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Sample records for 17b-hydroxysteroid dehydrogenase type

  1. Structural insight into the type-II mitochondrial NADH dehydrogenases.

    PubMed

    Feng, Yue; Li, Wenfei; Li, Jian; Wang, Jiawei; Ge, Jingpeng; Xu, Duo; Liu, Yanjing; Wu, Kaiqi; Zeng, Qingyin; Wu, Jia-Wei; Tian, Changlin; Zhou, Bing; Yang, Maojun

    2012-11-15

    The single-component type-II NADH dehydrogenases (NDH-2s) serve as alternatives to the multisubunit respiratory complex I (type-I NADH dehydrogenase (NDH-1), also called NADH:ubiquinone oxidoreductase; EC 1.6.5.3) in catalysing electron transfer from NADH to ubiquinone in the mitochondrial respiratory chain. The yeast NDH-2 (Ndi1) oxidizes NADH on the matrix side and reduces ubiquinone to maintain mitochondrial NADH/NAD(+) homeostasis. Ndi1 is a potential therapeutic agent for human diseases caused by complex I defects, particularly Parkinson's disease, because its expression restores the mitochondrial activity in animals with complex I deficiency. NDH-2s in pathogenic microorganisms are viable targets for new antibiotics. Here we solve the crystal structures of Ndi1 in its substrate-free, NADH-, ubiquinone- and NADH-ubiquinone-bound states, to help understand the catalytic mechanism of NDH-2s. We find that Ndi1 homodimerization through its carboxy-terminal domain is critical for its catalytic activity and membrane targeting. The structures reveal two ubiquinone-binding sites (UQ(I) and UQ(II)) in Ndi1. NADH and UQ(I) can bind to Ndi1 simultaneously to form a substrate-protein complex. We propose that UQ(I) interacts with FAD to act as an intermediate for electron transfer, and that NADH transfers electrons through this FAD-UQ(I) complex to UQ(II). Together our data reveal the regulatory and catalytic mechanisms of Ndi1 and may facilitate the development or targeting of NDH-2s for potential therapeutic applications.

  2. 11Beta-hydroxysteroid dehydrogenase-type 2 evolved from an ancestral 17beta-hydroxysteroid dehydrogenase-type 2.

    PubMed

    Baker, Michael E

    2010-08-20

    11Beta-hydroxysteroid dehydrogenase-type 2 (11beta-HSD2) regulates the local concentration of cortisol that can activate the glucocorticoid receptor and mineralocorticoid receptor, as well as the concentration of 11-keto-testosterone, the active androgen in fish. Similarly, 17beta-HSD2 regulates the levels of testosterone and estradiol that activate the androgen receptor and estrogen receptor, respectively. Interestingly, although human 11beta-HSD2 and 17beta-HSD2 act at different positions on different steroids, these enzymes are paralogs. Despite the physiological importance of 11beta-HSD2 and 17beta-HSD2, details of their origins and divergence from a common ancestor are not known. An opportunity to understand their evolution is presented by the recent sequencing of genomes from sea urchin, a basal deuterostome, and amphioxus, a basal chordate, and the availability of substantial sequence for acorn worm and elephant shark, which together provide a more complete dataset for analysis of the origins of 11beta-HSD2 and 17beta-HSD2. BLAST searches find an ancestral sequence of 17beta-HSD2 in sea urchin, acorn worm and amphioxus, while an ancestral sequence of 11beta-HSD2 first appears in sharks. Sequence analyses indicate that 17beta-HSD2 in sea urchin may have a non-enzymatic activity. Evolutionary analyses indicate that if acorn worm 17beta-HSD2 is catalytically active, then it metabolizes novel substrate(s). PMID:20654577

  3. Crystal structures of 11β-hydroxysteroid dehydrogenase type 1 and their use in drug discovery

    PubMed Central

    Thomas, Mark P; Potter, Barry VL

    2014-01-01

    Cortisol is synthesized by 11β-hydroxysteroid dehydrogenase type 1, inhibitors of which may treat disease associated with excessive cortisol levels. The crystal structures of 11β-hydroxysteroid dehydrogenase type 1 that have been released may aid drug discovery. The crystal structures have been analyzed in terms of the interactions between the protein and the ligands. Despite a variety of structurally different inhibitors the crystal structures of the proteins are quite similar. However, the differences are significant for drug discovery. The crystal structures can be of use in drug discovery, but care needs to be taken when selecting structures for use in virtual screening and ligand docking. PMID:21446847

  4. Alcohol and polyol dehydrogenases are both divided into two protein types, and structural properties cross-relate the different enzyme activities within each type.

    PubMed Central

    Jörnvall, H; Persson, M; Jeffery, J

    1981-01-01

    Sorbitol dehydrogenase from sheep liver shows similarities to mammalian and yeast alcohol dehydrogenases. Comparisons based on peptides from segments of sorbitol dehydrogenase reveal that homologous regions with 38% identity include two ligands to the active site zinc atom in liver alcohol dehydrogenase, as well as further important residues. Similarities in in other regions are less extensive, exactly as they are between different alcohol dehydrogenases. In all aspects, sorbitol dehydrogenase appears as a typical member of the alcohol dehydrogenase family. On the other hand, alcohol dehydrogenase from Drosophila, which has a shorter subunit, is not closely related to either of these enzymes, except for a region that probably corresponds to the first part of the coenzyme binding domain in many dehydrogenases. Instead, Drosophila alcohol dehydrogenase in its supposed catalytic region shows similarities toward Klebsiella ribitol dehydrogenase, which also has a small subunit. It may be concluded that both alcohol and polyol dehydrogenases show two types of protein subunit, reflecting an early subdivision of polypeptide types into "long" and "short" subunits rather than into different enzymatic specificities or quaternary structures. The relationships explain known properties of all these enzymes and provide insight into functional mechanisms and evolutionary interpretations. PMID:7027257

  5. Discovery of 2-Alkyl-1-arylsulfonylprolinamides as 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitors

    PubMed Central

    2012-01-01

    On the basis of scaffold hopping, a novel series of 2-alkyl-1-arylsulfonylprolinamides was discovered as 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1) inhibitors. A representative compound 4ek, obtained through SAR and structure optimization studies, demonstrates excellent in vitro potency against 11β-HSD-1 and dose-dependent in vivo inhibition of 11β-HSD-1 in a prednisone/prednisolone transformation biomarker study in mice. PMID:24900382

  6. XoxF Acts as the Predominant Methanol Dehydrogenase in the Type I Methanotroph Methylomicrobium buryatense

    PubMed Central

    Chu, Frances

    2016-01-01

    ABSTRACT Many methylotrophic taxa harbor two distinct methanol dehydrogenase (MDH) systems for oxidizing methanol to formaldehyde: the well-studied calcium-dependent MxaFI type and the more recently discovered lanthanide-containing XoxF type. MxaFI has traditionally been accepted as the major functional MDH in bacteria that contain both enzymes. However, in this study, we present evidence that, in a type I methanotroph, Methylomicrobium buryatense, XoxF is likely the primary functional MDH in the environment. The addition of lanthanides increases xoxF expression and greatly reduces mxa expression, even under conditions in which calcium concentrations are almost 100-fold higher than lanthanide concentrations. Mutations in genes encoding the MDH enzymes validate our finding that XoxF is the major functional MDH, as XoxF mutants grow more poorly than MxaFI mutants under unfavorable culturing conditions. In addition, mutant and transcriptional analyses demonstrate that the lanthanide-dependent MDH switch operating in methanotrophs is mediated in part by the orphan response regulator MxaB, whose gene transcription is itself lanthanide responsive. IMPORTANCE Aerobic methanotrophs, bacteria that oxidize methane for carbon and energy, require a methanol dehydrogenase enzyme to convert methanol into formaldehyde. The calcium-dependent enzyme MxaFI has been thought to primarily carry out methanol oxidation in methanotrophs. Recently, it was discovered that XoxF, a lanthanide-containing enzyme present in most methanotrophs, can also oxidize methanol. In a methanotroph with both MxaFI and XoxF, we demonstrate that lanthanides transcriptionally control genes encoding the two methanol dehydrogenases, in part by controlling expression of the response regulator MxaB. Lanthanides are abundant in the Earth's crust, and we demonstrate that micromolar amounts of lanthanides are sufficient to suppress MxaFI expression. Thus, we present evidence that XoxF acts as the predominant

  7. Chromosomal localization and structure of the human type II IMP dehydrogenase gene

    SciTech Connect

    Glesne, D.; Huberman, E. |; Collart, F.; Varkony, T.; Drabkin, H.

    1994-05-01

    We determined the chromosomal localization and structure of the gene encoding human type II inosine 5{prime}-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205), an enzyme associated with cellular proliferation, malignant transformation, and differentiation. Using polymerase chain reaction (PCR) primers specific for type II IMPDH, we screened a panel of human-Chinese hamster cell somatic hybrids and a separate deletion panel of chromosome 3 hybrids and localized the gene to 3p21.1{yields}p24.2. Two overlapping yeast artificial chromosome clones containing the full gene for type II IMPDH were isolated and a physical map of 117 kb of human genomic DNA in this region of chromosome 3 was constructed. The gene for type II IMPDH was localized and oriented on this map and found to span no more than 12.5 kb.

  8. Mutations in the genes encoding 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase interact to cause cortisone reductase deficiency.

    PubMed

    Draper, Nicole; Walker, Elizabeth A; Bujalska, Iwona J; Tomlinson, Jeremy W; Chalder, Susan M; Arlt, Wiebke; Lavery, Gareth G; Bedendo, Oliver; Ray, David W; Laing, Ian; Malunowicz, Ewa; White, Perrin C; Hewison, Martin; Mason, Philip J; Connell, John M; Shackleton, Cedric H L; Stewart, Paul M

    2003-08-01

    In cortisone reductase deficiency (CRD), activation of cortisone to cortisol does not occur, resulting in adrenocorticotropin-mediated androgen excess and a phenotype resembling polycystic ovary syndrome (PCOS; refs. 1,2). This suggests a defect in the gene HSD11B1 encoding 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), a primary regulator of tissue-specific glucocorticoid bioavailability. We identified intronic mutations in HSD11B1 that resulted in reduced gene transcription in three individuals with CRD. In vivo, 11beta-HSD1 catalyzes the reduction of cortisone to cortisol whereas purified enzyme acts as a dehydrogenase converting cortisol to cortisone. Oxo-reductase activity can be regained using a NADPH-regeneration system and the cytosolic enzyme glucose-6-phosphate dehydrogenase. But the catalytic domain of 11beta-HSD1 faces into the lumen of the endoplasmic reticulum (ER; ref. 6). We hypothesized that endolumenal hexose-6-phosphate dehydrogenase (H6PDH) regenerates NADPH in the ER, thereby influencing directionality of 11beta-HSD1 activity. Mutations in exon 5 of H6PD in individuals with CRD attenuated or abolished H6PDH activity. These individuals have mutations in both HSD11B1 and H6PD in a triallelic digenic model of inheritance, resulting in low 11beta-HSD1 expression and ER NADPH generation with loss of 11beta-HSD1 oxo-reductase activity. CRD defines a new ER-specific redox potential and establishes H6PDH as a potential factor in the pathogenesis of PCOS. PMID:12858176

  9. Molecular analysis of mutant and wild type alcohol dehydrogenase alleles from Drosophila

    SciTech Connect

    Batzer, M.A.

    1988-01-01

    Wild type alcohol dehydrogenase polypeptides (ADH) from Drosophila melanogaster transformants were examined using western blots and polyclonal antiserum specific for Drosophila melanogaster ADH. Mutants induced in Drosophila spermatozoa at the alcohol dehydrogenase (Adh) locus using X-rays, 1-ethyl-1-nitrosourea (ENU) or ethyl methanesulfonate (EMS) were characterized using genetic complementation tests, western blots, Southern blots, northern blots and enzymatic amplification of the Adh locus. Genetic complementation tests showed that 22/30 X-ray-induced mutants, and 3/13 ENU and EMS induced mutants were multi-locus deficiencies. Western blot analysis of the intragenic mutations showed that 4/7 X-ray-induced mutants produced detectable polypeptides, one of which was normal in molecular weight and charge. In contrast 8/10 intragenic ENU and EMS induced mutants produced normal polypeptides. Southern blot analysis showed that 5/7 intragenic X-ray induced mutants and all 10 of the intragenic ENU and EMS induced mutants were normal with respect to the alleles they were derived from.

  10. Glucose-6-phosphate dehydrogenase

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003671.htm Glucose-6-phosphate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) is a type of ...

  11. The dual targeting ability of type II NAD(P)H dehydrogenases arose early in land plant evolution

    PubMed Central

    2013-01-01

    Background Type II NAD(PH) dehydrogenases are located on the inner mitochondrial membrane of plants, fungi, protists and some primitive animals. However, recent observations have been made which identify several Arabidopsis type II dehydrogenases as dual targeted proteins. Targeting either mitochondria and peroxisomes or mitochondria and chloroplasts. Results Members of the ND protein family were identified in various plant species. Phylogenetic analyses and subcellular targeting predictions were carried out for all proteins. All ND proteins from three model plant species Arabidopsis, rice and Physcomitrella were cloned as N- and C-terminal GFP fusions and subcellular localisations were determined. Dual targeting of plant type II dehydrogenases was observed to have evolved early in plant evolution and to be widespread throughout different plant species. In all three species tested dual targeting to both mitochondria and peroxisomes was found for at least one NDA and NDB type protein. In addition two NDB type proteins from Physcomitrella were also found to target chloroplasts. The dual targeting of NDC type proteins was found to have evolved later in plant evolution. Conclusions The functions of type II dehydrogenases within plant cells will have to be re-evaluated in light of this newly identified subcellular targeting information. PMID:23841539

  12. The structure of retinal dehydrogenase type II at 2.7 A resolution: implications for retinal specificity.

    PubMed

    Lamb, A L; Newcomer, M E

    1999-05-11

    Retinoic acid, a hormonally active form of vitamin A, is produced in vivo in a two step process: retinol is oxidized to retinal and retinal is oxidized to retinoic acid. Retinal dehydrogenase type II (RalDH2) catalyzes this last step in the production of retinoic acid in the early embryo, possibly producing this putative morphogen to initiate pattern formation. The enzyme is also found in the adult animal, where it is expressed in the testis, lung, and brain among other tissues. The crystal structure of retinal dehydrogenase type II cocrystallized with nicotinamide adenine dinucleotide (NAD) has been determined at 2.7 A resolution. The structure was solved by molecular replacement using the crystal structure of a mitochondrial aldehyde dehydrogenase (ALDH2) as a model. Unlike what has been described for the structures of two aldehyde dehydrogenases involved in the metabolism of acetaldehyde, the substrate access channel is not a preformed cavity into which acetaldehyde can readily diffuse. Retinal dehydrogenase appears to utilize a disordered loop in the substrate access channel to discriminate between retinaldehyde and short-chain aldehydes.

  13. 11beta-Hydroxysteroid dehydrogenase type 1 inhibitors with oleanan and ursan scaffolds.

    PubMed

    Blum, Andreas; Favia, Angelo D; Maser, Edmund

    2009-03-25

    The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts cortisone to the active glucocorticoid cortisol, thereby acting as a cellular switch to mediate glucocorticoid action in many tissues. Several studies have indicated that 11beta-HSD1 plays a crucial role in the onset of type 2 diabetes and central obesity. As a consequence, selective inhibition of 11beta-HSD1 in humans might become a new and promising approach for lowering blood glucose concentrations and for counteracting the accumulation of visceral fat and its related metabolic abnormalities in type 2 diabetes. In this study, we present the synthesis and the biological evaluation of ursan or oleanan type triterpenoids which may act as selective 11beta-HSD1 inhibitors in liver as well as in peripheral tissues, like adipocytes and muscle cells. In order to rationalise the outcomes of the inhibition data, docking simulations of the ligands were performed on the experimentally determined structure of 11beta-HSD1. Furthermore, we discuss the structural determinants that confer enzymatic specificity. From our investigation, valuable information has been obtained to design selective 11beta-HSD1 blockers based on the oleanan and ursan scaffold.

  14. Abundant type 10 17 beta-hydroxysteroid dehydrogenase in the hippocampus of mouse Alzheimer's disease model.

    PubMed

    He, Xue Ying; Wen, Guang Yeong; Merz, George; Lin, Dawei; Yang, Ying Zi; Mehta, Penkaj; Schulz, Horst; Yang, Song Yu

    2002-02-28

    A full-length cDNA of mouse type 10 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD10) was cloned from brain, representing the accurate nucleotide sequence information that rendered possible an accurate deduction of the amino acid sequence of the wild-type enzyme. A comparison of sequences and three-dimensional models of this enzyme revealed that structures previously reported by other groups carry either a truncated or mutated amino-terminal sequence. Fusion of the first 11 residues of the wild-type enzyme to the green fluorescent protein directed the reporter protein into mitochondria. Thus, the N-terminus was identified as a mitochondrial targeting signal that accounts for the intracellular localization of the mouse enzyme. This enzyme is normally associated with mitochondria, not with the endoplasmic reticulum as suggested by its trivial name 'endoplasmic reticulum-associated amyloid-beta biding protein (ERAB)'. After its C-terminal region was used to raise rabbit anti-17 betaHSD10 antibodies, immunogold electron microscopy showed that an abundance of this enzyme could be found in hippocampal synaptic mitochondria of betaAPP transgenic mice, but not in normal controls. High levels of this enzyme may disrupt steroid hormone homeostasis in synapses and contribute to synapse loss in the hippocampus of the mouse Alzheimer's disease model. PMID:11869808

  15. Zebrafish 20β-Hydroxysteroid Dehydrogenase Type 2 Is Important for Glucocorticoid Catabolism in Stress Response

    PubMed Central

    Tokarz, Janina; Norton, William; Möller, Gabriele; Hrabé de Angelis, Martin; Adamski, Jerzy

    2013-01-01

    Stress, the physiological reaction to a stressor, is initiated in teleost fish by hormone cascades along the hypothalamus-pituitary-interrenal (HPI) axis. Cortisol is the major stress hormone and contributes to the appropriate stress response by regulating gene expression after binding to the glucocorticoid receptor. Cortisol is inactivated when 11β-hydroxysteroid dehydrogenase (HSD) type 2 catalyzes its oxidation to cortisone. In zebrafish, Danio rerio, cortisone can be further reduced to 20β-hydroxycortisone. This reaction is catalyzed by 20β-HSD type 2, recently discovered by us. Here, we substantiate the hypothesis that 20β-HSD type 2 is involved in cortisol catabolism and stress response. We found that hsd11b2 and hsd20b2 transcripts were up-regulated upon cortisol treatment. Moreover, a cortisol-independent, short-term physical stressor led to the up-regulation of hsd11b2 and hsd20b2 along with several HPI axis genes. The morpholino-induced knock down of hsd20b2 in zebrafish embryos revealed no developmental phenotype under normal culture conditions, but prominent effects were observed after a cortisol challenge. Reporter gene experiments demonstrated that 20β-hydroxycortisone was not a physiological ligand for the zebrafish glucocorticoid or mineralocorticoid receptor but was excreted into the fish holding water. Our experiments show that 20β-HSD type 2, together with 11β-HSD type 2, represents a short pathway in zebrafish to rapidly inactivate and excrete cortisol. Therefore, 20β-HSD type 2 is an important enzyme in stress response. PMID:23349977

  16. Cloning, characterization, and regulation of the human type II IMP dehydrogenase gene

    SciTech Connect

    Glesne, D.A.; Huberman, E. |

    1997-01-01

    Human type II inosine 5{prime}-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) is the rate-limiting enzyme in de novo guanine nucleotide biosynthesis. Regulated IMPDH activity is associated with cellular proliferation, transformation, and differentiation. The authors cloned and sequenced the entire gene for type II IMPDH and here provide details regarding the organization of the gene and the characterization of its promoter. The gene spans approximately 5 kb and is disrupted by 12 introns. The transcriptional start sites were determined by S1 nuclease mapping to be somewhat heterogeneous but predominated at 102 and 85 nucleotides from the translational initiation codon. Through the use of heterologous gene constructs and transient transfection assays, a minimal promoter from {minus}206 to {minus}85 was defined. This promoter is TATA-less and contains several transcription factor motifs including four potential Sp 1 binding sites. The minimal promoter is GC-rich (69%) and resembles a CpG island. Through the use of gel mobility shift assays, nuclear proteins were shown to specifically interact with this minimal promoter. Stable transfectants were used to demonstrate that the down-regulation of IMPDH gene expression in response to reduced cellular proliferation occurs by a transcriptional mechanism.

  17. 11beta-hydroxysteroid dehydrogenase type 1 inhibitors for metabolic syndrome.

    PubMed

    Schnackenberg, Christine G

    2008-03-01

    The metabolic syndrome is a constellation of interrelated metabolic risk factors that appear to promote the development of diabetes and cardiovascular disease. These risk factors include abdominal obesity, insulin resistance, hypertension and dyslipidemia. 11beta-Hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the interconversion of glucocorticoids through the activity of two isozymes: type 1 (11beta-HSD1) and type 2 (11beta-HSD2). 11beta-HSD1 converts inactive glucocorticoid to the active form, whereas 11beta-HSD2 converts active glucocorticoid to the inactive form. It is well established that reduced 11beta-HSD2 activity causes hypertension and electrolyte abnormalities. More recently, the pathophysiological role of 11beta-HSD1 has been explored and studies suggest that increased 11beta-HSD1 activity within target tissues may promote insulin resistance, obesity, hypertension and dyslipidemia. This review will discuss the evidence that inhibition of 11beta-HSD1 may be therapeutic in the treatment of the metabolic syndrome.

  18. XoxF-Type Methanol Dehydrogenase from the Anaerobic Methanotroph “Candidatus Methylomirabilis oxyfera”

    PubMed Central

    Wu, Ming L.; Wessels, Hans J. C. T.; Pol, Arjan; Op den Camp, Huub J. M.; Jetten, Mike S. M.; van Niftrik, Laura

    2014-01-01

    “Candidatus Methylomirabilis oxyfera” is a newly discovered anaerobic methanotroph that, surprisingly, oxidizes methane through an aerobic methane oxidation pathway. The second step in this aerobic pathway is the oxidation of methanol. In Gram-negative bacteria, the reaction is catalyzed by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH). The genome of “Ca. Methylomirabilis oxyfera” putatively encodes three different MDHs that are localized in one large gene cluster: one so-called MxaFI-type MDH and two XoxF-type MDHs (XoxF1 and XoxF2). MxaFI MDHs represent the canonical enzymes, which are composed of two PQQ-containing large (α) subunits (MxaF) and two small (β) subunits (MxaI). XoxF MDHs are novel, ecologically widespread, but poorly investigated types of MDHs that can be phylogenetically divided into at least five different clades. The XoxF MDHs described thus far are homodimeric proteins containing a large subunit only. Here, we purified a heterotetrameric MDH from “Ca. Methylomirabilis oxyfera” that consisted of two XoxF and two MxaI subunits. The enzyme was localized in the periplasm of “Ca. Methylomirabilis oxyfera” cells and catalyzed methanol oxidation with appreciable specific activity and affinity (Vmax of 10 μmol min−1 mg−1 protein, Km of 17 μM). PQQ was present as the prosthetic group, which has to be taken up from the environment since the known gene inventory required for the synthesis of this cofactor is lacking. The MDH from “Ca. Methylomirabilis oxyfera” is the first representative of type 1 XoxF proteins to be described. PMID:25527536

  19. Alternate pathways for NADH oxidation in Thermus thermophilus using type 2 NADH dehydrogenases.

    PubMed

    Venkatakrishnan, Padmaja; Lencina, Andrea M; Schurig-Briccio, Lici A; Gennis, Robert B

    2013-05-01

    Type 2 NADH dehydrogenase (NDH-2) is a single-subunit membrane-associated flavoenzyme that is part of the respiratory chain of many prokaryotes. The enzyme catalyzes the electron transfer from NADH to quinone but is not directly coupled to the generation of a proton motive force. The purpose of the current work is to compare two different NDH-2s that are encoded in strains of Thermus thermophilus. The aerobic T. thermophilus HB27 strain expresses one NDH-2 that has been previously isolated and characterized. In this work it is shown that a gene, which is misannotated as an NADH oxidase, encodes this enzyme. Unlike HB27, strain NAR1 of T. thermophilus is capable of partial denitrification, and in addition its genome contains the nrcN gene that encodes a second putative NDH-2. Of particular interest is the fact that nrcN is part of an operon (nrcDEFN) that is proposed to encode a protein complex specifically required for nitrate reduction. In this work, the nrcN gene has the activity expected of a NDH-2, and functions independently of other components of the putative Nrc complex. The biochemical properties of the two NDH-2 enzymes are compared. Efforts to demonstrate that NrcN is part of a multiprotein complex were not successful. However, the NrcE protein was expressed in Escherichia coli and shown to be a membrane-bound protein containing heme B. PMID:23370906

  20. Electronically type-sorted carbon nanotube-based electrochemical biosensors with glucose oxidase and dehydrogenase.

    PubMed

    Muguruma, Hitoshi; Hoshino, Tatsuya; Nowaki, Kohei

    2015-01-14

    An electrochemical enzyme biosensor with electronically type-sorted (metallic and semiconducting) single-walled carbon nanotubes (SWNTs) for use in aqueous media is presented. This research investigates how the electronic types of SWNTs influence the amperometric response of enzyme biosensors. To conduct a clear evaluation, a simple layer-by-layer process based on a plasma-polymerized nano thin film (PPF) was adopted because a PPF is an inactive matrix that can form a well-defined nanostructure composed of SWNTs and enzyme. For a biosensor with the glucose oxidase (GOx) enzyme in the presence of oxygen, the response of a metallic SWNT-GOx electrode was 2 times larger than that of a semiconducting SWNT-GOx electrode. In contrast, in the absence of oxygen, the response of the semiconducting SWNT-GOx electrode was retained, whereas that of the metallic SWNT-GOx electrode was significantly reduced. This indicates that direct electron transfer occurred with the semiconducting SWNT-GOx electrode, whereas the metallic SWNT-GOx electrode was dominated by a hydrogen peroxide pathway caused by an enzymatic reaction. For a biosensor with the glucose dehydrogenase (GDH; oxygen-independent catalysis) enzyme, the response of the semiconducting SWNT-GDH electrode was 4 times larger than that of the metallic SWNT-GDH electrode. Electrochemical impedance spectroscopy was used to show that the semiconducting SWNT network has less resistance for electron transfer than the metallic SWNT network. Therefore, it was concluded that semiconducting SWNTs are more suitable than metallic SWNTs for electrochemical enzyme biosensors in terms of direct electron transfer as a detection mechanism. This study makes a valuable contribution toward the development of electrochemical biosensors that employ sorted SWNTs and various enzymes. PMID:25522366

  1. Electronically type-sorted carbon nanotube-based electrochemical biosensors with glucose oxidase and dehydrogenase.

    PubMed

    Muguruma, Hitoshi; Hoshino, Tatsuya; Nowaki, Kohei

    2015-01-14

    An electrochemical enzyme biosensor with electronically type-sorted (metallic and semiconducting) single-walled carbon nanotubes (SWNTs) for use in aqueous media is presented. This research investigates how the electronic types of SWNTs influence the amperometric response of enzyme biosensors. To conduct a clear evaluation, a simple layer-by-layer process based on a plasma-polymerized nano thin film (PPF) was adopted because a PPF is an inactive matrix that can form a well-defined nanostructure composed of SWNTs and enzyme. For a biosensor with the glucose oxidase (GOx) enzyme in the presence of oxygen, the response of a metallic SWNT-GOx electrode was 2 times larger than that of a semiconducting SWNT-GOx electrode. In contrast, in the absence of oxygen, the response of the semiconducting SWNT-GOx electrode was retained, whereas that of the metallic SWNT-GOx electrode was significantly reduced. This indicates that direct electron transfer occurred with the semiconducting SWNT-GOx electrode, whereas the metallic SWNT-GOx electrode was dominated by a hydrogen peroxide pathway caused by an enzymatic reaction. For a biosensor with the glucose dehydrogenase (GDH; oxygen-independent catalysis) enzyme, the response of the semiconducting SWNT-GDH electrode was 4 times larger than that of the metallic SWNT-GDH electrode. Electrochemical impedance spectroscopy was used to show that the semiconducting SWNT network has less resistance for electron transfer than the metallic SWNT network. Therefore, it was concluded that semiconducting SWNTs are more suitable than metallic SWNTs for electrochemical enzyme biosensors in terms of direct electron transfer as a detection mechanism. This study makes a valuable contribution toward the development of electrochemical biosensors that employ sorted SWNTs and various enzymes.

  2. 3β-hydroxysteroid dehydrogenase type II deficiency on newborn screening test.

    PubMed

    Araújo, Vitor Guilherme Brito de; Oliveira, Renata Santarem de; Gameleira, Kallianna Paula Duarte; Cruz, Cátia Barbosa; Lofrano-Porto, Adriana

    2014-08-01

    3β-hydroxysteroid dehydrogenase II (3β-HSD) deficiency represents a rare CAH variant. Newborns affected with its classic form have salt wasting in early infancy and genital ambiguity in both sexes. High levels of 17-hydroxypregnenolone (Δ517OHP) are characteristic, but extra-adrenal conversion to 17-hydroxyprogesterone (17OHP) may lead to positive results on newborn screening tests. Filter paper 17OHP on newborn screening test was performed by immunofluorometric assay, and serum determinations of 17OHP and Δ517OHP, by radioimmunoassay. A 46,XY infant with genital ambiguity and adrenal crisis at three months of age presented a positive result on newborn screening for CAH. Serum determinations of 17OHP and Δ517OHP were elevated, and a high Δ517OHP/cortisol relation was compatible with the diagnosis of 3β-HSD deficiency. Molecular analysis of the HSD3B2 gene from the affected case revealed the presence of the homozygous p.P222Q mutation, whereas his parents were heterozygous for it. We present the first report of 3β-HSD type II deficiency genotype-proven detected at the Newborn Screening Program in Brazil. The case described herein corroborates the strong genotype-phenotype correlation associated with the HSD3B2 p.P222Q mutation, which leads to a classic salt-wasting 3β-HSD deficiency. Further evaluation of 17OHP assays used in newborn screening tests would aid in determining their reproducibility, as well as the potential significance of moderately elevated 17OHP levels as an early indicator to the diagnosis of other forms of classic CAH, beyond 21-hydroxylase deficiency.

  3. Reduction of glucocorticoid receptor ligand binding by the 11-beta hydroxysteroid dehydrogenase type 2 inhibitor, Thiram.

    PubMed

    Garbrecht, Mark R; Krozowski, Zygmunt S; Snyder, Jeanne M; Schmidt, Thomas J

    2006-10-01

    Endogenous and synthetic glucocorticoids (GCs), such as cortisol and dexamethasone (Dex), modulate airway inflammation, regulate the production of surfactant by lung epithelial cells, and influence fetal lung maturation. The 11-beta hydroxysteroid dehydrogenase type 2 (HSD2) enzyme catalyzes the oxidation of bioactive cortisol and Dex to their 11-keto metabolites. Thiram (tetramethylthiuram disulfide) specifically inhibits HSD2 activity by oxidizing cysteine residues located in the cofactor binding domain of the enzyme. During studies performed to define a potential role for HSD2 in modulating GC action in human lung epithelial cells, we observed that exposure of intact human lung epithelial cells (NCI-H441) to 50 microM Thiram significantly attenuated the down-stream effects of Dex (100 nM) on the expression of two GC-sensitive genes, pulmonary surfactant proteins A and B. This observation appeared to be inconsistent with simple inhibition of HSD2 activity. Although Thiram inhibited HSD2 oxidase activity in a dose-dependent manner without affecting HSD2 protein expression, Thiram also reduced specific binding of [3H]-Dex to the glucocorticoid receptor (GR). Pre-treatment of cells with 1 mM dithiothreitol (DTT), a thiol-reducing agent, completely blocked the inhibitory effect of Thiram on ligand binding. These results are suggestive that Thiram may alter the ligand-binding domain of the GR by oxidizing critical thiol-containing amino acid residues. Taken collectively, these data demonstrate that attenuated down-stream GC signaling, via decreased binding of ligand to the GR, is a novel cellular effect of Thiram exposure in human lung epithelial cells.

  4. A urine-concentrating defect in 11β-hydroxysteroid dehydrogenase type 2 null mice

    PubMed Central

    Evans, Louise C.; Livingstone, Dawn E.; Kenyon, Christopher J.; Jansen, Maurits A.; Dear, James W.; Mullins, John J.

    2012-01-01

    In aldosterone target tissues, 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) is coexpressed with mineralocorticoid receptors (MR) and protects the receptor from activation by glucocorticoids. Null mutations in the encoding gene, HSD11B2, cause apparent mineralocorticoid excess, in which hypertension is thought to reflect volume expansion secondary to sodium retention. Hsd11b2−/− mice are indeed hypertensive, but impaired natriuretic capacity is associated with significant volume contraction, suggestive of a urine concentrating defect. Water turnover and the urine concentrating response to a 24-h water deprivation challenge were therefore assessed in Hsd11b2−/− mice and controls. Hsd11b2−/− mice have a severe and progressive polyuric/polydipsic phenotype. In younger mice (∼2 mo of age), polyuria was associated with decreased abundance of aqp2 and aqp3 mRNA. The expression of other genes involved in water transport (aqp4, slc14a2, and slc12a2) was not changed. The kidney was structurally normal, and the concentrating response to water deprivation was intact. In older Hsd11b2−/− mice (>6 mo), polyuria was associated with a severe atrophy of the renal medulla and downregulation of aqp2, aqp3, aqp4, slc14a2, and slc12a2. The concentrating response to water deprivation was impaired, and the natriuretic effect of the loop diuretic bumetanide was lost. In older Hsd11b2−/− mice, the V2 receptor agonist desmopressin did not restore full urine concentrating capacity. We find that Hsd11b2−/− mice develop nephrogenic diabetes insipidus. Gross changes to renal structure are observed, but these were probably secondary to sustained polyuria, rather than of developmental origin. PMID:22622456

  5. Nutritional marginal zinc deficiency disrupts placental 11β-hydroxysteroid dehydrogenase type 2 modulation.

    PubMed

    Huang, Y L; Supasai, S; Kucera, H; Gaikwad, N W; Adamo, A M; Mathieu, P; Oteiza, P I

    2016-01-01

    This paper investigated if marginal zinc nutrition during gestation could affect fetal exposure to glucocorticoids as a consequence of a deregulation of placental 11βHSD2 expression. Placenta 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) plays a central role as a barrier protecting the fetus from the deleterious effects of excess maternal glucocorticoids. Rats were fed control (25 μg zinc per g diet) or marginal (10 μg zinc per g diet, MZD) zinc diets from day 0 through day 19 (GD19) of gestation. At GD19, corticosterone concentration in plasma, placenta, and amniotic fluid was similar in both groups. However, protein and mRNA levels of placenta 11βHSD2 were significantly higher (25% and 58%, respectively) in MZD dams than in controls. The main signaling cascades modulating 11βHSD2 expression were assessed. In MZD placentas the activation of ERK1/2 and of the downstream transcription factor Egr-1 was low, while p38 phosphorylation and SP-1-DNA binding were low compared to the controls. These results point to a central role of ERK1/Egr-1 in the regulation of 11βHSD2 expression under the conditions of limited zinc availability. In summary, results show that an increase in placenta 11βHSD2 expression occurs as a consequence of gestational marginal zinc nutrition. This seems to be due to a low tissue zinc-associated deregulation of ERK1/2 rather than to exposure to high maternal glucocorticoid exposure. The deleterious effects on brain development caused by diet-induced marginal zinc deficiency in rats do not seem to be due to fetal exposure to excess glucocorticoids.

  6. Inhibition of 11β-hydroxysteroid dehydrogenase type 1 ameliorates obesity-related insulin resistance.

    PubMed

    Shao, Shiying; Zhang, Xiaojie; Zhang, Muxun

    2016-09-01

    Excess 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) may be implicated in the development of obesity related metabolic disorders. The present study measured the expression level of 11β-HSD1 in visceral adipose tissues from 23 patients undergoing abdominal operation. Correlation of 11β-HSD1 expression with BMI, waist-to-hip ratio (WHR), HOMA-IR, and serum lipids was evaluated by spearman correlation analysis. High-fat diet-induced obese (DIO) rats were orally dosed with BVT.2733 for 4 weeks. Weight, plasma insulin, and lipids were detected at the end of the treatment. The effects of 11β-HSD1 inhibition on the key insulin-signaling cascade and adipocytokines were measured by western blot and ELISA respectively. 11β-HSD1 was increased in patients with central obesity, the expression level of which was closely related with WHR (r = 0.5851), BMI (r = 0.4952), and HOMA-IR (r = 0.4637). Obesity related insulin resistance in high-fat DIO rats, as reflected by a marked decrease in IRS-1, IRS-2, GLUT4, and PI3K, could be attenuated by 11β-HSD1 inhibition. Furthermore, the down-regulation of 11β-HSD1 could correct the disordered profiles of adipocytokines including adiponectin, IL-6, and TNF-α. These findings indicated that 11β-HSD1 inhibition can give a potential benefit in reducing obesity and lowering insulin resistance by modulating the insulin-signaling pathway and adipocytokine production. PMID:27268236

  7. 17{beta}-Hydroxysteroid dehydrogenase type 13 is a liver-specific lipid droplet-associated protein

    SciTech Connect

    Horiguchi, Yuka; Araki, Makoto; Motojima, Kiyoto

    2008-05-30

    17{beta}-Hydroxysteroid dehydrogenase (17{beta}HSD) type 13 is identified as a new lipid droplet-associated protein. 17{beta}HSD type 13 has an N-terminal sequence similar to that of 17{beta}HSD type 11, and both sequences function as an endoplasmic reticulum and lipid droplet-targeting signal. Localization of native 17{beta}HSD type 13 on the lipid droplets was confirmed by subcellular fractionation and Western blotting. In contrast to 17{beta}HSD type 11, however, expression of 17{beta}HSD type 13 is largely restricted to the liver and is not enhanced by peroxisome proliferator-activated receptor {alpha} and its ligand. Instead the expression level of 17{beta}HSD type 13 in the receptor-null mice was increased several-fold. 17{beta}HSD type 13 may have a distinct physiological role as a lipid droplet-associated protein in the liver.

  8. In vivo relationship between monoamine oxidase type B and alcohol dehydrogenase: effects of ethanol and phenylethylamine

    SciTech Connect

    Aliyu, S.U.; Upahi, L.

    1988-01-01

    The role of acute ethanol and phenylethylamine on the brain and platelet monoamine oxidase activities, hepatic cytosolic alcohol dehydrogenase, redox state and motor behavior were studied in male rats. Ethanol on its own decreased the redox couple ratio, as well as, alcohol dehydrogenase activity in the liver while at the same time it increased brain and platelet monoamine oxidase activity due to lower Km with no change in Vmax. The elevation in both brain and platelet MAO activity was associated with ethanol-induced hypomotility in the rats. Co-administration of phenylethylamine and ethanol to the animals, caused antagonism of the ethanol-induced effects described above. The effects of phenylethylamine alone, on the above mentioned biochemical and behavioral indices, are more complex. Phenylethylamine on its own, like ethanol, caused reduction of the cytosolic redox, ratio and elevation of monoamine oxidase activity in the brain and platelets. However, in contrast to ethanol, this monoamine produced hypermotility and activation of the hepatic cytosolic alcohol dehydrogenase activity in the animals.

  9. Cloning and characterization of sulfite dehydrogenase, two c-type cytochromes, and a flavoprotein of Paracoccus denitrificans GB17: essential role of sulfite dehydrogenase in lithotrophic sulfur oxidation.

    PubMed

    Wodara, C; Bardischewsky, F; Friedrich, C G

    1997-08-01

    A 13-kb genomic region of Paracoccus dentrificans GB17 is involved in lithotrophic thiosulfate oxidation. Adjacent to the previously reported soxB gene (C. Wodara, S. Kostka, M. Egert, D. P. Kelly, and C. G. Friedrich, J. Bacteriol. 176:6188-6191, 1994), 3.7 kb were sequenced. Sequence analysis revealed four additional open reading frames, soxCDEF. soxC coded for a 430-amino-acid polypeptide with an Mr of 47,339 that included a putative signal peptide of 40 amino acids (Mr of 3,599) with a RR motif present in periplasmic proteins with complex redox centers. The mature soxC gene product exhibited high amino acid sequence similarity to the eukaryotic molybdoenzyme sulfite oxidase and to nitrate reductase. We constructed a mutant, GBsoxC delta, carrying an in-frame deletion in soxC which covered a region possibly coding for the molybdenum cofactor binding domain. GBsoxC delta was unable to grow lithoautotrophically with thiosulfate but grew well with nitrate as a nitrogen source or as an electron acceptor. Whole cells and cell extracts of mutant GBsoxC delta contained 10% of the thiosulfate-oxidizing activity of the wild type. Only a marginal rate of sulfite-dependent cytochrome c reduction was observed from cell extracts of mutant GBsoxC delta. These results demonstrated that sulfite dehydrogenase was essential for growth with thiosulfate of P. dentrificans GB17. soxD coded for a periplasmic diheme c-type cytochrome of 384 amino acids (Mr of 39,983) containing a putative signal peptide with an Mr of 2,363. soxE coded for a periplasmic monoheme c-type cytochrome of 236 amino acids (Mr of 25,926) containing a putative signal peptide with an Mr of 1,833. SoxD and SoxE were highly identical to c-type cytochromes of P. denitrificans and other organisms. soxF revealed an incomplete open reading frame coding for a peptide of 247 amino acids with a putative signal peptide (Mr of 2,629). The deduced amino acid sequence of soxF was 47% identical and 70% similar to the sequence

  10. Crystal structures of type III{sub H} NAD-dependent D-3-phosphoglycerate dehydrogenase from two thermophiles

    SciTech Connect

    Kumar, S.M.; Pampa, K.J.; Manjula, M.; Hemantha Kumar, G.; Kunishima, Naoki; Lokanath, N.K.

    2014-08-15

    Highlights: • Determined the crystal structures of PGDH from two thermophiles. • Monomer is composed of nucleotide binding domain and substrate binding domain. • Crystal structures of type III{sub H} PGDH. - Abstract: In the L-Serine biosynthesis, D-3-phosphoglycerate dehydrogenase (PGDH) catalyzes the inter-conversion of D-3-phosphoglycerate to phosphohydroxypyruvate. PGDH belongs to 2-hydroxyacid dehydrogenases family. We have determined the crystal structures of PGDH from Sulfolobus tokodaii (StPGDH) and Pyrococcus horikoshii (PhPGDH) using X-ray diffraction to resolution of 1.77 Å and 1.95 Å, respectively. The PGDH protomer from both species exhibits identical structures, consisting of substrate binding domain and nucleotide binding domain. The residues and water molecules interacting with the NAD are identified. The catalytic triad residues Glu-His-Arg are highly conserved. The residues involved in the dimer interface and the structural features responsible for thermostability are evaluated. Overall, structures of PGDHs with two domains and histidine at the active site are categorized as type III{sub H} and such PGDHs structures having this type are reported for the first time.

  11. Identification of the 11 beta-hydroxysteroid dehydrogenase type 1 mRNA and protein in human mononuclear leukocytes.

    PubMed

    Fiore, C; Nardi, A; Dalla Valle, L; Pellati, D; Krozowski, Z; Colombo, L; Armanini, D

    2009-10-01

    The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion between inactive 11-ketoglucocorticoids and their active 11beta-hydroxy derivatives, such as cortisol and corticosterone. We have investigated the expression of 11beta-HSD1 in freshly isolated human peripheral mononuclear leukocytes (MNL). The presence of 11beta-HSD1 mRNA was demonstrated in total RNA by RT-PCR using specific primers designed on the 4th and 5th exons of the human 11beta-HSD1 gene. Fragments of the expected size were consistently detected on agarose gels, and sequencing showed complete identity with the corresponding sequence deposited in GenBank. The occurrence of 11beta-HSD1 protein was established by Western immunoblot analysis with a specific polyclonal antibody. Enzyme oxo-reductase activity was investigated by incubating 12 samples of MNL isolated from from 8 subjects with [3H]cortisone and formation of cortisol was established only in 4 subjects (yield range: 0.15-1.3%) after acetylation and TLC, blank subtraction and correction for losses. 18beta-Glycyrrhetinic acid, an inhibitor of 11 beta-HSD1, reduced cortisol production below detection limit. Dehydrogenase activity could not be demonstrated. It is suggested that, although enzyme activity of 11beta-HSD1 in circulating MNL is low, it is apparently ready for enhancement after MNL migration to sites of inflammation. PMID:19235128

  12. Structure of the bacterial type II NADH dehydrogenase: a monotopic membrane protein with an essential role in energy generation.

    PubMed

    Heikal, Adam; Nakatani, Yoshio; Dunn, Elyse; Weimar, Marion R; Day, Catherine L; Baker, Edward N; Lott, J Shaun; Sazanov, Leonid A; Cook, Gregory M

    2014-03-01

    Non-proton pumping type II NADH dehydrogenase (NDH-2) plays a central role in the respiratory metabolism of bacteria, and in the mitochondria of fungi, plants and protists. The lack of NDH-2 in mammalian mitochondria and its essentiality in important bacterial pathogens suggests these enzymes may represent a potential new drug target to combat microbial pathogens. Here, we report the first crystal structure of a bacterial NDH-2 enzyme at 2.5 Å resolution from Caldalkalibacillus thermarum. The NDH-2 structure reveals a homodimeric organization that has a unique dimer interface. NDH-2 is localized to the cytoplasmic membrane by two separated C-terminal membrane-anchoring regions that are essential for membrane localization and FAD binding, but not NDH-2 dimerization. Comparison of bacterial NDH-2 with the yeast NADH dehydrogenase (Ndi1) structure revealed non-overlapping binding sites for quinone and NADH in the bacterial enzyme. The bacterial NDH-2 structure establishes a framework for the structure-based design of small-molecule inhibitors.

  13. Type 2 NADH dehydrogenases in the cyanobacterium Synechocystis sp. strain PCC 6803 are involved in regulation rather than respiration.

