitol-inactivated. The enzymes fructose-1,6-diphosphate phosphatase (EC 3.1.3.11), sedoheptulose-1,7-diphosphate phosphatase, NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; EC 1.2.1.13) were not affected by light treatment of the intact algae, but sedoheptulose-diphosphate phosphatase and the glyceraldehyde-3-phosphate dehydrogenases were dithiothreitol-activated in crude extracts. Light apparently controls the activity of the reductive and oxidative pentose phosphate pathway in this photosynthetic procaryot as in higher plants, through a process which probably involves reductive modulation of enzyme activity.

  3. Functional assignment of gene AAC16202.1 from Rhodobacter capsulatus SB1003: new insights into the bacterial SDR sorbitol dehydrogenases family.

    PubMed

    Sola-Carvajal, Agustín; García-García, María Inmaculada; Sánchez-Carrón, Guiomar; García-Carmona, Francisco; Sánchez-Ferrer, Alvaro

    2012-11-01

    Short-chain dehydrogenases/reductases (SDR) constitute one of the largest enzyme superfamilies with over 60,000 non-redundant sequences in the database, many of which need a correct functional assignment. Among them, the gene AAC16202.1 (NCBI) from Rhodobacter capsulatus SB1003 has been assigned in Uniprot both as a sorbitol dehydrogenase (#D5AUY1) and, as an N-acetyl-d-mannosamine dehydrogenase (#O66112), both enzymes being of biotechnological interest. When the gene was overexpressed in Escherichia coli Rosetta (DE3)pLys, the purified enzyme was not active toward N-acetyl-d-mannosamine, whereas it was active toward d-sorbitol and d-fructose. However, the relative activities toward xylitol and l-iditol (0.45 and 6.9%, respectively) were low compared with that toward d-sorbitol. Thus, the enzyme could be considered sorbitol dehydrogenase (SDH) with very low activity toward xylitol, which could increase its biotechnological interest for determining sorbitol without the unspecific cross-determination of added xylitol in food and pharma compositions. The tetrameric enzyme (120 kDa) showed similar catalytic efficiency (2.2 × 10(3) M(-1) s(-1)) to other sorbitol dehydrogenases for d-sorbitol, with an optimum pH of 9.0 and an optimum temperature of 37 °C. The enzyme was also more thermostable than other reported SDH, ammonium sulfate being the best stabilizer in this respect, increasing the melting temperature (T(m)) up to 52.9 °C. The enzyme can also be considered as a new member of the Zn(2+) independent SDH family since no effect on activity was detected in the presence of divalent cations or chelating agents. Finally, its in silico analysis enabled the specific conserved sequence blocks that are the fingerprints of bacterial sorbitol dehydrogenases and mainly located at C-terminal of the protein, to be determined for the first time. This knowledge will facilitate future data curation of present databases and a better functional assignment of newly described

  4. Novel Xylose Dehydrogenase in the Halophilic Archaeon Haloarcula marismortui†

    PubMed Central

    Johnsen, Ulrike; Schönheit, Peter

    2004-01-01

    During growth of the halophilic archaeon Haloarcula marismortui on d-xylose, a specific d-xylose dehydrogenase was induced. The enzyme was purified to homogeneity. It constitutes a homotetramer of about 175 kDa and catalyzed the oxidation of xylose with both NADP+ and NAD+ as cosubstrates with 10-fold higher affinity for NADP+. In addition to d-xylose, d-ribose was oxidized at similar kinetic constants, whereas d-glucose was used with about 70-fold lower catalytic efficiency (kcat/Km). With the N-terminal amino acid sequence of the subunit, an open reading frame (ORF)—coding for a 39.9-kDA protein—was identified in the partially sequenced genome of H. marismortui. The function of the ORF as the gene designated xdh and coding for xylose dehydrogenase was proven by its functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione, and substrates. Xylose dehydrogenase showed the highest sequence similarity to glucose-fructose oxidoreductase from Zymomonas mobilis and other putative bacterial and archaeal oxidoreductases. Activities of xylose isomerase and xylulose kinase, the initial reactions of xylose catabolism of most bacteria, could not be detected in xylose-grown cells of H. marismortui, and the genes that encode them, xylA and xylB, were not found in the genome of H. marismortui. Thus, we propose that this first characterized archaeal xylose dehydrogenase catalyzes the initial step in xylose degradation by H. marismortui. PMID:15342590