    PubMed

    Howitt, C A; Udall, P K; Vermaas, W F

    1999-07-01

    Analysis of the genome of Synechocystis sp. strain PCC 6803 reveals three open reading frames (slr0851, slr1743, and sll1484) that may code for type 2 NAD(P)H dehydrogenases (NDH-2). The sequence similarity between the translated open reading frames and NDH-2s from other organisms is low, generally not exceeding 30% identity. However, NAD(P)H and flavin adenine dinucleotide binding motifs are conserved in all three putative NDH-2s in Synechocystis sp. strain PCC 6803. The three open reading frames were cloned, and deletion constructs were made for each. An expression construct containing one of the three open reading frames, slr1743, was able to functionally complement an Escherichia coli mutant lacking both NDH-1s and NDH-2s. Therefore, slr0851, slr1743, and sll1484 have been designated ndbA, ndbB, and ndbC, respectively. Strains that lacked one or more of the ndb genes were created in wild-type and photosystem (PS) I-less backgrounds. Deletion of ndb genes led to small changes in photoautotrophic growth rates and respiratory activities. Electron transfer rates into the plastoquinone pool in thylakoids in darkness were consistent with the presence of a small amount of NDH-2 activity in thylakoids. No difference was observed between wild-type and the Ndb-less strains in the banding patterns seen on native gels when stained for either NADH or NADPH dehydrogenase activity, indicating that the Ndb proteins do not accumulate to high levels. A striking phenotype of the PS I-less background strains lacking one or more of the NDH-2s is that they were able to grow at high light intensities that were lethal to the control strain but they retained normal PS II activity. We suggest that the Ndb proteins in Synechocystis sp. strain PCC 6803 are redox sensors and that they play a regulatory role responding to the redox state of the plastoquinone pool.

  14. Expression of gluconeogenic enzymes and 11β-hydroxysteroid dehydrogenase type 1 in liver of diabetic mice after acute exercise

    PubMed Central

    Brust, Korie B; Corbell, Kathryn A; Al-Nakkash, Layla; Babu, Jeganathan Ramesh; Broderick, Tom L

    2014-01-01

    During acute exercise, normoglycemia is maintained by a precise match between hepatic glucose production and its peripheral utilization. This is met by a complex interplay of hepatic responses and glucose uptake by muscle. However, the effect of a single bout of exercise on hepatic gluconeogenesis, corticosterone (CORT) secretion, and glucose homeostasis in the db/db mouse model of type 2 diabetes is poorly understood. Diabetic db/db and lean control littermates were subjected to a 30 minute session of treadmill running and sacrificed either immediately after exercise or 8 hours later. Plasma glucose levels were markedly increased in db/db mice after exercise, whereas no change in glucose was observed in lean mice. Post-exercise measurements revealed that plasma CORT levels were also significantly increased in db/db mice compared to lean mice. Plasma hypothalamic corticotropin releasing hormone and pituitary adrenocorticotropic hormone levels were reciprocally decreased in both db/db and lean mice after exercise, indicating intact feedback mechanisms. Protein expression, determined by Western blot analysis, of the glucocorticoid receptor in liver was significantly increased in db/db mice subjected to prior exercise. In liver of db/db mice, a significant increase in the expression of phosphoenolpyruvate carboxykinase was noted compared to lean mice after exercise. However, no change in the expression of glucose-6-phosphatase (G6Pase) α or β was observed in db/db mice. Expression of 11β-hydroxysteroid dehydrogenase type 1 was increased significantly in db/db mice compared to lean mice after exercise. Our results show differences in plasma glucose and protein expression of gluconeogenic enzymes after acute exercise between lean and diabetic db/db mice. The db/db diabetic mouse is hyperglycemic after acute exercise. This hyperglycemic state may be explained, in part, by enhanced endogenous CORT secretion and regulated hepatic phosphoenolpyruvate carboxykinase and 11

  15. Reciprocal Phosphorylation of Yeast Glycerol-3-Phosphate Dehydrogenases in Adaptation to Distinct Types of Stress

    PubMed Central

    Lee, Yong Jae; Jeschke, Grace R.; Roelants, Françoise M.; Thorner, Jeremy

    2012-01-01

    Eukaryotic cells have evolved mechanisms for ensuring growth and survival in the face of stress caused by a fluctuating environment. Saccharomyces cerevisiae has two homologous glycerol-3-phosphate dehydrogenases, Gpd1 and Gpd2, that are required to endure various stresses, including hyperosmotic shock and hypoxia. These enzymes are only partially redundant, and their unique functions were attributed previously to differential transcriptional regulation and localization. We find that Gpd1 and Gpd2 are negatively regulated through phosphorylation by distinct kinases under reciprocal conditions. Gpd2 is phosphorylated by the AMP-activated protein kinase Snf1 to curtail glycerol production when nutrients are limiting. Gpd1, in contrast, is a target of TORC2-dependent kinases Ypk1 and Ypk2. Inactivation of Ypk1 by hyperosmotic shock results in dephosphorylation and activation of Gpd1, accelerating recovery through increased glycerol production. Gpd1 dephosphorylation acts synergistically with its transcriptional upregulation, enabling long-term growth at high osmolarity. Phosphorylation of Gpd1 and Gpd2 by distinct kinases thereby enables rapid adaptation to specific stress conditions. Introduction of phosphorylation motifs targeted by distinct kinases provides a general mechanism for functional specialization of duplicated genes during evolution. PMID:22988299

  16. Comparison of fluorescence properties of wild type and the W15F mutant of horse liver alcohol dehydrogenase

    NASA Astrophysics Data System (ADS)

    Eftink, Maurice R.; Wong, Cing-Yuen; Park, Doo-Hong; Shearer, Gretchen L.; Plapp, Bryce V.

    1994-08-01

    Horse liver alcohol dehydrogenase is a homodimeric protein; each subunit has two tryptophan residues that are in distinctly different microenvironments. Trp-15 is located on the surface and Trp-314 is buried at the intersubunit interface. Steady-state and time-resolved fluorescence and phosphorescence studies have enabled the assignment of parameters, e.g., quantum yield, emission maximum, decay times, to the individual tryptophan residues of the protein. We have prepared, by site-directed mutagenesis, the mutated W15F protein and have characterized its fluorescence properties. We show that the Trp-314 of the mutant experiences an apolar microenvironment, but that the fluorescence decay and exposure to solute quenchers of the mutant are somewhat different than was expected from the assignments for the wild type.

  17. Screening Baccharin Analogs as Selective Inhibitors Against Type 5 17β-Hydroxysteroid Dehydrogenase (AKR1C3)

    PubMed Central

    Zang, Tianzhu; Verma, Kshitij; Chen, Mo; Jin, Yi; Trippier, Paul C.; Penning, Trevor M.

    2015-01-01

    Aldo-keto reductase 1C3 (AKR1C3), also known as type 5 17β-hydroxysteroid dehydrogenase, is a downstream steroidogenic enzyme and converts androgen precursors to the potent androgen receptor ligands: testosterone and 5α-dihydrotestosterone. Studies have shown that AKR1C3 is involved in the development of castration resistant prostate cancer (CRPC) and that it is a rational drug target for the treatment of CRPC. Baccharin, a component of Brazilian propolis, has been observed to exhibit a high inhibitory potency and selectivity for AKR1C3 over other AKR1C isoforms and is a promising lead compound for developing more potent and selective inhibitors. Here, we report the screening of fifteen baccharin analogs as selective inhibitors against AKR1C3 versus AKR1C2 (type 3 3α-hydroxysteroid dehydrogenase). Among these analogs, the inhibitory activity and selectivity of thirteen compounds were evaluated for the first time. The substitution of the 4-dihydrocinnamoyloxy group of baccharin by an acetate group displayed nanomolar inhibitory potency (IC50: 440 nM) and a 102-fold selectivity over AKR1C2. By contrast, when the cinnamic acid group of baccharin was esterified, there was a dramatic decrease in potency and selectivity for AKR1C3 in comparison to baccharin. Low or sub- micromolar inhibition was observed when the 3-prenyl group of baccharin was removed, and the selectivity over AKR1C2 was low. Although unsubstituted baccharin was still the most potent (IC50: 100 nM) and selective inhibitor for AKR1C3, these data provide structure-activity relationships required for the optimization of new baccharin analogs. They suggest that the carboxylate group on cinnamic acid, the prenyl group, and either retention of 4′-dihydrocinnamoyloxy group or acetate substituent on cinnamic acid are important to maintain the high potency and selectivity for AKR1C3. PMID:25555457

  18. Screening baccharin analogs as selective inhibitors against type 5 17β-hydroxysteroid dehydrogenase (AKR1C3).

    PubMed

    Zang, Tianzhu; Verma, Kshitij; Chen, Mo; Jin, Yi; Trippier, Paul C; Penning, Trevor M

    2015-06-01

    Aldo-keto reductase 1C3 (AKR1C3), also known as type 5 17β-hydroxysteroid dehydrogenase, is a downstream steroidogenic enzyme and converts androgen precursors to the potent androgen receptor ligands: testosterone and 5α-dihydrotestosterone. Studies have shown that AKR1C3 is involved in the development of castration resistant prostate cancer (CRPC) and that it is a rational drug target for the treatment of CRPC. Baccharin, a component of Brazilian propolis, has been observed to exhibit a high inhibitory potency and selectivity for AKR1C3 over other AKR1C isoforms and is a promising lead compound for developing more potent and selective inhibitors. Here, we report the screening of fifteen baccharin analogs as selective inhibitors against AKR1C3 versus AKR1C2 (type 3 3α-hydroxysteroid dehydrogenase). Among these analogs, the inhibitory activity and selectivity of thirteen compounds were evaluated for the first time. The substitution of the 4-dihydrocinnamoyloxy group of baccharin by an acetate group displayed nanomolar inhibitory potency (IC50: 440 nM) and a 102-fold selectivity over AKR1C2. By contrast, when the cinnamic acid group of baccharin was esterified, there was a dramatic decrease in potency and selectivity for AKR1C3 in comparison to baccharin. Low or sub-micromolar inhibition was observed when the 3-prenyl group of baccharin was removed, and the selectivity over AKR1C2 was low. Although unsubstituted baccharin was still the most potent (IC50: 100 nM) and selective inhibitor for AKR1C3, these data provide structure-activity relationships required for the optimization of new baccharin analogs. They suggest that the carboxylate group on cinnamic acid, the prenyl group, and either retention of 4-dihydrocinnamoyloxy group or acetate substituent on cinnamic acid are important to maintain the high potency and selectivity for AKR1C3.

  19. Genome Characteristics of Two Novel Type I Methanotrophs Enriched from North Sea Sediments Containing Exclusively a Lanthanide-Dependent XoxF5-Type Methanol Dehydrogenase.

    PubMed

    Vekeman, Bram; Speth, Daan; Wille, Jasper; Cremers, Geert; De Vos, Paul; Op den Camp, Huub J M; Heylen, Kim

    2016-10-01

    Microbial methane oxidizers play a crucial role in the oxidation of methane in marine ecosystems, as such preventing the escape of excessive methane to the atmosphere. Despite the important role of methanotrophs in marine ecosystems, only a limited number of isolates are described, with only four genomes available. Here, we report on two genomes of gammaproteobacterial methanotroph cultures, affiliated with the deep-sea cluster 2, obtained from North Sea sediment. Initial enrichments using methane as sole source of carbon and energy and mimicking the in situ conditions followed by serial subcultivations and multiple extinction culturing events over a period of 3 years resulted in a highly enriched culture. The draft genomes of the methane oxidizer in both cultures showed the presence of genes typically found in type I methanotrophs, including genes encoding particulate methane monooxygenase (pmoCAB), genes for tetrahydromethanopterin (H4MPT)- and tetrahydrofolate (H4F)-dependent C1-transfer pathways, and genes of the ribulose monophosphate (RuMP) pathway. The most distinctive feature, when compared to other available gammaproteobacterial genomes, is the absence of a calcium-dependent methanol dehydrogenase. Both genomes reported here only have a xoxF gene encoding a lanthanide-dependent XoxF5-type methanol dehydrogenase. Thus, these genomes offer novel insight in the genomic landscape of uncultured diversity of marine methanotrophs. PMID:27457652

  20. Specific combination of compound heterozygous mutations in 17β-hydroxysteroid dehydrogenase type 4 (HSD17B4) defines a new subtype of D-bifunctional protein deficiency

    PubMed Central

    2012-01-01

    Background D-bifunctional protein (DBP) deficiency is typically apparent within the first month of life with most infants demonstrating hypotonia, psychomotor delay and seizures. Few children survive beyond two years of age. Among patients with prolonged survival all demonstrate severe gross motor delay, absent language development, and severe hearing and visual impairment. DBP contains three catalytically active domains; an N-terminal dehydrogenase, a central hydratase and a C-terminal sterol carrier protein-2-like domain. Three subtypes of the disease are identified based upon the domain affected; DBP type I results from a combined deficiency of dehydrogenase and hydratase activity; DBP type II from isolated hydratase deficiency and DBP type III from isolated dehydrogenase deficiency. Here we report two brothers (16½ and 14 years old) with DBP deficiency characterized by normal early childhood followed by sensorineural hearing loss, progressive cerebellar and sensory ataxia and subclinical retinitis pigmentosa. Methods and results Biochemical analysis revealed normal levels of plasma VLCFA, phytanic acid and pristanic acid, and normal bile acids in urine; based on these results no diagnosis was made. Exome analysis was performed using the Agilent SureSelect 50Mb All Exon Kit and the Illumina HiSeq 2000 next-generation-sequencing (NGS) platform. Compound heterozygous mutations were identified by exome sequencing and confirmed by Sanger sequencing within the dehydrogenase domain (c.101C>T; p.Ala34Val) and hydratase domain (c.1547T>C; p.Ile516Thr) of the 17β-hydroxysteroid dehydrogenase type 4 gene (HSD17B4). These mutations have been previously reported in patients with severe-forms of DBP deficiency, however each mutation was reported in combination with another mutation affecting the same domain. Subsequent studies in fibroblasts revealed normal VLCFA levels, normal C26:0 but reduced pristanic acid beta-oxidation activity. Both DBP hydratase and dehydrogenase

  1. Hypertrophy in the Distal Convoluted Tubule of an 11β-Hydroxysteroid Dehydrogenase Type 2 Knockout Model.

    PubMed

    Hunter, Robert W; Ivy, Jessica R; Flatman, Peter W; Kenyon, Christopher J; Craigie, Eilidh; Mullins, Linda J; Bailey, Matthew A; Mullins, John J

    2015-07-01

    Na(+) transport in the renal distal convoluted tubule (DCT) by the thiazide-sensitive NaCl cotransporter (NCC) is a major determinant of total body Na(+) and BP. NCC-mediated transport is stimulated by aldosterone, the dominant regulator of chronic Na(+) homeostasis, but the mechanism is controversial. Transport may also be affected by epithelial remodeling, which occurs in the DCT in response to chronic perturbations in electrolyte homeostasis. Hsd11b2(-/-) mice, which lack the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) and thus exhibit the syndrome of apparent mineralocorticoid excess, provided an ideal model in which to investigate the potential for DCT hypertrophy to contribute to Na(+) retention in a hypertensive condition. The DCTs of Hsd11b2(-/-) mice exhibited hypertrophy and hyperplasia and the kidneys expressed higher levels of total and phosphorylated NCC compared with those of wild-type mice. However, the striking structural and molecular phenotypes were not associated with an increase in the natriuretic effect of thiazide. In wild-type mice, Hsd11b2 mRNA was detected in some tubule segments expressing Slc12a3, but 11βHSD2 and NCC did not colocalize at the protein level. Thus, the phosphorylation status of NCC may not necessarily equate to its activity in vivo, and the structural remodeling of the DCT in the knockout mouse may not be a direct consequence of aberrant corticosteroid signaling in DCT cells. These observations suggest that the conventional concept of mineralocorticoid signaling in the DCT should be revised to recognize the complexity of NCC regulation by corticosteroids.

  2. Association study of sorbitol dehydrogenase -888G>C polymorphism with type 2 diabetic retinopathy in Caucasian-Brazilians.

    PubMed

    Ferreira, Fábio Netto; Crispim, Daisy; Canani, Luís Henrique; Gross, Jorge Luiz; dos Santos, Kátia Gonçalves

    2013-10-01

    Diabetic retinopathy (DR) is a common chronic complication of diabetes and remains the leading cause of blindness in working-aged people. Hyperglycemia increases glucose flux through the polyol pathway, in which aldose reductase converts glucose into intracellular sorbitol, which is subsequently converted to fructose by sorbitol dehydrogenase (SDH). The accelerated polyol pathway triggers a cascade of events leading to retinal vascular endothelial dysfunction and the eventual development of DR. Polymorphisms in the gene encoding aldose reductase have been consistently associated with DR. However, only two studies have analyzed the relationship between polymorphisms in the gene encoding SDH (SORD) and DR. In this case-control study, we investigated whether the -888G > C polymorphism (rs3759890) in the SORD gene is associated with the presence or severity of DR in 446 Caucasian-Brazilians with type 2 diabetes (241 subjects with and 205 subjects without DR). The -888G > C polymorphism was also examined in 105 healthy Caucasian blood donors, and the genotyping of this polymorphism was carried out by real-time PCR. The genotype and allele frequencies of the -888G > C polymorphism in patients with type 2 diabetes were similar to those of blood donors (G allele frequency = 0.16 in both groups of subjects). Similarly, the genotype and allele frequencies in patients with DR or the proliferative form of DR were similar to those of patients without this complication (P > 0.05 for all comparisons). Thus, our findings suggest that the -888G > C polymorphism in the SORD gene is not involved in the pathogenesis of DR in type 2 diabetes.

  3. A Korean patient with glutaric aciduria type 1 with a novel mutation in the glutaryl CoA dehydrogenase gene.

    PubMed

    Kim, Hee Su; Yu, Hee Joon; Lee, Jeehun; Park, Hyung-Doo; Kim, Ji Hye; Shin, Hyung-Jin; Jin, Dong Kyu; Lee, Munhyang

    2014-01-01

    Mutations in the glutaryl-CoA dehydrogenase gene can result in Glutaric aciduria type 1(GA 1) by accumulation of glutaric acid, 3-hydroxyglutaric acid (3-OH-GA), and glutarylcarnitine (C5DC). GA 1 is characterized by macrocephaly, subdural hemorrhage (SDH), and dystonic movement disorder after acute encephalopathic crisis. We report a Korean patient with GA1 and a novel mutation. A 16-month-old boy presented with SDH, macrocephaly, and developmental delay. In the neurologic examination, the patient had mild axial hypotonia, but otherwise normal neurologic functions. The brain MRI showed large amounts of bilateral SDH and high signal intensity in both basal ganglia and thalamus. Metabolic screening tests detected highly elevated urinary GA levels but 3-OH-glutaric acid was normal. C5DC was 0.94 μM/L (reference range < 0.3 μM/L). The patient had compound heterozygous mutations of the GCDH gene: p.Arg257Gln (c.770G>A) and p.Cys308Arg (c.922T>C). p.Cys308Arg is a novel mutation; reports of p.Arg257Gln were also rare both in Caucasians and Asian populations. In summary, we hereby report one Korean patient with GA1 with clinical, biochemical, and radiologic characteristics confirmed by genetic analysis. PMID:24795062

  4. The Arabidopsis KS-type dehydrin recovers lactate dehydrogenase activity inhibited by copper with the contribution of His residues.

    PubMed

    Hara, Masakazu; Monna, Shuhei; Murata, Takae; Nakano, Taiyo; Amano, Shono; Nachbar, Markus; Wätzig, Hermann

    2016-04-01

    Dehydrin, which is one of the late embryogenesis abundant (LEA) proteins, is involved in the ability of plants to tolerate the lack of water. Although many reports have indicated that dehydrins bind heavy metals, the physiological role of this metal binding has not been well understood. Here, we report that the Arabidopsis KS-type dehydrin (AtHIRD11) recovered the lactate dehydrogenase (LDH) activity denatured by Cu(2+). The LDH activity was partially inhibited by 0.93 μM Cu(2+) but totally inactivated by 9.3 μM Cu(2+). AtHIRD11 recovered the activity of LDH treated with 9.3 μM Cu(2+) in a dose-dependent manner. The recovery activity of AtHIRD11 was significantly higher than those of serum albumin and lysozyme. The conversion of His residues to Ala in AtHIRD11 resulted in the loss of the Cu(2+) binding of the protein as well as the disappearance of the conformational change induced by Cu(2+) that is observed by circular dichroism spectroscopy. The mutant protein showed lower recovery activity than the original AtHIRD11. These results indicate that AtHIRD11 can reactivate LDH inhibited by Cu(2+) via the His residues. This function may prevent physiological damage to plants due to heavy-metal stress. PMID:26940498

  5. Chronic inhibition of 11 β -hydroxysteroid dehydrogenase type 1 activity decreases hypertension, insulin resistance, and hypertriglyceridemia in metabolic syndrome.

    PubMed

    Schnackenberg, Christine G; Costell, Melissa H; Krosky, Daniel J; Cui, Jianqi; Wu, Charlene W; Hong, Victor S; Harpel, Mark R; Willette, Robert N; Yue, Tian-Li

    2013-01-01

    Metabolic syndrome is a constellation of risk factors including hypertension, dyslipidemia, insulin resistance, and obesity that promote the development of cardiovascular disease. Metabolic syndrome has been associated with changes in the secretion or metabolism of glucocorticoids, which have important functions in adipose, liver, kidney, and vasculature. Tissue concentrations of the active glucocorticoid cortisol are controlled by the conversion of cortisone to cortisol by 11 β -hydroxysteroid dehydrogenase type 1 (11 β -HSD1). Because of the various cardiovascular and metabolic activities of glucocorticoids, we tested the hypothesis that 11 β -HSD1 is a common mechanism in the hypertension, dyslipidemia, and insulin resistance in metabolic syndrome. In obese and lean SHR/NDmcr-cp (SHR-cp), cardiovascular, metabolic, and renal functions were measured before and during four weeks of administration of vehicle or compound 11 (10 mg/kg/d), a selective inhibitor of 11 β -HSD1. Compound 11 significantly decreased 11 β -HSD1 activity in adipose tissue and liver of SHR-cp. In obese SHR-cp, compound 11 significantly decreased mean arterial pressure, glucose intolerance, insulin resistance, hypertriglyceridemia, and plasma renin activity with no effect on heart rate, body weight gain, or microalbuminuria. These results suggest that 11 β -HSD1 activity in liver and adipose tissue is a common mediator of hypertension, hypertriglyceridemia, glucose intolerance, and insulin resistance in metabolic syndrome.

  6. Chronic Inhibition of 11β-Hydroxysteroid Dehydrogenase Type 1 Activity Decreases Hypertension, Insulin Resistance, and Hypertriglyceridemia in Metabolic Syndrome

    PubMed Central

    Schnackenberg, Christine G.; Costell, Melissa H.; Krosky, Daniel J.; Cui, Jianqi; Wu, Charlene W.; Hong, Victor S.; Harpel, Mark R.; Willette, Robert N.; Yue, Tian-Li

    2013-01-01

    Metabolic syndrome is a constellation of risk factors including hypertension, dyslipidemia, insulin resistance, and obesity that promote the development of cardiovascular disease. Metabolic syndrome has been associated with changes in the secretion or metabolism of glucocorticoids, which have important functions in adipose, liver, kidney, and vasculature. Tissue concentrations of the active glucocorticoid cortisol are controlled by the conversion of cortisone to cortisol by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). Because of the various cardiovascular and metabolic activities of glucocorticoids, we tested the hypothesis that 11β-HSD1 is a common mechanism in the hypertension, dyslipidemia, and insulin resistance in metabolic syndrome. In obese and lean SHR/NDmcr-cp (SHR-cp), cardiovascular, metabolic, and renal functions were measured before and during four weeks of administration of vehicle or compound 11 (10 mg/kg/d), a selective inhibitor of 11β-HSD1. Compound 11 significantly decreased 11β-HSD1 activity in adipose tissue and liver of SHR-cp. In obese SHR-cp, compound 11 significantly decreased mean arterial pressure, glucose intolerance, insulin resistance, hypertriglyceridemia, and plasma renin activity with no effect on heart rate, body weight gain, or microalbuminuria. These results suggest that 11β-HSD1 activity in liver and adipose tissue is a common mediator of hypertension, hypertriglyceridemia, glucose intolerance, and insulin resistance in metabolic syndrome. PMID:23586038

  7. Distinct effect of stress on 11beta-hydroxysteroid dehydrogenase type 1 and corticosteroid receptors in dorsal and ventral hippocampus.

    PubMed

    Ergang, P; Kuželová, A; Soták, M; Klusoňová, P; Makal, J; Pácha, J

    2014-01-01

    Multiple lines of evidence suggest the participation of the hippocampus in the feedback inhibition of the hypothalamus-pituitary-adrenal axis during stress response. This inhibition is mediated by glucocorticoid feedback due to the sensitivity of the hippocampus to these hormones. The sensitivity is determined by the expression of glucocorticoid (GR) and mineralocorticoid (MR) receptors and 11beta-hydroxysteroid dehydrogenase type 1 (11HSD1), an enzyme that regulates the conversion of glucocorticoids from inactive to active form. The goal of our study was to assess the effect of stress on the expression of 11HSD1, GR and MR in the ventral and dorsal region of the CA1 hippocampus in three different rat strains with diverse responses to stress: Fisher 344, Lewis and Wistar. Stress stimulated 11HSD1 in the ventral but not dorsal CA1 hippocampus of Fisher 344 but not Lewis or Wistar rats. In contrast, GR expression following stress was decreased in the dorsal but not ventral CA1 hippocampus of all three strains. MR expression was not changed in either the dorsal or ventral CA1 region. These results indicate that (1) depending on the strain, stress stimulates 11HSD1 in the ventral hippocampus, which is known to be involved in stress and emotion reactions whereas (2) independent of strain, stress inhibits GR in the dorsal hippocampus, which is predominantly involved in cognitive functions.

  8. Prognosis and Clinicopathologic Features of Patients With Advanced Stage Isocitrate Dehydrogenase (IDH) Mutant and IDH Wild-Type Intrahepatic Cholangiocarcinoma

    PubMed Central

    Goyal, Lipika; Govindan, Aparna; Sheth, Rahul A.; Nardi, Valentina; Blaszkowsky, Lawrence S.; Faris, Jason E.; Clark, Jeffrey W.; Ryan, David P.; Kwak, Eunice L.; Allen, Jill N.; Murphy, Janet E.; Saha, Supriya K.; Hong, Theodore S.; Wo, Jennifer Y.; Ferrone, Cristina R.; Tanabe, Kenneth K.; Chong, Dawn Q.; Deshpande, Vikram; Borger, Darrell R.; Iafrate, A. John; Bardeesy, Nabeel; Zheng, Hui

    2015-01-01

    Background. Conflicting data exist regarding the prognostic impact of the isocitrate dehydrogenase (IDH) mutation in intrahepatic cholangiocarcinoma (ICC), and limited data exist in patients with advanced-stage disease. Similarly, the clinical phenotype of patients with advanced IDH mutant (IDHm) ICC has not been characterized. In this study, we report the correlation of IDH mutation status with prognosis and clinicopathologic features in patients with advanced ICC. Methods. Patients with histologically confirmed advanced ICC who underwent tumor mutational profiling as a routine part of their care between 2009 and 2014 were evaluated. Clinical and pathological data were collected by retrospective chart review for patients with IDHm versus IDH wild-type (IDHwt) ICC. Pretreatment tumor volume was calculated on computed tomography or magnetic resonance imaging. Results. Of the 104 patients with ICC who were evaluated, 30 (28.8%) had an IDH mutation (25.0% IDH1, 3.8% IDH2). The median overall survival did not differ significantly between IDHm and IDHwt patients (15.0 vs. 20.1 months, respectively; p = .17). The pretreatment serum carbohydrate antigen 19-9 (CA19-9) level in IDHm and IDHwt patients was 34.5 and 118.0 U/mL, respectively (p = .04). Age at diagnosis, sex, histologic grade, and pattern of metastasis did not differ significantly by IDH mutation status. Conclusion. The IDH mutation was not associated with prognosis in patients with advanced ICC. The clinical phenotypes of advanced IDHm and IDHwt ICC were similar, but patients with IDHm ICC had a lower median serum CA19-9 level at presentation. Implications for Practice: Previous studies assessing the prognostic impact of the isocitrate dehydrogenase (IDH) gene mutation in intrahepatic cholangiocarcinoma (ICC) mainly focused on patients with early-stage disease who have undergone resection. These studies offer conflicting results. The target population for clinical trials of IDH inhibitors is patients with

  9. A Novel Type II NAD+-Specific Isocitrate Dehydrogenase from the Marine Bacterium Congregibacter litoralis KT71

    PubMed Central

    Wu, Ming-Cai; Tian, Chang-Qing; Cheng, Hong-Mei; Xu, Lei; Wang, Peng; Zhu, Guo-Ping

    2015-01-01

    In most living organisms, isocitrate dehydrogenases (IDHs) convert isocitrate into ɑ-ketoglutarate (ɑ-KG). Phylogenetic analyses divide the IDH protein family into two subgroups: types I and II. Based on cofactor usage, IDHs are either NAD+-specific (NAD-IDH) or NADP+-specific (NADP-IDH); NADP-IDH evolved from NAD-IDH. Type I IDHs include NAD-IDHs and NADP-IDHs; however, no type II NAD-IDHs have been reported to date. This study reports a novel type II NAD-IDH from the marine bacterium Congregibacter litoralis KT71 (ClIDH, GenBank accession no. EAQ96042). His-tagged recombinant ClIDH was produced in Escherichia coli and purified; the recombinant enzyme was NAD+-specific and showed no detectable activity with NADP+. The Km values of the enzyme for NAD+ were 262.6±7.4 μM or 309.1±11.2 μM with Mg2+ or Mn2+ as the divalent cation, respectively. The coenzyme specificity of a ClIDH Asp487Arg/Leu488His mutant was altered, and the preference of the mutant for NADP+ was approximately 24-fold higher than that for NAD+, suggesting that ClIDH is an NAD+-specific ancestral enzyme in the type II IDH subgroup. Gel filtration and analytical ultracentrifugation analyses revealed the homohexameric structure of ClIDH, which is the first IDH hexamer discovered thus far. A 163-amino acid segment of CIIDH is essential to maintain its polymerization structure and activity, as a truncated version lacking this region forms a non-functional monomer. ClIDH was dependent on divalent cations, the most effective being Mn2+. The maximal activity of purified recombinant ClIDH was achieved at 35°C and pH 7.5, and a heat inactivation experiment showed that a 20-min incubation at 33°C caused a 50% loss of ClIDH activity. The discovery of a NAD+-specific, type II IDH fills a gap in the current classification of IDHs, and sheds light on the evolution of type II IDHs. PMID:25942017

  10. 11β-Hydroxysteroid Dehydrogenase Type 1 Is Expressed in Neutrophils and Restrains an Inflammatory Response in Male Mice

    PubMed Central

    Coutinho, Agnes E.; Kipari, Tiina M. J.; Zhang, Zhenguang; Esteves, Cristina L.; Lucas, Christopher D.; Gilmour, James S.; Webster, Scott P.; Walker, Brian R.; Hughes, Jeremy; Savill, John S.; Seckl, Jonathan R.; Rossi, Adriano G.

    2016-01-01

    Endogenous glucocorticoid action within cells is enhanced by prereceptor metabolism by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which converts intrinsically inert cortisone and 11-dehydrocorticosterone into active cortisol and corticosterone, respectively. 11β-HSD1 is highly expressed in immune cells elicited to the mouse peritoneum during thioglycollate-induced peritonitis and is down-regulated as the inflammation resolves. During inflammation, 11β-HSD1-deficient mice show enhanced recruitment of inflammatory cells and delayed acquisition of macrophage phagocytic capacity. However, the key cells in which 11β-HSD1 exerts these effects remain unknown. Here we have identified neutrophils (CD11b+,Ly6G+,7/4+ cells) as the thioglycollate-recruited cells that most highly express 11β-HSD1 and show dynamic regulation of 11β-HSD1 in these cells during an inflammatory response. Flow cytometry showed high expression of 11β-HSD1 in peritoneal neutrophils early during inflammation, declining at later states. In contrast, expression in blood neutrophils continued to increase during inflammation. Ablation of monocytes/macrophages by treatment of CD11b-diphtheria-toxin receptor transgenic mice with diphtheria toxin prior to thioglycollate injection had no significant effect on 11β-HSD1 activity in peritoneal cells, consistent with neutrophils being the predominant 11β-HSD1 expressing cell type at this time. Similar to genetic deficiency in 11β-HSD1, acute inhibition of 11β-HSD1 activity during thioglycollate-induced peritonitis augmented inflammatory cell recruitment to the peritoneum. These data suggest that neutrophil 11β-HSD1 increases during inflammation to contribute to the restraining effect of glucocorticoids upon neutrophil-mediated inflammation. In human neutrophils, lipopolysaccharide activation increased 11β-HSD1 expression, suggesting the antiinflammatory effects of 11β-HSD1 in neutrophils may be conserved in humans. PMID:27145012

  11. Glucose-6-Phosphate Dehydrogenase and NADPH Redox Regulates Cardiac Myocyte L-Type Calcium Channel Activity and Myocardial Contractile Function

    PubMed Central

    Rawat, Dhwajbahadur K.; Hecker, Peter; Watanabe, Makino; Chettimada, Sukrutha; Levy, Richard J.; Okada, Takao; Edwards, John G.; Gupte, Sachin A.

    2012-01-01

    We recently demonstrated that a 17-ketosteroid, epiandrosterone, attenuates L-type Ca2+ currents (ICa-L) in cardiac myocytes and inhibits myocardial contractility. Because 17-ketosteroids are known to inhibit glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, and to reduce intracellular NADPH levels, we hypothesized that inhibition of G6PD could be a novel signaling mechanism which inhibit ICa-L and, therefore, cardiac contractile function. We tested this idea by examining myocardial function in isolated hearts and Ca2+ channel activity in isolated cardiac myocytes. Myocardial function was tested in Langendorff perfused hearts and ICa-L were recorded in the whole-cell patch configuration by applying double pulses from a holding potential of −80 mV and then normalized to the peak amplitudes of control currents. 6-Aminonicotinamide, a competitive inhibitor of G6PD, increased pCO2 and decreased pH. Additionally, 6-aminonicotinamide inhibited G6PD activity, reduced NADPH levels, attenuated peak ICa-L amplitudes, and decreased left ventricular developed pressure and ±dp/dt. Finally, dialyzing NADPH into cells from the patch pipette solution attenuated the suppression of ICa-L by 6-aminonicotinamide. Likewise, in G6PD-deficient mice, G6PD insufficiency in the heart decreased GSH-to-GSSG ratio, superoxide, cholesterol and acetyl CoA. In these mice, M-mode echocardiographic findings showed increased diastolic volume and end-diastolic diameter without changes in the fraction shortening. Taken together, these findings suggest that inhibiting G6PD activity and reducing NADPH levels alters metabolism and leads to inhibition of L-type Ca2+ channel activity. Notably, this pathway may be involved in modulating myocardial contractility under physiological and pathophysiological conditions during which the pentose phosphate pathway-derived NADPH redox is modulated (e.g., ischemia-reperfusion and heart failure). PMID:23071515

  12. Effects of antisense-mediated inhibition of 11β-hydroxysteroid dehydrogenase type 1 on hepatic lipid metabolism[S

    PubMed Central

    Li, Guoping; Hernandez-Ono, Antonio; Crooke, Rosanne M.; Graham, Mark J.; Ginsberg, Henry N.

    2011-01-01

    11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) converts inactive 11-keto derivatives to active glucocorticoids within tissues and may play a role in the metabolic syndrome (MS). We used an antisense oligonucleotide (ASO) to knock down 11β-HSD1 in livers of C57BL/6J mice consuming a Western-type diet (WTD). 11β-HSD1 ASO-treated mice consumed less food, so we compared them to ad libitum-fed mice and to food-matched mice receiving control ASO. Knockdown of 11β-HSD1 directly protected mice from WTD-induced steatosis and dyslipidemia by reducing synthesis and secretion of triglyceride (TG) and increasing hepatic fatty acid oxidation. These changes in hepatic and plasma lipids were not associated with reductions in genes involved in de novo lipogenesis. However, protein levels of both sterol regulatory element-binding protein (SREBP) 1 and fatty acid synthase were significantly reduced in mice treated with 11β-HSD1 ASO. There was no change in hepatic secretion of apolipoprotein (apo)B, indicating assembly and secretion of smaller apoB-containing lipoproteins by the liver in the 11β-HSD1-treated mice. Our results indicate that inhibition of 11β-HSD1 by ASO treatment of WTD-fed mice resulted in improved plasma and hepatic lipid levels, reduced lipogenesis by posttranslational regulation, and secretion of similar numbers of apoB-containing lipoproteins containing less TG per particle. PMID:21364201

  13. Molecular alterations and expression of succinate dehydrogenase complex in wild-type KIT/PDGFRA/BRAF gastrointestinal stromal tumors

    PubMed Central

    Celestino, Ricardo; Lima, Jorge; Faustino, Alexandra; Vinagre, João; Máximo, Valdemar; Gouveia, António; Soares, Paula; Manuel Lopes, José

    2013-01-01

    Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract, disclosing somatic KIT, PDGFRA and BRAF mutations. Loss of function of succinate dehydrogenase (SDH) complex is an alternative molecular mechanism in GISTs, namely in carriers of germline mutations of the SDH complex that develop Carney–Stratakis dyad characterized by multifocal GISTs and multicentric paragangliomas (PGLs). We studied a series of 25 apparently sporadic primary wild-type (WT) KIT/PDGFRA/BRAF GISTs occurring in patients without personal or familial history of PGLs, re-evaluated clinicopathological features and analyzed molecular alterations and immunohistochemistry expression of SDH complex. As control, we used a series of well characterized 49 KIT/PDGFRA/BRAF-mutated GISTs. SDHB expression was absent in 20% and SDHB germline mutations were detected in 12% of WT GISTs. Germline SDHB mutations were significantly associated to younger age at diagnosis. A significant reduction in SDHB expression in WT GISTs was found when compared with KIT/PDGFRA/BRAF-mutated GISTs. No significant differences were found when comparing DOG-1 and c-KIT expression in WT, SDHB-mutated and KIT/PDGFRA/BRAF-mutated GISTs. Our results confirm the occurrence of germline SDH genes mutations in isolated, apparently sporadic WT GISTs. WT KIT/PDGFRA/BRAF GISTs without SDHB or SDHA/SDHB expression may correspond to Carney–Stratakis dyad or Carney triad. Most importantly, the possibility of PGLs (Carney–Stratakis dyad) and/or pulmonary chondroma (Carney triad) should be addressed in these patients and their kindred. PMID:22948025

  14. Molecular alterations and expression of succinate dehydrogenase complex in wild-type KIT/PDGFRA/BRAF gastrointestinal stromal tumors.

    PubMed

    Celestino, Ricardo; Lima, Jorge; Faustino, Alexandra; Vinagre, João; Máximo, Valdemar; Gouveia, António; Soares, Paula; Lopes, José Manuel

    2013-05-01

    Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract, disclosing somatic KIT, PDGFRA and BRAF mutations. Loss of function of succinate dehydrogenase (SDH) complex is an alternative molecular mechanism in GISTs, namely in carriers of germline mutations of the SDH complex that develop Carney-Stratakis dyad characterized by multifocal GISTs and multicentric paragangliomas (PGLs). We studied a series of 25 apparently sporadic primary wild-type (WT) KIT/PDGFRA/BRAF GISTs occurring in patients without personal or familial history of PGLs, re-evaluated clinicopathological features and analyzed molecular alterations and immunohistochemistry expression of SDH complex. As control, we used a series of well characterized 49 KIT/PDGFRA/BRAF-mutated GISTs. SDHB expression was absent in 20% and SDHB germline mutations were detected in 12% of WT GISTs. Germline SDHB mutations were significantly associated to younger age at diagnosis. A significant reduction in SDHB expression in WT GISTs was found when compared with KIT/PDGFRA/BRAF-mutated GISTs. No significant differences were found when comparing DOG-1 and c-KIT expression in WT, SDHB-mutated and KIT/PDGFRA/BRAF-mutated GISTs. Our results confirm the occurrence of germline SDH genes mutations in isolated, apparently sporadic WT GISTs. WT KIT/PDGFRA/BRAF GISTs without SDHB or SDHA/SDHB expression may correspond to Carney-Stratakis dyad or Carney triad. Most importantly, the possibility of PGLs (Carney-Stratakis dyad) and/or pulmonary chondroma (Carney triad) should be addressed in these patients and their kindred. PMID:22948025

  15. The Role of Placental 11-Beta Hydroxysteroid Dehydrogenase Type 1 and Type 2 Methylation on Gene Expression and Infant Birth Weight.

    PubMed

    Green, Benjamin B; Armstrong, David A; Lesseur, Corina; Paquette, Alison G; Guerin, Dylan J; Kwan, Lauren E; Marsit, Carmen J

    2015-06-01

    Maternal stress has been linked to infant birth weight outcomes, which itself may be associated with health later in life. The placenta acts as a master regulator for the fetal environment, mediating intrauterine exposures to stress through the activity of genes regulating glucocorticoids, including the 11beta-hydroxysteroid dehydrogenase (HSD11B) type 1 and 2 genes, and so we hypothesized that variation in these genes will be associated with infant birth weight. We investigated DNA methylation levels at six sites across the two genes, as well as mRNA expression for each, and the relationship to infant birth weight. Logistic regressions correcting for potential confounding factors revealed a significant association between methylation at a single CpG site within HSD11B1 and being born large for gestational age. In addition, our analysis identified correlations between methylation and gene expression, including sex-specific transcriptional regulation of HSD11B2. Our work is one of the first comprehensive views of DNA methylation and expression in the placenta for both HSD11B types 1 and 2, linking epigenetic alterations with the regulation of fetal stress and birth weight outcomes. PMID:25788665

  16. The oxidative pentose phosphate pathway in the haloarchaeon Haloferax volcanii involves a novel type of glucose-6-phosphate dehydrogenase--The archaeal Zwischenferment.

    PubMed

    Pickl, Andreas; Schönheit, Peter

    2015-04-28

    The oxidative pentose phosphate pathway (OPPP), catalyzing the oxidation of glucose-6-phosphate to ribulose-5-phosphate is ubiquitous in eukarya and bacteria but has not yet been reported in archaea. In haloarchaea a putative 6-phosphogluconate dehydrogenase (6PGDH) is annotated, whereas a gene coding for glucose-6-phosphate dehydrogenase (Glc6PDH) could not be identified. Here we report the purification and characterization of a novel type of Glc6PDH in Haloferax volcanii that is not related to bacterial and eukaryal Glc6PDHs and the encoding gene is designated as azf (archaeal zwischenferment). Further, recombinant H. volcanii 6PGDH was characterized. Deletion mutant analyses indicate that both, Glc6PDH and 6PGDH, are functionally involved in pentose phosphate formation in vivo. This is the first report on the operation of the OPPP in the domain of archaea.

  17. Synthesis, purification and crystallographic studies of the C-terminal sterol carrier protein type 2 (SCP-2) domain of human hydroxysteroid dehydrogenase-like protein 2.

    PubMed

    Cheng, Zhong; Li, Yao; Sui, Chun; Sun, Xiaobo; Xie, Yong

    2015-07-01

    Human hydroxysteroid dehydrogenase-like protein 2 (HSDL2) is a member of the short-chain dehydrogenase/reductase (SDR) subfamily of oxidoreductases and contains an N-terminal catalytic domain and a C-termianl sterol carrier protein type 2 (SCP-2) domain. In this study, the C-terminal SCP-2 domain of human HSDL2, including residues Lys318-Arg416, was produced in Escherichia coli, purified and crystallized. X-ray diffraction data were collected to 2.10 Å resolution. The crystal belonged to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a = b = 70.4, c = 60.6 Å, α = β = 90, γ = 120°. Two protein molecules are present in the asymmetric unit, resulting in a Matthews coefficient of 2.16 Å(3) Da(-1) and an approximate solvent content of 43%.

  18. L-Arabinose degradation pathway in the haloarchaeon Haloferax volcanii involves a novel type of L-arabinose dehydrogenase.

    PubMed

    Johnsen, Ulrike; Sutter, Jan-Moritz; Zaiß, Henning; Schönheit, Peter

    2013-11-01

    The pathway of L-arabinose degradation was studied in the haloarchaeon Haloferax volcanii. It is shown that L-arabinose is oxidatively degraded to α-ketoglutarate. During growth on L-arabinose, L-arabinose dehydrogenase (L-AraDH) was induced. The enzyme was purified as a 130 kDa homotetrameric protein catalyzing the oxidation of L-arabinose with both NADP(+) and NAD(+). The gene encoding L-AraDH was identified as HVO_B0032 and recombinant L-AraDH showed similar properties as the native enzyme. The L-AraDH deletion mutant did not grow on L-arabinose, but grew unaffected on glucose and D-xylose, indicating a specific involvement in L-arabinose degradation. Phylogenetic analyses attribute the first archaeal L-AraDH to the extended short-chain dehydrogenase/reductase (SDRe) family, where it is part of a novel cluster and thus differs from known archaeal and bacterial pentose dehydrogenases. Further, cell extracts of H. volcanii catalyzed the NADP(+)-dependent conversion of L-arabinoate to α-ketoglutarate. The genes involved in that conversion were identified by analyses of transcripts and deletion mutants as HVO_B0038A, HVO_B0027 and HVO_B0039 recently reported to be involved in D-xylonate conversion to α-ketoglutarate in H. volcanii (Johnsen et al. 2009).

  19. 4-Dihydromethyltrisporate dehydrogenase, an enzyme of the sex hormone pathway in Mucor mucedo, is constitutively transcribed but its activity is differently regulated in (+) and (-) mating types.

    PubMed

    Schimek, Christine; Petzold, Annett; Schultze, Kornelia; Wetzel, Jana; Wolschendorf, Frank; Burmester, Anke; Wöstemeyer, Johannes

    2005-09-01

    4-Dihydromethyltrisporate dehydrogenase (TDH) converts the (+) mating type sex pheromone 4-dihydromethyltrisporate into methyltrisporate. In Mucor mucedo, this conversion is required only in the (-) mating type. Expression of the TDH encoding TSP1 gene was analyzed qualitatively using reverse-transcribed PCR. TSP1 is constitutively transcribed in the (+) and in the (-) mating type, irrespective of the mating situation. By immunodetection, the translation product is also formed constitutively. In contrast to gene expression, TDH enzyme activity depends on the sexual status of the mycelium. Activity is restricted to the sexually stimulated (-) mating type. Non-stimulated (-), as well as stimulated and non-stimulated (+) mycelia exhibit no activity and do not influence activity in stimulated (-) mycelia. Time course analysis shows strongly increased enzyme activity at 80 min after stimulation. Low activity exists from the onset of stimulation, indicating that additional regulation mechanisms are involved in TDH function.

  20. Suppression of NDA-type alternative mitochondrial NAD(P)H dehydrogenases in arabidopsis thaliana modifies growth and metabolism, but not high light stimulation of mitochondrial electron transport.

    PubMed

    Wallström, Sabá V; Florez-Sarasa, Igor; Araújo, Wagner L; Escobar, Matthew A; Geisler, Daniela A; Aidemark, Mari; Lager, Ida; Fernie, Alisdair R; Ribas-Carbó, Miquel; Rasmusson, Allan G

    2014-05-01

    The plant respiratory chain contains several pathways which bypass the energy-conserving electron transport complexes I, III and IV. These energy bypasses, including type II NAD(P)H dehydrogenases and the alternative oxidase (AOX), may have a role in redox stabilization and regulation, but current evidence is inconclusive. Using RNA interference, we generated Arabidopsis thaliana plants simultaneously suppressing the type II NAD(P)H dehydrogenase genes NDA1 and NDA2. Leaf mitochondria contained substantially reduced levels of both proteins. In sterile culture in the light, the transgenic lines displayed a slow growth phenotype, which was more severe when the complex I inhibitor rotenone was present. Slower growth was also observed in soil. In rosette leaves, a higher NAD(P)H/NAD(P)⁺ ratio and elevated levels of lactate relative to sugars and citric acid cycle metabolites were observed. However, photosynthetic performance was unaffected and microarray analyses indicated few transcriptional changes. A high light treatment increased AOX1a mRNA levels, in vivo AOX and cytochrome oxidase activities, and levels of citric acid cycle intermediates and hexoses in all genotypes. However, NDA-suppressing plants deviated from the wild type merely by having higher levels of several amino acids. These results suggest that NDA suppression restricts citric acid cycle reactions, inducing a shift towards increased levels of fermentation products, but do not support a direct association between photosynthesis and NDA proteins.

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

    PubMed

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

    2014-09-01

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

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

    PubMed Central

    2015-01-01

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

  3. The Effects of Piper Sarmentosum Water Extract on the Expression and Activity of 11β-Hydroxysteroid Dehydrogenase Type 1 in the Bones with Excessive Glucocorticoids

    PubMed Central

    Suhana Mohd Ramli, Elvy; Nirwana Soelaiman, Ima; Othman, Faizah; Ahmad, Fairus; Nazrun Shuib, Ahmad; Mohamed, Norazlina; Muhammad, Norliza; Hj Suhaimi, Farihah

    2012-01-01

    Background: Long-term glucocorticoid therapy causes secondary osteoporosis leading to pathological fractures. Glucocorticoid action in bone is dependant upon the activity of 11β-hydroxysteroid dehydrogenase type 1 enzyme (11β-HSD1). Piper sarmentosum is a local herb that possesses the ability to inhibit 11-βHSD1 enzyme activity. We aimed to determine the effects of Piper sarmentosum water extract on 11-βHSD1 expressions and activity in the bones of glucocorticoid-treated adrenalectomized rats. Methods: Forty male Sprague–Dawley rats (200-250 g) were used. Twenty-four animals were adrenalectomized and received intramuscular injection of dexamethasone (120 μg/kg/day). They were simultaneously administered with either Piper sarmentosum water extract (125 mg/kg/day), GCA (120 mg/kg/day) or distilled water as vehicle by oral gavage for two months. Eight animals were sham-operated and given vehicle daily, i.e. intramuscular olive oil and oral distilled water. Results: Following two months treatment, dexamethasone-treated adrenalectomized rats had significantly lower 11β-HSD1 dehydrogenase activity and higher 11β-HSD1 expression in the femoral bones compared to the sham-operated and baseline group. The rats supplemented with Piper sarmentosum water extract had significantly higher 11β-HSD1 dehydrogenase activity and lower 11β-HSD1 expression in the bones. Conclusion: The results showed that Piper sarmentosum water extract had the ability to prevent glucocorcoticoid excess in the bones of glucocorticoid-treated adrenalectomized rats through the local modulation of 11β-HSD1 expression and activity, and may be used as prophylaxis for osteoporosis in patients on long-term glucocorticoid treatment. PMID:23115429

  4. A newborn lethal defect due to inactivation of retinaldehyde dehydrogenase type 3 is prevented by maternal retinoic acid treatment

    PubMed Central

    Dupé, Valérie; Matt, Nicolas; Garnier, Jean-Marie; Chambon, Pierre; Mark, Manuel; Ghyselinck, Norbert B.

    2003-01-01

    The retinoic acid (RA) signal, produced locally from vitamin A by retinaldehyde dehydrogenase (Raldh) and transduced by the nuclear receptors for retinoids (RA receptor and 9-cis-RA receptor), is indispensable for ontogenesis and homeostasis of numerous tissues. We demonstrate that Raldh3 knockout in mouse suppresses RA synthesis and causes malformations restricted to ocular and nasal regions, which are similar to those observed in vitamin A-deficient fetuses and/or in retinoid receptor mutants. Raldh3 knockout notably causes choanal atresia (CA), which is responsible for respiratory distress and death of Raldh3-null mutants at birth. CA is due to persistence of nasal fins, whose rupture normally allows the communication between nasal and oral cavities. This malformation, which is similar to isolated congenital CA in humans and may result from impaired RA-controlled down-regulation of Fgf8 expression in nasal fins, can be prevented by a simple maternal treatment with RA. PMID:14623956

  5. A non-enzymatic function of 17beta-hydroxysteroid dehydrogenase type 10 is required for mitochondrial integrity and cell survival.

    PubMed

    Rauschenberger, Katharina; Schöler, Katja; Sass, Jörn Oliver; Sauer, Sven; Djuric, Zdenka; Rumig, Cordula; Wolf, Nicole I; Okun, Jürgen G; Kölker, Stefan; Schwarz, Heinz; Fischer, Christine; Grziwa, Beate; Runz, Heiko; Nümann, Astrid; Shafqat, Naeem; Kavanagh, Kathryn L; Hämmerling, Günter; Wanders, Ronald J A; Shield, Julian P H; Wendel, Udo; Stern, David; Nawroth, Peter; Hoffmann, Georg F; Bartram, Claus R; Arnold, Bernd; Bierhaus, Angelika; Oppermann, Udo; Steinbeisser, Herbert; Zschocke, Johannes

    2010-02-01

    Deficiency of the mitochondrial enzyme 2-methyl-3-hydroxybutyryl-CoA dehydrogenase involved in isoleucine metabolism causes an organic aciduria with atypical neurodegenerative course. The disease-causing gene is HSD17B10 and encodes 17beta-hydroxysteroid dehydrogenase type 10 (HSD10), a protein also implicated in the pathogenesis of Alzheimer's disease. Here we show that clinical symptoms in patients are not correlated with residual enzymatic activity of mutated HSD10. Loss-of-function and rescue experiments in Xenopus embryos and cells derived from conditional Hsd17b10(-/-) mice demonstrate that a property of HSD10 independent of its enzymatic activity is essential for structural and functional integrity of mitochondria. Impairment of this function in neural cells causes apoptotic cell death whilst the enzymatic activity of HSD10 is not required for cell survival. This finding indicates that the symptoms in patients with mutations in the HSD17B10 gene are unrelated to accumulation of toxic metabolites in the isoleucine pathway and, rather, related to defects in general mitochondrial function. Therefore alternative therapeutic approaches to an isoleucine-restricted diet are required. PMID:20077426

  6. A non-enzymatic function of 17beta-hydroxysteroid dehydrogenase type 10 is required for mitochondrial integrity and cell survival.

    PubMed

    Rauschenberger, Katharina; Schöler, Katja; Sass, Jörn Oliver; Sauer, Sven; Djuric, Zdenka; Rumig, Cordula; Wolf, Nicole I; Okun, Jürgen G; Kölker, Stefan; Schwarz, Heinz; Fischer, Christine; Grziwa, Beate; Runz, Heiko; Nümann, Astrid; Shafqat, Naeem; Kavanagh, Kathryn L; Hämmerling, Günter; Wanders, Ronald J A; Shield, Julian P H; Wendel, Udo; Stern, David; Nawroth, Peter; Hoffmann, Georg F; Bartram, Claus R; Arnold, Bernd; Bierhaus, Angelika; Oppermann, Udo; Steinbeisser, Herbert; Zschocke, Johannes

    2010-02-01

    Deficiency of the mitochondrial enzyme 2-methyl-3-hydroxybutyryl-CoA dehydrogenase involved in isoleucine metabolism causes an organic aciduria with atypical neurodegenerative course. The disease-causing gene is HSD17B10 and encodes 17beta-hydroxysteroid dehydrogenase type 10 (HSD10), a protein also implicated in the pathogenesis of Alzheimer's disease. Here we show that clinical symptoms in patients are not correlated with residual enzymatic activity of mutated HSD10. Loss-of-function and rescue experiments in Xenopus embryos and cells derived from conditional Hsd17b10(-/-) mice demonstrate that a property of HSD10 independent of its enzymatic activity is essential for structural and functional integrity of mitochondria. Impairment of this function in neural cells causes apoptotic cell death whilst the enzymatic activity of HSD10 is not required for cell survival. This finding indicates that the symptoms in patients with mutations in the HSD17B10 gene are unrelated to accumulation of toxic metabolites in the isoleucine pathway and, rather, related to defects in general mitochondrial function. Therefore alternative therapeutic approaches to an isoleucine-restricted diet are required.

  7. Gene structure and chromosomal localization of the human HSD11K gene encoding the kidney (type 2) isozyme of 11{beta}-hydroxysteroid dehydrogenase

    SciTech Connect

    Agarwal, A.K.; Rogerson, F.M.; Mune, T.; White, P.C.

    1995-09-01

    11{beta}-hydroxysteroid dehydrogenase (11{beta}HSD) converts glucocorticoids to inactive products and is thus thought to confer specificity for aldosterone on the type I mineralocorticoid receptor in the kidney. Recent studies indicate the presence of at least two isozymes of 11{beta}HSD. In vitro, the NAD{sup +}-dependent kidney (type 2) isozyme catalyzes 11{beta}-dehydrogenase but not reductase reactions, whereas the NADP{sup +}-dependent liver (type 1) isozyme catalyzes both reactions. We have now characterized the human gene encoding kidney 11{beta}HSD (HSD11K). A bacteriophage P1 clone was isolated after screening a human genomic library by hybridization with sheep HSD11K cDNA. The gene consists of 5 exons spread over 6 kb. The nucleotide binding domain lies in the first exon are GC-rich (80%), suggesting that the gene may be transcriptionally regulated by factors that recognize GC-rich sequences. Fluorescence in situ hybridization of metaphase chromosomes with a positive P1 clone localized the gene to chromosome 16q22. In contrast, the HSD11L (liver isozyme) gene is located on chromosome 1 and contains 6 exons; the coding sequences of these genes are only 21% identical. HSD11K is expressed at high levels in the placenta and kidney of midgestation human fetuses and at lower levels in lung and testes. Different transcriptional start sites are utilized in kidney and placenta. These data should be applicable to genetic analysis of the syndrome of apparent mineralocorticoid excess, which may represent a deficiency of 11{beta}HSD. 25 refs., 5 figs.

  8. Annotated compound data for modulators of detergent-solubilised or lipid-reconstituted respiratory type II NADH dehydrogenase activity obtained by compound library screening

    PubMed Central

    Dunn, Elyse A.; Cook, Gregory M.; Heikal, Adam

    2015-01-01

    The energy-generating membrane protein NADH dehydrogenase (NDH-2), a proposed antibacterial drug target (see “Inhibitors of type II NADH:menaquinone oxidoreductase represent a class of antitubercular drugs” Weinstein et al. 2005 [1]), was screened for modulators of activity in either detergent-solublised or lipid reconstituted (proteolipsome) form. Here we present an annotated list of compounds identified in a small-scale screen against NDH-2. The dataset contains information regarding the libraries screened, the identities of hit compounds and the physicochemical properties governing solubility and permeability. The implications of these data for future antibiotic discovery are discussed in our associated report, “Comparison of lipid and detergent enzyme environments for identifying inhibitors of membrane-bound energy-transducing proteins” [2]. PMID:26862571

  9. Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia.

    PubMed

    Koszarska, Magdalena; Bors, Andras; Feczko, Angela; Meggyesi, Nora; Batai, Arpad; Csomor, Judit; Adam, Emma; Kozma, Andras; Orban, Tamas I; Lovas, Nora; Sipos, Andrea; Karaszi, Eva; Dolgos, Janos; Fekete, Sandor; Reichardt, Judit; Lehoczky, Eniko; Masszi, Tamas; Tordai, Attila; Andrikovics, Hajnalka

    2013-05-01

    Mutations of isocitrate dehydrogenase 1 and 2 (IDH1/2) are genetic alterations in acute myeloid leukemia (AML). The aim of our study was to investigate the frequency and prognostic effect of IDH1/2 mutations together followed by an individual analysis of each substitution in a Hungarian cohort consisting of 376 patients with AML. IDH1(mut) and IDH2(mut) were mutually exclusive, detected in 8.5% and 7.5% of cases, respectively. IDH1/2(mut) was associated with: older age (p = 0.001), higher average platelet count (p = 0.001), intermediate karyotype (p < 0.0001), NPM1(mut) (p = 0.022) and lower mRNA expression level of ABCG2 gene (p = 0.006). Overall survival (OS), remission and relapse rates were not different in IDH1(mut) or IDH2(mut) vs. IDH(neg). IDH1(mut) and IDH2(mut) were associated differently with NPM1(mut); co-occurrence was observed in 14.3% of IDH1 R132C vs. 70% of R132H carriers (p = 0.02) and in 47.4% of IDH2 R140Q vs. 0% of R172K carriers (p = 0.02). IDH1 R132H negatively influenced OS compared to IDH(neg) (p = 0.02) or R132C (p = 0.019). Particular amino acid changes affecting the same IDH1 codon influence the clinical characteristics and treatment outcome in AML.

  10. Misfolded forms of glyceraldehyde-3-phosphate dehydrogenase interact with GroEL and inhibit chaperonin-assisted folding of the wild-type enzyme.

    PubMed

    Polyakova, Oxana V; Roitel, Olivier; Asryants, Regina A; Poliakov, Alexei A; Branlant, Guy; Muronetz, Vladimir I

    2005-04-01

    We studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all SH-groups modified by 5,5'-dithiobis(2-nitrobenzoate); (2) O-R-type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O-P-type dimers of mutant GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G. It was shown that chemically modified GAPDH and the O-R-type mutant dimers bound to GroEL with 1:1 stoichiometry and dissociation constants K(d) of 0.4 and 0.9 muM, respectively. A striking feature of the resulting complexes with GroEL was their stability in the presence of Mg-ATP. Chemically modified GAPDH and the O-R-type mutant dimers inhibited GroEL-assisted refolding of urea-denatured wild-type GAPDH from B. stearothermophilus but did not affect its spontaneous reactivation. In contrast to the O-R-dimers, the O-P-type mutant dimers neither bound nor affected GroEL-assisted refolding of the wild-type GAPDH. Thus, we suggest that interaction of GroEL with certain types of misfolded proteins can result in the formation of stable complexes and the impairment of chaperonin activity. PMID:15741339

  11. Misfolded forms of glyceraldehyde-3-phosphate dehydrogenase interact with GroEL and inhibit chaperonin-assisted folding of the wild-type enzyme

    PubMed Central

    Polyakova, Oxana V.; Roitel, Olivier; Asryants, Regina A.; Poliakov, Alexei A.; Branlant, Guy; Muronetz, Vladimir I.

    2005-01-01

    We studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all SH-groups modified by 5,5′-dithiobis(2-nitrobenzoate); (2) O-R-type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O-P-type dimers of mutant GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G. It was shown that chemically modified GAPDH and the O-R-type mutant dimers bound to GroEL with 1:1 stoichiometry and dissociation constants Kd of 0.4 and 0.9 μM, respectively. A striking feature of the resulting complexes with GroEL was their stability in the presence of Mg-ATP. Chemically modified GAPDH and the O-R-type mutant dimers inhibited GroEL-assisted refolding of urea-denatured wild-type GAPDH from B. stearothermophilus but did not affect its spontaneous reactivation. In contrast to the O-R-dimers, the O-P-type mutant dimers neither bound nor affected GroEL-assisted refolding of the wild-type GAPDH. Thus, we suggest that interaction of GroEL with certain types of misfolded proteins can result in the formation of stable complexes and the impairment of chaperonin activity. PMID:15741339

  12. High fructose diets increase 11β-hydroxysteroid dehydrogenase type 1 in liver and visceral adipose in rats within 24-h exposure.

    PubMed

    London, Edra; Castonguay, Thomas W

    2011-05-01

    The increased prevalence of overweight and obesity in the United States during the past three decades coincides with a trend of increased sugar intake, especially fructose, leading to speculation that the two trends may be linked. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), that regulates intracellular tissue-specific glucocorticoid levels, is increased in adipose and suppressed in liver of obese humans and animals. Hexose-6-phosphate dehydrogenase (H6PDH) is colocalized with 11β-HSD1 and generates nicotinamide adenosine dinucleotide phosphate, the required cofactor for 11β-HSD1 reductase activity that converts inert glucocorticoid metabolite into active hormone. We examined the acute effects of ad lib access to 16% solutions of sucrose, fructose, or glucose and chow and water. Diets high in fructose, but not glucose or sucrose increased 11β-HSD1 mRNA within 24 h in liver and adipose by greater than two- and threefold, respectively (P ≤ 0.05). After 1 week, hepatic 11β-HSD1 mRNA and protein were suppressed by >60% in all sugar-fed groups, a phenomenon not previously reported in the absence of obesity. Sucrose- and fructose-fed rats had higher plasma triglycerides than did control or glucose-fed rats at both 24 h and 1 week (P ≤ 0.02), consistent with previously reported effects of fructose on lipid metabolism. We conclude that high-sugar diets initiate glucocorticoid dysregulation associated with obesity prior to the onset of phenotypic changes, and that high fructose diets specifically induce changes in 11β-HSD1 within 24-h exposure.

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

    PubMed Central

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

    2015-01-01

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

  14. The evolution of substrate specificity-associated residues and Ca(2+) -binding motifs in EF-hand-containing type II NAD(P)H dehydrogenases.

    PubMed

    Hao, Meng-Shu; Rasmusson, Allan G

    2016-07-01

    Most eukaryotic organisms, except some animal clades, have mitochondrial alternative electron transport enzymes that allow respiration to bypass the energy coupling in oxidative phosphorylation. The energy bypass enzymes in plants include the external type II NAD(P)H dehydrogenases (DHs) of the NDB family, which are characterized by an EF-hand domain for Ca(2+) binding. Here we investigate these plant enzymes by combining molecular modeling with evolutionary analysis. Molecular modeling of the Arabidopsis thaliana AtNDB1 with the yeast ScNDI1 as template revealed distinct similarities in the core catalytic parts, and highlighted the interaction between the pyridine nucleotide and residues correlating with NAD(P)H substrate specificity. The EF-hand domain of AtNDB1 has no counterpart in ScNDI1, and was instead modeled with Ca(2+) -binding signal transducer proteins. Combined models displayed a proximity of the AtNDB1 EF-hand domain to the substrate entrance side of the catalytic part. Evolutionary analysis of the eukaryotic NDB-type proteins revealed ancient and recent reversions between the motif observed in proteins specific for NADH (acidic type) and NADPH (non-acidic type), and that the clade of enzymes with acidic motifs in angiosperms derives from non-acidic-motif NDB-type proteins present in basal plants, fungi and protists. The results suggest that Ca(2+) -dependent external NADPH oxidation is an ancient process, indicating that it has a fundamental importance for eukaryotic cellular redox metabolism. In contrast, the external NADH DHs in plants are products of a recent expansion, mirroring the expansion of the alternative oxidase family.

  15. Transgenic expression of 11beta-hydroxysteroid dehydrogenase type 2 in osteoblasts reveals an anabolic role for endogenous glucocorticoids in bone.

    PubMed

    Sher, Lorin B; Woitge, Henning W; Adams, Douglas J; Gronowicz, Gloria A; Krozowski, Zygmunt; Harrison, John R; Kream, Barbara E

    2004-02-01

    Glucocorticoid excess leads to bone loss, primarily by decreasing bone formation. However, a variety of in vitro models show that glucocorticoids can promote osteogenesis. To elucidate the role of endogenous glucocorticoids in bone metabolism, we developed transgenic (TG) mice in which a 2.3-kb Col1a1 promoter fragment drives 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) expression in mature osteoblasts. 11beta-HSD2 should metabolically inactivate endogenous glucocorticoids in the targeted cells, thereby reducing glucocorticoid signaling. The inhibitory effect of 300 nm hydrocortisone on percent collagen synthesis was blunted in TG calvariae, demonstrating that the transgene was active. Collagen synthesis rates were lower in TG calvarial organ cultures compared with wild-type. Trabecular bone parameters measured by microcomputed tomography were reduced in L3 vertebrae, but not femurs, of 7- and 24-wk-old TG females. These changes were also not seen in males. In addition, histomorphometry showed that osteoid surface was increased in TG female vertebrae, suggesting that mineralization may be impaired. Our data demonstrate that endogenous glucocorticoid signaling is required for normal vertebral trabecular bone volume and architecture in female mice.

  16. Ginsenoside Rh1 eliminates the cytoprotective phenotype of human immunodeficiency virus type 1-transduced human macrophages by inhibiting the phosphorylation of pyruvate dehydrogenase lipoamide kinase isozyme 1.

    PubMed

    Jeong, Jin-Ju; Kim, Baek; Kim, Dong-Hyun

    2013-01-01

    Red ginseng (the steamed root of Panax ginseng C.A. MEYER, Araliaceae), which contains ginsenosides as its main constituents, is frequently used to treat tumor, inflammation, diabetes, stress and acquired immunodeficiency syndrome in Asian countries. Of these ginsenosides, only protopanaxadiol compound K has been reported to abolish the cytoprotective phenotype of human immunodeficiency virus type 1 (HIV-1)-transfected human macrophages. Here, we investigated the anti-cytoprotective effect of protopanaxatriol ginsenoside Rh1 on Tat-expressing cytoprotective CHME5 cells and D3-infected human primary macrophages. Treatment with ginsenoside Rh1 in the presence of lipopolysaccharide/cycloheximide (LPS/CHX) potently abolished the cytoprotective phenotype of Tat-transduced CHME5 cells as well as D3-infected human primary macrophages. Ginsenoside Rh1 significantly inhibited LPS/CHX-induced Akt phosphorylation, as well as mammalian target of rapamycin and Bcl-2-associated death promoter activation in both cell types. Furthermore, ginsenoside Rh1 inhibited pyruvate dehydrogenase lipoamide kinase isozyme 1 (PDK-1) phosphorylation. However, ginsenoside Rh1 did not inhibit phosphoinositide 3-kinase phosphorylation. Ginsenosides Rh1 in the presence of miltefosine (5 µM) additively increased the anti-cytoprotective activity against HIV-1 Tat-expressing macrophages. On the basis of these findings, we propose that ginsenoside Rh1 could possibly eliminate HIV-1 infected macrophages by inhibiting the PDK1/Akt pathway. PMID:23811558

  17. Fetal brain 11β-hydroxysteroid dehydrogenase type 2 selectively determines programming of adult depressive-like behaviors and cognitive function, but not anxiety behaviors in male mice.

    PubMed

    Wyrwoll, Caitlin; Keith, Marianne; Noble, June; Stevenson, Paula L; Bombail, Vincent; Crombie, Sandra; Evans, Louise C; Bailey, Matthew A; Wood, Emma; Seckl, Jonathan R; Holmes, Megan C

    2015-09-01

    Stress or elevated glucocorticoids during sensitive windows of fetal development increase the risk of neuropsychiatric disorders in adult rodents and humans, a phenomenon known as glucocorticoid programming. 11β-Hydroxysteroid dehydrogenase type 2 (11β-HSD2), which catalyses rapid inactivation of glucocorticoids in the placenta, controls access of maternal glucocorticoids to the fetal compartment, placing it in a key position to modulate glucocorticoid programming of behavior. However, the importance of the high expression of 11β-HSD2 within the midgestational fetal brain is unknown. To examine this, a brain-specific knockout of 11β-HSD2 (HSD2BKO) was generated and compared to wild-type littermates. HSD2BKO have markedly diminished fetal brain 11β-HSD2, but intact fetal body and placental 11β-HSD2 and normal fetal and placental growth. Despite normal fetal plasma corticosterone, HSD2BKO exhibit elevated fetal brain corticosterone levels at midgestation. As adults, HSD2BKO show depressive-like behavior and have cognitive impairments. However, unlike complete feto-placental deficiency, HSD2BKO show no anxiety-like behavioral deficits. The clear mechanistic separation of the programmed components of depression and cognition from anxiety implies distinct mechanisms of pathogenesis, affording potential opportunities for stratified interventions.

  18. Optimization of brain penetrant 11β-hydroxysteroid dehydrogenase type I inhibitors and in vivo testing in diet-induced obese mice.

    PubMed

    Goldberg, Frederick W; Dossetter, Alexander G; Scott, James S; Robb, Graeme R; Boyd, Scott; Groombridge, Sam D; Kemmitt, Paul D; Sjögren, Tove; Gutierrez, Pablo Morentin; deSchoolmeester, Joanne; Swales, John G; Turnbull, Andrew V; Wild, Martin J

    2014-02-13

    11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) has been widely considered by the pharmaceutical industry as a target to treat metabolic syndrome in type II diabetics. We hypothesized that central nervous system (CNS) penetration might be required to see efficacy. Starting from a previously reported pyrimidine compound, we removed hydrogen-bond donors to yield 3, which had modest CNS penetration. More significant progress was achieved by changing the core to give 40, which combines good potency and CNS penetration. Compound 40 was dosed to diet-induced obese (DIO) mice and gave excellent target engagement in the liver and high free exposures of drug, both peripherally and in the CNS. However, no body weight reduction or effects on glucose or insulin were observed in this model. Similar data were obtained with a structurally diverse thiazole compound 51. This work casts doubt on the hypothesis that localized tissue modulation of 11β-HSD1 activity alleviates metabolic syndrome.

  19. Proliferative responses to altered 17beta-hydroxysteroid dehydrogenase (17HSD) type 2 expression in human breast cancer cells are dependent on endogenous expression of 17HSD type 1 and the oestradiol receptors.

    PubMed

    Jansson, A; Gunnarsson, C; Stål, O

    2006-09-01

    The primary source of oestrogen in premenopausal women is the ovary but, after menopause, oestrogen biosynthesis in peripheral tissue is the exclusive site of formation. An enzyme group that affects the availability of active oestrogens is the 17beta-hydroxysteroid dehydrogenase (17HSD) family. In breast cancer, 17HSD type 1 and type 2 have been mostly investigated and seem to be the principal 17HSD enzymes involved thus far. The question whether 17HSD type 1 or type 2 is of greatest importance in breast tumour development is still not clear. The aim of this study was to investigate how the loss of 17HSD type 2 expression, using siRNA in the non-tumour breast epithelial cells HMEC (human mammal epithelial cells) and MCF10A, and gain of 17HSD type 2 expression, using transient transfection in the breast cancer derived cell lines MCF7 and T47D, affect oestradiol conversion and proliferation rate measured as S-phase fraction. We further investigated how this was related to the endogenous expression of 17HSD type 1 and oestradiol receptors in the examined cell lines. The oestradiol level in the medium changed significantly in the MCF7 transfected cells and the siRNA-treated HMEC cells, but not in T47D or MCF10A. The S-phase fraction decreased in the 17HSD type 2-transfected MCF7 cells and the siRNA-treated HMEC cells. The results seemed to be dependent on the endogenous expression of 17HSD type 1 and the oestradiol receptors. In conclusion, we found that high or low levels of 17HSD type 2 affected the oestradiol concentration significantly. However, the response was dependent on the endogenous expression of 17HSD type 1. Expression of 17HSD type 1 seems to be dominant to 17HSD type 2. Therefore, it may be important to investigate a ratio between 17HSD type 1 and 17HSD type 2.

  20. NdhV subunit regulates the activity of type-1 NAD(P)H dehydrogenase under high light conditions in cyanobacterium Synechocystis sp. PCC 6803

    PubMed Central

    Chen, Xin; He, Zhihui; Xu, Min; Peng, Lianwei; Mi, Hualing

    2016-01-01

    The cyanobacterial NAD(P)H dehydrogenase (NDH-1) complexes play crucial roles in variety of bioenergetic reactions. However, the regulative mechanism of NDH-1 under stressed conditions is still unclear. In this study, we detected that the NDH-1 activity is partially impaired, but the accumulation of NDH-1 complexes was little affected in the NdhV deleted mutant (ΔndhV) at low light in cyanobacterium Synechocystis sp. PCC 6803. ΔndhV grew normally at low light but slowly at high light under inorganic carbon limitation conditions (low pH or low CO2), meanwhile the activity of CO2 uptake was evidently lowered than wild type even at pH 8.0. The accumulation of NdhV in thylakoids strictly relies on the presence of the hydrophilic subcomplex of NDH-1. Furthermore, NdhV was co-located with hydrophilic subunits of NDH-1 loosely associated with the NDH-1L, NDH-1MS′ and NDH-1M complexes. The level of the NdhV was significantly increased at high light and deletion of NdhV suppressed the up-regulation of NDH-1 activity, causing the lowered the photosynthetic oxygen evolution at pH 6.5 and high light. These data indicate that NdhV is an intrinsic subunit of hydrophilic subcomplex of NDH-1, required for efficient operation of cyclic electron transport around photosystem I and CO2 uptake at high lights. PMID:27329499

  1. Multiple Acyl-CoA Dehydrogenation Deficiency (Glutaric Aciduria Type II) with a Novel Mutation of Electron Transfer Flavoprotein-Dehydrogenase in a Cat.

    PubMed

    Wakitani, Shoichi; Torisu, Shidow; Yoshino, Taiki; Hattanda, Kazuhisa; Yamato, Osamu; Tasaki, Ryuji; Fujita, Haruo; Nishino, Koichiro

    2014-01-01

    Multiple acyl-CoA dehydrogenation deficiency (MADD; also known as glutaric aciduria type II) is a human autosomal recessive disease classified as one of the mitochondrial fatty-acid oxidation disorders. MADD is caused by a defect in the electron transfer flavoprotein (ETF) or ETF dehydrogenase (ETFDH) molecule, but as yet, inherited MADD has not been reported in animals. Here we present the first report of MADD in a cat. The affected animal presented with symptoms characteristic of MADD including hypoglycemia, hyperammonemia, vomiting, diagnostic organic aciduria, and accumulation of medium- and long-chain fatty acids in plasma. Treatment with riboflavin and L-carnitine ameliorated the symptoms. To detect the gene mutation responsible for MADD in this case, we determined the complete cDNA sequences of feline ETFα, ETFβ, and ETFDH. Finally, we identified the feline patient-specific mutation, c.692T>G (p.F231C) in ETFDH. The affected animal only carries mutant alleles of ETFDH. p.F231 in feline ETFDH is completely conserved in eukaryotes, and is located on the apical surface of ETFDH, receiving electrons from ETF. This study thus identified the mutation strongly suspected to have been the cause of MADD in this cat. PMID:24142280

  2. Cognitive and Disease-Modifying Effects of 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibition in Male Tg2576 Mice, a Model of Alzheimer's Disease

    PubMed Central

    Sooy, Karen; Noble, June; McBride, Andrew; Binnie, Margaret; Yau, Joyce L. W.; Seckl, Jonathan R.; Walker, Brian R.

    2015-01-01

    Chronic exposure to elevated levels of glucocorticoids has been linked to age-related cognitive decline and may play a role in Alzheimer's disease. In the brain, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) amplifies intracellular glucocorticoid levels. We show that short-term treatment of aged, cognitively impaired C57BL/6 mice with the potent and selective 11β-HSD1 inhibitor UE2316 improves memory, including after intracerebroventricular drug administration to the central nervous system alone. In the Tg2576 mouse model of Alzheimer's disease, UE2316 treatment of mice aged 14 months for 4 weeks also decreased the number of β-amyloid (Aβ) plaques in the cerebral cortex, associated with a selective increase in local insulin-degrading enzyme (involved in Aβ breakdown and known to be glucocorticoid regulated). Chronic treatment of young Tg2576 mice with UE2316 for up to 13 months prevented cognitive decline but did not prevent Aβ plaque formation. We conclude that reducing glucocorticoid regeneration in the brain improves cognition independently of reduced Aβ plaque pathology and that 11β-HSD1 inhibitors have potential as cognitive enhancers in age-associated memory impairment and Alzheimer's dementia. PMID:26305888

  3. NdhV subunit regulates the activity of type-1 NAD(P)H dehydrogenase under high light conditions in cyanobacterium Synechocystis sp. PCC 6803.

    PubMed

    Chen, Xin; He, Zhihui; Xu, Min; Peng, Lianwei; Mi, Hualing

    2016-01-01

    The cyanobacterial NAD(P)H dehydrogenase (NDH-1) complexes play crucial roles in variety of bioenergetic reactions. However, the regulative mechanism of NDH-1 under stressed conditions is still unclear. In this study, we detected that the NDH-1 activity is partially impaired, but the accumulation of NDH-1 complexes was little affected in the NdhV deleted mutant (ΔndhV) at low light in cyanobacterium Synechocystis sp. PCC 6803. ΔndhV grew normally at low light but slowly at high light under inorganic carbon limitation conditions (low pH or low CO2), meanwhile the activity of CO2 uptake was evidently lowered than wild type even at pH 8.0. The accumulation of NdhV in thylakoids strictly relies on the presence of the hydrophilic subcomplex of NDH-1. Furthermore, NdhV was co-located with hydrophilic subunits of NDH-1 loosely associated with the NDH-1L, NDH-1MS' and NDH-1M complexes. The level of the NdhV was significantly increased at high light and deletion of NdhV suppressed the up-regulation of NDH-1 activity, causing the lowered the photosynthetic oxygen evolution at pH 6.5 and high light. These data indicate that NdhV is an intrinsic subunit of hydrophilic subcomplex of NDH-1, required for efficient operation of cyclic electron transport around photosystem I and CO2 uptake at high lights. PMID:27329499

  4. Mechanism of repression of 11β-hydroxysteroid dehydrogenase type 1 by growth hormone in 3T3-L1 adipocytes.

    PubMed

    Muraoka, Toko; Hizuka, Naomi; Fukuda, Izumi; Ishikawa, Yukiko; Ichihara, Atsuhiro

    2014-01-01

    11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is an NADPH-dependent reductase that converts cortisone to cortisol in adipose tissue. We previously reported that GH and IGF-I decrease 11β-HSD1 activity and mRNA levels in adipocytes. Hexose-6-phosphate dehydrogenase (H6PDH) is involved in the production of NADPH, which is a coenzyme for 11β-HSD1. The aim of the present study was to clarify further the mechanism of repression of 11β-HSD1 activity by GH using linsitinib, an IGF-I receptor inhibitor. The suppression of 11β-HSD1 mRNA by IGF-I was attenuated in the presence of 1 μM linsitinib (17.2% vs. 53.3% of basal level, P<0.05). 11β-HSD1 mRNA levels in cells treated with GH in the presence of 1 μM linsitinib were not different from those in absence of linsitinib (35.9% vs. 33.9%). The increase in IGF-I mRNA levels with GH and 1 μM linsitinib was not different from that in the absence of linsitinib (359% vs. 347%). H6PDH mRNA levels were significantly decreased in cells treated with IGF-I for 8 and 24 h (55.6% and 33.7%, P<0.05). In the presence of 1 μM linsitinib, there was no repression of H6PDH mRNA (111.4%). H6PDH mRNA levels were significantly decreased in cells treated with GH in the absence of linsitinib for 24 h (55.9%, P<0.05), but not for 8 h (89.5%). The presence of 1 μM linsitinib also prevented repression of H6PDH mRNA by GH over 24 h (107.8%). These results suggest that GH directly represses 11β-HSD1 mRNA rather than acting via the IGF-I receptor, and that GH represses H6PDH through locally produced IGF-I. PMID:24759003

  5. The Type II NADPH Dehydrogenase Facilitates Cyclic Electron Flow, Energy-Dependent Quenching, and Chlororespiratory Metabolism during Acclimation of Chlamydomonas reinhardtii to Nitrogen Deprivation.

    PubMed

    Saroussi, Shai I; Wittkopp, Tyler M; Grossman, Arthur R

    2016-04-01

    When photosynthetic organisms are deprived of nitrogen (N), the capacity to grow and assimilate carbon becomes limited, causing a decrease in the productive use of absorbed light energy and likely a rise in the cellular reduction state. Although there is a scarcity of N in many terrestrial and aquatic environments, a mechanistic understanding of how photosynthesis adjusts to low-N conditions and the enzymes/activities integral to these adjustments have not been described. In this work, we use biochemical and biophysical analyses of photoautotrophically grown wild-type and mutant strains of Chlamydomonas reinhardtii to determine the integration of electron transport pathways critical for maintaining active photosynthetic complexes even after exposure of cells to N deprivation for 3 d. Key to acclimation is the type II NADPH dehydrogenase, NDA2, which drives cyclic electron flow (CEF), chlororespiration, and the generation of an H(+) gradient across the thylakoid membranes. N deprivation elicited a doubling of the rate of NDA2-dependent CEF, with little contribution from PGR5/PGRL1-dependent CEF The H(+) gradient generated by CEF is essential to sustain nonphotochemical quenching, while an increase in the level of reduced plastoquinone would promote a state transition; both are necessary to down-regulate photosystem II activity. Moreover, stimulation of NDA2-dependent chlororespiration affords additional relief from the elevated reduction state associated with N deprivation through plastid terminal oxidase-dependent water synthesis. Overall, rerouting electrons through the NDA2 catalytic hub in response to photoautotrophic N deprivation sustains cell viability while promoting the dissipation of excess excitation energy through quenching and chlororespiratory processes. PMID:26858365

  6. The Type II NADPH Dehydrogenase Facilitates Cyclic Electron Flow, Energy-Dependent Quenching, and Chlororespiratory Metabolism during Acclimation of Chlamydomonas reinhardtii to Nitrogen Deprivation1[OPEN

    PubMed Central

    Grossman, Arthur R.

    2016-01-01

    When photosynthetic organisms are deprived of nitrogen (N), the capacity to grow and assimilate carbon becomes limited, causing a decrease in the productive use of absorbed light energy and likely a rise in the cellular reduction state. Although there is a scarcity of N in many terrestrial and aquatic environments, a mechanistic understanding of how photosynthesis adjusts to low-N conditions and the enzymes/activities integral to these adjustments have not been described. In this work, we use biochemical and biophysical analyses of photoautotrophically grown wild-type and mutant strains of Chlamydomonas reinhardtii to determine the integration of electron transport pathways critical for maintaining active photosynthetic complexes even after exposure of cells to N deprivation for 3 d. Key to acclimation is the type II NADPH dehydrogenase, NDA2, which drives cyclic electron flow (CEF), chlororespiration, and the generation of an H+ gradient across the thylakoid membranes. N deprivation elicited a doubling of the rate of NDA2-dependent CEF, with little contribution from PGR5/PGRL1-dependent CEF. The H+ gradient generated by CEF is essential to sustain nonphotochemical quenching, while an increase in the level of reduced plastoquinone would promote a state transition; both are necessary to down-regulate photosystem II activity. Moreover, stimulation of NDA2-dependent chlororespiration affords additional relief from the elevated reduction state associated with N deprivation through plastid terminal oxidase-dependent water synthesis. Overall, rerouting electrons through the NDA2 catalytic hub in response to photoautotrophic N deprivation sustains cell viability while promoting the dissipation of excess excitation energy through quenching and chlororespiratory processes. PMID:26858365

  7. Anti-Inflammatory Effects of Levalbuterol-Induced 11β-Hydroxysteroid Dehydrogenase Type 1 Activity in Airway Epithelial Cells

    PubMed Central

    Randall, Matthew J.; Kostin, Shannon F.; Burgess, Edward J.; Hoyt, Laura R.; Ather, Jennifer L.; Lundblad, Lennart K.; Poynter, Matthew E.

    2015-01-01

    production to an extent similar to that of dexamethasone. These results demonstrate that levalbuterol augments expression of 11β-HSD1 in airway epithelial cells, reducing LPS-induced NF-κB transcriptional activity and pro-inflammatory cytokine production through the conversion of inactive 11-keto corticosteroids into the active 11-hydroxy form in this cell type. PMID:25628603

  8. Gene expression patterns in the hippocampus during the development and aging of Glud1 (Glutamate Dehydrogenase 1) transgenic and wild type mice

    PubMed Central

    2014-01-01

    Background Extraneuronal levels of the neurotransmitter glutamate in brain rise during aging. This is thought to lead to synaptic dysfunction and neuronal injury or death. To study the effects of glutamate hyperactivity in brain, we created transgenic (Tg) mice in which the gene for glutamate dehydrogenase (Glud1) is over-expressed in neurons and in which such overexpression leads to excess synaptic release of glutamate. In this study, we analyzed whole genome expression in the hippocampus, a region important for learning and memory, of 10 day to 20 month old Glud1 and wild type (wt) mice. Results During development, maturation and aging, both Tg and wt exhibited decreases in the expression of genes related to neurogenesis, neuronal migration, growth, and process elongation, and increases in genes related to neuro-inflammation, voltage-gated channel activity, and regulation of synaptic transmission. Categories of genes that were differentially expressed in Tg vs. wt during development were: synaptic function, cytoskeleton, protein ubiquitination, and mitochondria; and, those differentially expressed during aging were: synaptic function, vesicle transport, calcium signaling, protein kinase activity, cytoskeleton, neuron projection, mitochondria, and protein ubiquitination. Overall, the effects of Glud1 overexpression on the hippocampus transcriptome were greater in the mature and aged than the young. Conclusions Glutamate hyperactivity caused gene expression changes in the hippocampus at all ages. Some of these changes may result in premature brain aging. The identification of these genomic expression differences is important in understanding the effects of glutamate dysregulation on neuronal function during aging or in neurodegenerative diseases. PMID:24593767

  9. Up-regulation of 11β-Hydroxysteroid Dehydrogenase Type 2 Expression by Hedgehog Ligand Contributes to the Conversion of Cortisol Into Cortisone.

    PubMed

    Zhu, Haibin; Zou, Chaochun; Fan, Xueying; Xiong, Wenyi; Tang, Lanfang; Wu, Ximei; Tang, Chao

    2016-09-01

    The cortisol-inactivating enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) that catalyzes the intracellular inactivation of glucocorticoids plays a pivotal role in human pregnant maintenance and normal fetal development. Given the fact that the main components of Hedgehog (HH) signaling pathway are predominantly expressed in syncytial layer of human placental villi where 11β-HSD2 is robustly expressed, in the present study, we have investigated the potential roles and underlying mechanisms of HH signaling in 11β-HSD2 expression. Activation of HH signaling by a variety of approaches robustly induced 11β-HSD2 expression as well as the 11β-HSD2 activity, whereas suppression of HH signaling significantly attenuated 11β-HSD2 expression as well as the 11β-HSD2 activity in both human primary cytotrophoblasts and trophoblast-like BeWo cells. Moreover, among glioma-associated oncogene (GLI) family transcriptional factors in HH signaling, knockdown of GLI2 but not GLI1 and GLI3 significantly attenuated HH-induced 11β-HSD2 expression and activity, and overexpression of GLI2 activator alone was sufficient to induce 11β-HSD2 expression and activity. Finally, GLI2 not only directly bound to the promoter region of gene hsd11b2 to transactivate hsd11b2 but also formed a heterodimer with RNA polymerase II, an enzyme that catalyzes the transcription of DNA to synthesize mRNAs, resulting in up-regulation of hsd11b2 gene transcription. Taken together, the present study has uncovered a hitherto uncharacterized role of HH/GLI2 signaling in 11β-HSD2 regulation, implicating that HH signaling through GLI2 could be required for the human pregnant maintenance and fetal development. PMID:27379371

  10. Molecular cloning and characterization of a steroidogenic enzyme, 17β-hydroxysteroid dehydrogenase type 14, from the stony coral Euphyllia ancora (Cnidaria, Anthozoa).

    PubMed

    Shikina, Shinya; Chung, Yi-Jou; Chiu, Yi-Ling; Huang, Yi-Jie; Lee, Yan-Horn; Chang, Ching-Fong

    2016-03-01

    Sex steroids play a fundamental role not only in reproduction but also in various other biological processes in vertebrates. Although the presence of sex steroids has been confirmed in cnidarians (e.g., coral, sea anemone, jellyfish, and hydra), which are basal metazoans, only a few studies to date have characterized steroidogenesis-related genes in cnidarians. Based on a transcriptomic analysis of the stony coral Euphyllia ancora, we identified the steroidogenic enzyme 17β-hydroxysteroid dehydrogenase type 14 (17beta-hsd 14), an oxidative enzyme that catalyzes the NAD(+)-dependent inactivation of estrogen/androgen (estradiol to estrone and testosterone to androstenedione) in mammals. Phylogenetic analysis showed that E. ancora 17beta-Hsd 14 (Ea17beta-Hsd 14) clusters with other animal 17beta-HSD 14s but not with other members of the 17beta-HSD family. Subsequent quantitative RT-PCR analysis revealed a lack of correlation of Ea17beta-hsd 14 transcript levels with the coral's reproductive cycle. In addition, Ea17beta-hsd 14 transcript and protein were detected in all tissues examined, such as the tentacles, mesenterial filaments, and gonads, at similar levels in both sexes, as determined by quantitative RT-PCR analysis and Western blotting with an anti-Ea17beta-Hsd 14 antibody. Immunohistochemical analysis revealed that Ea17beta-Hsd 14 is mainly distributed in the endodermal regions of the polyps, but the protein was also observed in all tissues examined. These results suggest that Ea17beta-Hsd 14 is involved in important functions that commonly occur in endodermal cells or has multiple functions in different tissues. Our data provide information for comparison with advanced animals as well as insight into the evolution of steroidogenesis-related genes in metazoans. PMID:26868454

  11. Short-term inhibition of 11β-hydroxysteroid dehydrogenase type 1 reversibly improves spatial memory but persistently impairs contextual fear memory in aged mice.

    PubMed

    Wheelan, Nicola; Webster, Scott P; Kenyon, Christopher J; Caughey, Sarah; Walker, Brian R; Holmes, Megan C; Seckl, Jonathan R; Yau, Joyce L W

    2015-04-01

    High glucocorticoid levels induced by stress enhance the memory of fearful events and may contribute to the development of anxiety and posttraumatic stress disorder. In contrast, elevated glucocorticoids associated with ageing impair spatial memory. We have previously shown that pharmacological inhibition of the intracellular glucocorticoid-amplifying enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) improves spatial memory in aged mice. However, it is not known whether inhibition of 11β-HSD1 will have any beneficial effects on contextual fear memories in aged mice. Here, we examined the effects of UE2316, a selective 11β-HSD1 inhibitor which accesses the brain, on both spatial and contextual fear memories in aged mice using a vehicle-controlled crossover study design. Short-term UE2316 treatment improved spatial memory in aged mice, an effect which was reversed when UE2316 was substituted with vehicle. In contrast, contextual fear memory induced by foot-shock conditioning was significantly reduced by UE2316 in a non-reversible manner. When the order of treatment was reversed following extinction of the original fear memory, and a second foot-shock conditioning was given in a novel context, UE2316 treated aged mice (previously on vehicle) now showed increased fear memory compared to vehicle-treated aged mice (previously on UE2316). Renewal of the original extinguished fear memory triggered by exposure to a new environmental context may explain these effects. Thus 11β-HSD1 inhibition reverses spatial memory impairments with ageing while reducing the strength and persistence of new contextual fear memories. Potentially this could help prevent anxiety-related disorders in vulnerable elderly individuals.