  5. Structural and biochemical insights into 7β-hydroxysteroid dehydrogenase stereoselectivity.

    PubMed

    Savino, Simone; Ferrandi, Erica Elisa; Forneris, Federico; Rovida, Stefano; Riva, Sergio; Monti, Daniela; Mattevi, Andrea

    2016-06-01

    Hydroxysteroid dehydrogenases are of great interest as biocatalysts for transformations involving steroid substrates. They feature a high degree of stereo- and regio-selectivity, acting on a defined atom with a specific configuration of the steroid nucleus. The crystal structure of 7β-hydroxysteroid dehydrogenase from Collinsella aerofaciens reveals a loop gating active-site accessibility, the bases of the specificity for NADP(+) , and the general architecture of the steroid binding site. Comparison with 7α-hydroxysteroid dehydrogenase provides a rationale for the opposite stereoselectivity. The presence of a C-terminal extension reshapes the substrate site of the β-selective enzyme, possibly leading to an inverted orientation of the bound substrate. Proteins 2016; 84:859-865. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  6. 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

  7. Glucose-6-phosphate dehydrogenase deficiency: disadvantages and possible benefits.

    PubMed

    Manganelli, Genesia; Masullo, Ugo; Passarelli, Stefania; Filosa, Stefania

    2013-03-01

    We review here some recent data about Glucose-6-phosphate dehydrogenase (G6PD), the housekeeping X-linked gene encoding the first enzyme of the pentose phosphate pathway (PPP), a NADPH-producing dehydrogenase. This enzyme has been popular among clinicians, biochemists, geneticists and molecular biologists because it is the most common form of red blood cell enzymopathy. G6PD deficient erythrocytes do not generate NADPH in any other way than through the PPP and for this reason they are more susceptible than any other cells to oxidative damage. Moreover, this enzyme has also been of crucial importance in many significant discoveries; indeed, G6PD polymorphisms have been instrumental in studying X-inactivation in the human species, as well as in establishing the clonal nature of certain tumors. G6PD deficiency, generally considered as a mild and benign condition, is significantly disadvantageous in certain environmental conditions like in presence of certain drugs. Nevertheless, G6PD deficiency has been positively selected by malaria, and recent knowledge seems to show that it also confers an advantage against the development of cancer, reduces the risk of coronary diseases and has a beneficial effect in terms of longevity.

  8. Subcellular distribution of 3 beta-hydroxysteroid dehydrogenase-isomerase in bovine and murine adrenocortical tissue: species differences in the localization of activity and immunoreactivity.

    PubMed

    Perry, J E; Ishii-Ohba, H; Stalvey, J R

    1991-06-01

    Key to the production of biologically active steroids is the enzyme 3 beta-hydroxysteroid dehydrogenase-isomerase. Some controversy has arisen concerning the subcellular distribution of this enzyme within steroidogenic cells. The distribution of 3 beta-hydroxysteroid dehydrogenase-isomerase was assessed in subcellular fractions obtained from homogenates of rat, bovine, and mouse adrenal glands in two ways. The activity of 3 beta-hydroxysteroid dehydrogenase-isomerase was quantitated by measuring the conversion of radiolabeled pregnenolone to radiolabeled progesterone in an aliquot of each of the fractions obtained. The presence of the enzyme was assessed by performing Western analyses on aliquots of each of the fractions obtained with the use of a specific polyclonal antiserum against 3 beta-hydroxysteroid dehydrogenase-isomerase, the characterization of which is described. In control experiments, the degree of contamination of the fractions was determined by assessing the presence of known subcellular fraction markers with Western analysis. In the bovine and mouse adrenal glands, 3 beta-hydroxysteroid dehydrogenase-isomerase appears to be localized solely in the microsomal fraction, while in the rat, 3 beta-hydroxysteroid dehydrogenase-isomerase appears to have dual subcellular distribution: the microsomes and the inner mitochondrial membrane. We conclude that there is a species difference in the subcellular distribution of this important steroidogenic enzyme and that this species difference may be related to the steroidogenic pathway preferred in that species.

  9. 40 CFR 721.639 - Amine aldehyde condensate.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Amine aldehyde condensate. 721.639... Substances § 721.639 Amine aldehyde condensate. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as an amine aldehyde condensate (PMN P-94-1810...

  10. 40 CFR 721.639 - Amine aldehyde condensate.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Amine aldehyde condensate. 721.639... Substances § 721.639 Amine aldehyde condensate. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as an amine aldehyde condensate (PMN P-94-1810...