  12. Repeated maternal dexamethasone treatments in late gestation increases 11beta-hydroxysteroid dehydrogenase type 1 expression in the hippocampus of the newborn rat.

    PubMed

    Wan, Shunlun; Hao, Rusong; Sun, Kang

    This study was designed to investigate the effect of repeated maternal injections of dexamethasone in late gestation on the expression of newborn hippocampal 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), the enzyme amplifying glucocorticoids' action by converting biologically inactive 11-ketone metabolites into active glucocorticoids. Daily dexamethasone treatments (0.10 mg/kg body weight) in the last week of gestation were carried out in the pregnant rat. The expression of 11beta-HSD1 in the newborn hippocampal tissue was analyzed with Western blot and real-time polymerase chain reaction (PCR). The effect of corticosterone on the expression of 11beta-HSD1 was studied in cultured hippocampal neurons derived from newborn offspring received prenatal dexamethasone treatments. Both body and brain weights of the offspring were reduced significantly by repeated dexamethasone treatments in the last week of gestation. Western blot and real-time PCR analysis showed that both 11beta-HSD1 protein and mRNA expressions were increased significantly in the hippocampus of the newborn offspring on the first and seventh days after birth. Corticosterone could induce 11beta-HSD1 expression in cultured hippocampal neurons prepared from newborns received prenatal dexamethasone treatments, which was blocked by glucocorticoid receptor antagonist RU38486. The above findings suggest that repeated prenatal dexamethasone treatments at the end of gestation increase 11beta-HSD1 expression in the hippocampal tissue of the offspring, which may trigger a positive feedback pathway for the generation of biologically active glucocorticoids in the hippocampal tissue of the newborns.

  13. Elevation of 11β-hydroxysteroid dehydrogenase type 2 activity in Holocaust survivor offspring: evidence for an intergenerational effect of maternal trauma exposure

    PubMed Central

    Bierer, Linda M.; Bader, Heather N.; Daskalakis, Nikolaos P.; Lehrner, Amy; Makotkine, Iouri; Seckl, Jonathan R.; Yehuda, Rachel

    2014-01-01

    Background Adult offspring of Holocaust survivors comprise an informative cohort in which to study intergenerational transmission of the effects of trauma exposure. Lower cortisol and enhanced glucocorticoid sensitivity have been previously demonstrated in Holocaust survivors with PTSD, and in offspring of Holocaust survivors in association with maternal PTSD. In other work, reduction in the activity of the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD-2), which inactivates cortisol, was identified in Holocaust survivors in comparison to age-matched, unexposed Jewish controls. Therefore, we investigated glucocorticoid metabolism in offspring of Holocaust survivors to evaluate if similar enzymatic decrements would be observed that might help to explain glucocorticoid alterations previously shown for Holocaust offspring. Methods Holocaust offspring (n=85) and comparison subjects (n=27) were evaluated with clinical diagnostic interview and self-rating scales, and asked to collect a 24-hr urine sample from which concentrations of cortisol and glucocorticoid metabolites were assayed by GCMS. 11β-HSD-2 activity was determined as the ratio of urinary cortisone to cortisol. Results Significantly reduced cortisol excretion was observed in Holocaust offspring compared to controls (p=.046), as had been shown for Holocaust survivors. However, 11β-HSD-2 activity was elevated for offspring compared to controls (p=.008), particularly among those whose mothers had been children, rather than adolescents or adults, during World War II (p=.032). The effect of paternal Holocaust exposure could not be reliably investigated in the current sample. Conclusions The association of offspring 11β-HSD-2 activity with maternal age at Holocaust exposure is consistent with the influence of glucocorticoid programming. Whereas a long standing reduction in 11β-HSD-2 activity among survivors is readily interpreted in the context of Holocaust related deprivation, understanding the

  14. Promoter Methylation Status of Breast Cancer Susceptibility Gene 1 and 17 Beta Hydroxysteroid Dehydrogenase Type 1 Gene in Sporadic Breast Cancer Patients

    PubMed Central

    Hosny, Marwa M.; Sabek, Nagwan A.; El-Abaseri, Taghrid B.; Hassan, Fathalla M.; Farrag, Sherif H.

    2016-01-01

    Epigenetic modifications are involved in breast carcinogenesis. Identifying genes that are epigenetically silenced via methylation could select target patients for diagnostic as well as therapeutic potential. We assessed promoter methylation of breast cancer susceptibility gene 1 (BRCA1) and 17 Beta Hydroxysteroid Dehydrogenase Type 1 (17βHSD-1) in normal and cancer breast tissues of forty sporadic breast cancer (BC) cases using restriction enzyme based methylation-specific PCR (REMS-PCR). In cancerous tissues, BRCA1 and 17βHSD-1 were methylated in 42.5% and 97.5%, respectively, while normal tissues had 35% and 95% methylation, respectively. BRCA1 methylation in normal tissues was 12.2-fold more likely to associate with methylation in cancer tissues (p < 0.001). It correlated significantly with increased age at menopause, mitosis, the negative status of Her2, and the molecular subtype “luminal A” (p = 0.048, p = 0.042, p = 0.007, and p = 0.049, resp.). Methylation of BRCA1 and 17βHSD-1 related to luminal A subtype of breast cancer. Since a small proportion of normal breast epithelial cells had BRCA1 methylation, our preliminary findings suggest that methylation of BRCA1 may be involved in breast tumors initiation and progression; therefore, it could be used as a biomarker for the early detection of sporadic breast cancer. Methylation of 17βHSD-1 in normal and cancer tissue could save patients the long term use of adjuvant antiestrogen therapies. PMID:27413552

  15. Plastidial Expression of Type II NAD(P)H Dehydrogenase Increases the Reducing State of Plastoquinones and Hydrogen Photoproduction Rate by the Indirect Pathway in Chlamydomonas reinhardtii1.

    PubMed

    Baltz, Anthony; Dang, Kieu-Van; Beyly, Audrey; Auroy, Pascaline; Richaud, Pierre; Cournac, Laurent; Peltier, Gilles

    2014-05-12

    Biological conversion of solar energy into hydrogen is naturally realized by some microalgae species due to a coupling between the photosynthetic electron transport chain and a plastidial hydrogenase. While promising for the production of clean and sustainable hydrogen, this process requires improvement to be economically viable. Two pathways, called direct and indirect photoproduction, lead to sustained hydrogen production in sulfur-deprived Chlamydomonas reinhardtii cultures. The indirect pathway allows an efficient time-based separation of O2 and H2 production, thus overcoming the O2 sensitivity of the hydrogenase, but its activity is low. With the aim of identifying the limiting step of hydrogen production, we succeeded in overexpressing the plastidial type II NAD(P)H dehydrogenase (NDA2). We report that transplastomic strains overexpressing NDA2 show an increased activity of nonphotochemical reduction of plastoquinones (PQs). While hydrogen production by the direct pathway, involving the linear electron flow from photosystem II to photosystem I, was not affected by NDA2 overexpression, the rate of hydrogen production by the indirect pathway was increased in conditions, such as nutrient limitation, where soluble electron donors are not limiting. An increased intracellular starch was observed in response to nutrient deprivation in strains overexpressing NDA2. It is concluded that activity of the indirect pathway is limited by the nonphotochemical reduction of PQs, either by the pool size of soluble electron donors or by the PQ-reducing activity of NDA2 in nutrient-limited conditions. We discuss these data in relation to limitations and biotechnological improvement of hydrogen photoproduction in microalgae. PMID:24820024

  16. 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

  17. 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

  18. Detection of 11 beta-hydroxysteroid dehydrogenase type 1, the glucocorticoid and mineralocorticoid receptor in various adipose tissue depots of dairy cows supplemented with conjugated linoleic acids.

    PubMed

    Friedauer, K; Dänicke, S; Schulz, K; Sauerwein, H; Häussler, S

    2015-10-01

    Early lactating cows mobilize adipose tissue (AT) to provide energy for milk yield and maintenance and are susceptible to metabolic disorders and impaired immune response. Conjugated linoleic acids (CLA), mainly the trans-10, cis-12 isomer, reduce milk fat synthesis and may attenuate negative energy balance. Circulating glucocorticoids (GC) are increased during parturition in dairy cows and mediate differentiating and anti-inflammatory effects via glucocorticoid (GR) and mineralocorticoid receptors (MR) in the presence of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1). Activated GC are the main ligands for both receptors in AT; therefore, we hypothesized that tissue-specific GC metabolism is effected by varying amounts of GR, MR and 11βHSD1 and/or their localization within AT depots. Furthermore, the lipolytic and antilipogenic effects of CLA might influence the GC/GR/MR system in AT. Therefore, we aimed to localize GR and MR as well as the expression pattern and activity of 11βHSD1 in different AT depots during early lactation in dairy cows and to identify potential effects of CLA. Primiparous German Holstein cows were divided into a control (CON) and a CLA group. From day 1 post-partum (p.p.) until sample collection, the CLA group was fed with 100 g/d CLA (contains 10 g each of the cis-9, trans-11 and the trans-10, cis-12-CLA isomers). CON cows (n = 5 each) were slaughtered on day 1, 42 and 105 p.p., while CLA cows (n = 5 each) were slaughtered on day 42 and 105 p.p. Subcutaneous fat from tailhead, withers and sternum, and visceral fat from omental, mesenteric and retroperitoneal depots were sampled. The localization of GR and 11βHSD1 in mature adipocytes - being already differentiated - indicates that GC promote other effects via GR than differentiation. Moreover, MR were observed in the stromal vascular cell fraction and positively related to the pre-adipocyte marker Pref-1. However, only marginal CLA effects were observed in this study.

  19. Detection of 11 beta-hydroxysteroid dehydrogenase type 1, the glucocorticoid and mineralocorticoid receptor in various adipose tissue depots of dairy cows supplemented with conjugated linoleic acids.

    PubMed

    Friedauer, K; Dänicke, S; Schulz, K; Sauerwein, H; Häussler, S

    2015-10-01

    Early lactating cows mobilize adipose tissue (AT) to provide energy for milk yield and maintenance and are susceptible to metabolic disorders and impaired immune response. Conjugated linoleic acids (CLA), mainly the trans-10, cis-12 isomer, reduce milk fat synthesis and may attenuate negative energy balance. Circulating glucocorticoids (GC) are increased during parturition in dairy cows and mediate differentiating and anti-inflammatory effects via glucocorticoid (GR) and mineralocorticoid receptors (MR) in the presence of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1). Activated GC are the main ligands for both receptors in AT; therefore, we hypothesized that tissue-specific GC metabolism is effected by varying amounts of GR, MR and 11βHSD1 and/or their localization within AT depots. Furthermore, the lipolytic and antilipogenic effects of CLA might influence the GC/GR/MR system in AT. Therefore, we aimed to localize GR and MR as well as the expression pattern and activity of 11βHSD1 in different AT depots during early lactation in dairy cows and to identify potential effects of CLA. Primiparous German Holstein cows were divided into a control (CON) and a CLA group. From day 1 post-partum (p.p.) until sample collection, the CLA group was fed with 100 g/d CLA (contains 10 g each of the cis-9, trans-11 and the trans-10, cis-12-CLA isomers). CON cows (n = 5 each) were slaughtered on day 1, 42 and 105 p.p., while CLA cows (n = 5 each) were slaughtered on day 42 and 105 p.p. Subcutaneous fat from tailhead, withers and sternum, and visceral fat from omental, mesenteric and retroperitoneal depots were sampled. The localization of GR and 11βHSD1 in mature adipocytes - being already differentiated - indicates that GC promote other effects via GR than differentiation. Moreover, MR were observed in the stromal vascular cell fraction and positively related to the pre-adipocyte marker Pref-1. However, only marginal CLA effects were observed in this study. PMID

  20. The M405V allele of the glutaryl-CoA dehydrogenase gene is an important marker for glutaric aciduria type I (GA-I) low excretors.

    PubMed

    Schillaci, Lori-Anne P; Greene, Carol L; Strovel, Erin; Rispoli-Joines, Jessica; Spector, Elaine; Woontner, Michael; Scharer, Gunter; Enns, Gregory M; Gallagher, Renata; Zinn, Arthur B; McCandless, Shawn E; Hoppel, Charles L; Goodman, Stephen I; Bedoyan, Jirair K

    2016-09-01

    Glutaric aciduria type I (GA-I) is an autosomal recessive organic aciduria resulting from a functional deficiency of glutaryl-CoA dehydrogenase, encoded by GCDH. Two clinically indistinguishable diagnostic subgroups of GA-I are known; low and high excretors (LEs and HEs, respectively). Early medical and dietary interventions can result in significantly better outcomes and improved quality of life for patients with GA-I. We report on nine cases of GA-I LE patients all sharing the M405V allele with two cases missed by newborn screening (NBS) using tandem mass spectrometry (MS/MS). We describe a novel case with the known pathogenic M405V variant and a novel V133L variant, and present updated and previously unreported clinical, biochemical, functional and molecular data on eight other patients all sharing the M405V allele. Three of the nine patients are of African American ancestry, with two as siblings. GCDH activity was assayed in six of the nine patients and varied from 4 to 25% of the control mean. We support the use of urine glutarylcarnitine as a biochemical marker of GA-I by demonstrating that glutarylcarnitine is efficiently cleared by the kidney (50-90%) and that plasma and urine glutarylcarnitine follow a linear relationship. We report the allele frequencies for three known GA-I LE GCDH variants (M405V, V400M and R227P) and note that both the M405V and V400M variants are significantly more common in the population of African ancestry compared to the general population. This report highlights the M405V allele as another important molecular marker in patients with the GA-I LE phenotype. Therefore, the incorporation into newborn screening of molecular screening for the M405V and V400M variants in conjunction with MS/MS could help identify asymptomatic at-risk GA-I LE patients that could potentially be missed by current NBS programs.

  1. Sustained photoevolution of molecular hydrogen in a mutant of Synechocystis sp. strain PCC 6803 deficient in the type I NADPH-dehydrogenase complex.

    PubMed

    Cournac, Laurent; Guedeney, Geneviève; Peltier, Gilles; Vignais, Paulette M

    2004-03-01

    The interaction between hydrogen metabolism, respiration, and photosynthesis was studied in vivo in whole cells of Synechocystis sp. strain PCC 6803 by continuously monitoring the changes in gas concentrations (H2, CO2, and O2) with an online mass spectrometer. The in vivo activity of the bidirectional [NiFe]hydrogenase [H2:NAD(P) oxidoreductase], encoded by the hoxEFUYH genes, was also measured independently by the proton-deuterium (H-D) exchange reaction in the presence of D2. This technique allowed us to demonstrate that the hydrogenase was insensitive to light, was reversibly inactivated by O2, and could be quickly reactivated by NADH or NADPH (+H2). H2 was evolved by cells incubated anaerobically in the dark, after an adaptation period. This dark H2 evolution was enhanced by exogenously added glucose and resulted from the oxidation of NAD(P)H produced by fermentation reactions. Upon illumination, a short (less than 30-s) burst of H2 output was observed, followed by rapid H2 uptake and a concomitant decrease in CO2 concentration in the cyanobacterial cell suspension. Uptake of both H2 and CO2 was linked to photosynthetic electron transport in the thylakoids. In the ndhB mutant M55, which is defective in the type I NADPH-dehydrogenase complex (NDH-1) and produces only low amounts of O2 in the light, H2 uptake was negligible during dark-to-light transitions, allowing several minutes of continuous H2 production. A sustained rate of photoevolution of H2 corresponding to 6 micro mol of H2 mg of chlorophyll(-1) h(-1) or 2 ml of H2 liter(-1) h(-1) was observed over a longer time period in the presence of glucose and was slightly enhanced by the addition of the O2 scavenger glucose oxidase. By the use of the inhibitors DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] and DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), it was shown that two pathways of electron supply for H2 production operate in M55, namely photolysis of water at the level of photosystem II and

  2. Regulation of human 3-beta-hydroxysteroid dehydrogenase type-2 (3βHSD2) by molecular chaperones and the mitochondrial environment affects steroidogenesis.

    PubMed

    Thomas, James L; Bose, Himangshu S

    2015-07-01

    Human 3-β-hydroxysteroid dehydrogenase/isomerase types 1 and 2 (3βHSD1 and 3βHSD2, respectively) are expressed in a tissue-specific pattern by different genes. Site-directed mutagenesis studies have confirmed the function of the catalytic amino acids (Tyr154, Lys 158, Ser124 in both isoenzymes), substrate/inhibitor isoform-specific residues (His156 and Arg195 in 3βHSD1) and cofactor binding residues (Asp36 provides NAD(+) specificity in both isoenzymes). However, detailed analysis of isoform-specific organelle localization and characterization is difficult due to the 93% amino acid identity between the two isoforms. With recent advances in the knowledge of mitochondrial architecture and localization of the various translocases, our laboratory has studied the mechanisms regulating mitochondrial 3βHSD2 localization. The mitochondrial N-terminal leader sequence of 3βHSD2 directs its entry into the mitochondria where it is localized to the intermembrane space (IMS). Unlike other mitochondrial proteins, the N-terminal signal sequence of 3βHSD2 is not cleaved upon mitochondrial import. 3βHSD2 interacts with the mitochondrial translocase, Tim50, to regulate progesterone and androstenedione formation. Our studies suggest that its activity at the IMS is facilitated in a partially unfolded "molten globule" conformation by the proton pump between the matrix and IMS. The unfolded protein is refolded by the mitochondrial chaperones. The protons at the IMS are absorbed by the lipid vesicles, to maintain the proton pump and recycle 3βHSD2. As a result, one molecule of 3βHSD2 may participate in multiple catalytic reactions. In summary, the steroidogenic cell recycles 3βHSD2 to catalyze the reactions needed to produce androstenedione, progesterone and 17α-hydroxyprogesterone on demand in coordination with the mitochondrial translocase, Tim50. This article is part of a Special Issue entitled 'Steroid/Sterol signaling'. PMID:25448736

  3. Plant Formate Dehydrogenase

    SciTech Connect

    John Markwell

    2005-01-10

    The research in this study identified formate dehydrogenase, an enzyme that plays a metabolic role on the periphery of one-carbon metabolism, has an unusual localization in Arabidopsis thaliana and that the enzyme has an unusual kinetic plasticity. These properties make it possible that this enzyme could be engineered to attempt to engineer plants with an improved photosynthetic efficiency. We have produced transgenic Arabidopsis and tobacco plants with increased expression of the formate dehydrogenase enzyme to initiate further studies.

  4. The co-immobilization of P450-type nitric oxide reductase and glucose dehydrogenase for the continuous reduction of nitric oxide via cofactor recycling.

    PubMed

    Garny, Seike; Beeton-Kempen, Natasha; Gerber, Isak; Verschoor, Jan; Jordaan, Justin

    2016-04-01

    The co-immobilization of enzymes on target surfaces facilitates the development of self-contained, multi-enzyme biocatalytic platforms. This generally entails the co-immobilization of an enzyme with catalytic value in combination with another enzyme that performs a complementary function, such as the recycling of a critical cofactor. In this study, we co-immobilized two enzymes from different biological sources for the continuous reduction of nitric oxide, using epoxide- and carboxyl-functionalized hyper-porous microspheres. Successful co-immobilization of a fungal nitric oxide reductase (a member of the cytochrome P450 enzyme family) and a bacterial glucose dehydrogenase was obtained with the carboxyl-functionalized microspheres, with enzyme activity maintenance of 158% for nitric oxide reductase and 104% for glucose dehydrogenase. The optimal stoichiometric ratio of these two enzymes was subsequently determined to enable the two independent chemical reactions to be catalyzed concomitantly, allowing for near-synchronous cofactor conversion rates. This dual-enzyme system provides a novel research tool with potential for in vitro investigations of nitric oxide, and further demonstrates the successful immobilization of a P450 enzyme with potential application towards the immobilization of other cytochrome P450 enzymes.

  5. NAD + -dependent Formate Dehydrogenase from Plants

    PubMed Central

    Alekseeva, A.A.; Savin, S.S.; Tishkov, V.I.

    2011-01-01

    NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) widely occurs in nature. FDH consists of two identical subunits and contains neither prosthetic groups nor metal ions. This type of FDH was found in different microorganisms (including pathogenic ones), such as bacteria, yeasts, fungi, and plants. As opposed to microbiological FDHs functioning in cytoplasm, plant FDHs localize in mitochondria. Formate dehydrogenase activity was first discovered as early as in 1921 in plant; however, until the past decade FDHs from plants had been considerably less studied than the enzymes from microorganisms. This review summarizes the recent results on studying the physiological role, properties, structure, and protein engineering of plant formate dehydrogenases. PMID:22649703

  6. 11β-Hydroxysteroid dehydrogenase type 1 contributes to the balance between 7-keto- and 7-hydroxy-oxysterols in vivo

    PubMed Central

    Mitić, Tijana; Shave, Steven; Semjonous, Nina; McNae, Iain; Cobice, Diego F.; Lavery, Gareth G.; Webster, Scott P.; Hadoke, Patrick W.F.; Walker, Brian R.; Andrew, Ruth

    2013-01-01

    11β-Hydroxysteroid dehydrogenase 1 (11βHSD1; EC 1.1.1.146) generates active glucocorticoids from inert 11-keto metabolites. However, it can also metabolize alternative substrates, including 7β-hydroxy- and 7-keto-cholesterol (7βOHC, 7KC). This has been demonstrated in vitro but its consequences in vivo are uncertain. We used genetically modified mice to investigate the contribution of 11βHSD1 to the balance of circulating levels of 7KC and 7βOHC in vivo, and dissected in vitro the kinetics of the interactions between oxysterols and glucocorticoids for metabolism by the mouse enzyme. Circulating levels of 7KC and 7βOHC in mice were 91.3 ± 22.3 and 22.6 ± 5.7 nM respectively, increasing to 1240 ± 220 and 406 ± 39 nM in ApoE−/− mice receiving atherogenic western diet. Disruption of 11βHSD1 in mice increased (p < 0.05) the 7KC/7βOHC ratio in plasma (by 20%) and also in isolated microsomes (2 fold). The 7KC/7βOHC ratio was similarly increased when NADPH generation was restricted by disruption of hexose-6-phosphate dehydrogenase. Reduction and oxidation of 7-oxysterols by murine 11βHSD1 proceeded more slowly and substrate affinity was lower than for glucocorticoids. in vitro 7βOHC was a competitive inhibitor of oxidation of corticosterone (Ki = 0.9 μM), whereas 7KC only weakly inhibited reduction of 11-dehydrocorticosterone. However, supplementation of 7-oxysterols in cultured cells, secondary to cholesterol loading, preferentially slowed reduction of glucocorticoids, rather than oxidation. Thus, in mouse, 11βHSD1 influenced the abundance and balance of circulating and tissue levels of 7βOHC and 7KC, promoting reduction of 7KC. In health, 7-oxysterols are unlikely to regulate glucocorticoid metabolism. However, in hyperlipidaemia, 7-oxysterols may inhibit glucocorticoid metabolism and modulate signaling through corticosteroid receptors. PMID:23415904

  7. Opine dehydrogenases in marine invertebrates.

    PubMed

    Harcet, Matija; Perina, Drago; Pleše, Bruna

    2013-10-01

    It is well known today that opine production anaerobic pathways are analogs to the classical glycolytic pathway (lactate production pathway). These pathways, catalyzed by a group of enzymes called opine dehydrogenases (OpDHs), ensure continuous flux of glycolysis and a constant supply of ATP by maintaining the NADH/NAD(+) ratio during exercise and hypoxia, thus regulating the cytosolic redox balance in glycolysis under anoxia. OpDHs are distributed in a wide range of marine invertebrate phyla, including sponges (Porifera). Phylogenetic analyses supported with enzymatic assays strongly indicate that sponge OpDHs constitute an enzyme class unrelated to other OpDHs. Therefore, OpDHs in marine invertebrates are divided into two groups, a mollusk/annelid type and a sponge type, which belongs to the OCD/mu-crystallin family.

  8. Dengue Virus Type 2 (DENV2)-Induced Oxidative Responses in Monocytes from Glucose-6-Phosphate Dehydrogenase (G6PD)-Deficient and G6PD Normal Subjects

    PubMed Central

    Al-alimi, Abdullah Ahmed; Ali, Syed A.; Al-Hassan, Faisal Muti; Idris, Fauziah Mohd; Teow, Sin-Yeang; Mohd Yusoff, Narazah

    2014-01-01

    Background Dengue virus is endemic in peninsular Malaysia. The clinical manifestations vary depending on the incubation period of the virus as well as the immunity of the patients. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is prevalent in Malaysia where the incidence is 3.2%. It has been noted that some G6PD-deficient individuals suffer from more severe clinical presentation of dengue infection. In this study, we aim to investigate the oxidative responses of DENV2-infected monocytes from G6PD-deficient individuals. Methodology Monocytes from G6PD-deficient individuals were infected with DENV2 and infection rate, levels of oxidative species, nitric oxide (NO), superoxide anions (O2−), and oxidative stress were determined and compared with normal controls. Principal Findings Monocytes from G6PD-deficient individuals exhibited significantly higher infection rates compared to normal controls. In an effort to explain the reason for this enhanced susceptibility, we investigated the production of NO and O2− in the monocytes of individuals with G6PD deficiency compared with normal controls. We found that levels of NO and O2− were significantly lower in the DENV-infected monocytes from G6PD-deficient individuals compared with normal controls. Furthermore, the overall oxidative stress in DENV-infected monocytes from G6PD-deficient individuals was significantly higher when compared to normal controls. Correlation studies between DENV-infected cells and oxidative state of monocytes further confirmed these findings. Conclusions/Significance Altered redox state of DENV-infected monocytes from G6PD-deficient individuals appears to augment viral replication in these cells. DENV-infected G6PD-deficient individuals may contain higher viral titers, which may be significant in enhanced virus transmission. Furthermore, granulocyte dysfunction and higher viral loads in G6PD-deificient individuals may result in severe form of dengue infection. PMID:24625456

  9. Insulin, CCAAT/Enhancer-Binding Proteins and Lactate Regulate the Human 11β-Hydroxysteroid Dehydrogenase Type 2 Gene Expression in Colon Cancer Cell Lines

    PubMed Central

    Alikhani-Koupaei, Rasoul; Ignatova, Irena D.; Guettinger, Andreas; Frey, Felix J.; Frey, Brigitte M.

    2014-01-01

    11β-Hydroxysteroid dehydrogenases (11beta-HSD) modulate mineralocorticoid receptor transactivation by glucocorticoids and regulate access to the glucocorticoid receptor. The isozyme 11beta-HSD2 is selectively expressed in mineralocorticoid target tissues and its activity is reduced in various disease states with abnormal sodium retention and hypertension, including the apparent mineralocorticoid excess. As 50% of patients with essential hypertension are insulin resistant and hyperinsulinemic, we hypothesized that insulin downregulates the 11beta-HSD2 activity. In the present study we show that insulin reduced the 11beta-HSD2 activity in cancer colon cell lines (HCT116, SW620 and HT-29) at the transcriptional level, in a time and dose dependent manner. The downregulation was reversible and required new protein synthesis. Pathway analysis using mRNA profiling revealed that insulin treatment modified the expression of the transcription factor family C/EBPs (CCAAT/enhancer-binding proteins) but also of glycolysis related enzymes. Western blot and real time PCR confirmed an upregulation of C/EBP beta isoforms (LAP and LIP) with a more pronounced increase in the inhibitory isoform LIP. EMSA and reporter gene assays demonstrated the role of C/EBP beta isoforms in HSD11B2 gene expression regulation. In addition, secretion of lactate, a byproduct of glycolysis, was shown to mediate insulin-dependent HSD11B2 downregulation. In summary, we demonstrate that insulin downregulates HSD11B2 through increased LIP expression and augmented lactate secretion. Such mechanisms are of interest and potential significance for sodium reabsorption in the colon. PMID:25133511

  10. Cyanobacterial NADPH dehydrogenase complexes

    SciTech Connect

    Ogawa, Teruo; Mi, Hualing

    2007-07-01

    Cyanobacteria possess functionally distinct multiple NADPH dehydrogenase (NDH-1) complexes that are essential to CO2 uptake, photosystem-1 cyclic electron transport and respiration. The unique nature of cyanobacterial NDH-1 complexes is the presence of subunits involved in CO2 uptake. Other than CO2 uptake, chloroplastic NDH-1 complex has similar role as cyanobacterial NDH-1 complexes in photosystem-1 cyclic electron transport and respiration (chlororespiration). In this mini-review we focus on the structure and function of cyanobacterial NDH-1 complexes and their phylogeny. The function of chloroplastic NDH-1 complex and characteristics of plants defective in NDH-1 are also described forcomparison.

  11. AB073. Mutations in the type II 3β-hydroxysteroid dehydrogenase gene caused primary adrenal insufficiency & 46,XY disorders of sex development

    PubMed Central

    Dung, Vu Chi; Thao, Bui Phuong; Khanh, Nguyen Ngoc; Ngoc, Can Thi Bich; Morel, Yves

    2015-01-01

    Congenital adrenal hyperplasia (CAH) is one of the most common inherited metabolic disorders. It includes a group of autosomal recessive disorders caused by the deficiency of one of the enzymes involved in one of the various steps of adrenal steroid synthesis. 3β-hydroxysteroid dehydrogenase (3β-HSD) deficiency is a rare cause of CAH caused by inactivating mutations in the HSD3B2 gene. Most mutations are located within domains regarded crucial for enzyme function. Our aim is to describe phenotype and to identify mutations of HSD3B2 in two classic β-HSD deficient patients belonging to two apparently unrelated pedigrees. This is a case series study. Family history and clinical manifestations were described. Genomic DNA from these patients was extracted using standard procedures from the peripheral blood leukocytes. Mutation analysis of HSD3B2 was performed using polymerase chain reaction (PCR) and DNA direct sequencing. Vietnamese 46,XY newborn referred at 2.5th month of life with salt loss associated with hyponatremia (123 nmol/L) and hyperpigmentation. The testes were palpable in the scrotum but associated with a severe hypospadias (micropenis 0.5 cm; posterior). At 4 months of age, a second adrenal crisis has occurred with hyponatremia 127 nmol/L and increased 17OH-Progesterone (26.8 ng/mL) in this 46,XY DSD. This clinical and biological data associated with a sibling with female phenotype deceased at 18 months old after adrenal crisis (1st occurred at 7 days of life) suggest the diagnosis of 3β-HSD deficiency. The sequencing of HSD3B2 confirms the diagnosis because he is homozygous for a missense mutation, pAla161Pro. This mutation affects an amino acid conserved in all species and is located in one two alpha-helix involved in the dimerization of the two sub-units of the enzyme. The changing from Alanine to proline could break the alpha-helix. The same mutation has been found in the other Vietnamese family. The 46,XY newborn referred at 3th month of life with

  12. A new compound heterozygous frameshift mutation in the type II 3{beta}-hydroxysteroid dehydrogenase 3{beta}-HSD gene causes salt-wasting 3{beta}-HSD deficiency congenital adrenal hyperplasia

    SciTech Connect

    Zhang, L.; Sakkal-Alkaddour, S.; Chang, Ying T.; Yang, Xiaojiang; Songya Pang

    1996-01-01

    We report a new compound heterozygous frameshift mutation in the type II 3{Beta}-hydroxysteroid dehydrogenase (3{beta}-HSD) gene in a Pakistanian female child with the salt-wasting form of 3{Beta}-HSD deficiency congenital adrenal hyperplasia. The etiology for her congenital adrenal hyperplasia was not defined. Although the family history suggested possible 3{beta}-HSd deficiency disorder, suppressed adrenal function caused by excess glucocorticoid therapy in this child at 7 yr of age did not allow hormonal diagnosis. To confirm 3{beta}-HSD deficiency, we sequenced the type II 3{beta}-HSD gene in the patient, her family, and the parents of her deceased paternal cousins. The type II 3{beta}-HSD gene region of a putative promotor, exons I, II, III, and IV, and exon-intron boundaries were amplified by PCR and sequenced in all subjects. The DNA sequence of the child revealed a single nucleotide deletion at codon 318 [ACA(Thr){r_arrow}AA] in exon IV in one allele, and two nucleotide deletions at codon 273 [AAA(Lys){r_arrow}A] in exon IV in the other allele. The remaining gene sequences were normal. The codon 318 mutation was found in one allele from the father, brother, and parents of the deceased paternal cousins. The codon 273 mutation was found in one allele of the mother and a sister. These findings confirmed inherited 3{beta}-HSD deficiency in the child caused by the compound heterozygous type II 3{beta}-HSD gene mutation. Both codons at codons 279 and 367, respectively, are predicted to result in an altered and truncated type II 3{beta}-HSD protein, thereby causing salt-wasting 3{beta}-HSD deficiency in the patient. 21 refs., 2 figs., 1 tab.

  13. Multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase causing excessive acetaldehyde production from ethanol by oral streptococci.

    PubMed

    Pavlova, Sylvia I; Jin, Ling; Gasparovich, Stephen R; Tao, Lin

    2013-07-01

    Ethanol consumption and poor oral hygiene are risk factors for oral and oesophageal cancers. Although oral streptococci have been found to produce excessive acetaldehyde from ethanol, little is known about the mechanism by which this carcinogen is produced. By screening 52 strains of diverse oral streptococcal species, we identified Streptococcus gordonii V2016 that produced the most acetaldehyde from ethanol. We then constructed gene deletion mutants in this strain and analysed them for alcohol and acetaldehyde dehydrogenases by zymograms. The results showed that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol and ethanol, respectively. Two additional dehydrogenases, S-AdhA and TdhA, were identified with specificities to the secondary alcohol 2-propanol and threonine, respectively, but not to ethanol. S. gordonii V2016 did not show a detectable acetaldehyde dehydrogenase even though its adhE gene encodes a putative bifunctional acetaldehyde/alcohol dehydrogenase. Mutants with adhE deletion showed greater tolerance to ethanol in comparison with the wild-type and mutant with adhA or adhB deletion, indicating that AdhE is the major alcohol dehydrogenase in S. gordonii. Analysis of 19 additional strains of S. gordonii, S. mitis, S. oralis, S. salivarius and S. sanguinis showed expressions of up to three alcohol dehydrogenases, but none showed detectable acetaldehyde dehydrogenase, except one strain that showed a novel ALDH. Therefore, expression of multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase may contribute to excessive production of acetaldehyde from ethanol by certain oral streptococci.

  14. Genetics Home Reference: pyruvate dehydrogenase deficiency

    MedlinePlus

    ... control the activity of the complex: pyruvate dehydrogenase phosphatase turns on (activates) the complex, while pyruvate dehydrogenase ... binding protein (the PDHX gene), and pyruvate dehydrogenase phosphatase (the PDP1 gene) have been identified in people ...

  15. Analysis of 17beta-hydroxysteroid dehydrogenase types 5, 7, and 12 genetic sequence variants in breast cancer cases from French Canadian Families with high risk of breast and ovarian cancer.

    PubMed

    Plourde, Marie; Ferland, Alexandra; Soucy, Penny; Hamdi, Yosr; Tranchant, Martine; Durocher, Francine; Sinilnikova, Olga; Luu The, Van; Simard, Jacques

    2009-09-01

    A family history and estrogen exposure are well-known risk factors for breast cancer. Members of the 17beta-hydroxysteroid dehydrogenase family are responsible for important steps in the metabolism of androgens and estrogens in peripheral tissues, including the mammary gland. The crucial biological function of 17beta-HSDs renders these genes good candidates for being involved in breast cancer etiology. This study screened for mutations in HSD17B7 and HSD17B12 genes, which encode enzymes involved in estradiol biosynthesis and in AKR1C3, which codes for 17beta-HSD type 5 enzyme involved in androgen and progesterone metabolism, to assess whether high penetrance allelic variants in these genes could be involved in breast cancer susceptibility. Mutation screening of 50 breast cancer cases from non-BRCA1/2 high-risk French Canadian families failed to identify germline likely high-risk mutations in HSD17B7, HSD17B12 and AKR1C3 genes. However, 107 sequence variants were identified, including seven missense variants. Assessment of the impact of missense variants on enzymatic activity of the corresponding enzymes revealed no difference in catalytic properties between variants of 17beta-HSD types 7 and 12 and wild-type enzymes, while variants p.Glu77Gly and p.Lys183Arg in 17beta-HSD type 5 showed a slightly decreased activity. Finally, a haplotype-based approach was used to determine tagging SNPs providing valuable information for studies investigating associations of common variants in these genes with breast cancer risk.

  16. Gene structure and mutations of glutaryl-coenzyme A dehydrogenase: Impaired association of enzyme subunits that is due to an A421V substitution causes glutaric acidemia type I in the Amish

    SciTech Connect

    Biery, B.J.; Stein, D.E.; Goodman, S.I.

    1996-11-01

    The structure of the human glutaryl coenzyme A dehydrogenase (GCD) gene was determined to contain 11 exons and to span {approximately}7 kb. Fibroblast DNA from 64 unrelated glutaric academia type I (GA1) patients was screened for mutations by PCR amplification and analysis of SSCP. Fragments with altered electrophoretic mobility were subcloned and sequenced to detect mutations that caused GA1. This report describes the structure of the GCD gene, as well as point mutations and polymorphisms found in 7 of its 11 exons. Several mutations were found in more than one patient, but no one prevalent mutation was detected in the general population. As expected from pedigree analysis, a single mutant allele causes GA1 in the Old Order Amish of Lancaster County, Pennsylvania. Several mutations have been expressed in Escherichia coli, and all produce diminished enzyme activity. Reduced activity in GCD encoded by the A421V mutation in the Amish may be due to impaired association of enzyme subunits. 13 refs., 5 figs., 3 tabs.

  17. Development of Potent and Selective Inhibitors of Aldo-Keto Reductase 1C3 (type 5 17β-Hydroxysteroid Dehydrogenase) Based on N-Phenyl-Aminobenzoates and Their Structure Activity Relationships

    PubMed Central

    Adeniji, Adegoke O.; Twenter, Barry M.; Byrns, Michael C.; Jin, Yi; Chen, Mo; Winkler, Jeffrey D.; Penning, Trevor M.

    2012-01-01

    Aldo-keto reductase 1C3 (AKR1C3; type 5 17β-hydroxysteroid dehydrogenase) is overexpressed in castrate resistant prostate cancer (CRPC) and is implicated in the intratumoral biosynthesis of testosterone and 5α-dihydrotestosterone. Selective AKR1C3 inhibitors are required since compounds should not inhibit the highly related AKR1C1 and AKR1C2 isoforms which are involved in the inactivation of 5α-dihydrotestosterone. NSAIDs, N-phenylanthranilates in particular are potent but non-selective AKR1C3 inhibitors. Using flufenamic acid, 2-{[3-(trifluoromethyl)phenyl]amino}benzoic acid as lead compound, five classes of structural analogs were synthesized and evaluated for AKR1C3 inhibitory potency and selectivity. Structure activity relationship (SAR) studies revealed that a meta-carboxylic acid group relative to the amine conferred pronounced AKR1C3 selectivity without loss of potency, while electron withdrawing groups on the phenylamino B-ring were optimal for AKR1C3 inhibition. Lead compounds did not inhibit COX-1 or COX-2 but blocked the AKR1C3 mediated production of testosterone in LNCaP-AKR1C3 cells. These compounds offer promising leads towards new therapeutics for CRPC. PMID:22263837

  18. A point mutation in the putative TATA box, detected in nondiseased individuals and patients with hereditary breast cancer, decreases promoter activity of the 17{beta}-hydroxysteroid dehydrogenase type 1 gene 2 (EDH17B2) in vitro

    SciTech Connect

    Peltoketo, H.; Piao, Y.; Isomaa, V.

    1994-09-01

    EDH17B2, the gene encoding 17{beta}-hydroxysteroid dehydrogenase type 1, has been suggested as a candidate for the familial breast cancer gene, BRCA1, located on 17q12-q21. We analyzed the promoter region of EDH17B2 in DNA from 20 control individuals and 40 patients with familial breast cancer. Two frequent (designated vI and vIII) and two rare (vII and vIV) nucleotide variations were present in both the breast cancer patients and the controls, except the alteration vII, which was found only in one patient. Although the data do not support the identification of EDH17B2 as the BRCA1 gene, it is of interest that point mutation vIV (A {yields} C) was located in the putative TATA box of the EDH17B2 gene. Reporter gene analysis showed that the mutation vIV decreases EDH17B2 promoter activity by an average of 45% in in vitro assays, suggesting that nucleotide A at position -27 is significant for efficient transcription. 12 refs., 2 figs., 1 tab.

  19. [The regulation of glucose-6-phosphate dehydrogenase and glycogen synthase activities by insulin superfamily peptides in myometrium of pregnant women and its impairments under different types of diabetes mellitus].

    PubMed

    Kuznetsova, L A; Chistiakova, O V

    2009-01-01

    The regulatory effects of insulin, insulin-like growth factor 1 (IGF-1), and relaxin on glucose-6-phosphate dehydrogenase (G6PDH) and glycogen synthase (GS) activities have been studied in myometrium of pregnant women of control group and with diabetes mellitus of different etiology. In patients with type 1 diabetes G6PDH activity did not differ from the control group, but the enzyme activity was sharply decreased in pregnant women with type 2 diabetes and gestational diabetes. In the control group maximal stimulation of G6PDH activity was observed at 10(-9) M of peptides and their stimulating effect decreased in the following order: insulin > relaxin > IGF-1. In pregnant women with types 1 diabetes insulin effect on the enzyme activity was lower than in the control, and the effects of IGF-1 and relaxin were absent. In the group of pregnant women with type 2 diabetes and gestational diabetes the effects of insulin and IGF-1 were decreased, but the effect of relaxin was somewhat higher thus giving the following order in their efficiency relaxin > IGF-1 = insulin. At 10(-9) M peptides exhibited similar stimulating effects on the active form of GS-I, but had no influence on the total enzyme activity in the control group of pregnant women. In patients with type 1 diabetes GS activity remained unchanged (versus control), and peptides did not stimulate the enzyme activity. In patients with type 2 diabetes a significant decrease in GS activity was accompanied by the decrease in the effect of peptides, giving the following order of their efficiency: insulin = IGF-1 > relaxin. In myometrium of pregnant women with gestational (treated and untreated) diabetes GS activity decreased, the effect of insulin was weaker, whereas the effects of relaxin and IGF-1 increased thus giving the following order of their efficiency: relaxin > IGF-1 > insulin. Insulin therapy of type 1 diabetes incompletely restored sensitivity of the enzymes to the peptide actions. At the same time, in women

  20. Green Tea and One of Its Constituents, Epigallocatechine-3-gallate, Are Potent Inhibitors of Human 11β-hydroxysteroid Dehydrogenase Type 1

    PubMed Central

    Hintzpeter, Jan; Stapelfeld, Claudia; Loerz, Christine; Martin, Hans-Joerg; Maser, Edmund

    2014-01-01

    The microsomal enzyme 11β-hydroxysteroid deydrogenase type 1 (11β-HSD1) catalyzes the interconversion of glucocorticoid receptor-inert cortisone to receptor- active cortisol, thereby acting as an intracellular switch for regulating the access of glucocorticoid hormones to the glucocorticoid receptor. There is strong evidence for an important aetiological role of 11β-HSD1 in various metabolic disorders including insulin resistance, diabetes type 2, hypertension, dyslipidemia and obesity. Hence, modulation of 11β-HSD1 activity with selective inhibitors is being pursued as a new therapeutic approach for the treatment of the metabolic syndrome. Since tea has been associated with health benefits for thousands of years, we sought to elucidate the active principle in tea with regard to diabetes type 2 prevention. Several teas and tea specific polyphenolic compounds were tested for their possible inhibition of cortisone reduction with human liver microsomes and purified human 11β-HSD1. Indeed we found that tea extracts inhibited 11β-HSD1 mediated cortisone reduction, where green tea exhibited the highest inhibitory potency with an IC50 value of 3.749 mg dried tea leaves per ml. Consequently, major polyphenolic compounds from green tea, in particular catechins were tested with the same systems. (−)-Epigallocatechin gallate (EGCG) revealed the highest inhibition of 11β-HSD1 activity (reduction: IC50 = 57.99 µM; oxidation: IC50 = 131.2 µM). Detailed kinetic studies indicate a direct competition mode of EGCG, with substrate and/or cofactor binding. Inhibition constants of EGCG on cortisone reduction were Ki = 22.68 µM for microsomes and Ki = 18.74 µM for purified 11β-HSD1. In silicio docking studies support the view that EGCG binds directly to the active site of 11β-HSD1 by forming a hydrogen bond with Lys187 of the catalytic triade. Our study is the first to provide evidence that the health benefits of green tea and its polyphenolic compounds

  1. Green tea and one of its constituents, Epigallocatechine-3-gallate, are potent inhibitors of human 11β-hydroxysteroid dehydrogenase type 1.

    PubMed

    Hintzpeter, Jan; Stapelfeld, Claudia; Loerz, Christine; Martin, Hans-Joerg; Maser, Edmund

    2014-01-01

    The microsomal enzyme 11β-hydroxysteroid deydrogenase type 1 (11β-HSD1) catalyzes the interconversion of glucocorticoid receptor-inert cortisone to receptor- active cortisol, thereby acting as an intracellular switch for regulating the access of glucocorticoid hormones to the glucocorticoid receptor. There is strong evidence for an important aetiological role of 11β-HSD1 in various metabolic disorders including insulin resistance, diabetes type 2, hypertension, dyslipidemia and obesity. Hence, modulation of 11β-HSD1 activity with selective inhibitors is being pursued as a new therapeutic approach for the treatment of the metabolic syndrome. Since tea has been associated with health benefits for thousands of years, we sought to elucidate the active principle in tea with regard to diabetes type 2 prevention. Several teas and tea specific polyphenolic compounds were tested for their possible inhibition of cortisone reduction with human liver microsomes and purified human 11β-HSD1. Indeed we found that tea extracts inhibited 11β-HSD1 mediated cortisone reduction, where green tea exhibited the highest inhibitory potency with an IC50 value of 3.749 mg dried tea leaves per ml. Consequently, major polyphenolic compounds from green tea, in particular catechins were tested with the same systems. (-)-Epigallocatechin gallate (EGCG) revealed the highest inhibition of 11β-HSD1 activity (reduction: IC50 = 57.99 µM; oxidation: IC50 = 131.2 µM). Detailed kinetic studies indicate a direct competition mode of EGCG, with substrate and/or cofactor binding. Inhibition constants of EGCG on cortisone reduction were Ki = 22.68 µM for microsomes and Ki = 18.74 µM for purified 11β-HSD1. In silicio docking studies support the view that EGCG binds directly to the active site of 11β-HSD1 by forming a hydrogen bond with Lys187 of the catalytic triade. Our study is the first to provide evidence that the health benefits of green tea and its polyphenolic compounds may

  2. Peroxisomal multifunctional enzyme type 2 from the fruitfly: dehydrogenase and hydratase act as separate entities, as revealed by structure and kinetics.

    PubMed

    Haataja, Tatu J K; Koski, M Kristian; Hiltunen, J Kalervo; Glumoff, Tuomo

    2011-05-01

    All of the peroxisomal β-oxidation pathways characterized thus far house at least one MFE (multifunctional enzyme) catalysing two out of four reactions of the spiral. MFE type 2 proteins from various species display great variation in domain composition and predicted substrate preference. The gene CG3415 encodes for Drosophila melanogaster MFE-2 (DmMFE-2), complements the Saccharomyces cerevisiae MFE-2 deletion strain, and the recombinant protein displays both MFE-2 enzymatic activities in vitro. The resolved crystal structure is the first one for a full-length MFE-2 revealing the assembly of domains, and the data can also be transferred to structure-function studies for other MFE-2 proteins. The structure explains the necessity of dimerization. The lack of substrate channelling is proposed based on both the structural features, as well as by the fact that hydration and dehydrogenation activities of MFE-2, if produced as separate enzymes, are equally efficient in catalysis as the full-length MFE-2.

  3. 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) Inhibitors Still Improve Metabolic Phenotype in Male 11β-HSD1 Knockout Mice Suggesting Off-Target Mechanisms

    PubMed Central

    Harno, Erika; Cottrell, Elizabeth C.; Yu, Alice; DeSchoolmeester, Joanne; Gutierrez, Pablo Morentin; Denn, Mark; Swales, John G.; Goldberg, Fred W.; Bohlooly-Y, Mohammad; Andersén, Harriet; Wild, Martin J.; Turnbull, Andrew V.; Leighton, Brendan

    2013-01-01

    The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a target for novel type 2 diabetes and obesity therapies based on the premise that lowering of tissue glucocorticoids will have positive effects on body weight, glycemic control, and insulin sensitivity. An 11β-HSD1 inhibitor (compound C) inhibited liver 11β-HSD1 by >90% but led to only small improvements in metabolic parameters in high-fat diet (HFD)–fed male C57BL/6J mice. A 4-fold higher concentration produced similar enzyme inhibition but, in addition, reduced body weight (17%), food intake (28%), and glucose (22%). We hypothesized that at the higher doses compound C might be accessing the brain. However, when we developed male brain-specific 11β-HSD1 knockout mice and fed them the HFD, they had body weight and fat pad mass and glucose and insulin responses similar to those of HFD-fed Nestin-Cre controls. We then found that administration of compound C to male global 11β-HSD1 knockout mice elicited improvements in metabolic parameters, suggesting “off-target” mechanisms. Based on the patent literature, we synthesized another 11β-HSD1 inhibitor (MK-0916) from a different chemical series and showed that it too had similar off-target body weight and food intake effects at high doses. In summary, a significant component of the beneficial metabolic effects of these 11β-HSD1 inhibitors occurs via 11β-HSD1–independent pathways, and only limited efficacy is achievable from selective 11β-HSD1 inhibition. These data challenge the concept that inhibition of 11β-HSD1 is likely to produce a “step-change” treatment for diabetes and/or obesity. PMID:24169553

  4. Succinate dehydrogenase-deficient gastrointestinal stromal tumors

    PubMed Central

    Wang, Ya-Mei; Gu, Meng-Li; Ji, Feng

    2015-01-01

    Most gastrointestinal stromal tumors (GISTs) are characterized by KIT or platelet-derived growth factor alpha (PDGFRA) activating mutations. However, there are still 10%-15% of GISTs lacking KIT and PDGFRA mutations, called wild-type GISTs (WT GISTs). Among these so-called WT GISTs, a small subset is associated with succinate dehydrogenase (SDH) deficiency, known as SDH-deficient GISTs. In addition, GISTs that occur in Carney triad and Carney-Stratakis syndrome represent specific examples of SDH-deficient GISTs. SDH-deficient GISTs locate exclusively in the stomach, showing predilection for children and young adults with female preponderance. The tumor generally pursues an indolent course and exhibits primary resistance to imatinib therapy in most cases. Loss of succinate dehydrogenase subunit B expression and overexpression of insulin-like growth factor 1 receptor (IGF1R) are common features of SDH-deficient GISTs. In WT GISTs without succinate dehydrogenase activity, upregulation of hypoxia-inducible factor 1α may lead to increased growth signaling through IGF1R and vascular endothelial growth factor receptor (VEGFR). As a result, IGF1R and VEGFR are promising to be the novel therapeutic targets of GISTs. This review will update the current knowledge on characteristics of SDH-deficient GISTs and further discuss the possible mechanisms of tumorigenesis and clinical management of SDH-deficient GISTs. PMID:25741136

  5. Alcohol Dehydrogenase from Methylobacterium organophilum

    PubMed Central

    Wolf, H. J.; Hanson, R. S.

    1978-01-01

    The alcohol dehydrogenase from Methylobacterium organophilum, a facultative methane-oxidizing bacterium, has been purified to homogeneity as indicated by sodium dodecyl sulfate-gel electrophoresis. It has several properties in common with the alcohol dehydrogenases from other methylotrophic bacteria. The active enzyme is a dimeric protein, both subunits having molecular weights of about 62,000. The enzyme exhibits broad substrate specificity for primary alcohols and catalyzes the two-step oxidation of methanol to formate. The apparent Michaelis constants of the enzyme are 2.9 × 10−5 M for methanol and 8.2 × 10−5 M for formaldehyde. Activity of the purified enzyme is dependent on phenazine methosulfate. Certain characteristics of this enzyme distinguish it from the other alcohol dehydrogenases of other methylotrophic bacteria. Ammonia is not required for, but stimulates the activity of newly purified enzyme. An absolute dependence on ammonia develops after storage of the purified enzyme. Activity is not inhibited by phosphate. The fluorescence spectrum of the enzyme indicates that it and the cofactor associated with it may be chemically different from the alcohol dehydrogenases from other methylotrophic bacteria. The alcohol dehydrogenases of Hyphomicrobium WC-65, Pseudomonas methanica, Methylosinus trichosporium, and several facultative methylotrophs are serologically related to the enzyme purified in this study. The enzymes of Rhodopseudomonas acidophila and of organisms of the Methylococcus group did not cross-react with the antiserum prepared against the alcohol dehydrogenase of M. organophilum. Images PMID:80974

  6. Re-engineering the discrimination between the oxidized coenzymes NAD+ and NADP+ in clostridial glutamate dehydrogenase and a thorough reappraisal of the coenzyme specificity of the wild-type enzyme.

    PubMed

    Capone, Marina; Scanlon, David; Griffin, Joanna; Engel, Paul C

    2011-07-01

    Clostridial glutamate dehydrogenase mutants, designed to accommodate the 2'-phosphate of disfavoured NADPH, showed the expected large specificity shifts with NAD(P)H. Puzzlingly, similar assays with oxidized cofactors initially revealed little improvement with NADP(+) , although rates with NAD(+) were markedly diminished. This article reveals that the enzyme's discrimination in favour of NAD(+) and against NADP(+) had been greatly underestimated and has indeed been abated by a factor of > 16,000 by the mutagenesis. Initially, stopped-flow studies of the wild-type enzyme showed a burst increase of A(340) with NADP(+) but not NAD(+), with amplitude depending on the concentration of the coenzyme, rather than enzyme. Amplitude also varied with the commercial source of the NADP(+). FPLC, HPLC and mass spectrometry identified NAD(+) contamination ranging from 0.04 to 0.37% in different commercial samples. It is now clear that apparent rates of NADP(+) utilization mainly reflected the reduction of contaminating NAD(+), creating an entirely false view of the initial coenzyme specificity and also of the effects of mutagenesis. Purification of the NADP(+) eliminated the burst. With freshly purified NADP(+), the NAD(+) : NADP(+) activity ratio under standard conditions, previously estimated as 300 : 1, is 11,000. The catalytic efficiency ratio is even higher at 80,000. Retested with pure cofactor, mutants showed marked specificity shifts in the expected direction, for example, 16 200 fold change in catalytic efficiency ratio for the mutant F238S/P262S, confirming that the key structural determinants of specificity have been successfully identified. Of wider significance, these results underline that, without purification, even the best commercial coenzyme preparations are inadequate for such studies.

  7. Anti-inflammatory effect of a selective 11β-hydroxysteroid dehydrogenase type 1 inhibitor via the stimulation of heme oxygenase-1 in LPS-activated mice and J774.1 murine macrophages.

    PubMed

    Park, Sung Bum; Park, Ji Seon; Jung, Won Hoon; Kim, Hee Youn; Kwak, Hyun Jung; Ahn, Jin Hee; Choi, Kyoung-Jin; Na, Yoon-Ju; Choi, Sunhwa; Dal Rhee, Sang; Kim, Ki Young

    2016-08-01

    11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts inactive cortisone to the active cortisol. 11β-HSD1 may be involved in the resolution of inflammation. In the present study, we investigate the anti-inflammatory effects of 2-(3-benzoyl)-4-hydroxy-1,1-dioxo-2H-1,2-benzothiazine-2-yl-1-phenylethanone (KR-66344), a selective 11β-HSD1 inhibitor, in lipopolysaccharide (LPS)-activated C57BL/6J mice and macrophages. LPS increased 11β-HSD1 activity and expression in macrophages, which was inhibited by KR-66344. In addition, KR-66344 increased survival rate in LPS treated C57BL/6J mice. HO-1 mRNA expression level was increased by KR-66344, and this effect was reversed by the HO competitive inhibitor, ZnPP, in macrophages. Moreover, ZnPP reversed the suppression of ROS formation and cell death induced by KR-66344. ZnPP also suppressed animal survival rate in LPS plus KR-66344 treated C57BL/6J mice. In the spleen of LPS-treated mice, KR-66344 prevented cell death via suppression of inflammation, followed by inhibition of ROS, iNOS and COX-2 expression. Furthermore, LPS increased NFκB-p65 and MAPK phosphorylation, and these effects were abolished by pretreatment with KR-66344. Taken together, KR-66344 protects against LPS-induced animal death and spleen injury by inhibition of inflammation via induction of HO-1 and inhibition of 11β-HSD1 activity. Thus, we concluded that the selective 11β-HSD1 inhibitor may provide a novel strategy in the prevention/treatment of inflammatory disorders in patients. PMID:27523796

  8. Immune response to lactate dehydrogenase-elevating virus: isolation of infectious virus-immunoglobulin G complexes and quantitation of specific antiviral immunoglobulin G response in wild-type and nude mice.

    PubMed Central

    Cafruny, W A; Plagemann, P G

    1982-01-01

    Lactate dehydrogenase-elevating virus (LDV) causes a normally benign persistent infection of mice, resulting in a life-long viremia characterized by the presence of circulating infectious immune complexes, impaired clearance of certain enzymes from the blood, and modification of the host immune response to various heterologous antigens. In this study, we isolated infectious immunoglobulin G (IgG)-LDV complexes in the plasma of persistently infected mice by adsorption to and elution from protein A-Sepharose CL-4B. We found that practically all infectious LDV in the plasma of persistently infected mice is complexed to IgG. LDV infectivity in these complexes was partially neutralized, but could be reactivated by treatment with 2-mercaptoethanol. We also quantitated total plasma IgG and anti-LDV IgG in wild-type and nude Swiss and BALB/c mice as a function of the time after infection with LDV by radial immunodiffusion and an enzyme-linked immunosorbent assay, respectively. Total plasma IgG levels nearly doubled in BALB/c mice during 150 days of infection. IgG levels in uninfected nude mice were only 20% of those in uninfected BALB/c mice, but during infection with LDV increased to approximately those found in uninfected BALB/c mice. Anti-LDV IgG levels were almost as high in nude mice as in normal BALB/c mice. Isoelectric focusing of purified IgG from BALB/c mice showed that LDV infection resulted in the enhanced synthesis of all 16 normal IgG fractions that we could separate by this method, which suggests that LDV infection results in polyclonal activation of IgG-producing lymphocytes. PMID:7129626

  9. Michael hydratase alcohol dehydrogenase or just alcohol dehydrogenase?

    PubMed Central

    2014-01-01

    The Michael hydratase – alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans was previously identified as a bi-functional enzyme performing a hydration of α,β-unsaturated ketones and subsequent oxidation of the formed alcohols. The investigations of the bi-functionality were based on a spectrophotometric assay and an activity staining in a native gel of the dehydrogenase. New insights in the recently discovered organocatalytic Michael addition of water led to the conclusion that the previously performed experiments to identify MhyADH as a bi-functional enzyme and their results need to be reconsidered and the reliability of the methodology used needs to be critically evaluated. PMID:24949265

  10. The lactate dehydrogenase of the icefish heart: biochemical adaptations to hypoxia tolerance.

    PubMed

    Feller, G; Pauly, J P; Smal, A; O'Carra, P; Gerday, C

    1991-09-20

    Cardiac lactate dehydrogenase from the hemoglobin- and myoglobin-free antarctic icefish has been purified by affinity chromatography. Structural and kinetic properties of the enzyme were found close or identical to those of its skeletal muscle counterpart and other M-type lactate dehydrogenases. A model involving a dual oxidative-anaerobic metabolism of the icefish heart is proposed. PMID:1911860

  11. Relationships between the H and A-O blood types, phosphohexose isomerase and 6-phosphogluconate dehydrogenase red cell enzyme systems and halothane sensitivity, and economic traits in a superior and an inferior selection line of swiss landrace pigs.

    PubMed

    Vögeli, P; Stranzinger, G; Schneebeli, H; Hagger, C; Künzi, N; Gerwig, C

    1984-12-01

    Associations between production traits and the genes for halothane sensitivity (HAL), S, A and H blood group systems and phosphohexose isomerase (PHI) and 6-phosphogluconate dehydrogenase (6-PGD) enzyme systems were investigated in two lines of pigs selected for an index. The phenotypic variance-covariance matrix of the index included backfat thickness and daily gain, whereas the genetic variance-covariance matrix included daily gain, feed conversion and percentage of lean meat. The experiment was conducted at the experimental station of the Institute of Animal Production and has been underway since 1973. The same index was applied but in two opposite directions to give a superior and inferior line in relation to the production traits. One hundred twenty-nine animals of the superior line in the seventh generation and 88 animals of the inferior line in the sixth generation were studied. Forty-two percent (54/129) of the animals of the superior line were halothane-positive. No animals in the inferior line were halothane reactors. Of the halothane-positive pigs, 70.4% (38/54) in the superior line had the HaHa and 94.4% (51/54) had the SsSs genotype, whereas only 4% (3/75) of the HaHa and 12% (9/75) of the SsSs pigs were halothane-negative. By practicing selection at the H and S loci, it seems possible to efficiently reduce halothane sensitivity in Swiss Landrace pigs. In pigs of the superior line, there were significant differences in percentage of lean meat, carcass length, pH1 (pH value at 45 min to 1 h postmortem, M. longissimus) and reflectance values among genotypes of the HAL, S and H systems and among some genotypes of the 6-PGD system. Poorest meat quality, highest percentage of lean meat and shortest carcass length were observed in pigs homozygous for the alleles HALn, Ss, Ha, PHIB and 6-PGDA. In the inferior line, these associations were absent. As the HAL locus is associated with the above mentioned production traits, linkage disequilibria may explain the

  12. 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 ...

  13. Aminotransferase and glutamate dehydrogenase activities in lactobacilli and streptococci.

    PubMed

    Peralta, Guillermo Hugo; Bergamini, Carina Viviana; Hynes, Erica Rut

    2016-01-01

    Aminotransferases and glutamate dehydrogenase are two main types of enzymes involved in the initial steps of amino acid catabolism, which plays a key role in the cheese flavor development. In the present work, glutamate dehydrogenase and aminotransferase activities were screened in twenty one strains of lactic acid bacteria of dairy interest, either cheese-isolated or commercial starters, including fifteen mesophilic lactobacilli, four thermophilic lactobacilli, and two streptococci. The strains of Streptococcus thermophilus showed the highest glutamate dehydrogenase activity, which was significantly elevated compared with the lactobacilli. Aspartate aminotransferase prevailed in most strains tested, while the levels and specificity of other aminotransferases were highly strain- and species-dependent. The knowledge of enzymatic profiles of these starter and cheese-isolated cultures is helpful in proposing appropriate combinations of strains for improved or increased cheese flavor. PMID:27266631

  14. Aminotransferase and glutamate dehydrogenase activities in lactobacilli and streptococci.

    PubMed

    Peralta, Guillermo Hugo; Bergamini, Carina Viviana; Hynes, Erica Rut

    2016-01-01

    Aminotransferases and glutamate dehydrogenase are two main types of enzymes involved in the initial steps of amino acid catabolism, which plays a key role in the cheese flavor development. In the present work, glutamate dehydrogenase and aminotransferase activities were screened in twenty one strains of lactic acid bacteria of dairy interest, either cheese-isolated or commercial starters, including fifteen mesophilic lactobacilli, four thermophilic lactobacilli, and two streptococci. The strains of Streptococcus thermophilus showed the highest glutamate dehydrogenase activity, which was significantly elevated compared with the lactobacilli. Aspartate aminotransferase prevailed in most strains tested, while the levels and specificity of other aminotransferases were highly strain- and species-dependent. The knowledge of enzymatic profiles of these starter and cheese-isolated cultures is helpful in proposing appropriate combinations of strains for improved or increased cheese flavor.

  15. 11β-Hydroxysteroid dehydrogenase type 1 contributes to the regulation of 7-oxysterol levels in the arterial wall through the inter-conversion of 7-ketocholesterol and 7β-hydroxycholesterol

    PubMed Central

    Mitić, Tijana; Andrew, Ruth; Walker, Brian R.; Hadoke, Patrick W.F.

    2013-01-01

    The atherogenic 7-oxysterols, 7-ketocholesterol (7-KC) and 7β-hydroxycholesterol (7βOHC), can directly impair arterial function. Inter-conversion of 7-KC and 7βOHC has recently been shown as a novel role for the glucocorticoid-metabolizing enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). Since this enzyme is expressed in vascular smooth muscle cells, we addressed the hypothesis that inter-conversion of 7-KC and 7βOHC by 11β-HSD1 may contribute to regulation of arterial function. Incubation (4–24 h) of aortic rings with either 7-KC (25 μM) or 7βOHC (20 μM) had no effect on endothelium-dependent (acetylcholine) or -independent (sodium nitroprusside) relaxation. In contrast, exposure to 7-KC (but not to 7βOHC) attenuated noradrenaline-induced contraction (Emax) after 4 h (0.78 ± 0.28 vs 0.40 ± 0.08 mN/mm; p < 0.05) and 24 h (2.28 ± 0.34 vs 1.56 ± 0.48 mN/mm; p < 0.05). Both 7-oxysterols were detected by GCMS in the aortic wall of chow-fed C57Bl6/J mice, with concentrations of 7-KC (1.41 ± 0.81 ng/mg) higher (p = 0.05) than 7βOHC (0.16 ± 0.06 ng/mg). In isolated mouse aortic rings 11β-HSD1 was shown to act as an oxo-reductase, inter-converting 7-KC and 7βOHC. This activity was lost in aorta from 11β-HSD1−/− mice, which had low oxysterol levels. Renal homogenates from 11β-HSD1−/− mice were used to confirm that the type 2 isozyme of 11β-HSD does not inter-convert 7-KC and 7βOHC. These results demonstrate that 7-KC has greater effects than 7βOHC on vascular function, and that 11β-HSD1 can inter-convert 7-KC and 7βOHC in the arterial wall, contributing to the regulation of 7-oxysterol levels and potentially influencing vascular function. This mechanism may be important in the cardioprotective effects of 11β-HSD1 inhibitors. PMID:22940536

  16. Convergent evolution of Trichomonas vaginalis lactate dehydrogenase from malate dehydrogenase

    PubMed Central

    Wu, Gang; Fiser, András; ter Kuile, Benno; Šali, Andrej; Müller, Miklós

    1999-01-01

    Lactate dehydrogenase (LDH) is present in the amitochondriate parasitic protist Trichomonas vaginalis and some but not all other trichomonad species. The derived amino acid sequence of T. vaginalis LDH (TvLDH) was found to be more closely related to the cytosolic malate dehydrogenase (MDH) of the same species than to any other LDH. A key difference between the two T. vaginalis sequences was that Arg91 of MDH, known to be important in coordinating the C-4 carboxyl of oxalacetate/malate, was replaced by Leu91 in LDH. The change Leu91Arg by site-directed mutagenesis converted TvLDH into an MDH. The reverse single amino acid change Arg91Leu in TvMDH, however, gave a product with no measurable LDH activity. Phylogenetic reconstructions indicate that TvLDH arose from an MDH relatively recently. PMID:10339579

  17. Activity of select dehydrogenases with Sepharose-immobilized N6-carboxymethyl-NAD

    PubMed Central

    Beauchamp, Justin; Vieille, Claire

    2015-01-01

    N6-carboxymethyl-NAD (N6-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N6-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 N6-CM-NAD. The products of all enzymes but 6-phospho-D-glucono-1,5-lactone were formed when sepharose-immobilized N6-CM-NAD was recycled by T. maritima glycerol dehydrogenase, indicating that N6-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 N6-amine group on NAD. PMID:25611453

  18. 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.

  19. Sorbitol dehydrogenase is a zinc enzyme.

    PubMed Central

    Jeffery, J; Chesters, J; Mills, C; Sadler, P J; Jörnvall, H

    1984-01-01

    Evidence is given that tetrameric sorbitol dehydrogenase from sheep liver contains one zinc atom per subunit, most probably located at the active site, and no other specifically bound zinc or iron atom. In alcohol dehydrogenases that are structurally related to sorbitol dehydrogenase, more than one zinc atom per subunit can complicate investigations of zinc atom function. Therefore, sorbitol dehydrogenase will be particularly valuable for defining the precise roles of zinc in alcohol and polyol dehydrogenases, and for establishing correlations of structure and function with other important zinc-containing proteins. PMID:6370679

  20. Interactions between heparinoids and alcohol dehydrogenase.

    PubMed

    Paulíková, H; Valusová, E; Antalík, M

    1997-07-01

    The interaction between polysulfated polysaecharides (low-molecular-weight heparin LMWH, dextran sulfate DS and pentosan sulfate PS) and yeast alcohol dehydrogenase (YADH) was investigated. The fluorescence and UV spectra of YADH after adding the tested polysaccharides have confirmed the interaction between the enzyme and these compounds. Kinetic studies have shown that LMWH, DS and PS are inhibitors of YADH (mixed type with respect to NAD). The most potent inhibitor is PS (ID50=37.5 ng/ml, Ki=0.6 muM). The inhibition effect depends on the ionic strength (the inhibition decreased by about 50% in the presence of 100 mM Na2SO4) and pH value (the inhibition decreased at pH>7). The results indicate that the inhibition effect of these polyanions is caused by their electrostatic interactions with the NAD-binding region of YADH.

  1. Molybdenum and tungsten-dependent formate dehydrogenases.

    PubMed

    Maia, Luisa B; Moura, José J G; Moura, Isabel

    2015-03-01

    The prokaryotic formate metabolism is considerably diversified. Prokaryotes use formate in the C1 metabolism, but also evolved to exploit the low reduction potential of formate to derive energy, by coupling its oxidation to the reduction of numerous electron acceptors. To fulfil these varied physiological roles, different types of formate dehydrogenase (FDH) enzymes have evolved to catalyse the reversible 2-electron oxidation of formate to carbon dioxide. This review will highlight our present knowledge about the diverse physiological roles of FDH in prokaryotes, their modular structural organisation and active site structures and the mechanistic strategies followed to accomplish the formate oxidation. In addition, the ability of FDH to catalyse the reverse reaction of carbon dioxide reduction, a potentially relevant reaction for carbon dioxide sequestration, will also be addressed.

  2. Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase

    SciTech Connect

    Mat-Jan, F.; Alam, K.Y.; Clark, D.P. )

    1989-01-01

    Mutants of Escherichia coli deficient in the fermentative NAD-linked lactate dehydrogenase (ldh) have been isolated. These mutants showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl). Double mutants (pfl ldh) were unable to grow anaerobically on glucose or other sugars even when supplemented with acetate, whereas pfl mutants can do so. The ldh mutation was found to map at 30.5 min on the E. coli chromosome. The ldh mutant FMJ39 showed no detectable lactate dehydrogenase activity and produced no lactic acid from glucose under anaerobic conditions as estimated by in vivo nuclear magnetic resonance measurements. We also found that in wild-type strains the fermentative lactate dehydrogenase was conjointly induced by anaerobic conditions and an acidic pH. Despite previous findings that phosphate concentrations affect the proportion of lactic acid produced during fermentation, we were unable to find any intrinsic effect of phosphate on lactate dehydrogenase activity, apart from the buffering effect of this ion.

  3. Crystal structure of quinone-dependent alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes. A versatile dehydrogenase oxidizing alcohols and carbohydrates.

    PubMed

    Rozeboom, Henriëtte J; Yu, Shukun; Mikkelsen, Rene; Nikolaev, Igor; Mulder, Harm J; Dijkstra, Bauke W

    2015-12-01

    The quinone-dependent alcohol dehydrogenase (PQQ-ADH, E.C. 1.1.5.2) from the Gram-negative bacterium Pseudogluconobacter saccharoketogenes IFO 14464 oxidizes primary alcohols (e.g. ethanol, butanol), secondary alcohols (monosaccharides), as well as aldehydes, polysaccharides, and cyclodextrins. The recombinant protein, expressed in Pichia pastoris, was crystallized, and three-dimensional (3D) structures of the native form, with PQQ and a Ca(2+) ion, and of the enzyme in complex with a Zn(2+) ion and a bound substrate mimic were determined at 1.72 Å and 1.84 Å resolution, respectively. PQQ-ADH displays an eight-bladed β-propeller fold, characteristic of Type I quinone-dependent methanol dehydrogenases. However, three of the four ligands of the Ca(2+) ion differ from those of related dehydrogenases and they come from different parts of the polypeptide chain. These differences result in a more open, easily accessible active site, which explains why PQQ-ADH can oxidize a broad range of substrates. The bound substrate mimic suggests Asp333 as the catalytic base. Remarkably, no vicinal disulfide bridge is present near the PQQ, which in other PQQ-dependent alcohol dehydrogenases has been proposed to be necessary for electron transfer. Instead an associated cytochrome c can approach the PQQ for direct electron transfer.

  4. Spatial variability of the dehydrogenase activity in forest soils

    NASA Astrophysics Data System (ADS)

    Błońska, Ewa; Lasota, Jarosław

    2014-05-01

    The aim of this study was to assess the spatial variability of the dehydrogenase activity (DH) in forest soils using geostatistics. We have studied variability soil dehydrogenase and their relationship with variability of some physic-chemical properties. Two study areas (A and B) were set up in southern Poland in the Zlotoryja Forest District. Study areas were covered by different types of vegetation (A- broadleaf forest with beech, ash and sycamore), B- coniferous forest with Norway spruce). The soils were classified as Dystric Cambisols (WRB 2006). The samples for laboratory testing were collected from 49 places on each areas. 15 cm of surface horizon of soil were taken (with previously removed litter). Dehydrogenase activity was marked with Lenhard's method according to the Casida procedure. Soil pH, nitrogen (N) and soil organic carbon (C) content (by LECO CNS 2000 carbon analyzer) was marked. C/N ratio was calculated. Particle size composition was determined using laser diffraction. Statistical analysis were performed using STATISTICA 10 software. Geostatistical analysis and mapping were done by application of GS 9+ (Gamma Design) and Surfer 11 (Golden Software). The activity of DH ranged between 5,02 and 71,20 mg TPP• kg-1 •24 h-1 on the A area and between 0,94 and 16,47 mg TPP• kg-1 •24 h-1. Differences in spatial variability of the analised features were noted. The variability of dehydrogenase activity on the A study area was described by an exponential model, whereas on the B study area the spatial correlation has not been noted. The relationship of dehydrogenase activity with the remaining parameters of soil was noted only in the case of A study area. The variability of organic carbon content on the A and B study areas were described by an exponential model. The variability of nitrogen content on both areas were described by an spherical model.

  5. SAXS fingerprints of aldehyde dehydrogenase oligomers

    PubMed Central

    Tanner, John J.

    2015-01-01

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

  6. SAXS fingerprints of aldehyde dehydrogenase oligomers.

    PubMed

    Tanner, John J

    2015-12-01

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

  7. Malic dehydrogenase locus of Paramecium tetraurelia.

    PubMed

    Williams, T J; Smith-Sonneborn, J

    1980-04-01

    A search was undertaken for naturally occurring genetic markers for use in clonal aging studies of Paramecium tetraurelia. Clonal age is defined as the number of cell divisions since the last sexual process. Autogamy (self-fertilization) is a sexual process which can occur in aging lines, resulting in homozygosity and initiation of the next generation. Such "illicit" autogamies must be detected and eliminated from the aged clone. With codominant alleles, heterozygous aging lines can be established which will express a phenotype distinguishable from that of either parental type and autogamy can then be monitored by the appearance of either segregant homozygous phenotype. However, very few codominant alleles are available in this species. Electrophoretic mobilities of malic dehydrogenase (MDH) were assayed in 11 stocks of Paramecium tetraurelia by polyacrylamide gel electrophoresis. Nine stocks showed a single-banded "stock 51" type, while stock 174 and stock 29 each exhibited unique mobility. Crosses between stock 51 and the deviant stocks revealed distinct three-banded patterns indicative of heterozygosity of the F1 generation. In the autogamous F2 generation, 1:1 segregation of the parental types were recovered. The pattern of inheritance is consistent with codominant alleles and Mendelian inheritance. These naturally occurring biochemical markers are stable with increasing clonal age and are therefore useful genetic markers for studies of cellular aging. PMID:6934772

  8. Regulation of human dihydrodiol dehydrogenase by Michael acceptor xenobiotics.

    PubMed

    Ciaccio, P J; Jaiswal, A K; Tew, K D

    1994-06-01

    A human oxidoreductase (H-37) that is overexpressed in ethacrynic acid-resistant HT29 colon cells (Ciaccio, P. J., Stuart, J.E., and Tew, K.D. (1993) Mol. Pharmacol. 43, 845-853) has been identified as a dihydrodiol dehydrogenase. Translated protein from a dihydrodiol dehydrogenase cDNA isolated from a library prepared from ethacrynic acid-resistant HT29 cell poly(A+) RNA was recognized by anti-H-37 IgG and was identical in molecular weight with H-37. The isolated cDNA was identical in both nucleotide and amino acid sequences with the recently cloned liver dihydrodiol dehydrogenase (Stolz, A., Hammond, L., Lou, H., Takikawa, H., Ronk, M., and Shively, J.E. (1993) J. Biol. Chem. 268, 10448-10457). Using this cDNA as probe, we have examined its induction by Michael acceptors. The steady state dihydrodiol dehydrogenase mRNA level in the ethacrynic acid-resistant line was increased 30-fold relative to that of wild-type cells. Twenty-four hour treatment of wild-type cells with ethacrynic acid or dimethyl maleate increased mRNA 10-fold and 5-fold, respectively. These changes are accompanied by both increased protein expression and increased NADP-dependent 1-acenaphthenol oxidative activity in cell cytosol. In gel shift assays, compared to wild type controls, increased binding of NAD(P)H quinone oxidoreductase human antioxidant response element (hARE) DNA to redox labile protein complexes present in treated and resistant cell nuclear extract was observed. Ethacrynic acid induced CAT activity 2-fold in Hepa1 cells stably transfected with NAD(P)H quinone oxidoreductase hARE-tk-CAT chimeric gene construct. Thus, dihydrodiol dehydrogenase protein is inducible by de novo synthesis from mRNA by structurally related monofunctional inducer Michael acceptors. Altered in vitro binding of nuclear protein to the hARE is indirect evidence for the involvement of an element similar to hARE in the regulation of dihydrodiol dehydrogenase by these agents. PMID:7515059

  9. Isocitrate dehydrogenases and oxoglutarate dehydrogenase activities of baker's yeast grown in a variety of hypoxic conditions.

    PubMed

    Machado, A; Nuñez de Castro, I; Mayor, F

    1975-02-28

    The activities of isocitrate dehydrogenase (NAD), isocitrate dehydrogenase (NADP) and oxoglutarate dehydrogenase have been investigated in Saccharomyces cerevisiae grown in a variety of aerobic and hypoxic conditions, the latter including oxygen deprivation, high glucose concentration, addition of inhibitors of mitochondrial protein synthesis, respiratory inhibition by azide, and impaired respiration mutants. All hypoxic conditions led to a marked decrease of oxoglutarate dehydrogenase and significant decreases of the two isocitrate dehydrogenases. According to its kinetic properties, the NAD-isocitrate dehydrogenase will not be operative in hypoxia "in vivo". From these and other related facts it is concluded that hypoxic conditions in yeast generally lead to a splitting of the tricarboxylic acid cycle and that glutamate synthesis in these conditions takes place through the coupling of the NADP-linked isocitrate and glutamate dehydrogenases.

  10. [The PQQ-dehydrogenases. A novel example of bacterial quinoproteins].

    PubMed

    Flores-Encarnación, Marcos; Sánchez-Cuevas, Mariano; Ortiz-Gutiérrez, Felipe

    2004-01-01

    The word "quinoprotein" describes four groups of different enzymes which have cofactors containing o-quinones. Pyrrolo-quinoline quinone (PQQ) is not covalently attached. PQQ is the cofactor of several quinoprotein bacterial dehydrogenases including glucose dehydrogenase (G-DH), alcohol dehydrogenase (A-DH) and aldehyde dehydrogenase (AL-DH). These dehydrogenases are located in the periplasm of Gram-negative bacteria. This report summarises the structural properties of quinoprotein dehydrogenases, such as the biological functions and biotechnological aspects more important.

  11. Strategy for the isolation of native dehydrogenases with potential for biosensor development from the organism Hyphomicrobium zavarzinii ZV580.

    PubMed

    Hilbrig, Frank; Jérôme, Valérie; Salzig, Mark; Freitag, Ruth

    2009-04-17

    Dehydrogenases are interesting candidates for the development of electrochemical biosensors. Most dehydrogenases are characterised by a comparatively broad substrate spectrum, yet highly specific enzymes exist as well. A specific formaldehyde dehydrogenase has, e.g., been described for the organism Hyphomicrobium zavarzinii ZV580. Isolation of enzymes from their natural source instead of a recombinant expression renders the isolation more challenging, as common tools such as affinity tags are no longer available. In this contribution, we develop chromatographic procedures for such isolation tasks. The previously described formaldehyde dehydrogenase was isolated by two procedures, one based on affinity chromatography, the other on hydroxyapatite. Neither procedure yielded an active enzyme. In addition two dehydrogenases, a formaldehyde and a methylamine dehydrogenase, were found in the cell free extract, which had not been described previously. Both enzymes could be isolated to near purity by a sequence of hydroxyapatite and anion exchange chromatography. The new formaldehyde dehydrogenase requires reconstitution with calcium and pyrroloquinoline quinone in order to become active. The enzyme shows no cross-reactivity with methylamine or methanol. The methylamine dehydrogenase catalyses the conversion of methylamine into formaldehyde, hence it could become a technical catalyst for the inverse reaction. This enzyme consists of two types of subunit and may be one of the rare alpha,beta-methylamine dehydrogenases. PMID:18835606

  12. Strategy for the isolation of native dehydrogenases with potential for biosensor development from the organism Hyphomicrobium zavarzinii ZV580.

    PubMed

    Hilbrig, Frank; Jérôme, Valérie; Salzig, Mark; Freitag, Ruth

    2009-04-17

    Dehydrogenases are interesting candidates for the development of electrochemical biosensors. Most dehydrogenases are characterised by a comparatively broad substrate spectrum, yet highly specific enzymes exist as well. A specific formaldehyde dehydrogenase has, e.g., been described for the organism Hyphomicrobium zavarzinii ZV580. Isolation of enzymes from their natural source instead of a recombinant expression renders the isolation more challenging, as common tools such as affinity tags are no longer available. In this contribution, we develop chromatographic procedures for such isolation tasks. The previously described formaldehyde dehydrogenase was isolated by two procedures, one based on affinity chromatography, the other on hydroxyapatite. Neither procedure yielded an active enzyme. In addition two dehydrogenases, a formaldehyde and a methylamine dehydrogenase, were found in the cell free extract, which had not been described previously. Both enzymes could be isolated to near purity by a sequence of hydroxyapatite and anion exchange chromatography. The new formaldehyde dehydrogenase requires reconstitution with calcium and pyrroloquinoline quinone in order to become active. The enzyme shows no cross-reactivity with methylamine or methanol. The methylamine dehydrogenase catalyses the conversion of methylamine into formaldehyde, hence it could become a technical catalyst for the inverse reaction. This enzyme consists of two types of subunit and may be one of the rare alpha,beta-methylamine dehydrogenases.

  13. 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...

  14. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 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...

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

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 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...

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

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 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...

  17. 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...

  18. Formate dehydrogenase from Pseudomonas oxalaticus.

    PubMed

    Müller, U; Willnow, P; Ruschig, U; Höpner, T

    1978-02-01

    Formate dehydrogenase (EC 1.2.1.2) from Pseudomonas oxalaticus has been isolated and characterized. The enzyme (molecular weight 315000) is a complex flavoprotein containing 2 FMN, 18--25 non-heme iron atoms and 15--20 acid-labile sulphides. In the last step of the purification, a sucrose gradient centrifugation, a second catalytically active species has been found apparently originating from a dissociation of the enzyme into two equal subunits. The enzyme is specific toward its natural substrate formate. It transfers electrons to NAD+, oxygen, ferricyanide, and a lot of nonphysiological acceptors (dyes). In addition electrons are transferred from NADH to these acceptors. The (reversible) removal of FMN requires a reduction step. Reincorporation has been followed by the reappearance of the reactivity against formate and by fluorescence titration. The deflavo enzyme also binds FAD and riboflavin. The resulting enzyme species show characteristic catalytic abilities. Activity against formate is peculiar to the FMN species. PMID:631130

  19. Histidine 51 facilitates proton transfer in alcohol dehydrogenase

    SciTech Connect

    Gould, R.M.; Plapp, B.V.

    1987-05-01

    Operating through a proton relay system, His-51 has been proposed to serve as a base during ethanol oxidation by alcohol dehydrogenase. This residue is highly conserved in alcohol dehydrogenases. They have used mutamer directed mutagenesis to change this residue to Gln-51. Diethyl pyrocarbonate treatment decreases the activity of the wild type enzyme 60-fold, whereas the Gln-51 enzyme is inactivated by only 5-fold. The rate of inactivation is also much slower with the mutant enzyme. They conclude that His-51 is the reactive residue in yeast alcohol dehydrogenase. The mutation also alters the Km for acetaldehyde and the pH dependence of several kinetic constants. At pH 7.0 the Km for acetaldehyde is 18-fold higher in the Gln-51 enzyme, whereas Vmax for acetaldehyde reduction is the same as with the wild type enzyme. For ethanol oxidation the pH dependence of the log of Vmax and V/K shows a linear dependence with a slope of 0.5 and no discernible pK. They propose a mechanism that can explain these data. For the Gln-51 enzyme, after the ternary complex has formed in an Ordered Bi mechanism, a random component for proton release and hydride transfer occurs. With histidine at position 51, serving as a base, a more rapid proton release from the enzyme-NAD-ethanol complex precedes product formation.

  20. Molecular characterization of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II of Acinetobacter calcoaceticus.

    PubMed Central

    Gillooly, D J; Robertson, A G; Fewson, C A

    1998-01-01

    The nucleotide sequences of xylB and xylC from Acinetobacter calcoaceticus, the genes encoding benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II, were determined. The complete nucleotide sequence indicates that these two genes form part of an operon and this was supported by heterologous expression and physiological studies. Benzaldehyde dehydrogenase II is a 51654 Da protein with 484 amino acids per subunit and it is typical of other prokaryotic and eukaryotic aldehyde dehydrogenases. Benzyl alcohol dehydrogenase has a subunit Mr of 38923 consisting of 370 amino acids, it stereospecifically transfers the proR hydride of NADH, and it is a member of the family of zinc-dependent long-chain alcohol dehydrogenases. The enzyme appears to be more similar to animal and higher-plant alcohol dehydrogenases than it is to most other microbial alcohol dehydrogenases. Residue His-51 of zinc-dependent alcohol dehydrogenases is thought to be necessary as a general base for catalysis in this category of alcohol dehydrogenases. However, this residue was found to be replaced in benzyl alcohol dehydrogenase from A. calcoaceticus by an isoleucine, and the introduction of a histidine residue in this position did not alter the kinetic coefficients, pH optimum or substrate specificity of the enzyme. Other workers have shown that His-51 is also absent from the TOL-plasmid-encoded benzyl alcohol dehydrogenase of Pseudomonas putida and so these two closely related enzymes presumably have a catalytic mechanism that differs from that of the archetypal zinc-dependent alcohol dehydrogenases. PMID:9494109

  1. X-linked glucose-6-phosphate dehydrogenase (G6PD) and autosomal 6-phosphogluconate dehydrogenase (6PGD) polymorphisms in baboons

    SciTech Connect

    VandeBerg, J.L.; Aivaliotis, M.J.; Samollow, P.B. )

    1992-12-01

    Electrophoretic polymorphisms of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were examined in captive colonies of five subspecies of baboons (Papio hamadryas). Phenotype frequencies and family data verified the X-linked inheritance of the G6PD polymorphism. Insufficient family data were available to confirm autosomal inheritance of the 6PGD polymorphism, but the electrophoretic patterns of variant types (putative heterozygotes) suggested the codominant expression of alleles at an autosomal locus. Implications of the G6PD polymorphism are discussed with regard to its utility as a marker system for research on X-chromosome inactivation during baboon development and for studies of clonal cell proliferation and/or cell selection during the development of atherosclerotic lesions in the baboon model. 61 refs., 1 fig., 4 tabs.

  2. Shikimate dehydrogenase from Pinu sylvestris L. needles

    SciTech Connect

    Osipov, V.I.; Shein, I.V.

    1986-07-10

    Shikimate dehydrogenase was isolated by extraction from pine needles and partially purified by fractionation with ammonium sulfate. In conifers, in contrast to other plants, all three isoenzymes of shikimate dehydrogenase exhibit activity not only with NADP/sup +/, but also with NAD/sup +/. The values of K/sub m/ for shikimate, when NADP/sup +/ and NAD/sup +/ are used as cofactors, are 0.22 and 1.13 mM, respectively. The enzyme is maximally active at pH 10 with both cofactors. It is suggested that NAD-dependent shikimate dehydrogenase catalyzes the initial reaction of the alternative pathway of the conversion of shikimic acid to hydroxybenzoic acid. The peculiarities of the organization and regulation of the initial reactions of the shikimate pathway in conifers and in plants with shikimate dehydrogenase absolutely specific for NADP are discussed.

  3. Phosphorylation site on yeast pyruvate dehydrogenase complex

    SciTech Connect

    Uhlinger, D.J.

    1986-01-01

    The pyruvate dehydrogenase complex was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). Yeast cells were disrupted in a Manton-Gaulin laboratory homogenizer. The pyruvate dehydrogenase complex was purified by fractionation with polyethylene glycol, isoelectric precipitation, ultracentrifugation and chromatography on hydroxylapatite. Final purification of the yeast pyruvate dehydrogenase complex was achieved by cation-exchange high pressure liquid chromatography (HPLC). No endogenous pyruvate dehydrogenase kinase activity was detected during the purification. However, the yeast pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. Tryptic digestion of the /sup 32/P-labeled complex yielded a single phosphopeptide which was purified to homogeniety. The tryptic digest was subjected to chromatography on a C-18 reverse phase HPLC column with a linear gradient of acetonitrile. Radioactive fractions were pooled, concentrated, and subjected to anion-exchange HPLC. The column was developed with a linear gradient of ammonium acetate. Final purification of the phosphopeptide was achieved by chromatography on a C-18 reverse phase HPLC column developed with a linear gradient of acetonitrile. The amino acid sequence of the homogeneous peptide was determined by manual modified Edman degradation.

  4. Characterization of interactions of dihydrolipoamide dehydrogenase with its binding protein in the human pyruvate dehydrogenase complex

    SciTech Connect

    Park, Yun-Hee; Patel, Mulchand S.

    2010-05-07

    Unlike pyruvate dehydrogenase complexes (PDCs) from prokaryotes, PDCs from higher eukaryotes have an additional structural component, E3-binding protein (BP), for binding of dihydrolipoamide dehydrogenase (E3) in the complex. Based on the 3D structure of the subcomplex of human (h) E3 with the di-domain (L3S1) of hBP, the amino acid residues (H348, D413, Y438, and R447) of hE3 for binding to hBP were substituted singly by alanine or other residues. These substitutions did not have large effects on hE3 activity when measured in its free form. However, when these hE3 mutants were reconstituted in the complex, the PDC activity was significantly reduced to 9% for Y438A, 20% for Y438H, and 18% for D413A. The binding of hE3 mutants with L3S1 determined by isothermal titration calorimetry revealed that the binding affinities of the Y438A, Y438H, and D413A mutants to L3S1 were severely reduced (1019-, 607-, and 402-fold, respectively). Unlike wild-type hE3 the binding of the Y438A mutant to L3S1 was accompanied by an unfavorable enthalpy change and a large positive entropy change. These results indicate that hE3-Y438 and hE3-D413 play important roles in binding of hE3 to hBP.

  5. Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids

    PubMed Central

    Gallego, Oriol; Belyaeva, Olga V.; Porté, Sergio; Ruiz, F. Xavier; Stetsenko, Anton V.; Shabrova, Elena V.; Kostereva, Natalia V.; Farrés, Jaume; Parés, Xavier; Kedishvili, Natalia Y.

    2006-01-01

    Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low Km values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low Km values for retinoids (0.12–1.1 μM), whilst they strongly differ in their kcat values, which range from 0.35 min−1 for AKR1B1 to 302 min−1 for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo. PMID:16787387

  6. Alteration of substrate specificity of alanine dehydrogenase

    PubMed Central

    Fernandes, Puja; Aldeborgh, Hannah; Carlucci, Lauren; Walsh, Lauren; Wasserman, Jordan; Zhou, Edward; Lefurgy, Scott T.; Mundorff, Emily C.