  11. 40 CFR 721.639 - Amine aldehyde condensate.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Amine aldehyde condensate. 721.639... Substances § 721.639 Amine aldehyde condensate. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as an amine aldehyde condensate (PMN P-94-1810...

  12. 40 CFR 721.639 - Amine aldehyde condensate.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Amine aldehyde condensate. 721.639... Substances § 721.639 Amine aldehyde condensate. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as an amine aldehyde condensate (PMN P-94-1810...

  13. 40 CFR 721.639 - Amine aldehyde condensate.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Amine aldehyde condensate. 721.639... Substances § 721.639 Amine aldehyde condensate. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as an amine aldehyde condensate (PMN P-94-1810...

  14. Discovery of piperonal-converting oxidase involved in the metabolism of a botanical aromatic aldehyde

    PubMed Central

    Doi, Shiori; Hashimoto, Yoshiteru; Tomita, Chiaki; Kumano, Takuto; Kobayashi, Michihiko

    2016-01-01

    Piperonal-catabolizing microorganisms were isolated from soil, the one (strain CT39-3) exhibiting the highest activity being identified as Burkholderia sp. The piperonal-converting enzyme involved in the initial step of piperonal metabolism was purified from strain CT39-3. Gene cloning of the enzyme and a homology search revealed that the enzyme belongs to the xanthine oxidase family, which comprises molybdoenzymes containing a molybdopterin cytosine dinucleotide cofactor. We found that the piperonal-converting enzyme acts on piperonal in the presence of O2, leading to formation of piperonylic acid and H2O2. The growth of strain CT39-3 was inhibited by higher concentrations of piperonal in the culture medium. Together with this finding, the broad substrate specificity of this enzyme for various aldehydes suggests that it would play an important role in the defense mechanism against antimicrobial compounds derived from plant species. PMID:27905507

  15. Threonine-Insensitive Homoserine Dehydrogenase From Soybean: Genomic Organization, Kinetic Mechanism, and In vivo Activity

    USDA-ARS?s Scientific Manuscript database

    Aspartate kinase (AK) and homoserine dehydrogenase (HSD) functions as key regulatory enzymes at branch points in the aspartate amino acid pathway and are feedback inhibited by threonine. In plants, the biochemical properties of AK and bifunctional AK-HSD enzymes have been characterized, but the mol...

  16. Comparative Studies of Enzymes Related to Serine Metabolism in Higher Plants 1

    PubMed Central

    Cheung, Geoffrey P.; Rosenblum, I. Y.; Sallach, H. J.

    1968-01-01

    The following enzymes related to serine metabolism in higher plants have been investigated: 1) d-3-phosphoglycerate dehydrogenase, 2) phosphohydroxypyruvate:l-glutamate transaminase, 3) d-glycerate dehydrogenase, and 4) hydroxypyruvate:l-alanine transaminase. Comparative studies on the distribution of the 2 dehydrogenases in seeds and leaves from various plants revealed that d-3-phosphoglycerate dehydrogenase is widely distributed in seeds in contrast to d-glycerate dehydrogenase, which is either absent or present at low levels, and that the reverse pattern is observed in green leaves. The levels of activity of the 4 enzymes listed above were followed in different tissues of the developing pea (Pisum sativum, var. Alaska). In the leaf, from the tenth to seventeenth day of germination, the specific activity of d-glycerate dehydrogenase increased markedly and was much higher than d-3-phosphoglycerate dehydrogenase which remained relatively constant during this time period. Etiolation resulted in a decrease in d-glycerate dehydrogenase and an increase in d-3-phosphoglycerate dehydrogenase activities. In apical meristem, on the other hand, the level of d-3-phosphoglycerate dehydrogenase exceeded that of d-glycerate dehydrogenase at all time periods studied. Low and decreasing levels of both dehydrogenases were found in epicotyl and cotyledon. The specific activities of the 2 transaminases remained relatively constant during development in both leaf and apical meristem. In general, however, the levels of phosphohydroxypyruvate:l-glutamate transaminase were comparable to those of d-3-phosphoglycerate dehydrogenase in a given tissue as were those for hydroxypyruvate: l-alanine transaminase and d-glycerate dehydrogenase. PMID:5699148

  17. Effects of folic acid deficiency in pregnant Wistar rats on the activities of D5-3 beta hydroxysteroid dehydrogenase and glucose-6 phosphate dehydrogenase in the ovaries of their litters.