    2015-01-01

    The l-alanine dehydrogenase (AlaDH) has a natural history that suggests it would not be a promising candidate for expansion of substrate specificity by protein engineering: it is the only amino acid dehydrogenase in its fold family, it has no sequence or structural similarity to any known amino acid dehydrogenase, and it has a strong preference for l-alanine over all other substrates. By contrast, engineering of the amino acid dehydrogenase superfamily members has produced catalysts with expanded substrate specificity; yet, this enzyme family already contains members that accept a broad range of substrates. To test whether the natural history of an enzyme is a predictor of its innate evolvability, directed evolution was carried out on AlaDH. A single mutation identified through molecular modeling, F94S, introduced into the AlaDH from Mycobacterium tuberculosis (MtAlaDH) completely alters its substrate specificity pattern, enabling activity toward a range of larger amino acids. Saturation mutagenesis libraries in this mutant background additionally identified a double mutant (F94S/Y117L) showing improved activity toward hydrophobic amino acids. The catalytic efficiencies achieved in AlaDH are comparable with those that resulted from similar efforts in the amino acid dehydrogenase superfamily and demonstrate the evolvability of MtAlaDH specificity toward other amino acid substrates. PMID:25538307

  7. Benzene toxicity: emphasis on cytosolic dihydrodiol dehydrogenases

    SciTech Connect

    Bolcsak, L.E.

    1982-01-01

    Blood dyscrasias such as leukopenia and anemia have been clearly identified as consequences of chronic benzene exposure. The metabolites, phenol, catechol, and hydroquinone produced inhibition of /sup 59/Fe uptake in mice which followed the same time course as that produced by benzene. The inhibitor of benzene oxidation, 3-amino-1,2,4-triazole, mitigated the inhibitory effects of benzene and phenol only. These data support the contention that benzene toxicity is mediated by a metabolite and suggest that the toxicity of phenol is a consequence of its metabolism to hydroquinone and that the route of metabolism to catechol may also contribute to the production of toxic metabolite(s). The properties of mouse liver cytosolic dihydrodiol dehydrogenases were examined. These enzymes catalyze the NADP/sup +/-dependent oxidation of trans-1,2-dihydro-1,2-dihydroxybenzene (BDD) to catechol, a possible toxic metabolite of benzene produced via this metabolic route. Four distinct dihydrodiol dehydrogenases (DD1, DD2, DD3, and DD4) were purified to apparent homogeneity as judged by SDS polyacrylamide gel electrophoresis and isoelectric focusing. DD1 appeared to be identical to the major ketone reductase and 17..beta..-hydroxysteroid dehydrogenase activity in the liver. DD2 exhibited aldehyde reductase activity. DD3 and DD4 oxidized 17..beta..-hydroxysteroids, but no carbonyl reductase activity was detected. These relationships between BDD dehydrogenases and carbonyl reductase and/or 17..beta..-hydroxysteroid dehydrogenase activities were supported by several lines of evidence.

  8. Identification of the iron-sulfur center in trimethylamine dehydrogenase.

    PubMed

    Hill, C L; Steenkamp, D J; Holm, R H; Singer, T P

    1977-02-01

    Trimethylamine dehydrogenase [trimethylamine:(acceptor) oxidoreductase (demethylating), EC 1.5.99.7] from a facultative methylotroph bacterium has a molecular weight of 147,000 and contains two types of prosthetic groups, one a covalently bound organic chromophore of uncertain structure and the other containing iron and labile sulfur (S*). The structure of the Fe-S* center has been investigated by reactions of the enzyme with sodium mersalyl, o-xylyl-alpha,alpha'-dithiol, and p-methoxybenzenethiol in a 4:1 vol/vol hexamethylphosphoramide/water reaction medium, which destabilizes tertiary structure. Mersalyl treatment results in reduction of visible absorbance consistent with the presence of a 4-Fe center of the ferredoxin type. Reaction with thiols effects partial bleaching of the organic chromophore, as established by separate studies of a detached chromophore peptide, and results in removal (extrusion) of the core unit of the Fe-s* center in the form of the complexes [Fe4S*4(S2-o-xylyl)2]n2n- and [Fe4S*4(SC6H4OMe)4]2-, which were identified by absorption spectra. These results, in conjunction with control extrusion reactions of oxidized ferredoxins from spinach and Clostridium pasteurianum, establish that trimethylamine dehydrogenase contains one Fe4S*4 core unit most probably present as a ferredoxin-type, cysteinate-ligated cluster [Fe4S*4(S-Cys)4].

  9. Affinity chromatography of bacterial lactate dehydrogenases.

    PubMed Central

    Kelly, N; Delaney, M; O'Carra, P

    1978-01-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  10. Affinity chromatography of bacterial lactate dehydrogenases.

    PubMed

    Kelly, N; Delaney, M; O'Carra, P

    1978-06-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  11. Molybdopterin cofactor from Methanobacterium formicicum formate dehydrogenase.

    PubMed Central

    May, H D; Schauer, N L; Ferry, J G

    1986-01-01

    The molybdopterin cofactor from the formate dehydrogenase of Methanobacterium formicicum was studied. The cofactor was released by guanidine denaturation of homogeneous enzyme, which also released greater than 80% of the molybdenum present in the enzyme. The anoxically isolated cofactor was nonfluorescent, but after exposure to air it fluoresced with spectra similar to those of described molybdopterin cofactors. Aerobic release from acid-denatured formate dehydrogenase in the presence of I2 and potassium iodide produced a mixture of fluorescent products. Alkaline permanganate oxidation of the mixture yielded pterin-6-carboxylic acid as the only detectable fluorescent product. The results showed that the cofactor from formate dehydrogenase contained a pterin nucleus with a 6-alkyl side chain of unknown structure. Covalently bound phosphate was also present. The isolated cofactor was unable to complement the cofactor-deficient nitrate reductase of the Neurospora crassa nit-1 mutant. PMID:3700335

  12. Monoterpene metabolism. Cloning, expression, and characterization of (-)-isopiperitenol/(-)-carveol dehydrogenase of peppermint and spearmint.

    PubMed

    Ringer, Kerry L; Davis, Edward M; Croteau, Rodney

    2005-03-01

    The essential oils of peppermint (Mentha x piperita) and spearmint (Mentha spicata) are distinguished by the oxygenation position on the p-menthane ring of the constitutive monoterpenes that is conferred by two regiospecific cytochrome P450 limonene-3- and limonene-6-hydroxylases. Following hydroxylation of limonene, an apparently similar dehydrogenase oxidizes (-)-trans-isopiperitenol to (-)-isopiperitenone in peppermint and (-)-trans-carveol to (-)-carvone in spearmint. Random sequencing of a peppermint oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes, including dehydrogenases. Full-length dehydrogenase clones were screened by functional expression in Escherichia coli using a recently developed in situ assay. A single full-length acquisition encoding (-)-trans-isopiperitenol dehydrogenase (ISPD) was isolated. The (-)-ISPD cDNA has an open reading frame of 795 bp that encodes a 265-residue enzyme with a calculated molecular mass of 27,191. Nondegenerate primers were designed based on the (-)-trans-ISPD cDNA sequence and employed to screen a spearmint oil gland secretory cell cDNA library from which a 5'-truncated cDNA encoding the spearmint homolog, (-)-trans-carveol-dehydrogenase, was isolated. Reverse transcription-PCR amplification and RACE were used to acquire the remaining 5'-sequence from RNA isolated from oil gland secretory cells of spearmint leaf. The full-length spearmint dehydrogenase shares >99% amino acid identity with its peppermint homolog and both dehydrogenases are capable of utilizing (-)-trans-isopiperitenol and (-)-trans-carveol. These isopiperitenol/carveol dehydrogenases are members of the short-chain dehydrogenase/reductase superfamily and are related to other plant short-chain dehydrogenases/reductases involved in secondary metabolism (lignan biosynthesis), stress responses, and phytosteroid biosynthesis, but they are quite dissimilar (approximately 13% identity) to the monoterpene

  13. Monoterpene metabolism. Cloning, expression, and characterization of (-)-isopiperitenol/(-)-carveol dehydrogenase of peppermint and spearmint.

    PubMed

    Ringer, Kerry L; Davis, Edward M; Croteau, Rodney

    2005-03-01

    The essential oils of peppermint (Mentha x piperita) and spearmint (Mentha spicata) are distinguished by the oxygenation position on the p-menthane ring of the constitutive monoterpenes that is conferred by two regiospecific cytochrome P450 limonene-3- and limonene-6-hydroxylases. Following hydroxylation of limonene, an apparently similar dehydrogenase oxidizes (-)-trans-isopiperitenol to (-)-isopiperitenone in peppermint and (-)-trans-carveol to (-)-carvone in spearmint. Random sequencing of a peppermint oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes, including dehydrogenases. Full-length dehydrogenase clones were screened by functional expression in Escherichia coli using a recently developed in situ assay. A single full-length acquisition encoding (-)-trans-isopiperitenol dehydrogenase (ISPD) was isolated. The (-)-ISPD cDNA has an open reading frame of 795 bp that encodes a 265-residue enzyme with a calculated molecular mass of 27,191. Nondegenerate primers were designed based on the (-)-trans-ISPD cDNA sequence and employed to screen a spearmint oil gland secretory cell cDNA library from which a 5'-truncated cDNA encoding the spearmint homolog, (-)-trans-carveol-dehydrogenase, was isolated. Reverse transcription-PCR amplification and RACE were used to acquire the remaining 5'-sequence from RNA isolated from oil gland secretory cells of spearmint leaf. The full-length spearmint dehydrogenase shares >99% amino acid identity with its peppermint homolog and both dehydrogenases are capable of utilizing (-)-trans-isopiperitenol and (-)-trans-carveol. These isopiperitenol/carveol dehydrogenases are members of the short-chain dehydrogenase/reductase superfamily and are related to other plant short-chain dehydrogenases/reductases involved in secondary metabolism (lignan biosynthesis), stress responses, and phytosteroid biosynthesis, but they are quite dissimilar (approximately 13% identity) to the monoterpene

  14. Anti-diabetic and anti-adipogenic effects of a novel selective 11β-hydroxysteroid dehydrogenase type 1 inhibitor, 2-(3-benzoyl)-4-hydroxy-1,1-dioxo-2H-1,2-benzothiazine-2-yl-1-phenylethanone (KR-66344).

    PubMed

    Park, Ji Seon; Rhee, Sang Dal; Kang, Nam Sook; Jung, Won Hoon; Kim, Hee Youn; Kim, Jun Hyoung; Kang, Seung Kyu; Cheon, Hyae Gyeong; Ahn, Jin Hee; Kim, Ki Young

    2011-04-15

    The selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) have considerable potential for treating type 2 diabetes mellitus and metabolic syndrome. In the present study, we investigated the anti-diabetic and anti-adipogenic effects of 2-(3-benzoyl)-4-hydroxy-1,1-dioxo-2H-1,2-benzothiazine-2-yl-1-phenylethanone (KR-66344), as a 11β-HSD1 inhibitor; we also investigated the underlying molecular mechanisms in the cortisone-induced 3T3-L1 adipogenesis model system and C57BL/6-Lep(ob/ob) mice. KR-66344 concentration-dependently inhibited 11β-HSD1 activity in human liver microsome, mouse C2C12 myotube and human SW982 cells. In the C57BL/6-Lep(ob/ob) mice study, the administration of KR-66344 (200mg/kg/d, orally for 5 days) improved the glucose intolerance as determined by the oral glucose tolerance test, in which the area under the curve (AUC) of the plasma glucose concentration was significantly reduced by 27% compared with the vehicle treated group. Further, KR-66344 suppressed adipocyte differentiation on cortisone-induced adipogenesis in 3T3-L1 cells is associated with the suppression of the cortisone-induced mRNA levels of FABP4, G3PD, PPARγ2 and Glut4, and 11β-HSD1 expression and activity. Our results additionally demonstrate evidence showing that KR-66344 improved glycemic control and inhibited adipogenesis via 11β-HSD1 enzyme activity. Taken together, these results may provide evidence of the therapeutic potential of KR-66344, as a 11β-HSD1 inhibitor, in obesity and type 2 diabetes patients with metabolic syndrome.

  15. The first step in polyethylene glycol degradation by sphingomonads proceeds via a flavoprotein alcohol dehydrogenase containing flavin adenine dinucleotide.

    PubMed

    Sugimoto, M; Tanabe, M; Hataya, M; Enokibara, S; Duine, J A; Kawai, F

    2001-11-01

    Several Sphingomonas spp. utilize polyethylene glycols (PEGs) as a sole carbon and energy source, oxidative PEG degradation being initiated by a dye-linked dehydrogenase (PEG-DH) that oxidizes the terminal alcohol groups of the polymer chain. Purification and characterization of PEG-DH from Sphingomonas terrae revealed that the enzyme is membrane bound. The gene encoding this enzyme (pegA) was cloned, sequenced, and expressed in Escherichia coli. The purified recombinant enzyme was vulnerable to aggregation and inactivation, but this could be prevented by addition of detergent. It is as a homodimeric protein with a subunit molecular mass of 58.8 kDa, each subunit containing 1 noncovalently bound flavin adenine dinucleotide but not Fe or Zn. PEG-DH recognizes a broad variety of primary aliphatic and aromatic alcohols as substrates. Comparison with known sequences revealed that PEG-DH belongs to the group of glucose-methanol-choline (GMC) flavoprotein oxidoreductases and that it is a novel type of flavoprotein alcohol dehydrogenase related (percent identical amino acids) to other, so far uncharacterized bacterial, membrane-bound, dye-linked dehydrogenases: alcohol dehydrogenase from Pseudomonas oleovorans (46%); choline dehydrogenase from E. coli (40%); L-sorbose dehydrogenase from Gluconobacter oxydans (38%); and 4-nitrobenzyl alcohol dehydrogenase from a Pseudomonas species (35%). PMID:11673442

  16. Yeast surface display of dehydrogenases in microbial fuel-cells.

    PubMed

    Gal, Idan; Schlesinger, Orr; Amir, Liron; Alfonta, Lital

    2016-12-01

    Two dehydrogenases, cellobiose dehydrogenase from Corynascus thermophilus and pyranose dehydrogenase from Agaricus meleagris, were displayed for the first time on the surface of Saccharomyces cerevisiae using the yeast surface display system. Surface displayed dehydrogenases were used in a microbial fuel cell and generated high power outputs. Surface displayed cellobiose dehydrogenase has demonstrated a midpoint potential of -28mV (vs. Ag/AgCl) at pH=6.5 and was used in a mediator-less anode compartment of a microbial fuel cell producing a power output of 3.3μWcm(-2) using lactose as fuel. Surface-displayed pyranose dehydrogenase was used in a microbial fuel cell and generated high power outputs using different substrates, the highest power output that was achieved was 3.9μWcm(-2) using d-xylose. These results demonstrate that surface displayed cellobiose dehydrogenase and pyranose dehydrogenase may successfully be used in microbial bioelectrochemical systems.

  17. Yeast surface display of dehydrogenases in microbial fuel-cells.

    PubMed

    Gal, Idan; Schlesinger, Orr; Amir, Liron; Alfonta, Lital

    2016-12-01

    Two dehydrogenases, cellobiose dehydrogenase from Corynascus thermophilus and pyranose dehydrogenase from Agaricus meleagris, were displayed for the first time on the surface of Saccharomyces cerevisiae using the yeast surface display system. Surface displayed dehydrogenases were used in a microbial fuel cell and generated high power outputs. Surface displayed cellobiose dehydrogenase has demonstrated a midpoint potential of -28mV (vs. Ag/AgCl) at pH=6.5 and was used in a mediator-less anode compartment of a microbial fuel cell producing a power output of 3.3μWcm(-2) using lactose as fuel. Surface-displayed pyranose dehydrogenase was used in a microbial fuel cell and generated high power outputs using different substrates, the highest power output that was achieved was 3.9μWcm(-2) using d-xylose. These results demonstrate that surface displayed cellobiose dehydrogenase and pyranose dehydrogenase may successfully be used in microbial bioelectrochemical systems. PMID:27459246

  18. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the activity of the lactic dehydrogenase enzyme in serum. Increased levels of lactic dehydrogenase...

  19. Determination of the Subunit Molecular Mass and Composition of Alcohol Dehydrogenase by SDS-PAGE

    ERIC Educational Resources Information Center

    Nash, Barbara T.

    2007-01-01

    SDS-PAGE is a simple, rapid technique that has many uses in biochemistry and is readily adaptable to the undergraduate laboratory. It is, however, a technique prone to several types of procedural pitfalls. This article describes the use of SDS-PAGE to determine the subunit molecular mass and composition of yeast alcohol dehydrogenase employing…

  20. Glucose-6-phosphate dehydrogenase alloenzymes and their relationship to pigmentation in Serratia marcescens.

    PubMed

    Gargallo, D; Lorén, J G; Guinea, J; Viñas, M

    1987-08-01

    A comparative study of environmental and clinical isolates of Serratia marcescens was undertaken with regard to glucose-6-phosphate dehydrogenase (G6PD) electrophoretic mobility and the production of prodigiosin. Two electromorphs of G6PD with electrophoretic mobilities of 0.22 and 0.30 were detected. G6PD electrophoretic type showed a good correlation with the ability to produce prodigiosin.

  1. Effects of herbal infusions, tea and carbonated beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity.

    PubMed

    Li, Sha; Gan, Li-Qin; Li, Shu-Ke; Zheng, Jie-Cong; Xu, Dong-Ping; Li, Hua-Bin

    2014-01-01

    Various alcoholic beverages containing different concentrations of ethanol are widely consumed, and excessive alcohol consumption may result in serious health problems. The consumption of alcoholic beverages is often accompanied by non-alcoholic beverages, such as herbal infusions, tea and carbonated beverages to relieve drunk symptoms. The aim of this study was to supply new information on the effects of these beverages on alcohol metabolism for nutritionists and the general public, in order to reduce problems associated with excessive alcohol consumption. The effects of 57 kinds of herbal infusions, tea and carbonated beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity were evaluated. Generally, the effects of these beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity are very different. The results suggested that some beverages should not be drank after excessive alcohol consumption, and several beverages may be potential dietary supplements for the prevention and treatment of problems related to excessive alcohol consumption.

  2. Multiple retinoid dehydrogenases in testes cytosol from alcohol dehydrogenase negative or positive deermice.

    PubMed

    Posch, K C; Napoli, J L

    1992-05-28

    Retinoic acid syntheses from retinol by cytosol from testes of alcohol dehydrogenase negative or positive deermice were similar in specific activity and in their insensitivity to 1 M ethanol or 100 mM 4-methylpyrazole. Anion-exchange followed by size-exclusion chromatography revealed multiple and similarly migrating peaks in each cytosol that had both retinol and retinal dehydrogenase activities. Thus, the effects of ethanol on testes cannot be caused by direct inhibition of cytosolic retinoic acid synthesis because retinoid dehydrogenases distinct from mouse class A2 alcohol dehydrogenases, which corresponds to human class I, occurred in testes and they were not inhibited by ethanol. These data also demonstrate the occurrence of multiple cytosolic retinoic acid synthesis activities and indicate that the two reactions of cytosolic retinoic acid synthesis, retinol and retinal dehydrogenation, may be catalyzed by enzymes that occur as complexes. PMID:1599517

  3. The physiological role of liver alcohol dehydrogenase.

    PubMed

    Krebs, H A; Perkins, J R

    1970-07-01

    1. Yeast alcohol dehydrogenase was used to determine ethanol in the portal and hepatic veins and in the contents of the alimentary canal of rats given a diet free from ethanol. Measurable amounts of a substance behaving like ethanol were found. Its rate of interaction with yeast alcohol dehydrogenase and its volatility indicate that the substance measured was in fact ethanol. 2. The mean alcohol concentration in the portal blood of normal rats was 0.045mm. In the hepatic vein, inferior vena cava and aorta it was about 15 times lower. 3. The contents of all sections of the alimentary canal contained measurable amounts of ethanol. The highest values (average 3.7mm) were found in the stomach. 4. Infusion of pyrazole (an inhibitor of alcohol dehydrogenase) raised the alcohol concentration in the portal vein 10-fold and almost removed the difference between portal and hepatic venous blood. 5. Addition of antibiotics to the food diminished the ethanol concentration of the portal blood to less than one-quarter and that of the stomach contents to less than one-fortieth. 6. The concentration of alcohol in the alimentary canal and in the portal blood of germ-free rats was much decreased, to less than one-tenth in the alimentary canal and to one-third in the portal blood, but detectable quantities remained. These are likely to arise from acetaldehyde formed by the normal pathways of degradation of threonine, deoxyribose phosphate and beta-alanine. 7. The results indicate that significant amounts of alcohol are normally formed in the gastro-intestinal tract. The alcohol is absorbed into the circulation and almost quantitatively removed by the liver. Thus the function, or a major function, of liver alcohol dehydrogenase is the detoxication of ethanol normally present. 8. The alcohol concentration in the stomach of alloxan-diabetic rats was increased about 8-fold. 9. The activity of liver alcohol dehydrogenase is generally lower in carnivores than in herbivores and omnivores

  4. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120.

    PubMed

    Frébortová, Jitka; Greplová, Marta; Seidl, Michael F; Heyl, Alexander; Frébort, Ivo

    2015-01-01

    Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants. PMID:26376297

  5. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5560 Lactic dehydrogenase immunological test system. (a) Identification. A lactic dehydrogenase... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Lactic dehydrogenase immunological test...

  6. Properties of formate dehydrogenase in Methanobacterium formicicum.

    PubMed Central

    Schauer, N L; Ferry, J G

    1982-01-01

    Soluble formate dehydrogenase from Methanobacterium formicicum was purified 71-fold with a yield of 35%. Purification was performed anaerobically in the presence of 10 mM sodium azide which stabilized the enzyme. The purified enzyme reduced, with formate, 50 mumol of methyl viologen per min per mg of protein and 8.2 mumol of coenzyme F420 per min per mg of protein. The apparent Km for 7,8-didemethyl-8-hydroxy-5-deazariboflavin, a hydrolytic derivative of coenzyme F420, was 10-fold greater (63 microM) than for coenzyme F420 (6 microM). The purified enzyme also reduced flavin mononucleotide (Km = 13 microM) and flavin adenine dinucleotide (Km = 25 microM) with formate, but did not reduce NAD+ or NADP+. The reduction of NADP+ with formate required formate dehydrogenase, coenzyme F420, and coenzyme F420:NADP+ oxidoreductase. The formate dehydrogenase had an optimal pH of 7.9 when assayed with the physiological electron acceptor coenzyme F420. The optimal reaction rate occurred at 55 degrees C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (Ki = 6 microM), azide (Ki = 39 microM), alpha,alpha-dipyridyl, and 1,10-phenanthroline. Denaturation of the purified formate dehydrogenase with sodium dodecyl sulfate under aerobic conditions revealed a fluorescent compound. Maximal excitation occurred at 385 nm, with minor peaks at 277 and 302 nm. Maximal fluorescence emission occurred at 455 nm. Images PMID:7061389

  7. Characterization of xylitol dehydrogenase from Debaryomyces hansenii

    SciTech Connect

    Girio, F.M.; Amaral-Collaco, M.T.; Pelica, F.

    1996-01-01

    The xylitol dehydrogenase (EC 1.1.1.9) from xylose-grown cells of Debaryomyces hansenii was partially purified in two chromatographic steps, and characterization studies were carried out in order to investigate the role of the xylitol dehydrogenase-catalyzed step in the regulation of D-xylose metabolism. The enzyme was most active at pH 9.0-9.5, and exhibited a broad polyol specificity. The Michaelis constants for xylitol and NAD{sup +} were 16.5 and 0.55 mM, respectively. Ca{sup 2+}, Mg{sup 2+}, and Mn{sup 2+} did not affect the enzyme activity. Conversely, Zn{sup 2+}, Cd{sup 2+}, and Co{sup 2+} strongly inhibited the enzyme activity. It was concluded that NAD{sup +}-xylitol dehydrogenase from D. hansenii has similarities with other xylose-fermenting yeasts in respect to optimal pH, substrate specificity, and K{sub m} value for xylitol, and therefore should be named L-iditol:NAD{sup +}-5-oxidoreductase (EC 1.1.1.14). The reason D. hansenii is a good xylitol producer is not because of its value of K for xylitol, which is low enough to assure its fast oxidation by NAD{sup +}-xylitol dehydrogenase. However, a higher K{sub m} value of xylitol dehydrogenase for NAD{sup +} compared to the K{sub m} values of other xylose-fermenting yeasts may be responsible for the higher xylitol yields. 22 refs., 4 figs., 2 tabs.

  8. Measuring the Impact of Microenvironmental Conditions on Mitochondrial Dehydrogenase Activity in Cultured Cells.

    PubMed

    Sun, Ramon C; Koong, Albert; Giaccia, Amato; Denko, Nicholas C

    2016-01-01

    Mitochondria are powerhouses of a cell, producing much of the cellular ATP. However, mitochondrial enzymes also participate in many cellular biosynthetic processes. They are responsible for helping to maintain NAD(P)/H and redox balance, supplying metabolic intermediates for cell growth, and regulating several types of programed cell death. Several mitochondrial enzymes have even been shown to participate in the oncogenic process such as isocitrate dehydrogenase, succinate dehydrogenase, and fumarate hydratase. Recent advances have identified significant metabolic changes in the mitochondria that are regulated by malignant transformation and environmental stimuli. Understanding the biological activity and regulation of mitochondrial enzymes can provide insight into how they participate in the process of oncogenic transformation and work to sustain malignant growth. This chapter describes a technique to measure mitochondrial dehydrogenase activities that is faster and more cost effective which can also be scaled up for high throughput. PMID:27325264

  9. Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

    PubMed Central

    Henningsen, Brooks M.; Hon, Shuen; Covalla, Sean F.; Sonu, Carolina; Argyros, D. Aaron; Barrett, Trisha F.; Wiswall, Erin; Froehlich, Allan C.

    2015-01-01

    Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter−1 acetate during fermentation of 114 g liter−1 glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter−1, this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter−1 and raised the ethanol yield to 7% above the wild-type level. PMID:26386051

  10. Increasing anaerobic acetate consumption and ethanol yields in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase.

    PubMed

    Henningsen, Brooks M; Hon, Shuen; Covalla, Sean F; Sonu, Carolina; Argyros, D Aaron; Barrett, Trisha F; Wiswall, Erin; Froehlich, Allan C; Zelle, Rintze M

    2015-12-01

    Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level.

  11. Proline dehydrogenase 2 (PRODH2) is a hydroxyproline dehydrogenase (HYPDH) and molecular target for treating primary hyperoxaluria.

    PubMed

    Summitt, Candice B; Johnson, Lynnette C; Jönsson, Thomas J; Parsonage, Derek; Holmes, Ross P; Lowther, W Todd

    2015-03-01

    The primary hyperoxalurias (PH), types 1-3, are disorders of glyoxylate metabolism that result in increased oxalate production and calcium oxalate stone formation. The breakdown of trans-4-hydroxy-L-proline (Hyp) from endogenous and dietary sources of collagen makes a significant contribution to the cellular glyoxylate pool. Proline dehydrogenase 2 (PRODH2), historically known as hydroxyproline oxidase, is the first step in the hydroxyproline catabolic pathway and represents a drug target to reduce the glyoxylate and oxalate burden of PH patients. This study is the first report of the expression, purification, and biochemical characterization of human PRODH2. Evaluation of a panel of N-terminal and C-terminal truncation variants indicated that residues 157-515 contain the catalytic core with one FAD molecule. The 12-fold higher k(cat)/K(m) value of 0.93 M⁻¹·s⁻¹ for Hyp over Pro demonstrates the preference for Hyp as substrate. Moreover, an anaerobic titration determined a K(d) value of 125 μM for Hyp, a value ~1600-fold lower than the K(m) value. A survey of ubiquinone analogues revealed that menadione, duroquinone, and CoQ₁ reacted more efficiently than oxygen as the terminal electron acceptor during catalysis. Taken together, these data and the slow reactivity with sodium sulfite support that PRODH2 functions as a dehydrogenase and most likely utilizes CoQ₁₀ as the terminal electron acceptor in vivo. Thus, we propose that the name of PRODH2 be changed to hydroxyproline dehydrogenase (HYPDH). Three Hyp analogues were also identified to inhibit the activity of HYPDH, representing the first steps toward the development of a novel approach to treat all forms of PH. PMID:25697095

  12. The pivotal role of pyruvate dehydrogenase kinases in metabolic flexibility.

    PubMed

    Zhang, Shuai; Hulver, Matthew W; McMillan, Ryan P; Cline, Mark A; Gilbert, Elizabeth R

    2014-01-01

    Metabolic flexibility is the capacity of a system to adjust fuel (primarily glucose and fatty acids) oxidation based on nutrient availability. The ability to alter substrate oxidation in response to nutritional state depends on the genetically influenced balance between oxidation and storage capacities. Competition between fatty acids and glucose for oxidation occurs at the level of the pyruvate dehydrogenase complex (PDC). The PDC is normally active in most tissues in the fed state, and suppressing PDC activity by pyruvate dehydrogenase (PDH) kinase (PDK) is crucial to maintain energy homeostasis under some extreme nutritional conditions in mammals. Conversely, inappropriate suppression of PDC activity might promote the development of metabolic diseases. This review summarizes PDKs' pivotal role in control of metabolic flexibility under various nutrient conditions and in different tissues, with emphasis on the best characterized PDK4. Understanding the regulation of PDC and PDKs and their roles in energy homeostasis could be beneficial to alleviate metabolic inflexibility and to provide possible therapies for metabolic diseases, including type 2 diabetes (T2D). PMID:24520982

  13. Differing roles of pyruvate dehydrogenase kinases during mouse oocyte maturation

    PubMed Central

    Hou, Xiaojing; Zhang, Liang; Han, Longsen; Ge, Juan; Ma, Rujun; Zhang, Xuesen; Moley, Kelle; Schedl, Tim; Wang, Qiang

    2015-01-01

    ABSTRACT Pyruvate dehydrogenase kinases (PDKs) modulate energy homeostasis in multiple tissues and cell types, under various nutrient conditions, through phosphorylation of the α subunit (PDHE1α, also known as PDHA1) of the pyruvate dehydrogenase (PDH) complex. However, the roles of PDKs in meiotic maturation are currently unknown. Here, by undertaking knockdown and overexpression analysis of PDK paralogs (PDK1–PDK4) in mouse oocytes, we established the site-specificity of PDKs towards the phosphorylation of three serine residues (Ser232, Ser293 and Ser300) on PDHE1α. We found that PDK3-mediated phosphorylation of Ser293-PDHE1α results in disruption of meiotic spindle morphology and chromosome alignment and decreased total ATP levels, probably through inhibition of PDH activity. Unexpectedly, we discovered that PDK1 and PDK2 promote meiotic maturation, as their knockdown disturbs the assembly of the meiotic apparatus, without significantly altering ATP content. Moreover, phosphorylation of Ser232-PDHE1α was demonstrated to mediate PDK1 and PDK2 action in meiotic maturation, possibly through a mechanism that is distinct from PDH inactivation. These findings reveal that there are divergent roles of PDKs during oocyte maturation and indicate a new mechanism controlling meiotic structure. PMID:25991547

  14. Differing roles of pyruvate dehydrogenase kinases during mouse oocyte maturation.

    PubMed

    Hou, Xiaojing; Zhang, Liang; Han, Longsen; Ge, Juan; Ma, Rujun; Zhang, Xuesen; Moley, Kelle; Schedl, Tim; Wang, Qiang

    2015-07-01

    Pyruvate dehydrogenase kinases (PDKs) modulate energy homeostasis in multiple tissues and cell types, under various nutrient conditions, through phosphorylation of the α subunit (PDHE1α, also known as PDHA1) of the pyruvate dehydrogenase (PDH) complex. However, the roles of PDKs in meiotic maturation are currently unknown. Here, by undertaking knockdown and overexpression analysis of PDK paralogs (PDK1-PDK4) in mouse oocytes, we established the site-specificity of PDKs towards the phosphorylation of three serine residues (Ser232, Ser293 and Ser300) on PDHE1α. We found that PDK3-mediated phosphorylation of Ser293-PDHE1α results in disruption of meiotic spindle morphology and chromosome alignment and decreased total ATP levels, probably through inhibition of PDH activity. Unexpectedly, we discovered that PDK1 and PDK2 promote meiotic maturation, as their knockdown disturbs the assembly of the meiotic apparatus, without significantly altering ATP content. Moreover, phosphorylation of Ser232-PDHE1α was demonstrated to mediate PDK1 and PDK2 action in meiotic maturation, possibly through a mechanism that is distinct from PDH inactivation. These findings reveal that there are divergent roles of PDKs during oocyte maturation and indicate a new mechanism controlling meiotic structure. PMID:25991547

  15. Peroxisomal lactate dehydrogenase is generated by translational readthrough in mammals

    PubMed Central

    Schueren, Fabian; Lingner, Thomas; George, Rosemol; Hofhuis, Julia; Dickel, Corinna; Gärtner, Jutta; Thoms, Sven

    2014-01-01

    Translational readthrough gives rise to low abundance proteins with C-terminal extensions beyond the stop codon. To identify functional translational readthrough, we estimated the readthrough propensity (RTP) of all stop codon contexts of the human genome by a new regression model in silico, identified a nucleotide consensus motif for high RTP by using this model, and analyzed all readthrough extensions in silico with a new predictor for peroxisomal targeting signal type 1 (PTS1). Lactate dehydrogenase B (LDHB) showed the highest combined RTP and PTS1 probability. Experimentally we show that at least 1.6% of the total cellular LDHB is targeted to the peroxisome by a conserved hidden PTS1. The readthrough-extended lactate dehydrogenase subunit LDHBx can also co-import LDHA, the other LDH subunit, into peroxisomes. Peroxisomal LDH is conserved in mammals and likely contributes to redox equivalent regeneration in peroxisomes. DOI: http://dx.doi.org/10.7554/eLife.03640.001 PMID:25247702

  16. Engineering of pyranose dehydrogenase for increased oxygen reactivity.

    PubMed

    Krondorfer, Iris; Lipp, Katharina; Brugger, Dagmar; Staudigl, Petra; Sygmund, Christoph; Haltrich, Dietmar; Peterbauer, Clemens K

    2014-01-01

    Pyranose dehydrogenase (PDH), a member of the GMC family of flavoproteins, shows a very broad sugar substrate specificity but is limited to a narrow range of electron acceptors and reacts extremely slowly with dioxygen as acceptor. The use of substituted quinones or (organo)metals as electron acceptors is undesirable for many production processes, especially of food ingredients. To improve the oxygen reactivity, site-saturation mutagenesis libraries of twelve amino acids around the active site of Agaricus meleagris PDH were expressed in Saccharomyces cerevisiae. We established high-throughput screening assays for oxygen reactivity and standard dehydrogenase activity using an indirect Amplex Red/horseradish peroxidase and a DCIP/D-glucose based approach. The low number of active clones confirmed the catalytic role of H512 and H556. Only one position was found to display increased oxygen reactivity. Histidine 103, carrying the covalently linked FAD cofactor in the wild-type, was substituted by tyrosine, phenylalanine, tryptophan and methionine. Variant H103Y was produced in Pichia pastoris and characterized and revealed a five-fold increase of the oxygen reactivity. PMID:24614932

  17. In Silico Analysis of Arabidopsis thaliana Peroxisomal 6-Phosphogluconate Dehydrogenase

    PubMed Central

    Fernández-Fernández, Álvaro D.; Corpas, Francisco J.

    2016-01-01

    NADPH, whose regeneration is critical for reductive biosynthesis and detoxification pathways, is an essential component in cell redox homeostasis. Peroxisomes are subcellular organelles with a complex biochemical machinery involved in signaling and stress processes by molecules such as hydrogen peroxide (H2O2) and nitric oxide (NO). NADPH is required by several peroxisomal enzymes involved in β-oxidation, NO, and glutathione (GSH) generation. Plants have various NADPH-generating dehydrogenases, one of which is 6-phosphogluconate dehydrogenase (6PGDH). Arabidopsis contains three 6PGDH genes that probably are encoded for cytosolic, chloroplastic/mitochondrial, and peroxisomal isozymes, although their specific functions remain largely unknown. This study focuses on the in silico analysis of the biochemical characteristics and gene expression of peroxisomal 6PGDH (p6PGDH) with the aim of understanding its potential function in the peroxisomal NADPH-recycling system. The data show that a group of plant 6PGDHs contains an archetypal type 1 peroxisomal targeting signal (PTS), while in silico gene expression analysis using affymetrix microarray data suggests that Arabidopsis p6PGDH appears to be mainly involved in xenobiotic response, growth, and developmental processes. PMID:27034898

  18. Peafowl lactate dehydrogenase: problem of isoenzyme identification.

    PubMed

    Rose, R G; Wilson, A C

    1966-09-16

    Peafowl, like other vertebrates, contain multiple forms of lactate dehydrogenase. The electrophoretic properties of the peafowl isoenzymes are unusual in that the isoenzyme from heart tissue can be either more or less anodic than that of muscle, depending on the pH. This finding focuses attention on the problem of isoenzyme identification. It is suggested that isoenzymes be identified on the basis of properties that are chemically and biologically more significant than electrophoretic mobility.

  19. Peafowl lactate dehydrogenase: problem of isoenzyme identification.

    PubMed

    Rose, R G; Wilson, A C

    1966-09-16

    Peafowl, like other vertebrates, contain multiple forms of lactate dehydrogenase. The electrophoretic properties of the peafowl isoenzymes are unusual in that the isoenzyme from heart tissue can be either more or less anodic than that of muscle, depending on the pH. This finding focuses attention on the problem of isoenzyme identification. It is suggested that isoenzymes be identified on the basis of properties that are chemically and biologically more significant than electrophoretic mobility. PMID:5917779

  20. Prenatal presentation of pyruvate dehydrogenase complex deficiency.

    PubMed

    Natarajan, Niranjana; Tully, Hannah M; Chapman, Teresa

    2016-08-01

    We present the case of a female infant referred for prenatal MR evaluation of ventriculomegaly, which had been attributed by the referring obstetrician to aqueductal stenosis. Fetal MR confirmed ventriculomegaly but also demonstrated cerebral volume loss and white matter abnormalities. After birth, the infant developed persistent lactic acidosis. A diagnosis of pyruvate dehydrogenase complex deficiency was made on the basis of metabolic and molecular genetic studies. Ventriculomegaly is a common referral reason for fetal MR, yet there are few published reports of the radiographic findings that accompany inborn errors of metabolism, one potentially under-recognized cause of enlarged ventricles. This case contributes to this small body of literature on the imaging features of pyruvate dehydrogenase complex deficiency by describing pre- and postnatal MR findings and key clinical details. Our report emphasizes the necessity of considering pyruvate dehydrogenase complex deficiency and other metabolic disorders as potential etiologies for fetal ventriculomegaly since prompt diagnosis may allow for early initiation of treatment and improve outcome. PMID:27026023

  1. Dihydrodiol dehydrogenase and polycyclic aromatic hydrocarbon metabolism

    SciTech Connect

    Smithgall, T.E.

    1986-01-01

    Carcinogenic activation of polycyclic aromatic hydrocarbons by microsomal monoxygenases proceeds through trans-dihydrodiol metabolites to diol-epoxide ultimate carcinogens. This thesis directly investigated the role of dihydrodiol dehydrogenase, a cytosolic NAD(P)-linked oxidoreductase, in the detoxification of polycyclic aromatic trans-dihydrodiols. A wide variety of non-K-region trans-dihydrodiols were synthesized and shown to be substrates for the homogeneous rat liver dehydrogenase, including several potent proximate carcinogens derived from 7,12-dimethylbenz(a)anthracene, 5-methylchrysene, and benzo(a)pyrene. Since microsomal activation of polycyclic aromatic hydrocarbons is highly stereospecific, the stereochemical course of enzymatic trans-dihydrodiol oxidation was monitored using circular dichroism spectropolarimetry. The major product formed from the dehydrogenase-catalyzed oxidation of the trans-1,2-dihydrodiol of naphthalene was characterized using UV, IR, NMR, and mass spectroscopy, and appears to be 4-hydroxy-1,2-naphthoquinone. Mass spectral analysis suggests that an analogous hydroxylated o-quinone is formed as the major product of benzo(a)pyrene-7,8-dihydrodiol oxidation. Enzymatic oxidation of trans-dihydrodiols was shown to be potently inhibited by all of the major classes of the nonsteroidal antiinflammatory drugs. Enhancement of trans-dihydrodiol proximate carcinogen oxidation may protect against possible adverse effects of the aspirin-like drugs, and help maintain the balance between activation and detoxification of polycyclic aromatic hydrocarbons.

  2. Relationships within the aldehyde dehydrogenase extended family.

    PubMed

    Perozich, J; Nicholas, H; Wang, B C; Lindahl, R; Hempel, J

    1999-01-01

    One hundred-forty-five full-length aldehyde dehydrogenase-related sequences were aligned to determine relationships within the aldehyde dehydrogenase (ALDH) extended family. The alignment reveals only four invariant residues: two glycines, a phenylalanine involved in NAD binding, and a glutamic acid that coordinates the nicotinamide ribose in certain E-NAD binary complex crystal structures, but which may also serve as a general base for the catalytic reaction. The cysteine that provides the catalytic thiol and its closest neighbor in space, an asparagine residue, are conserved in all ALDHs with demonstrated dehydrogenase activity. Sixteen residues are conserved in at least 95% of the sequences; 12 of these cluster into seven sequence motifs conserved in almost all ALDHs. These motifs cluster around the active site of the enzyme. Phylogenetic analysis of these ALDHs indicates at least 13 ALDH families, most of which have previously been identified but not grouped separately by alignment. ALDHs cluster into two main trunks of the phylogenetic tree. The largest, the "Class 3" trunk, contains mostly substrate-specific ALDH families, as well as the class 3 ALDH family itself. The other trunk, the "Class 1/2" trunk, contains mostly variable substrate ALDH families, including the class 1 and 2 ALDH families. Divergence of the substrate-specific ALDHs occurred earlier than the division between ALDHs with broad substrate specificities. A site on the World Wide Web has also been devoted to this alignment project.

  3. Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas putida Fu1: two molybdenum-containing dehydrogenases of novel structural composition.

    PubMed Central

    Koenig, K; Andreesen, J R

    1990-01-01

    The constitutive xanthine dehydrogenase and the inducible 2-furoyl-coenzyme A (CoA) dehydrogenase could be labeled with [185W]tungstate. This labeling was used as a reporter to purify both labile proteins. The radioactivity cochromatographed predominantly with the residual enzymatic activity of both enzymes during the first purification steps. Both radioactive proteins were separated and purified to homogeneity. Antibodies raised against the larger protein also exhibited cross-reactivity toward the second smaller protein and removed xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase activity up to 80 and 60% from the supernatant of cell extracts, respectively. With use of cell extract, Western immunoblots showed only two bands which correlated exactly with the activity stains for both enzymes after native polyacrylamide gel electrophoresis. Molybdate was absolutely required for incorporation of 185W, formation of cross-reacting material, and enzymatic activity. The latter parameters showed a perfect correlation. This evidence proves that the radioactive proteins were actually xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase. The apparent molecular weight of the native xanthine dehydrogenase was about 300,000, and that of 2-furoyl-CoA dehydrogenase was 150,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both enzymes revealed two protein bands corresponding to molecular weights of 55,000 and 25,000. The xanthine dehydrogenase contained at least 1.6 mol of molybdenum, 0.9 ml of cytochrome b, 5.8 mol of iron, and 2.4 mol of labile sulfur per mol of enzyme. The composition of the 2-furoyl-CoA dehydrogenase seemed to be similar, although the stoichiometry was not determined. The oxidation of furfuryl alcohol to furfural and further to 2-furoic acid by Pseudomonas putida Fu1 was catalyzed by two different dehydrogenases. Images PMID:2170335

  4. Improved Production of Propionic Acid in Propionibacterium jensenii via Combinational Overexpression of Glycerol Dehydrogenase and Malate Dehydrogenase from Klebsiella pneumoniae

    PubMed Central

    Liu, Long; Zhuge, Xin; Shin, Hyun-dong; Chen, Rachel R.; Li, Jianghua

    2015-01-01

    Microbial production of propionic acid (PA), an important chemical building block used as a preservative and chemical intermediate, has gained increasing attention for its environmental friendliness over traditional petrochemical processes. In previous studies, we constructed a shuttle vector as a useful tool for engineering Propionibacterium jensenii, a potential candidate for efficient PA synthesis. In this study, we identified the key metabolites for PA synthesis in P. jensenii by examining the influence of metabolic intermediate addition on PA synthesis with glycerol as a carbon source under anaerobic conditions. We also further improved PA production via the overexpression of the identified corresponding enzymes, namely, glycerol dehydrogenase (GDH), malate dehydrogenase (MDH), and fumarate hydratase (FUM). Compared to those in wild-type P. jensenii, the activities of these enzymes in the engineered strains were 2.91- ± 0.17- to 8.12- ± 0.37-fold higher. The transcription levels of the corresponding enzymes in the engineered strains were 2.85- ± 0.19- to 8.07- ± 0.63-fold higher than those in the wild type. The coexpression of GDH and MDH increased the PA titer from 26.95 ± 1.21 g/liter in wild-type P. jensenii to 39.43 ± 1.90 g/liter in the engineered strains. This study identified the key metabolic nodes limiting PA overproduction in P. jensenii and further improved PA titers via the coexpression of GDH and MDH, making the engineered P. jensenii strain a potential industrial producer of PA. PMID:25595755

  5. Idiopathic intracranial hypertension, hormones, and 11β-hydroxysteroid dehydrogenases

    PubMed Central

    Markey, Keira A; Uldall, Maria; Botfield, Hannah; Cato, Liam D; Miah, Mohammed A L; Hassan-Smith, Ghaniah; Jensen, Rigmor H; Gonzalez, Ana M; Sinclair, Alexandra J

    2016-01-01

    Idiopathic intracranial hypertension (IIH) results in raised intracranial pressure (ICP) leading to papilledema, visual dysfunction, and headaches. Obese females of reproductive age are predominantly affected, but the underlying pathological mechanisms behind IIH remain unknown. This review provides an overview of pathogenic factors that could result in IIH with particular focus on hormones and the impact of obesity, including its role in neuroendocrine signaling and driving inflammation. Despite occurring almost exclusively in obese women, there have been a few studies evaluating the mechanisms by which hormones and adipokines exert their effects on ICP regulation in IIH. Research involving 11β-hydroxysteroid dehydrogenase type 1, a modulator of glucocorticoids, suggests a potential role in IIH. Improved understanding of the complex interplay between adipose signaling factors such as adipokines, steroid hormones, and ICP regulation may be key to the understanding and future management of IIH. PMID:27186074

  6. On dihydroorotate dehydrogenases and their inhibitors and uses.

    PubMed

    Munier-Lehmann, Hélène; Vidalain, Pierre-Olivier; Tangy, Frédéric; Janin, Yves L

    2013-04-25

    Proper nucleosides availability is crucial for the proliferation of living entities (eukaryotic cells, parasites, bacteria, and virus). Accordingly, the uses of inhibitors of the de novo nucleosides biosynthetic pathways have been investigated in the past. In the following we have focused on dihydroorotate dehydrogenase (DHODH), the fourth enzyme in the de novo pyrimidine nucleosides biosynthetic pathway. We first described the different types of enzyme in terms of sequence, structure, and biochemistry, including the reported bioassays. In a second part, the series of inhibitors of this enzyme along with a description of their potential or actual uses were reviewed. These inhibitors are indeed used in medicine to treat autoimmune diseases such as rheumatoid arthritis or multiple sclerosis (leflunomide and teriflunomide) and have been investigated in treatments of cancer, virus, and parasite infections (i.e., malaria) as well as in crop science. PMID:23452331

  7. A comparison of potato and vertebrate lactate dehydrogenases.

    PubMed Central

    Poerio, E; Davies, D D

    1980-01-01

    A 2000-fold purification of L(+)-lactate dehydrogenase from potatoes is reported. Five isoenzymes of lactate dehydrogenase can be detected in crude extracts of potato, and three of these are present in the purified preparation. The enzyme (mol.wt. 150 000), which is composed of four subunits (mol.wt. 37 500), is active with the same oxo acids and hydroxy acids that have been reported as substrates with the same oxo acids and hydroxy acids that have been reported as substrates for vertebrate lactate dehydrogenases. These similarities between potato and vertebrate lactate dehydrogenases contrast sharply with some other reports on potato lactate dehydrogenase. These discrepancies are discussed in relation to the proposition that vertebrate and potato lactate dehydrogenases share a common evolutionary origin. PMID:7236200

  8. Partial Similarities Between Yeast and Liver Alcohol Dehydrogenases

    PubMed Central

    Jörnvall, Hans

    1973-01-01

    The primary structure of about half of the protein chain of yeast alcohol dehydrogenase has been determined and compared with the amino-acid sequences of other dehydrogenases. The enzyme is found to be distantly related to horse-liver alcohol dehydrogenase, although these two proteins have different quaternary structures and subunit sizes. Some regions show no significant similarities, but long segments within the N-terminal parts of the molecules are homologous, suggesting a common and important function for these segments. Ancestral connections between some different dehydrogenases can be concluded and the degree of evolutionary changes may be estimated. PMID:4599620

  9. 21 CFR 862.1500 - Malic dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... plasma. Malic dehydrogenase measurements are used in the diagnosis and treatment of muscle and liver diseases, myocardial infarctions, cancer, and blood disorders such as myelogenous (produced in the...

  10. 21 CFR 862.1500 - Malic dehydrogenase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... plasma. Malic dehydrogenase measurements are used in the diagnosis and treatment of muscle and liver diseases, myocardial infarctions, cancer, and blood disorders such as myelogenous (produced in the...

  11. 21 CFR 862.1500 - Malic dehydrogenase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... plasma. Malic dehydrogenase measurements are used in the diagnosis and treatment of muscle and liver diseases, myocardial infarctions, cancer, and blood disorders such as myelogenous (produced in the...

  12. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... and plasma. Isocitric dehydrogenase measurements are used in the diagnosis and treatment of liver disease such as viral hepatitis, cirrhosis, or acute inflammation of the biliary tract; pulmonary...

  13. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... and plasma. Isocitric dehydrogenase measurements are used in the diagnosis and treatment of liver disease such as viral hepatitis, cirrhosis, or acute inflammation of the biliary tract; pulmonary...

  14. Separate physiological roles for two isozymes of pyridine nucleotide-linked glycerol-3-phosphate dehydrogenase in chicken.

    NASA Technical Reports Server (NTRS)

    White, H. B., III; Kaplan, N. O.

    1972-01-01

    The isozymes considered are designated 'liver type' and 'muscle type' based on the tissue of highest concentration. Electrophoretic analysis shows that the liver type is found in small amounts or is undetectable in all tissues studied except liver. The muscle type is found in skeletal muscles and kidney. Presumptive hybrid enzymes occur at low levels in chicken liver and kidney. The tissue distribution of glyceron-3-P dehydrogenase in several birds capable of sustained flight is different than in chicken.

  15. Phosphorylation-dephosphorylation of yeast pyruvate dehydrogenase

    SciTech Connect

    Uhlinger, D.J.; Reed, L.J.

    1986-05-01

    Pyruvate dehydrogenase complex (PDC) was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). No pyruvate dehydrogenase (PDH) kinase activity was detected at any stage of the purification. However, the purified PDC was phosphorylated and inactivated by purified PDH kinase from bovine kidney mitochondria, Mg/sup 2 +/, and (..gamma..-/sup 32/P)ATP. The protein-bound radioactivity was localized in the PDH ..cap alpha.. subunit. The phosphorylated, inactivated PDC was dephosphorylated and reactivated with purified bovine PDH phosphatase, Mg/sup 2 +/, and Ca/sup 2 +/. From a tryptic digest of phosphorylated yeast PDC a radioactive peptide was isolated by anion and reverse phase HPLC. The sequence of this tetradecapeptide is Tyr-Gly-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Thr-Tyr-Arg. This sequence is very similar to the sequence of a tryptic phosphopeptide derived from the ..cap alpha.. subunit of bovine kidney and heart PDH: Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser-Tyr-Arg.

  16. Transcriptional regulation of pyruvate dehydrogenase kinase.

    PubMed

    Jeong, Ji Yun; Jeoung, Nam Ho; Park, Keun-Gyu; Lee, In-Kyu

    2012-10-01

    The pyruvate dehydrogenase complex (PDC) activity is crucial to maintains blood glucose and ATP levels, which largely depends on the phosphorylation status by pyruvate dehydrogenase kinase (PDK) isoenzymes. Although it has been reported that PDC is phosphorylated and inactivated by PDK2 and PDK4 in metabolically active tissues including liver, skeletal muscle, heart, and kidney during starvation and diabetes, the precise mechanisms by which expression of PDK2 and PDK4 are transcriptionally regulated still remains unclear. Insulin represses the expression of PDK2 and PDK4 via phosphorylation of FOXO through PI3K/Akt signaling pathway. Several nuclear hormone receptors activated due to fasting or increased fat supply, including peroxisome proliferator-activated receptors, glucocorticoid receptors, estrogen-related receptors, and thyroid hormone receptors, also participate in the up-regulation of PDK2 and PDK4; however, the endogenous ligands that bind those nuclear receptors have not been identified. It has been recently suggested that growth hormone, adiponectin, epinephrine, and rosiglitazone also control the expression of PDK4 in tissue-specific manners. In this review, we discuss several factors involved in the expressional regulation of PDK2 and PDK4, and introduce current studies aimed at providing a better understanding of the molecular mechanisms that underlie the development of metabolic diseases such as diabetes. PMID:23130316

  17. Expression of 11β-hydroxysteroid dehydrogenase isoforms in canine adrenal glands treated with trilostane.

    PubMed

    Teshima, Takahiro; Matsumoto, Hirotaka; Kumagai, Takayuki; Kurano, Mai; Koyama, Hidekazu

    2014-06-01

    Trilostane, a competitive inhibitor of 3β-hydroxysteroid dehydrogenase, is often used to treat canine hyperadrenocorticism. In some species, trilostane has been shown to have additional effects on steroid biosynthesis, and it has been postulated that trilostane might have effects on 11β-hydroxysteroid dehydrogenase (11β-HSD) in dogs. To investigate the effect of trilostane on 11β-HSD in canine adrenal glands, healthy Beagle dogs were treated with trilostane for 8 weeks. Trilostane treatment resulted in a significant decrease of the cortisol/cortisone ratio in the serum. The adrenal gland mRNA and protein expression levels of 11β-HSD type 1 and 11β-HSD type 2 were significantly higher and significantly lower respectively in dogs treated with trilostane compared to those in control healthy Beagle dogs. These findings suggest that trilostane may have an effect on 11β-HSD activity in canine adrenal glands.

  18. Physicochemical Characterization of a Thermostable Alcohol Dehydrogenase from Pyrobaculum aerophilum

    PubMed Central

    Vitale, Annalisa; Thorne, Natasha; Lovell, Scott; Battaile, Kevin P.; Hu, Xin; Shen, Min; D'Auria, Sabato; Auld, Douglas S.

    2013-01-01

    In this work we characterize an alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII). We have previously found that PyAeADHII has no activity when standard ADH substrates are used but is active when α-tetralone is used as substrate. Here, to gain insights into enzyme function, we screened several chemical libraries for enzymatic modulators using an assay employing α-tetralone. The results indicate that PyAeADHII activity in the presence of α-tetralone was inhibited by compounds such as flunarizine. We also examined metal coordination of the enzyme in solution by performing metal substitution of the enzyme-bound zinc (Zn2+) with cobalt. The solution-based absorption spectra for cobalt substituted PyAeADHII supports substitution at the structural Zn2+ site. To gain structural insight, we obtained the crystal structure of both wild-type and cobalt-substituted PyAeADHII at 1.75 Å and 2.20 Å resolution, respectively. The X-ray data confirmed one metal ion per monomer present only at the structural site with otherwise close conservation to other ADH enzymes. We next determined the co-crystal structure of the NADPH-bound form of the enzyme at 2.35 Å resolution to help define the active site region of the enzyme and this data shows close structural conservation with horse ADH, despite the lack of a catalytic Zn2+ ion in PyAeADHII. Modeling of α-tetralone into the NADPH bound structure suggests an arginine as a possible catalytic residue. The data presented here can yield a better understanding of alcohol dehydrogenases lacking the catalytic zinc as well as the structural features inherent to thermostable enzymes. PMID:23755111

  19. NADP-dehydrogenases from pepper fruits: effect of maturation.

    PubMed

    Mateos, Rosa M; Bonilla-Valverde, Daniel; del Río, Luis A; Palma, José M; Corpas, Francisco J

    2009-02-01

    NADPH is an important molecule in the redox balance of the cell. Pepper fruits are the second worldwide consumable vegetables and exhibit different phenotypes after maturation. In this paper, two pepper cultivars were studied: Vergasa whose fruits shift from green to red after maturation, and Biela that shifts to yellow. Using fresh fruits from the same plants of the two cultivars at distinct maturation stages, the activity and gene expression of the main NADPH-generating dehydrogenases was studied. The activity analysis of the main NADP-dehydrogenases, glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-isocitrate dehydrogenase (NADP-ICDH) and NADP-malic enzyme (NADP-ME), showed that, except for the G6PDH, all the activities were enhanced (54-100%) in the mature pepper fruits from both cultivars (red or yellow) with respect to green pepper fruits. The content of NADPH and NADP in the mature fruits of both cultivars showed a noteworthy increase with respect to green fruits. For the transcript analysis, a partial cDNA of each NADP-dehydrogenase was obtained, and the NADP-ME was the only NADP-dehydrogenase that showed a significant induction. The increase in the content of NADPH in mature fruits because of the enhanced activity of NADP-dehydrogenases suggests that these NADPH-generating enzymes could be involved in the maturation of pepper fruits.

  20. 21 CFR 862.1670 - Sorbitol dehydrogenase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Sorbitol dehydrogenase test system. 862.1670 Section 862.1670 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1670 Sorbitol dehydrogenase...

  1. Clostridium beijerinckii and Clostridium difficile Detoxify Methylglyoxal by a Novel Mechanism Involving Glycerol Dehydrogenase

    PubMed Central

    Liyanage, Hemachandra; Kashket, Shelby; Young, Michael; Kashket, Eva R.

    2001-01-01

    In contrast to gram-negative bacteria, little is known about the mechanisms by which gram-positive bacteria degrade the toxic metabolic intermediate methylglyoxal (MG). Clostridium beijerinckii BR54, a Tn1545 insertion mutant of the NCIMB 8052 strain, formed cultures that contained significantly more (free) MG than wild-type cultures. Moreover, BR54 was more sensitive to growth inhibition by added MG than the wild type, suggesting that it has a reduced ability to degrade MG. The single copy of Tn1545 in this strain lies just downstream from gldA, encoding glycerol dehydrogenase. As a result of antisense RNA production, cell extracts of BR54 possess significantly less glycerol dehydrogenase activity than wild-type cell extracts (H. Liyanage, M. Young, and E. R. Kashket, J. Mol. Microbiol. Biotechnol. 2:87–93, 2000). Inactivation of gldA in both C. beijerinckii and Clostridium difficile gave rise to pinpoint colonies that could not be subcultured, indicating that glycerol dehydrogenase performs an essential function in both organisms. We propose that this role is detoxification of MG. To our knowledge, this is the first report of targeted gene disruption in the C. difficile chromosome. PMID:11319074

  2. Conformations of Diphosphopyridine Coenzymes upon Binding to Dehydrogenases

    PubMed Central

    Lee, Chi-Yu; Eichner, Ronald D.; Kaplan, Nathan O.

    1973-01-01

    The binding of oxidized as well as reduced coenzyme to some dehydrogenases has been studied under different concentration ratios and temperatures by nuclear magnetic resonance spectroscopy. A significant difference in the spectral behavior between DPN+ and DPNH upon binding is interpreted in terms of fast and slow on-off rates relative to the nuclear magnetic resonance time scale in the binding of these two coenzymes. Significant downfield shifts of DPN+ were observed upon binding, comparable in magnitude to those expected upon opening (destacking) of the coenzymes in the case of chicken-muscle and lobster-tail lactate dehydrogenase (EC 1.1.1.27) and yeast alchol dehydrogenase (EC 1.1.1.1.). A preliminary survey of several other dehydrogenases is consistent with these findings. In the case of 3-phosphoglyceraldehyde dehydrogenase, there is a possibility that the coenzyme exists in the folded form. PMID:4351183

  3. Origins of the high catalytic activity of human alcohol dehydrogenase 4 studied with horse liver A317C alcohol dehydrogenase.

    PubMed

    Herdendorf, Timothy J; Plapp, Bryce V

    2011-05-30

    The turnover numbers and other kinetic constants for human alcohol dehydrogenase (ADH) 4 ("stomach" isoenzyme) are substantially larger (10-100-fold) than those for human class I and horse liver alcohol dehydrogenases. Comparison of the primary amino acid sequences (69% identity) and tertiary structures of these enzymes led to the suggestion that residue 317, which makes a hydrogen bond with the nicotinamide amide nitrogen of the coenzyme, may account for these differences. Ala-317 in the class I enzymes is substituted with Cys in human ADH4, and locally different conformations of the peptide backbones could affect coenzyme binding. This hypothesis was tested by making the A317C substitution in horse liver ADH1E and comparisons to the wild-type ADH1E. The steady-state kinetic constants for the oxidation of benzyl alcohol and the reduction of benzaldehyde catalyzed by the A317C enzyme were very similar (up to about 2-fold differences) to those for the wild-type enzyme. Transient kinetics showed that the rate constants for binding of NAD(+) and NADH were also similar. Transient reaction data were fitted to the full Ordered Bi Bi mechanism and showed that the rate constants for hydride transfer decreased by about 2.8-fold with the A317C substitution. The structure of A317C ADH1E complexed with NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol at 1.2 Å resolution is essentially identical to the structure of the wild-type enzyme, except near residue 317 where the additional sulfhydryl group displaces a water molecule that is present in the wild-type enzyme. ADH is adaptable and can tolerate internal substitutions, but the protein dynamics apparently are affected, as reflected in rates of hydride transfer. The A317C substitution is not solely responsible for the larger kinetic constants in human ADH4; thus, the differences in catalytic activity must arise from one or more of the other hundred substitutions in the enzyme.

  4. 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...

  5. The Aldehyde Dehydrogenase Gene Superfamily Resource Center

    PubMed Central

    2009-01-01

    The website http://www.aldh.org is a publicly available database for nomenclature and functional and molecular sequence information for members of the aldehyde dehydrogenase (ALDH) gene superfamily for animals, plants, fungi and bacteria. The site has organised gene-specific records. It provides synopses of ALDH gene records, marries trivial terms to correct nomenclature and links global accession identifiers with source data. Server-side alignment software characterises the integrity of each sequence relative to the latest genomic assembly and provides identifier-specific detail reports, including a graphical presentation of the transcript's exon - intron structure, its size, coding sequence, genomic strand and locus. Also included are a summary of substrates, inhibitors and enzyme kinetics. The site provides reference lists and is designed to facilitate data mining by interested investigators. PMID:20038501

  6. Mitochondrial aldehyde dehydrogenase and cardiac diseases

    PubMed Central

    Chen, Che-Hong; Sun, Lihan; Mochly-Rosen, Daria

    2010-01-01

    Numerous conditions promote oxidative stress, leading to the build-up of reactive aldehydes that cause cell damage and contribute to cardiac diseases. Aldehyde dehydrogenases (ALDHs) are important enzymes that eliminate toxic aldehydes by catalysing their oxidation to non-reactive acids. The review will discuss evidence indicating a role for a specific ALDH enzyme, the mitochondrial ALDH2, in combating oxidative stress by reducing the cellular ‘aldehydic load’. Epidemiological studies in humans carrying an inactive ALDH2, genetic models in mice with altered ALDH2 levels, and small molecule activators of ALDH2 all highlight the role of ALDH2 in cardioprotection and suggest a promising new direction in cardiovascular research and the development of new treatments for cardiovascular diseases. PMID:20558439

  7. Untangling the glutamate dehydrogenase allosteric nightmare.

    PubMed

    Smith, Thomas J; Stanley, Charles A

    2008-11-01

    Glutamate dehydrogenase (GDH) is found in all living organisms, but only animal GDH is regulated by a large repertoire of metabolites. More than 50 years of research to better understand the mechanism and role of this allosteric network has been frustrated by its sheer complexity. However, recent studies have begun to tease out how and why this complex behavior evolved. Much of GDH regulation probably occurs by controlling a complex ballet of motion necessary for catalytic turnover and has evolved concomitantly with a long antenna-like feature of the structure of the enzyme. Ciliates, the 'missing link' in GDH evolution, might have created the antenna to accommodate changing organelle functions and was refined in humans to, at least in part, link amino acid catabolism with insulin secretion.

  8. Fast internal dynamics in alcohol dehydrogenase.

    PubMed

    Monkenbusch, M; Stadler, A; Biehl, R; Ollivier, J; Zamponi, M; Richter, D

    2015-08-21

    Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in the fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D2O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains. PMID:26298156

  9. NADH electrochemical sensor coupled with dehydrogenase enzymes

    SciTech Connect

    Yamanaka, Hideko; Mascini, Marco )

    1992-06-01

    A graphite electrode assembled in a flow cell has shown to be a good detector for NADH. Current is linearly dependent on concentration in the range 10{sup {minus}7}-10{sup {minus}3} M without any mediator at the potential applied of 300 mV vs Ag/AgCl. Lactate and alcohol dehydrogenases were immobilized near to the electrode surface or in a reactor to obtain an NADH-based biosensor for lactate or ethanol. With lactate the authors succeeded to obtain a response only if the reactor was used and for alcohol a current proportional to the concentration was obtained either if the enzyme was immobilized in a membrane and placed near the electrode surface or when the enzyme was immobilized in a reactor form. By FIA procedures fast responses and recoveries were obtained, but with a short linear range.

  10. Crystal structure of Arabidopsis thaliana cytokinin dehydrogenase

    SciTech Connect

    Bae, Euiyoung; Bingman, Craig A.; Bitto, Eduard; Aceti, David J.; Phillips, Jr., George N.

    2008-08-13

    Since first discovered in Zea mays, cytokinin dehydrogenase (CKX) genes have been identified in many plants including rice and Arabidopsis thaliana, which possesses CKX homologues (AtCKX1-AtCKX7). So far, the three-dimensional structure of only Z. mays CKX (ZmCKX1) has been determined. The crystal structures of ZmCKX1 have been solved in the native state and in complex with reaction products and a slowly reacting substrate. The structures revealed four glycosylated asparagine residues and a histidine residue covalently linked to FAD. Combined with the structural information, recent biochemical analyses of ZmCKX1 concluded that the final products of the reaction, adenine and a side chain aldehyde, are formed by nonenzymatic hydrolytic cleavage of cytokinin imine products resulting directly from CKX catalysis. Here, we report the crystal structure of AtCKX7 (gene locus At5g21482.1, UniProt code Q9FUJ1).

  11. Fast internal dynamics in alcohol dehydrogenase

    SciTech Connect

    Monkenbusch, M.; Stadler, A. Biehl, R.; Richter, D.; Ollivier, J.; Zamponi, M.

    2015-08-21

    Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in the fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D{sub 2}O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains.

  12. Betaine aldehyde dehydrogenase isozymes of spinach

    SciTech Connect

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

    1986-04-01

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

  13. Structure-Function Relationships in Lactate Dehydrogenase

    PubMed Central

    Adams, Margaret J.; Buehner, Manfred; Chandrasekhar, K.; Ford, Geoffrey C.; Hackert, Marvin L.; Liljas, Anders; Rossmann, Michael G.; Smiley, Ira E.; Allison, William S.; Everse, Johannes; Kaplan, Nathan O.; Taylor, Susan S.

    1973-01-01

    The binding of coenzyme and substrate are considered in relation to the known primary and tertiary structure of lactate dehydrogenase (EC 1.1.1.27). The adenine binds in a hydrophobic crevice, and the two coenzyme phosphates are oriented by interactions with the protein. The positively charged guanidinium group of arginine 101 then folds over the negatively charged phosphates, collapsing the loop region over the active center and positioning the unreactive B side of the nicotinamide in a hydrophobic protein environment. Collapse of the loop also introduces various charged groups into the vicinity of the substrate binding site. The substrate is situated between histidine 195 and the C4 position on the nicotinamide ring, and is partially oriented by interactions between its carboxyl group and arginine 171. The spatial arrangements of these groups may provide the specificity for the L-isomer of lactate. PMID:4146647

  14. Regulation of hepatic branched-chain alpha-keto acid dehydrogenase complex in rats fed a high-fat diet

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: Branched-chain alpha-keto acid dehydrogenase complex (BCKDC) regulates branched-chain amino acid (BCAA) metabolism at the level of branched chain alpha-ketoacid (BCKA) catabolism. It has been demonstrated that the activity of hepatic BCKDC is markedly decreased in type 2 diabetic animal...

  15. Decreased succinate dehydrogenase activity of gamma and alpha motoneurons in mouse spinal cords following 13 weeks of exposure to microgravity.

    PubMed

    Ishihara, Akihiko; Nagatomo, Fumiko; Fujino, Hidemi; Kondo, Hiroyo; Ohira, Yoshinobu

    2013-10-01

    Cell body size and succinate dehydrogenase activity of motoneurons in the dorsolateral region of the ventral horn in the lumbar and cervical segments of the mouse spinal cord were assessed after long-term exposure to microgravity and compared with those of ground-based controls. Mice were housed in a mouse drawer system on the International Space Station for 13 weeks. The mice were transported to the International Space Station by the Space Shuttle Discovery and returned to Earth by the Space Shuttle Atlantis. No changes in the cell body size of motoneurons were observed in either segment after exposure to microgravity, but succinate dehydrogenase activity of small-sized (<300 μm(2)) gamma and medium-sized (300-700 μm(2)) alpha motoneurons, which have higher succinate dehydrogenase activity than large-sized (>700 μm(2)) alpha motoneurons, in both segments was lower than that of ground-based controls. We concluded that exposure to microgravity for longer than 3 months induced decreased succinate dehydrogenase activity of both gamma and slow-type alpha motoneurons. In particular, the decreased succinate dehydrogenase activity of gamma motoneurons was observed only after long-term exposure to microgravity. PMID:23943522

  16. Cytoplasm-to-myonucleus ratios and succinate dehydrogenase activities in adult rat slow and fast muscle fibers

    NASA Technical Reports Server (NTRS)

    Tseng, B. S.; Kasper, C. E.; Edgerton, V. R.

    1994-01-01

    The relationship between myonuclear number, cellular size, succinate dehydrogenase activity, and myosin type was examined in single fiber segments (n = 54; 9 +/- 3 mm long) mechanically dissected from soleus and plantaris muscles of adult rats. One end of each fiber segment was stained for DNA before quantitative photometric analysis of succinate dehydrogenase activity; the other end was double immunolabeled with fast and slow myosin heavy chain monoclonal antibodies. Mean +/- S.D. cytoplasmic volume/myonucleus ratio was higher in fast and slow plantaris fibers (112 +/- 69 vs. 34 +/- 21 x 10(3) microns3) than fast and slow soleus fibers (40 +/- 20 vs. 30 +/- 14 x 10(3) microns3), respectively. Slow fibers always had small volumes/myonucleus, regardless of fiber diameter, succinate dehydrogenase activity, or muscle of origin. In contrast, smaller diameter (< 70 microns) fast soleus and plantaris fibers with high succinate dehydrogenase activity appeared to have low volumes/myonucleus while larger diameter (> 70 microns) fast fibers with low succinate dehydrogenase activity always had large volume/myonucleus. Slow soleus fibers had significantly greater numbers of myonuclei/mm than did either fast soleus or fast plantaris fibers (116 +/- 51 vs. 55 +/- 22 and 44 +/- 23), respectively. These data suggest that the myonuclear domain is more limited in slow than fast fibers and in the fibers with a high, compared to a low, oxidative metabolic capability.

  17. Decreased succinate dehydrogenase activity of gamma and alpha motoneurons in mouse spinal cords following 13 weeks of exposure to microgravity.

    PubMed

    Ishihara, Akihiko; Nagatomo, Fumiko; Fujino, Hidemi; Kondo, Hiroyo; Ohira, Yoshinobu

    2013-10-01

    Cell body size and succinate dehydrogenase activity of motoneurons in the dorsolateral region of the ventral horn in the lumbar and cervical segments of the mouse spinal cord were assessed after long-term exposure to microgravity and compared with those of ground-based controls. Mice were housed in a mouse drawer system on the International Space Station for 13 weeks. The mice were transported to the International Space Station by the Space Shuttle Discovery and returned to Earth by the Space Shuttle Atlantis. No changes in the cell body size of motoneurons were observed in either segment after exposure to microgravity, but succinate dehydrogenase activity of small-sized (<300 μm(2)) gamma and medium-sized (300-700 μm(2)) alpha motoneurons, which have higher succinate dehydrogenase activity than large-sized (>700 μm(2)) alpha motoneurons, in both segments was lower than that of ground-based controls. We concluded that exposure to microgravity for longer than 3 months induced decreased succinate dehydrogenase activity of both gamma and slow-type alpha motoneurons. In particular, the decreased succinate dehydrogenase activity of gamma motoneurons was observed only after long-term exposure to microgravity.

  18. Cloning and sequencing of the gene encoding the 72-kilodalton dehydrogenase subunit of alcohol dehydrogenase from Acetobacter aceti.

    PubMed

    Inoue, T; Sunagawa, M; Mori, A; Imai, C; Fukuda, M; Takagi, M; Yano, K

    1989-06-01

    A genomic library of Acetobacter aceti DNA was constructed by using a broad-host-range cosmid vector. Complementation of a spontaneous alcohol dehydrogenase-deficient mutant resulted in the isolation of a plasmid designated pAA701. Subcloning and deletion analysis of pAA701 limited the region that complemented the deficiency in alcohol dehydrogenase activity of the mutant. The nucleotide sequence of this region was determined and showed that this region contained the full structural gene for the 72-kilodalton dehydrogenase subunit of the alcohol dehydrogenase enzyme complex. The predicted amino acid sequence of the gene showed homology with sequences of methanol dehydrogenase structural genes of Paracoccus denitrificans and Methylobacterium organophilum.

  19. Effect of 15-hydroxyprostaglandin dehydrogenase inhibitor on wound healing.

    PubMed

    Seo, Seung Yong; Han, Song-Iy; Bae, Chun Sik; Cho, Hoon; Lim, Sung Chul

    2015-06-01

    PGE2 is an important mediator of wound healing. It is degraded and inactivated by 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Various growth factors, type IV collagen, TIMP-2 and PGE2 are important mediators of inflammation involving wound healing. Overproduction of TGF-β and suppression of PGE2 are found in excessive wound scarring. If we make the condition downregulating growth factors and upregulating PGE2, the wound will have a positive effect which results in little scar formation after healing. TD88 is a 15-PGDH inhibitor based on thiazolinedione structure. We evaluated the effect of TD88 on wound healing. In 10 guinea pigs (4 control and 6 experimental groups), we made four 1cm diameter-sized circular skin defects on each back. TD88 and vehicle were applicated on the wound twice a day for 4 days in the experimental and control groups, respectively. Tissue samples were harvested for qPCR and histomorphometric analyses on the 2nd and 4th day after treatment. Histomorphometric analysis showed significant reepithelization in the experimental group. qPCR analysis showed significant decrease of PDGF, CTGF and TIMP-2, but significant increase of type IV collagen in the experimental group. Taken together TD88 could be a good effector on wound healing, especially in the aspects of prevention of scarring.

  20. Characteristics of aldehyde dehydrogenase 2 (Aldh2) knockout mice.

    PubMed

    Yu, Hsu-Sheng; Oyama, Tsunehiro; Isse, Toyohi; Kitakawa, Kyoko; Ogawa, Masanori; Pham, Thi-Thu-Phuong; Kawamoto, Toshihiro

    2009-11-01

    Acetaldehyde is an intermediate of ethanol oxidation. It covalently binds to DNA, and is known as a carcinogen. Aldehyde dehydrogenase 2 (ALDH2) is an important enzyme that oxidizes acetaldehyde. Approximately 45% of Chinese and Japanese individuals have the inactive ALDH2 genotypes (ALDH2*2/*2 and ALDH2*1/*2), and Aldh2 knockout mice appear to be a valid animal model for humans with inactive ALDH2. This review gives an overview of published studies on Aldh2 knockout mice, which were treated with ethanol or acetaldehyde. According to these studies, it was found that Aldh2 -/- mice (Aldh2 knockout mice) are more susceptible to ethanol and acetaldehyde-induced toxicity than Aldh2 +/+ mice (wild type mice). When mice were fed with ethanol, the mortality was increased. When they were exposed to atmospheres containing acetaldehyde, the Aldh2 -/- mice showed more severe toxic symptoms, like weight loss and higher blood acetaldehyde levels, as compared with the Aldh2 +/+ mice. Thus, ethanol and acetaldehyde treatment affects Aldh2 knockout mice more than wild type mice. Based on these findings, it is suggested that ethanol consumption and acetaldehyde inhalation are inferred to pose a higher risk to ALDH2-inactive humans. These results also support that ALDH2-deficient humans who habitually consume alcohol have a higher rate of cancer than humans with functional ALDH2. PMID:19874182

  1. [Succinate dehydrogenase (SDH)-deficient renal cell carcinoma].

    PubMed

    Agaimy, A

    2016-03-01

    Succinate dehydrogenase (SDH) represents a type II mitochondrial complex related to the respiratory chain and Krebs cycle. The complex is composed of four major subunits, SDHA, SDHB, SDHC and SDHD. The oncogenic role of this enzyme complex has only recently been recognized and the complex is currently considered an important oncogenic signaling pathway with tumor suppressor properties. In addition to the familial paraganglioma syndromes (types 1-5) as prototypical SDH-related diseases, many other tumors have been defined as SDH-deficient, in particular a subset of gastrointestinal stromal tumors (GIST), rare hypophyseal adenomas, a subset of pancreatic neuroendocrine neoplasms (recently added) and a variety of other tumor entities, the latter mainly described as rare case reports. As a central core subunit responsible for the integrity of the SDH complex, the expression of SDHB is lost in all SDH-deficient neoplasms irrespective of the specific SDH subunit affected by a genetic mutation in addition to concurrent loss of the subunit specifically affected by genetic alteration. Accordingly, all SDH-deficient neoplasms are by definition SDHB-deficient. The SDH-deficient renal cell carcinoma (RCC) has only recently been well-characterized and it is included as a specific subtype of RCC in the new World Health Organization (WHO) classification published in 2016. In this review, the major clinicopathological, immunohistochemical and genetic features of this rare disease entity are presented and discussed in the context of the broad differential diagnosis. PMID:26979428

  2. On the mechanism underlying tellurite reduction by Aeromonas caviae ST dihydrolipoamide dehydrogenase.

    PubMed

    Arenas, F A; Leal, C A; Pinto, C A; Arenas-Salinas, M A; Morales, W A; Cornejo, F A; Díaz-Vásquez, W A; Vásquez, C C

    2014-07-01

    The dihydrolipoamide dehydrogenase (LpdA) from the tellurite-resistant bacterium Aeromonas caviae ST reduces tellurite to elemental tellurium. To characterize this NADH-dependent activity, the A. caviae lpdA gene was subjected to site-directed mutagenesis and genes containing C45A, H322Y and E354K substitutions were individually transformed into Escherichia coli Δlpd. Cells expressing the modified genes exhibited decreased pyruvate dehydrogenase, dihydrolipoamide dehydrogenase and TR activity regarding that observed with the wild type A. caviae lpdA gene. In addition, cells expressing the altered lpdA genes showed increased oxidative stress levels and tellurite sensitivity than those carrying the wild type counterpart. The involvement of Cys residues in LpdA's TR activity was analyzed using specific inhibitors that interact with catalytic cysteines and/or disulfide bridges such as aurothiomalate, zinc or nickel. TR activity of purified LpdA was drastically affected by these compounds. Since LpdA belongs to the flavoprotein family, the involvement of the FAD/NAD(P)(+)-binding domain in TR activity was determined. FAD removal from purified LpdA results in loss of TR activity, which was restored with exogenously added FAD. Substitutions in E354, involved in FAD/NADH binding, resulted in low TR activity because of flavin loss. Finally, changing H322 (involved in NAD(+)/NADH binding) by tyrosine also resulted in altered TR activity.

  3. Acute overexpression of lactate dehydrogenase-A perturbs beta-cell mitochondrial metabolism and insulin secretion.

    PubMed

    Ainscow, E K; Zhao, C; Rutter, G A

    2000-07-01

    Islet beta-cells express low levels of lactate dehydrogenase and have high glycerol phosphate dehydrogenase activity. To determine whether this configuration favors oxidative glucose metabolism via mitochondria in the beta-cell and is important for beta-cell metabolic signal transduction, we have determined the effects on glucose metabolism and insulin secretion of acute overexpression of the skeletal muscle isoform of lactate dehydrogenase (LDH)-A. Monitored in single MIN6 beta-cells, LDH hyperexpression (achieved by intranuclear cDNA microinjection or adenoviral infection) diminished the response to glucose of both phases of increases in mitochondrial NAD(P)H, as well as increases in mitochondrial membrane potential, cytosolic free ATP, and cystolic free Ca2+. These effects were observed at all glucose concentrations, but were most pronounced at submaximal glucose levels. Correspondingly, adenoviral vector-mediated LDH-A overexpression reduced insulin secretion stimulated by 11 mmol/l glucose and the subsequent response to stimulation with 30 mmol/l glucose, but it was without significant effect when the concentration of glucose was raised acutely from 3 to 30 mmol/l. Thus, overexpression of LDH activity interferes with normal glucose metabolism and insulin secretion in the islet beta-cell type, and it may therefore be directly responsible for insulin secretory defects in some forms of type 2 diabetes. The results also reinforce the view that glucose-derived pyruvate metabolism in the mitochondrion is critical for glucose-stimulated insulin secretion in the beta-cell.

  4. On the mechanism underlying tellurite reduction by Aeromonas caviae ST dihydrolipoamide dehydrogenase.

    PubMed

    Arenas, F A; Leal, C A; Pinto, C A; Arenas-Salinas, M A; Morales, W A; Cornejo, F A; Díaz-Vásquez, W A; Vásquez, C C

    2014-07-01

    The dihydrolipoamide dehydrogenase (LpdA) from the tellurite-resistant bacterium Aeromonas caviae ST reduces tellurite to elemental tellurium. To characterize this NADH-dependent activity, the A. caviae lpdA gene was subjected to site-directed mutagenesis and genes containing C45A, H322Y and E354K substitutions were individually transformed into Escherichia coli Δlpd. Cells expressing the modified genes exhibited decreased pyruvate dehydrogenase, dihydrolipoamide dehydrogenase and TR activity regarding that observed with the wild type A. caviae lpdA gene. In addition, cells expressing the altered lpdA genes showed increased oxidative stress levels and tellurite sensitivity than those carrying the wild type counterpart. The involvement of Cys residues in LpdA's TR activity was analyzed using specific inhibitors that interact with catalytic cysteines and/or disulfide bridges such as aurothiomalate, zinc or nickel. TR activity of purified LpdA was drastically affected by these compounds. Since LpdA belongs to the flavoprotein family, the involvement of the FAD/NAD(P)(+)-binding domain in TR activity was determined. FAD removal from purified LpdA results in loss of TR activity, which was restored with exogenously added FAD. Substitutions in E354, involved in FAD/NADH binding, resulted in low TR activity because of flavin loss. Finally, changing H322 (involved in NAD(+)/NADH binding) by tyrosine also resulted in altered TR activity. PMID:24680738

  5. 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.

  6. Pyruvate Dehydrogenase Complex from Chloroplasts of Pisum sativum L 1

    PubMed Central

    Williams, Michael; Randall, Douglas D.

    1979-01-01

    Pyruvate dehydrogenase complex is associated with intact chloroplasts and mitochondria of 9-day-old Pisum sativum L. seedlings. The ratio of the mitochondrial complex to the chloroplast complex activities is about 3 to 1. Maximal rates observed for chloroplast pyruvate dehydrogenase complex activity ranged from 6 to 9 micromoles of NADH produced per milligram of chlorophyll per hour. Osmotic rupture of pea chloroplasts released 88% of the complex activity, indicating that chloroplast pyruvate dehydrogenase complex is a stromal complex. The pH optimum for chloroplast pyruvate dehydrogenase complex was between 7.8 and 8.2, whereas the mitochondrial pyruvate dehydrogenase complex had a pH optimum between 7.3 and 7.7. Chloroplast pyruvate dehydrogenase complex activity was specific for pyruvate, dependent upon coenzyme A and NAD and partially dependent upon Mg2+ and thiamine pyrophosphate. Chloroplast-associated pyruvate dehydrogenase complex provides a direct link between pyruvate metabolism and chloroplast fatty acid biosynthesis by providing the substrate, acetyl-CoA, necessary for membrane development in young plants. Images PMID:16661100

  7. Pyruvate dehydrogenase complex from higher plant mitochondria and proplastids.

    PubMed

    Reid, E E; Thompson, P; Lyttle, C R; Dennis, D T

    1977-05-01

    The pyruvate dehydrogenase complex from pea (Pisum sativum L.) mitochondria was purified 23-fold by high speed centrifugation and glycerol gradient fractionation. The complex had a s(20,w) of 47.5S but this is a minimal value since the complex is unstable. The complex is specific for NAD(+) and pyruvate; NADP(+) and other keto acids give no reaction. Mg(2+), thiamine pyrophosphate, and cysteine are also required for maximal activity. The pH optimum for the complex was between 6.5 and 7.5.Continuous sucrose density gradients were used to separate castor bean (Ricinus communis L.) endosperm proplastids from mitochondria. Pyruvate dehydrogenase complex activity was found to be coincident with the proplastid peak on all of the gradients. Some separation of proplastids and mitochondria could be achieved by differential centrifugation and the ratios of the activities of the pyruvate dehydrogenase complex to succinic dehydrogenase and acetyl-CoA carboxylase to succinic dehydrogenase were consistent with both the pyruvate dehydrogenase complex and acetyl-CoA carboxylase being present in the proplastid. The proplastid fraction has to be treated with a detergent, Triton X-100, before maximal activity of the pyruvate dehydrogenase complex activity is expressed, indicating that it is bound in the organelle. The complex had a sharp pH optimum of 7.5. The complex required added Mg(2+), cysteine, and thiamine pyrophosphate for maximal activity but thiamine pyrophosphate was inhibitory at higher concentrations.

  8. 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. PMID:25542170

  9. Deletion of the structural gene for the NADH-dehydrogenase subunit 4 of Synechocystis 6803 alters respiratory properties.

    PubMed Central

    Dzelzkalns, V A; Obinger, C; Regelsberger, G; Niederhauser, H; Kamensek, M; Peschek, G A; Bogorad, L

    1994-01-01

    Chloroplasts and cyanobacteria contain genes encoding polypeptides homologous to some subunits of the mitochondrial respiratory NADH-ubiquinol oxidoreductase complex (NADH dehydrogenase). Nothing is known of the role of the NADH dehydrogenase complex in photosynthesis, respiration, or other functions in chloroplasts, and little is known about the specific roles of the perhaps 42 subunits of this complex in the mitochondrion. Inactivation of a gene for subunit 4 (ndhD-2, ndh4) of this complex in the cyanobacterium Synechocystis 6803 has no effect on photosynthesis, judging from the rate of photoautotrophic growth of mutant cells, but the mutant's respiratory rate is about 6 times greater than that of wild-type cells. Respiratory electron transport activity in cyanobacteria is associated both with photosynthetic thylakoid membranes and with the outer cytoplasmic membrane of the cell. Cytoplasmic membranes of mutant cells have much greater NADH-dependent cytochrome reductase activity than preparations from wild-type cells; this activity remains at wild-type levels in isolated thylakoid membranes. It is suggested that the 56.6-kD product of ndhD-2 is not essential for the activity of a cytoplasmic membrane-bound NADH dehydrogenase but that it regulates the rate of electron flow through the complex, establishing a link between this ndh gene and respiration. The activity of the molecularly distinct thylakoid-bound NADH dehydrogenase is apparently unaffected by the loss of ndhD-2. PMID:7846157

  10. Biospecific affinity chromatographic purification of octopine dehydrogenase from molluscs.

    PubMed

    Mulcahy, P; Griffin, T; O'Carra, P

    1997-02-01

    The development of a biospecific affinity chromatographic method for the purification of octopine dehydrogenase from molluscs is described. The method utilizes immobilized NAD+ derivatives in conjunction with soluble specific substrates to promote binding. Using this method, octopine dehydrogenase has been purified to electrophoretic homogeneity in a single chromatographic step from three different marine invertebrate sources [the queen scallop, Chlamys opercularis (adductor muscle), the great scallop, Pecten maximus (adductor muscle), and the squid Loligo vulgaris (mantle muscle)]. However, the system is not applicable to the purification of octopine dehydrogenase from some other marine invertebrate sources investigated (the mussel Mytilus edulis and the topshell Monodonta lineata). PMID:9116492

  11. Role of quinate dehydrogenase in quinic acid metabolism in conifers

    SciTech Connect

    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.

  12. Structural and Thermodynamic Basis for Weak Interactions between Dihydrolipoamide Dehydrogenase and Subunit-binding Domain of the Branched-chain [alpha]-Ketoacid Dehydrogenase Complex

    SciTech Connect

    Brautigam, Chad A.; Wynn, R. Max; Chuang, Jacinta L.; Naik, Mandar T.; Young, Brittany B.; Huang, Tai-huang; Chuang, David T.

    2012-02-27

    The purified mammalian branched-chain {alpha}-ketoacid dehydrogenase complex (BCKDC), which catalyzes the oxidative decarboxylation of branched-chain {alpha}-keto acids, is essentially devoid of the constituent dihydrolipoamide dehydrogenase component (E3). The absence of E3 is associated with the low affinity of the subunit-binding domain of human BCKDC (hSBDb) for hE3. In this work, sequence alignments of hSBDb with the E3-binding domain (E3BD) of the mammalian pyruvate dehydrogenase complex show that hSBDb has an arginine at position 118, where E3BD features an asparagine. Substitution of Arg-118 with an asparagine increases the binding affinity of the R118N hSBDb variant (designated hSBDb*) for hE3 by nearly 2 orders of magnitude. The enthalpy of the binding reaction changes from endothermic with the wild-type hSBDb to exothermic with the hSBDb* variant. This higher affinity interaction allowed the determination of the crystal structure of the hE3/hSBDb* complex to 2.4-{angstrom} resolution. The structure showed that the presence of Arg-118 poses a unique, possibly steric and/or electrostatic incompatibility that could impede E3 interactions with the wild-type hSBDb. Compared with the E3/E3BD structure, the hE3/hSBDb* structure has a smaller interfacial area. Solution NMR data corroborated the interactions of hE3 with Arg-118 and Asn-118 in wild-type hSBDb and mutant hSBDb*, respectively. The NMR results also showed that the interface between hSBDb and hE3 does not change significantly from hSBDb to hSBDb*. Taken together, our results represent a starting point for explaining the long standing enigma that the E2b core of the BCKDC binds E3 far more weakly relative to other {alpha}-ketoacid dehydrogenase complexes.

  13. 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...

  14. A novel glutamate dehydrogenase from bovine brain: purification and characterization.

    PubMed

    Lee, J; Kim, S W; Cho, S W

    1995-08-01

    A soluble form of novel glutamate dehydrogenase has been purified from bovine brain. The preparation was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and composed of six identical subunits having a subunit size of 57,500 Da. The biochemical properties of glutamate dehydrogenase such as N-terminal amino acids sequences, kinetic parameters, amino acids analysis, and optimum pH were examined in both reductive amination of alpha-ketoglutarate and oxidative deamination of glutamate. N-terminal amino acid sequences of the bovine brain enzyme showed the significant differences in the first 5 amino acids compared to other glutamate dehydrogenases from various sources. These results indicate that glutamate dehydrogenase isolated from bovine brain is a novel polypeptide.

  15. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  16. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  17. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  18. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  19. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  20. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dehydrogenase measurements are used in the diagnosis and treatment of liver diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial...

  1. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... dehydrogenase measurements are used in the diagnosis and treatment of liver diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial...

  2. Genetics Home Reference: 3-beta-hydroxysteroid dehydrogenase deficiency

    MedlinePlus

    ... not by hormone test. Clin Endocrinol (Oxf). 2003 Mar;58(3):323-31. Citation on PubMed Pang S, ... dehydrogenase deficiency. Endocrinol Metab Clin North Am. 2001 Mar;30(1):81-99, vi-vii. Review. Citation ...

  3. Mammalian class IV alcohol dehydrogenase (stomach alcohol dehydrogenase): structure, origin, and correlation with enzymology.