    PubMed

    Uche-Nwachi, E O; Caxton-Martins, A E

    1997-06-01

    Histochemical studies of the activities of glucose-6-phosphate dehydrogenase (G-6-PD) and D5-3 beta-hydroxysteroid dehydrogenase (D5-3 beta-HSD) in the ovaries of 40 day old litters of Wistar rats whose mothers were folic acid deficient from the 13th day of gestation showed very weak or no enzyme activity. Biochemical estimations of these enzymes showed that the specific activity of 3 beta-HSD in the experimental animal was 20% that of control while that of G-6-PD in the experimental animals was 14% that of control. This implies that folic acid deficiency instituted at a critical period in gestation in Wistar rats adversely affects steroidogenesis in the ovaries of their litters.

  18. Acute and chronic effects of clofibrate and clofibric acid on the enzymes acetylcholinesterase, lactate dehydrogenase and catalase of the mosquitofish, Gambusia holbrooki.

    PubMed

    Nunes, B; Carvalho, F; Guilhermino, L

    2004-12-01

    The objective of this study was to investigate both acute and chronic effects of clofibrate and clofibric acid on the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH) and catalase (CAT) of the mosquitofish (Gambusia holbrooki). AChE, commonly used as a biomarker of neurotoxicity, was determined in the total head. LDH, an important enzyme of anaerobic metabolism, was quantified in dorsal muscle, and CAT, enzyme which has been used as indicative parameter of peroxisome proliferation, was determined in the liver. Furthermore, alterations of body and liver weight were also determined, through the calculation of the ratios final body weight/initial body weight, liver weight/final body weight, liver weight/gills weight and liver weight/head weight. Acute exposure of G. holbrooki to both clofibrate and clofibric acid induced a decrease in liver CAT activity, an increase in muscle LDH activity, while no effects were observed on AChE activity. However, chronic exposure did not alter significantly the enzymatic activities, suggesting reduced or null effects over these pathways, relative to effects reported in other species. No effects were observed for the calculated ratios, except a significant weight reduction for males chronically exposed to clofibrate.

  19. Effects of microgravity and tail suspension on enzymes of individual soleus and tibialis anterior fibers

    NASA Technical Reports Server (NTRS)

    Chi, Maggie M.-Y.; Choski, Rati; Nemeth, Patti; Krasnov, Igor'; Il'ina-Kakueva, E. I.; Manchester, Jill K.; Lowry, Oliver H.

    1992-01-01

    Selected enzymes of energy metabolism were measured in random individual fibers of soleus and tibialis anterior (TA) muscles from rats exposed for 2 wk to spaceflight (F) aboard Cosmos 2044 or tail suspension (T) and from synchronous controls. Average size of soleus fibers (dry weight per unit length) was reduced 37 percent in F and T fibers; there was little change in Ta fibers. Enzyme changes were more pronounced in soleus than in TA fibers. Three enzymes characteristic of fast-twitch muscles, pyruvate kinase, glycerol-3-phosphate dehydrogenase, and 1-phosphofructokinase, were elevated in F and T soleus fibers, but changes in phosphofructokinase were not statistically significant. In TA fibers analyzed for hexokinase, malate dehydrogenase, phosphohexoisomerase, and pyruvate kinase, only hexokinase and malate dehydrogenase showed significant changes. Hexokinase incresed 83 percent in one of two T muscles. Enzyme data for TA fibers typed by myosin adenosinetriphosphatase were more informative: phosphofructokinase, phosphorylase, and glycerol-3-phosphate dehydrogenase were increased in type IIn fibers of either F or T muscles or both. Malate dehydrogenase was not changed in fibers of any type in either F or T muscle.

  20. Glucose-6-Phosphate Dehydrogenase Deficiency.

    PubMed

    Luzzatto, Lucio; Nannelli, Caterina; Notaro, Rosario

    2016-04-01

    G6PD is a housekeeping gene expressed in all cells. Glucose-6-phosphate dehydrogenase (G6PD) is part of the pentose phosphate pathway, and its main physiologic role is to provide NADPH. G6PD deficiency, one of the commonest inherited enzyme abnormalities in humans, arises through one of many possible mutations, most of which reduce the stability of the enzyme and its level as red cells age. G6PD-deficient persons are mostly asymptomatic, but they can develop severe jaundice during the neonatal period and acute hemolytic anemia when they ingest fava beans or when they are exposed to certain infections or drugs. G6PD deficiency is a global health issue. Copyright © 2016 Elsevier Inc. All rights reserved.