    PubMed Central

    Parés, X; Cederlund, E; Moreno, A; Hjelmqvist, L; Farrés, J; Jörnvall, H

    1994-01-01

    The structure of a mammalian class IV alcohol dehydrogenase has been determined by peptide analysis of the protein isolated from rat stomach. The structure indicates that the enzyme constitutes a separate alcohol dehydrogenase class, in agreement with the distinct enzymatic properties; the class IV enzyme is somewhat closer to class I (the "classical" liver alcohol dehydrogenase; approximately 68% residue identities) than to the other classes (II, III, and V; approximately 60% residue identities), suggesting that class IV might have originated through duplication of an early vertebrate class I gene. The activity of the class IV protein toward ethanol is even higher than that of the classical liver enzyme. Both Km and kcat values are high, the latter being the highest of any class characterized so far. Structurally, these properties are correlated with replacements at the active site, affecting both substrate and coenzyme binding. In particular, Ala-294 (instead of valine) results in increased space in the middle section of the substrate cleft, Gly-47 (instead of a basic residue) results in decreased charge interactions with the coenzyme pyrophosphate, and Tyr-363 (instead of a basic residue) may also affect coenzyme binding. In combination, these exchanges are compatible with a promotion of the off dissociation and an increased turnover rate. In contrast, residues at the inner part of the substrate cleft are bulky, accounting for low activity toward secondary alcohols and cyclohexanol. Exchanges at positions 259-261 involve minor shifts in glycine residues at a reverse turn in the coenzyme-binding fold. Clearly, class IV is distinct in structure, ethanol turnover, stomach expression, and possible emergence from class I. PMID:8127901

  4. Elusive transition state of alcohol dehydrogenase unveiled

    PubMed Central

    Roston, Daniel; Kohen, Amnon

    2010-01-01

    For several decades the hydride transfer catalyzed by alcohol dehydrogenase has been difficult to understand. Here we add to the large corpus of anomalous and paradoxical data collected for this reaction by measuring a normal (> 1) 2° kinetic isotope effect (KIE) for the reduction of benzaldehyde. Because the relevant equilibrium effect is inverse (< 1), this KIE eludes the traditional interpretation of 2° KIEs. It does, however, enable the development of a comprehensive model for the “tunneling ready state” (TRS) of the reaction that fits into the general scheme of Marcus-like models of hydrogen tunneling. The TRS is the ensemble of states along the intricate reorganization coordinate, where H tunneling between the donor and acceptor occurs (the crossing point in Marcus theory). It is comparable to the effective transition state implied by ensemble-averaged variational transition state theory. Properties of the TRS are approximated as an average of the individual properties of the donor and acceptor states. The model is consistent with experimental findings that previously appeared contradictory; specifically, it resolves the long-standing ambiguity regarding the location of the TRS (aldehyde-like vs. alcohol-like). The new picture of the TRS for this reaction identifies the principal components of the collective reaction coordinate and the average structure of the saddle point along that coordinate. PMID:20457944

  5. Optimization of adsorptive immobilization of alcohol dehydrogenases.

    PubMed

    Trivedi, Archana; Heinemann, Matthias; Spiess, Antje C; Daussmann, Thomas; Büchs, Jochen

    2005-04-01

    In this work, a systematic examination of various parameters of adsorptive immobilization of alcohol dehydrogenases (ADHs) on solid support is performed and the impact of these parameters on immobilization efficiency is studied. Depending on the source of the enzymes, these parameters differently influence the immobilization efficiency, expressed in terms of residual activity and protein loading. Residual activity of 79% was achieved with ADH from bakers' yeast (YADH) after optimizing the immobilization parameters. A step-wise drying process has been found to be more effective than one-step drying. A hypothesis of deactivation through bubble nucleation during drying of the enzyme/glass bead suspension at low drying pressure (<45 kPa) is experimentally verified. In the case of ADH from Lactobacillus brevis (LBADH), >300% residual activity was found after drying. Hyperactivation of the enzyme is probably caused by structural changes in the enzyme molecule during the drying process. ADH from Thermoanaerobacter species (ADH T) is found to be stable under drying conditions (>15 kPa) in contrast to LBADH and YADH.

  6. Targeting Aldehyde Dehydrogenase 2: New Therapeutic Opportunities

    PubMed Central

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

    2014-01-01

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

  7. Targeting isocitrate dehydrogenase (IDH) in cancer.

    PubMed

    Fujii, Takeo; Khawaja, Muhammad Rizwan; DiNardo, Courtney D; Atkins, Johnique T; Janku, Filip

    2016-05-01

    Isocitrate dehydrogenase (IDH) is an essential enzyme for cellular respiration in the tricarboxylic acid (TCA) cycle. Recurrent mutations in IDH1 or IDH2 are prevalent in several cancers including glioma, acute myeloid leukemia (AML), cholangiocarcinoma and chondrosarcoma. The mutated IDH1 and IDH2 proteins have a gain-of-function, neomorphic activity, catalyzing the reduction of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG) by NADPH. Cancer-associated IDH mutations block normal cellular differentiation and promote tumorigenesis via the abnormal production of the oncometabolite 2-HG. High levels of 2-HG have been shown to inhibit α-KG dependent dioxygenases, including histone and deoxyribonucleic acid (DNA) demethylases, which play a key role in regulating the epigenetic state of cells. Current targeted inhibitors of IDH1 (AG120, IDH305), IDH2 (AG221), and pan-IDH1/2 (AG881) selectively inhibit mutant IDH protein and induce cell differentiation in in vitro and in vivo models. Preliminary results from phase I clinical trials with IDH inhibitors in patients with advanced hematologic malignancies have demonstrated an objective response rate ranging from 31% to 40% with durable responses (>1 year) observed. Furthermore, the IDH inhibitors have demonstrated early signals of activity in solid tumors with IDH mutations, including cholangiocarcinomas and low grade gliomas. PMID:27355333

  8. Lactic dehydrogenase and cancer: an overview.

    PubMed

    Gallo, Monica; Sapio, Luigi; Spina, Annamaria; Naviglio, Daniele; Calogero, Armando; Naviglio, Silvio

    2015-01-01

    Despite the intense scientific efforts made, there are still many tumors that are difficult to treat and the percentage of patient survival in the long-term is still too low. Thus, new approaches to the treatment of cancer are needed. Cancer is a highly heterogeneous and complex disease, whose development requires a reorganization of cell metabolism. Most tumor cells downregulate mitochondrial oxidative phosphorylation and increase the rate of glucose consumption and lactate release, independently of oxygen availability (Warburg effect). This metabolic rewiring is largely believed to favour tumor growth and survival, although the underlying molecular mechanisms are not completely understood. Importantly, the correlation between the aerobic glycolysis and cancer is widely regarded as a useful biochemical basis for the development of novel anticancer strategies. Among the enzymes involved in glycolysis, lactate dehydrogenase (LDH) is emerging as a very attractive target for possible pharmacological approaches in cancer therapy. This review addresses the state of the art and the perspectives concerning LDH both as a useful diagnostic marker and a relevant molecular target in cancer therapy and management.

  9. Vascular Bioactivation of Nitroglycerin by Aldehyde Dehydrogenase-2

    PubMed Central

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

    2012-01-01

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

  10. Sorbitol dehydrogenase from bovine lens: purification and properties.

    PubMed

    Marini, I; Bucchioni, L; Borella, P; Del Corso, A; Mura, U

    1997-04-15

    Bovine lens sorbitol dehydrogenase (L-iditol:NAD+ 2-oxidoreductase, EC 1.1.1.14) (SDH) was purified to electrophoretic homogeneity (51 U/mg of protein) and characterized for both kinetic and some structural properties. The enzyme proves to be a homotetramer of 156 kDa containing one equivalent of zinc ion per subunit. Metal chelators such as EDTA and 1,10-phenanthroline determine a loss of enzyme activity which can be specifically recovered by addition of either zinc or manganese ions. Inactivation induced not only by metal chelators but also by thiol reagents is effectively prevented by the pyridine cofactor. Bovine lens SDH is active on polyalcohols and keto-sugars with more than three carbon atoms, and also requires special steric constraints for substrate recognition. Of the polyols, xylitol is the most effective substrate (kcat/KM of 8.1 s-1 mM-1), followed by sorbitol (kcat/KM of 1.59 s-1 mM-1); fructose, the most effective carbonyl substrate, displays a kcat/KM of only 0.9 s-1 mM-1. Analysis at the steady state of initial velocities as a function of the concentration of different substrates and cofactors and studies of product inhibition indicate for both fructose reduction and sorbitol oxidation a Theorell and Chance-type kinetic mechanism of action.

  11. Malate dehydrogenase: a useful phylogenetic marker for the genus Aeromonas.

    PubMed

    Farfán, Maribel; Miñana-Galbis, David; Garreta, Albert; Lorén, J Gaspar; Fusté, M Carmen

    2010-12-01

    The reconstruction of correct genealogies among biological entities, the estimation of the divergence time between organisms or the study of the different events that occur along evolutionary lineages are not always based on suitable genes. For reliable results, it is necessary to look at full-length sequences of genes under stabilizing selection (neutral or purifying) and behaving as good molecular clocks. In bacteria it has been proved that the malate dehydrogenase gene (mdh) can be used to determine the inter- and intraspecies divergence, and hence this gene constitutes a potential marker for phylogeny and bacterial population genetics. We have sequenced the full-length mdh gene in 36 type and reference strains of Aeromonas. The species grouping obtained in the phylogenetic tree derived from mdh sequences was in agreement with that currently accepted for the genus Aeromonas. The maximum likelihood models applied to our sequences indicated that the mdh gene is highly conserved among the Aeromonas species and the main evolutionary force acting on it is purifying selection. Only two sites under potential diversifying selection were identified (T 108 and S 193). In order to determine if these two residues could have an influence on the MDH structure, we mapped them in a three-dimensional model constructed from the sequence of A. hydrophila using the human mitochondrial MDH as a template. The presence of purifying selection together with the linear relationship between substitutions and gene divergence makes the mdh an excellent candidate gene for a phylogeny of Aeromonas and probably for other bacterial groups.

  12. Aldehyde dehydrogenase 1A1 in stem cells and cancer

    PubMed Central

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

    2016-01-01

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

  13. Lactate Dehydrogenase in Hepatocellular Carcinoma: Something Old, Something New

    PubMed Central

    Faloppi, Luca; Bianconi, Maristella; Memeo, Riccardo; Casadei Gardini, Andrea; Giampieri, Riccardo; Bittoni, Alessandro; Andrikou, Kalliopi; Del Prete, Michela; Cascinu, Stefano; Scartozzi, Mario

    2016-01-01

    Hepatocellular carcinoma (HCC) is the most common primary liver tumour (80–90%) and represents more than 5.7% of all cancers. Although in recent years the therapeutic options for these patients have increased, clinical results are yet unsatisfactory and the prognosis remains dismal. Clinical or molecular criteria allowing a more accurate selection of patients are in fact largely lacking. Lactic dehydrogenase (LDH) is a glycolytic key enzyme in the conversion of pyruvate to lactate under anaerobic conditions. In preclinical models, upregulation of LDH has been suggested to ensure both an efficient anaerobic/glycolytic metabolism and a reduced dependence on oxygen under hypoxic conditions in tumour cells. Data from several analyses on different tumour types seem to suggest that LDH levels may be a significant prognostic factor. The role of LDH in HCC has been investigated by different authors in heterogeneous populations of patients. It has been tested as a potential biomarker in retrospective, small, and nonfocused studies in patients undergoing surgery, transarterial chemoembolization (TACE), and systemic therapy. In the major part of these studies, high LDH serum levels seem to predict a poorer outcome. We have reviewed literature in this setting trying to resume basis for future studies validating the role of LDH in this disease. PMID:27314036

  14. Improvement of the soy formate dehydrogenase properties by rational design.

    PubMed

    Kargov, I S; Kleimenov, S Y; Savin, S S; Tishkov, V I; Alekseeva, A A

    2015-06-01

    Previous experiments on substitution of the residue Phe290 to Asp, Asn and Ser in NAD(+)-dependent formate dehydrogenase from soya Glycine max (SoyFDH) showed important role of the residue in enzyme thermal stability and catalytic properties (Alekseeva et al. Prot. Eng. Des. Sel., 2012a; 25: :781-88). In this work, we continued site-directed mutagenesis experiments of the Phe290 and the residue was changed to Ala, Thr, Tyr, Glu and Gln. All amino acid changes resulted in increase of catalytic constant from 2.9 to 3.5-4.7 s(-1). The substitution Phe290Ala led to KM (NAD+) decrease from 13.3 to 8.6 μM, and substitutions Phe290Tyr and Phe290Glu resulted in decrease and increase of KM (HCOO-) from 1.5 to 0.9 and -2.9 mM, respectively. The highest improvement of catalytic properties was observed for SoyFDH Phe290Ala which showed 2-fold higher catalytic efficiency with both substrates. Stability of mutants was examined by study of thermal inactivation kinetics and differential scanning calorimetry (DSC). All five amino acids provided increase of thermal stability of mutant SoyFDH in comparison with wild-type enzyme. Mutant SoyFDH Phe290Glu showed the highest improvement-the stabilization effect was 43 at 56°C. The DSC data agree with results of thermal inactivation kinetics. Substitutions Phe290Tyr, Phe290Thr, Phe290Gln and Phe290Glu provided Tm value increase 0.6°-6.6°. SoyFDH Phe290Glu and previously prepared SoyFDH Phe290Asp show similar thermal stability as enzymes from Candida boidinii and Mycobacterium vaccae N10 and have higher catalytic efficiency with NAD(+) compared with all described FDHs. Therefore, these mutants are very perspective enzymes for coenzyme regeneration in processes of chiral synthesis with dehydrogenases.

  15. Succinate Dehydrogenase Loss in Familial Paraganglioma: Biochemistry, Genetics, and Epigenetics

    PubMed Central

    Her, Yeng F.; Maher, L. James

    2015-01-01

    It is counterintuitive that metabolic defects reducing ATP production can cause, rather than protect from, cancer. Yet this is precisely the case for familial paraganglioma, a form of neuroendocrine malignancy caused by loss of succinate dehydrogenase in the tricarboxylic acid cycle. Here we review biochemical, genetic, and epigenetic considerations in succinate dehydrogenase loss and present leading models and mysteries associated with this fascinating and important tumor. PMID:26294907

  16. The Occurrence of Glycolate Dehydrogenase and Glycolate Oxidase in Green Plants

    PubMed Central

    Frederick, Sue Ellen; Gruber, Peter J.; Tolbert, N. E.

    1973-01-01

    Homogenates of various lower land plants, aquatic angiosperms, and green algae were assayed for glycolate oxidase, a peroxisomal enzyme present in green leaves of higher plants, and for glycolate dehydrogenase, a functionally analogous enzyme characteristic of certain green algae. Green tissues of all lower land plants examined (including mosses, liverworts, ferns, and fern allies), as well as three freshwater aquatic angiosperms, contained an enzyme resembling glycolate oxidase, in that it oxidized l- but not d-lactate in addition to glycolate, and was insensitive to 2 mm cyanide. Many of the green algae (including Chlorella vulgaris, previously claimed to have glycolate oxidase) contained an enzyme resembling glycolate dehydrogenase, in that it oxidized d- but not l-lactate, and was inhibited by 2 mm cyanide. Other green algae had activity characteristic of glycolate oxidase and, accordingly, showed a substantial glycolate-dependent O2 uptake. It is pointed out that this distribution pattern of glycolate oxidase and glycolate dehydrogenase among the green plants may have phylogenetic significance. Activities of catalase, a marker enzyme for peroxisomes, were also determined and were generally lower in the algae than in the land plants or aquatic angiosperms. Among the algae, however, there were no consistent correlations between levels of catalase and the type of enzyme which oxidized glycolate. PMID:16658555

  17. 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.

  18. Structure-guided development of specific pyruvate dehydrogenase kinase inhibitors targeting the ATP-binding pocket.

    PubMed

    Tso, Shih-Chia; Qi, Xiangbing; Gui, Wen-Jun; Wu, Cheng-Yang; Chuang, Jacinta L; Wernstedt-Asterholm, Ingrid; Morlock, Lorraine K; Owens, Kyle R; Scherer, Philipp E; Williams, Noelle S; Tambar, Uttam K; Wynn, R Max; Chuang, David T

    2014-02-14

    Pyruvate dehydrogenase kinase isoforms (PDKs 1-4) negatively regulate activity of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation. PDK isoforms are up-regulated in obesity, diabetes, heart failure, and cancer and are potential therapeutic targets for these important human diseases. Here, we employed a structure-guided design to convert a known Hsp90 inhibitor to a series of highly specific PDK inhibitors, based on structural conservation in the ATP-binding pocket. The key step involved the substitution of a carbonyl group in the parent compound with a sulfonyl in the PDK inhibitors. The final compound of this series, 2-[(2,4-dihydroxyphenyl)sulfonyl]isoindoline-4,6-diol, designated PS10, inhibits all four PDK isoforms with IC50 = 0.8 μM for PDK2. The administration of PS10 (70 mg/kg) to diet-induced obese mice significantly augments pyruvate dehydrogenase complex activity with reduced phosphorylation in different tissues. Prolonged PS10 treatments result in improved glucose tolerance and notably lessened hepatic steatosis in the mouse model. The results support the pharmacological approach of targeting PDK to control both glucose and fat levels in obesity and type 2 diabetes. PMID:24356970

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

    PubMed Central

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

    2015-01-01

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

  20. Virulence of Mycobacterium tuberculosis depends on lipoamide dehydrogenase, a member of three multi-enzyme complexes

    PubMed Central

    Venugopal, Aditya; Bryk, Ruslana; Shi, Shuangping; Rhee, Kyu; Rath, Poonam; Schnappinger, Dirk; Ehrt, Sabine; Nathan, Carl

    2011-01-01

    SUMMARY Mycobacterium tuberculosis (Mtb) adapts to persist in a nutritionally limited macrophage compartment. Lipoamide dehydrogenase (Lpd), the third enzyme (E3) in Mtb’s pyruvate dehydrogenase complex (PDH), also serves as E1 of peroxynitrite reductase/peroxidase (PNR/P), which helps Mtb resist host reactive nitrogen intermediates. In contrast to Mtb lacking dihydrolipoamide acyltransferase (DlaT), the E2 of PDH and PNR/P, Lpd-deficient Mtb is severely attenuated in wild type and immunodeficient mice. This suggests that Lpd has a function that DlaT does not share. When DlaT is absent, Mtb upregulates an Lpd-dependent branched chain keto-acid dehydrogenase (BCKADH) encoded by pdhA, pdhB, pdhC and lpdC. Without Lpd, Mtb cannot metabolize branched chain amino acids and potentially toxic branched chain intermediates accumulate. Mtb deficient in both DlaT and PdhC phenocopies Lpd-deficient Mtb. Thus, Mtb critically requires BCKADH along with PDH and PNR/P for pathogenesis. These findings position Lpd as a potential target for anti-infectives against Mtb. PMID:21238944

  1. The structure of the quinoprotein alcohol dehydrogenase of Acetobacter aceti modelled on that of methanol dehydrogenase from Methylobacterium extorquens.

    PubMed

    Cozier, G E; Giles, I G; Anthony, C

    1995-06-01

    The 1.94 A structure of methanol dehydrogenase has been used to provide a model structure for part of a membrane quinohaemoprotein alcohol dehydrogenase. The basic superbarrel structure and the active-site region are retained, indicating essentially similar mechanisms of action, but there are considerable differences in the external loops, particularly those involved in formation of the shallow funnel leading to the active site.

  2. ald of Mycobacterium tuberculosis encodes both the alanine dehydrogenase and the putative glycine dehydrogenase.

    PubMed

    Giffin, Michelle M; Modesti, Lucia; Raab, Ronald W; Wayne, Lawrence G; Sohaskey, Charles D

    2012-03-01

    The putative glycine dehydrogenase of Mycobacterium tuberculosis catalyzes the reductive amination of glyoxylate to glycine but not the reverse reaction. The enzyme was purified and identified as the previously characterized alanine dehydrogenase. The Ald enzyme was expressed in Escherichia coli and had both pyruvate and glyoxylate aminating activities. The gene, ald, was inactivated in M. tuberculosis, which resulted in the loss of all activities. Both enzyme activities were found associated with the cell and were not detected in the extracellular filtrate. By using an anti-Ald antibody, the protein was localized to the cell membrane, with a smaller fraction in the cytosol. None was detected in the extracellular medium. The ald knockout strain grew without alanine or glycine and was able to utilize glycine but not alanine as a nitrogen source. Transcription of ald was induced when alanine was the sole nitrogen source, and higher levels of Ald enzyme were measured. Ald is proposed to have several functions, including ammonium incorporation and alanine breakdown.

  3. Rapid inhibition of pyruvate dehydrogenase: an initiating event in high dietary fat-induced loss of metabolic flexibility in the heart.

    PubMed

    Crewe, Clair; Kinter, Michael; Szweda, Luke I

    2013-01-01

    Cardiac function depends on the ability to switch between fatty acid and glucose oxidation for energy production in response to changes in substrate availability and energetic stress. In obese and diabetic individuals, increased reliance on fatty acids and reduced metabolic flexibility are thought to contribute to the development of cardiovascular disease. Mechanisms by which cardiac mitochondria contribute to diet-induced metabolic inflexibility were investigated. Mice were fed a high fat or low fat diet for 1 d, 1 wk, and 20 wk. Cardiac mitochondria isolated from mice fed a high fat diet displayed a diminished ability to utilize the glycolytically derived substrate pyruvate. This response was rapid, occurring within the first day on the diet, and persisted for up to 20 wk. A selective increase in the expression of pyruvate dehydrogenase kinase 4 and inhibition of pyruvate dehydrogenase are responsible for the rapid suppression of pyruvate utilization. An important consequence is that pyruvate dehydrogenase is sensitized to inhibition when mitochondria respire in the presence of fatty acids. Additionally, increased expression of pyruvate dehydrogenase kinase 4 preceded any observed diet-induced reductions in the levels of glucose transporter type 4 and glycolytic enzymes and, as judged by Akt phosphorylation, insulin signaling. Importantly, diminished insulin signaling evident at 1 wk on the high fat diet did not occur in pyruvate dehydrogenase kinase 4 knockout mice. Dietary intervention leads to a rapid decline in pyruvate dehydrogenase kinase 4 levels and recovery of pyruvate dehydrogenase activity indicating an additional form of regulation. Finally, an overnight fast elicits a metabolic response similar to that induced by high dietary fat obscuring diet-induced metabolic changes. Thus, our data indicate that diet-induced inhibition of pyruvate dehydrogenase may be an initiating event in decreased oxidation of glucose and increased reliance of the heart on

  4. Complete knockout of the lactate dehydrogenase A gene is lethal in pyruvate dehydrogenase kinase 1, 2, 3 down-regulated CHO cells.

    PubMed

    Yip, Shirley S M; Zhou, Meixia; Joly, John; Snedecor, Bradley; Shen, Amy; Crawford, Yongping

    2014-09-01

    Accumulation of high level of lactate can negatively impact cell growth during fed-batch culture process. In this study, we attempted to knockout the lactate dehydrogenase A (LDHA) gene in CHO cells in order to attenuate the lactate level. To prevent the potential deleterious effect of pyruvate accumulation, consequent to LDHA knockout, on cell culture, we chose a pyruvate dehydrogenase kinase 1, 2, and 3 (PDHK1, 2, and 3) knockdown cell line in which to knock out LDHA alleles. Around 3,000 clones were screened to obtain 152 mutants. Only heterozygous mutants were identified. An attempt to knockout the remaining wild-type allele from one such heterozygote yielded only two mutants after screening 567 clones. One had an extra valine. Another evidenced a duplication event, possessing at lease one wild-type and two different frameshifted alleles. Both mutants still retained LDH activity. Together, our data strongly suggest that a complete knockout of LDHA is lethal in CHO cells, despite simultaneous down-regulation of PDHK1, 2, and 3. PMID:24841241

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

    SciTech Connect

    Boernke, W. E.; Millard, C. S.; Stevens, P. W.; Kakar, S. N.; Stevens, F. J.; Donnelly, M. I.; Nebraska Wesleyan Univ.

    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 native 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 mutant

  6. Yeast Alcohol Dehydrogenase Structure and Catalysis

    PubMed Central

    2015-01-01

    Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (ADH1) is the constitutive enzyme that reduces acetaldehyde to ethanol during the fermentation of glucose. ADH1 is a homotetramer of subunits with 347 amino acid residues. A structure for ADH1 was determined by X-ray crystallography at 2.4 Å resolution. The asymmetric unit contains four different subunits, arranged as similar dimers named AB and CD. The unit cell contains two different tetramers made up of “back-to-back” dimers, AB:AB and CD:CD. The A and C subunits in each dimer are structurally similar, with a closed conformation, bound coenzyme, and the oxygen of 2,2,2-trifluoroethanol ligated to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. In contrast, the B and D subunits have an open conformation with no bound coenzyme, and the catalytic zinc has an alternative, inverted coordination with Cys-43, Cys-153, His-66, and the carboxylate of Glu-67. The asymmetry in the dimeric subunits of the tetramer provides two structures that appear to be relevant for the catalytic mechanism. The alternative coordination of the zinc may represent an intermediate in the mechanism of displacement of the zinc-bound water with alcohol or aldehyde substrates. Substitution of Glu-67 with Gln-67 decreases the catalytic efficiency by 100-fold. Previous studies of structural modeling, evolutionary relationships, substrate specificity, chemical modification, and site-directed mutagenesis are interpreted more fully with the three-dimensional structure. PMID:25157460

  7. Structural Studies of Human Pyruvate Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Ciszak, Ewa; Korotchkina, Lioubov G.; Dominiak, Paulina; Sidhu, Sukhdeep; Patel, Mulchand S.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    Human pyruvate dehydrogenase (E1) catalyzes the irreversible decarboxylation of pyruvate in the presence of Mg(2+) and thiamin pyrophosphate (TPP) followed by the rate-limiting reductive acetylation of the lipoyl moiety linked to dihydrolipoamide acetyltransferase. The three-dimensional structure of human E1 is elucidated using the methods of macromolecular X-ray crystallography. The structure is an alpha, alpha', beta and beta' tetramer with the protein units being in the tetrahedral arrangement. Each 361-residue alpha-subunit and 329-residue beta-subunit is composed of a beta-sheet core surrounded by alpha-helical domains. Each subunit is in extensive contact with all the three subunits involving TPP and magnesium cofactors, and potassium ions. The two binding sites for TPP are at the alpha-beta' and alpha'-beta interfaces, each involving a magnesium ion and Phe6l, His63, Tyr89, and Met200 from the alpha-subunit (or alpha'-subunit), and Met81 Phe85, His128 from the beta-subunit (or beta'-subunit). K+ ions are nestled between two beta-sheets and the end of an alpha-helix in each beta-subunit, where they are coordinated by four carbonyl oxygen groups from Ile12, Ala160, Asp163, and Asnl65, and a water molecule. The catalytic C2 carbon of thiazolium ring in this structure forms a 3.2 A contact with a water molecule involved in a series of H-bonds with other water molecules, and indirectly with amino acids including those involved in the catalysis and regulation of the enzyme.

  8. Thiosulfate Dehydrogenase (TsdA) from Allochromatium vinosum

    PubMed Central

    Brito, José A.; Denkmann, Kevin; Pereira, Inês A. C.; Archer, Margarida; Dahl, Christiane

    2015-01-01

    Although the oxidative condensation of two thiosulfate anions to tetrathionate constitutes a well documented and significant part of the natural sulfur cycle, little is known about the enzymes catalyzing this reaction. In the purple sulfur bacterium Allochromatium vinosum, the reaction is catalyzed by the periplasmic diheme c-type cytochrome thiosulfate dehydrogenase (TsdA). Here, we report the crystal structure of the “as isolated” form of A. vinosum TsdA to 1.98 Å resolution and those of several redox states of the enzyme to different resolutions. The protein contains two typical class I c-type cytochrome domains wrapped around two hemes axially coordinated by His53/Cys96 and His164/Lys208. These domains are very similar, suggesting a gene duplication event during evolution. A ligand switch from Lys208 to Met209 is observed upon reduction of the enzyme. Cys96 is an essential residue for catalysis, with the specific activity of the enzyme being completely abolished in several TsdA-Cys96 variants. TsdA-K208N, K208G, and M209G variants were catalytically active in thiosulfate oxidation as well as in tetrathionate reduction, pointing to heme 2 as the electron exit point. In this study, we provide spectroscopic and structural evidence that the TsdA reaction cycle involves the transient presence of heme 1 in the high-spin state caused by movement of the Sγ atom of Cys96 out of the iron coordination sphere. Based on the presented data, we draw important conclusions about the enzyme and propose a possible reaction mechanism for TsdA. PMID:25673691

  9. 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.

  10. Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein-protein interactions by the MDM2 ligand nutlin-3.

    PubMed

    Way, Luke; Faktor, Jakub; Dvorakova, Petra; Nicholson, Judith; Vojtesek, Borek; Graham, Duncan; Ball, Kathryn L; Hupp, Ted

    2016-09-01

    Drugs targeting MDM2's hydrophobic pocket activate p53. However, these agents act allosterically and have agonist effects on MDM2's protein interaction landscape. Dominant p53-independent MDM2-drug responsive-binding proteins have not been stratified. We used as a variable the differential expression of MDM2 protein as a function of cell density to identify Nutlin-3 responsive MDM2-binding proteins that are perturbed independent of cell density using SWATH-MS. Dihydrolipoamide dehydrogenase, the E3 subunit of the mitochondrial pyruvate dehydrogenase complex, was one of two Nutlin-3 perturbed proteins identified fours hour posttreatment at two cell densities. Immunoblotting confirmed that dihydrolipoamide dehydrogenase was induced by Nutlin-3. Depletion of MDM2 using siRNA also elevated dihydrolipoamide dehydrogenase in Nutlin-3 treated cells. Mitotracker confirmed that Nutlin-3 inhibits mitochondrial activity. Enrichment of mitochondria using TOM22+ immunobeads and TMT labeling defined key changes in the mitochondrial proteome after Nutlin-3 treatment. Proximity ligation identified rearrangements of cellular protein-protein complexes in situ. In response to Nutlin-3, a reduction of dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase protein complexes highlighted a disruption of the pyruvate dehydrogenase complex. This coincides with an increase in MDM2/dihydrolipoamide dehydrogenase complexes in the nucleus that was further enhanced by the nuclear export inhibitor Leptomycin B. The data suggest one therapeutic impact of MDM2 drugs might be on the early perturbation of specific protein-protein interactions within the mitochondria. This methodology forms a blueprint for biomarker discovery that can identify rearrangements of MDM2 protein-protein complexes in drug-treated cells. PMID:27273042

  11. Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

    PubMed Central

    Way, Luke; Faktor, Jakub; Dvorakova, Petra; Nicholson, Judith; Vojtesek, Borek; Graham, Duncan; Ball, Kathryn L.

    2016-01-01

    Drugs targeting MDM2's hydrophobic pocket activate p53. However, these agents act allosterically and have agonist effects on MDM2's protein interaction landscape. Dominant p53‐independent MDM2‐drug responsive‐binding proteins have not been stratified. We used as a variable the differential expression of MDM2 protein as a function of cell density to identify Nutlin‐3 responsive MDM2‐binding proteins that are perturbed independent of cell density using SWATH‐MS. Dihydrolipoamide dehydrogenase, the E3 subunit of the mitochondrial pyruvate dehydrogenase complex, was one of two Nutlin‐3 perturbed proteins identified fours hour posttreatment at two cell densities. Immunoblotting confirmed that dihydrolipoamide dehydrogenase was induced by Nutlin‐3. Depletion of MDM2 using siRNA also elevated dihydrolipoamide dehydrogenase in Nutlin‐3 treated cells. Mitotracker confirmed that Nutlin‐3 inhibits mitochondrial activity. Enrichment of mitochondria using TOM22+ immunobeads and TMT labeling defined key changes in the mitochondrial proteome after Nutlin‐3 treatment. Proximity ligation identified rearrangements of cellular protein–protein complexes in situ. In response to Nutlin‐3, a reduction of dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase protein complexes highlighted a disruption of the pyruvate dehydrogenase complex. This coincides with an increase in MDM2/dihydrolipoamide dehydrogenase complexes in the nucleus that was further enhanced by the nuclear export inhibitor Leptomycin B. The data suggest one therapeutic impact of MDM2 drugs might be on the early perturbation of specific protein–protein interactions within the mitochondria. This methodology forms a blueprint for biomarker discovery that can identify rearrangements of MDM2 protein–protein complexes in drug‐treated cells. PMID:27273042

  12. [Succinate dehydrogenase-deficient tumors--a novel mechanism of tumor formation].

    PubMed

    Miettinen, Markku

    2015-01-01

    Succinate dehydrogenase (SDH) is a heterotetrameric enzyme complex participating in the Krebs cycle and electron transfer of oxidative phosphorylation. These tumors, discovered during the past 15 years, often occur in young patients and include 15% of paragangliomas, 7% of gastric gastrointestinal stromal tumors (GISTs), and <1% of renal cell carcinomas and pituitary adenomas. SDH-deficient tumors have lost SDH complex activity via bi-allelic genomic losses or epigenetic silencing. This deficiency is oncogenic, activating pseudohypoxia signaling. SDH deficiency has to be suspected in the above-cited tumor types presenting at a young age. Immunohistochemical testing of tumor tissue for SDHB loss is diagnostic. PMID:26749909

  13. Dehydrogenase activity of forest soils depends on the assay used

    NASA Astrophysics Data System (ADS)

    Januszek, Kazimierz; Długa, Joanna; Socha, Jarosław

    2015-01-01

    Dehydrogenases are exclusively intracellular enzymes, which play an important role in the initial stages of oxidation of soil organic matter. One of the most frequently used methods to estimate dehydrogenase activity in soil is based on the use of triphenyltetrazolium chloride as an artificial electron acceptor. The purpose of this study was to compare the activity of dehydrogenases of forest soils with varied physicochemical properties using different triphenyltetrazolium chloride assays. The determination was carried out using the original procedure by Casida et al., a modification of the procedure which involves the use of Ca(OH)2 instead of CaCO3, the Thalmann method, and the assay by Casida et al. without addition of buffer or any salt. Soil dehydrogenase activity depended on the assay used. Dehydrogenase determined by the Casida et al. method without addition of buffer or any salt correlated with the pH values of soils. The autoclaved strongly acidic samples of control soils showed high concentrations of triphenylformazan, probably due to chemical reduction of triphenyltetrazolium chloride. There is, therefore, a need for a sterilization method other than autoclaving, ie a process that results in significant changes in soil properties, thus helping to increase the chemical reduction of triphenyltetrazolium chloride.

  14. Characterization and purification of carbon monoxide dehydrogenase from Methanosarcina barkeri.

    PubMed Central

    Krzycki, J A; Zeikus, J G

    1984-01-01

    Carbon monoxide-dependent production of H2, CO2, and CH4 was detected in crude cell extracts of acetate-grown Methanosarcina barkeri. This metabolic transformation was associated with an active methyl viologen-linked CO dehydrogenase activity (5 to 10 U/mg of protein). Carbon monoxide dehydrogenase activity was inhibited 85% by 10 microM KCN and was rapidly inactivated by O2. The enzyme was nearly homogeneous after 20-fold purification, indicating that a significant proportion of soluble cell protein was CO dehydrogenase (ca. 5%). The native purified enzyme displayed a molecular weight of 232,000 and a two-subunit composition of 92,000 and 18,000 daltons. The enzyme was shown to contain nickel by isolation of radioactive CO dehydrogenase from cells grown in 63Ni. Analysis of enzyme kinetic properties revealed an apparent Km of 5 mM for CO and a Vmax of 1,300 U/mg of protein. The spectral properties of the enzyme were similar to those published for CO dehydrogenase from acetogenic anaerobes. The physiological functions of the enzyme are discussed. Images PMID:6425262

  15. Interaction of carbohydrates with alcohol dehydrogenase: Effect on enzyme activity.

    PubMed

    Jadhav, Swati B; Bankar, Sandip B; Granström, Tom; Ojamo, Heikki; Singhal, Rekha S; Survase, Shrikant A

    2015-09-01

    Alcohol dehydrogenase was covalently conjugated with three different oxidized carbohydrates i.e., glucose, starch and pectin. All the carbohydrates inhibited the enzyme. The inhibition was studied with respect to the inhibition rate constant, involvement of thiol groups in the binding, and structural changes in the enzyme. The enzyme activity decreased to half of its original activity at the concentration of 2 mg/mL of pectin, 4 mg/mL of glucose and 10 mg/mL of starch within 10 min at pH 7. This study showed oxidized pectin to be a potent inhibitor of alcohol dehydrogenase followed by glucose and starch. Along with the aldehyde-amino group interaction, thiol groups were also involved in the binding between alcohol dehydrogenase and carbohydrates. The structural changes occurring on binding of alcohol dehydrogenase with oxidized carbohydrates was also confirmed by fluorescence spectrophotometry. Oxidized carbohydrates could thus be used as potential inhibitors of alcohol dehydrogenase.

  16. Determination of Dehydrogenase Activities Involved in D-Glucose Oxidation in Gluconobacter and Acetobacter Strains

    PubMed Central

    Sainz, Florencia; Jesús Torija, María; Matsutani, Minenosuke; Kataoka, Naoya; Yakushi, Toshiharu; Matsushita, Kazunobu; Mas, Albert

    2016-01-01

    Acetic acid bacteria (AAB) are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane-bound dehydrogenases. In the present study, the enzyme activity of the membrane-bound dehydrogenases [membrane-bound PQQ-glucose dehydrogenase (mGDH), D-gluconate dehydrogenase (GADH) and membrane-bound glycerol dehydrogenase (GLDH)] involved in the oxidation of D-glucose and D-gluconic acid (GA) was determined in six strains of three different species of AAB (three natural and three type strains). Moreover, the effect of these activities on the production of related metabolites [GA, 2-keto-D-gluconic acid (2KGA) and 5-keto-D-gluconic acid (5KGA)] was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the Acetobacter malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h), which coincided with D-glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of Gluconobacter oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24 h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition. Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter

  17. Determination of Dehydrogenase Activities Involved in D-Glucose Oxidation in Gluconobacter and Acetobacter Strains.

    PubMed

    Sainz, Florencia; Jesús Torija, María; Matsutani, Minenosuke; Kataoka, Naoya; Yakushi, Toshiharu; Matsushita, Kazunobu; Mas, Albert

    2016-01-01

    Acetic acid bacteria (AAB) are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane-bound dehydrogenases. In the present study, the enzyme activity of the membrane-bound dehydrogenases [membrane-bound PQQ-glucose dehydrogenase (mGDH), D-gluconate dehydrogenase (GADH) and membrane-bound glycerol dehydrogenase (GLDH)] involved in the oxidation of D-glucose and D-gluconic acid (GA) was determined in six strains of three different species of AAB (three natural and three type strains). Moreover, the effect of these activities on the production of related metabolites [GA, 2-keto-D-gluconic acid (2KGA) and 5-keto-D-gluconic acid (5KGA)] was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the Acetobacter malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h), which coincided with D-glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of Gluconobacter oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24 h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition. Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter

  18. Determination of Dehydrogenase Activities Involved in D-Glucose Oxidation in Gluconobacter and Acetobacter Strains.

    PubMed

    Sainz, Florencia; Jesús Torija, María; Matsutani, Minenosuke; Kataoka, Naoya; Yakushi, Toshiharu; Matsushita, Kazunobu; Mas, Albert

    2016-01-01

    Acetic acid bacteria (AAB) are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane-bound dehydrogenases. In the present study, the enzyme activity of the membrane-bound dehydrogenases [membrane-bound PQQ-glucose dehydrogenase (mGDH), D-gluconate dehydrogenase (GADH) and membrane-bound glycerol dehydrogenase (GLDH)] involved in the oxidation of D-glucose and D-gluconic acid (GA) was determined in six strains of three different species of AAB (three natural and three type strains). Moreover, the effect of these activities on the production of related metabolites [GA, 2-keto-D-gluconic acid (2KGA) and 5-keto-D-gluconic acid (5KGA)] was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the Acetobacter malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h), which coincided with D-glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of Gluconobacter oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24 h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition. Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter

  19. Determination of Dehydrogenase Activities Involved in D-Glucose Oxidation in Gluconobacter and Acetobacter Strains

    PubMed Central

    Sainz, Florencia; Jesús Torija, María; Matsutani, Minenosuke; Kataoka, Naoya; Yakushi, Toshiharu; Matsushita, Kazunobu; Mas, Albert

    2016-01-01

    Acetic acid bacteria (AAB) are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane-bound dehydrogenases. In the present study, the enzyme activity of the membrane-bound dehydrogenases [membrane-bound PQQ-glucose dehydrogenase (mGDH), D-gluconate dehydrogenase (GADH) and membrane-bound glycerol dehydrogenase (GLDH)] involved in the oxidation of D-glucose and D-gluconic acid (GA) was determined in six strains of three different species of AAB (three natural and three type strains). Moreover, the effect of these activities on the production of related metabolites [GA, 2-keto-D-gluconic acid (2KGA) and 5-keto-D-gluconic acid (5KGA)] was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the Acetobacter malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h), which coincided with D-glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of Gluconobacter oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24 h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition. Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter

  20. p53 negatively regulates transcription of the pyruvate dehydrogenase kinase Pdk2.

    PubMed

    Contractor, Tanupriya; Harris, Chris R

    2012-01-15

    In cancer cells, the aberrant conversion of pyruvate into lactate instead of acetyl-CoA in the presence of oxygen is known as the Warburg effect. The consequences and mechanisms of this metabolic peculiarity are incompletely understood. Here we report that p53 status is a key determinant of the Warburg effect. Wild-type p53 expression decreased levels of pyruvate dehydrogenase kinase-2 (Pdk2) and the product of its activity, the inactive form of the pyruvate dehydrogenase complex (P-Pdc), both of which are key regulators of pyruvate metabolism. Decreased levels of Pdk2 and P-Pdc in turn promoted conversion of pyruvate into acetyl-CoA instead of lactate. Thus, wild-type p53 limited lactate production in cancer cells unless Pdk2 could be elevated. Together, our results established that wild-type p53 prevents manifestation of the Warburg effect by controlling Pdk2. These findings elucidate a new mechanism by which p53 suppresses tumorigenesis acting at the level of cancer cell metabolism. PMID:22123926

  1. Aldehyde Dehydrogenases in Cellular Responses to Oxidative/electrophilic Stress

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-03-01

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

  3. Induced fit and the catalytic mechanism of isocitrate dehydrogenase.

    PubMed

    Gonçalves, Susana; Miller, Stephen P; Carrondo, Maria A; Dean, Anthony M; Matias, Pedro M

    2012-09-11

    NADP(+) dependent isocitrate dehydrogenase (IDH; EC 1.1.1.42) belongs to a large family of α-hydroxyacid oxidative β-decarboxylases that catalyze similar three-step reactions, with dehydrogenation to an oxaloacid intermediate preceding β-decarboxylation to an enol intermediate followed by tautomerization to the final α-ketone product. A comprehensive view of the induced fit needed for catalysis is revealed on comparing the first "fully closed" crystal structures of a pseudo-Michaelis complex of wild-type Escherichia coli IDH (EcoIDH) and the "fully closed" reaction product complex of the K100M mutant with previously obtained "quasi-closed" and "open" conformations. Conserved catalytic residues, binding the nicotinamide ring of NADP(+) and the metal-bound substrate, move as rigid bodies during domain closure by a hinge motion that spans the central β-sheet in each monomer. Interactions established between Thr105 and Ser113, which flank the "phosphorylation loop", and the nicotinamide mononucleotide moiety of NADP(+) establish productive coenzyme binding. Electrostatic interactions of a Lys100-Leu103-Asn115-Glu336 tetrad play a pivotal role in assembling a catalytically competent active site. As predicted, Lys230* is positioned to deprotonate/reprotonate the α-hydroxyl in both reaction steps and Tyr160 moves into position to protonate C3 following β-decarboxylation. A proton relay from the catalytic triad Tyr160-Asp307-Lys230* connects the α-hydroxyl of isocitrate to the bulk solvent to complete the picture of the catalytic mechanism. PMID:22891681

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

    PubMed

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

    2013-03-01

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

  5. Functional Specialization of Maize Mitochondrial Aldehyde Dehydrogenases1

    PubMed Central

    Liu, Feng; Schnable, Patrick S.

    2002-01-01

    The maize (Zea mays) rf2a and rf2b genes both encode homotetrameric aldehyde dehydrogenases (ALDHs). The RF2A protein was shown previously to accumulate in the mitochondria. In vitro import experiments and ALDH assays on mitochondrial extracts from rf2a mutant plants established that the RF2B protein also accumulates in the mitochondria. RNA gel-blot analyses and immunohistolocation experiments revealed that these two proteins have only partially redundant expression patterns in organs and cell types. For example, RF2A, but not RF2B, accumulates to high levels in the tapetal cells of anthers. Kinetic analyses established that RF2A and RF2B have quite different substrate specificities; although RF2A can oxidize a broad range of aldehydes, including aliphatic aldehydes and aromatic aldehydes, RF2B can oxidize only short-chain aliphatic aldehydes. These two enzymes also have different pH optima and responses to changes in substrate concentration. In addition, RF2A, but not RF2B or any other natural ALDHs, exhibits positive cooperativity. These functional specializations may explain why many species have two mitochondrial ALDHs. This study provides data that serve as a basis for identifying the physiological pathway by which the rf2a gene participates in normal anther development and the restoration of Texas cytoplasm-based male sterility. For example, the observations that Texas cytoplasm anthers do not accumulate elevated levels of reactive oxygen species or lipid peroxidation and the kinetic features of RF2A make it unlikely that rf2a restores fertility by preventing premature programmed cell death. PMID:12481049

  6. MAPPING OF SUCCINATE DEHYDROGENASE LOSSES IN 2258 EPITHELIAL NEOPLASMS

    PubMed Central

    Miettinen, Markku; Sarlomo-Rikala, Maarit; Cue, Peter Mc.; Czapiewski, Piotr; Langfor, Renata; Waloszczyk, Piotr; Wazny, Krzysztof; Biernat, Wojciech; Lasota, Jerzy; Wang, Zengfeng

    2013-01-01

    Losses in the succinate dehydrogenase (SDH) complex characterize 20–30% of extra-adrenal paragangliomas and 7–8% of gastric GISTs, and rare renal cell carcinomas. This loss is reflected as lack of the normally ubiquitous immunohistochemical expression of the SDH subunit B (SDHB). In paragangliomas, SDHB loss correlates with homozygous loss of any of the SDH subunits, typically by loss-of-function mutations. The occurrence of SDHB losses in other epithelial malignancies is unknown. In this study, we immunohistochemically examined 2258 epithelial, mostly malignant neoplasms including common carcinomas of all sites. Among renal cell carcinomas, SDHB loss was observed in 4 of 711 cases (0.6%) including a patient with an SDHB-deficient GIST. Histologically the SDHB-negative renal carcinomas varied. There was one clear cell carcinoma with a high nuclear grade, one papillary carcinoma type 2, one unclassified carcinoma with a glandular pattern, and one oncocytoid low-grade carcinoma as previously described for SDHB-negative renal carcinoma. None of these patients was known to have paragangliomas or had loss of SDHA expression in the tumor. Three of these patients had metastases at presentation (2 in the adrenal, one in the retroperitoneal lymph nodes). There were no cases with SDHB-loss among 64 renal oncocytomas. SDHB-losses were not seen in other carcinomas, except in one prostatic adenocarcinoma (1/57), one lymphoepithelial carcinoma of the stomach, and one (1/40) seminoma. Based on this study, SDHB-losses occur in 0.6% of renal cell carcinomas and extremely rarely in other carcinomas. Some of these renal carcinomas may be clinically aggressive. The clinical significance and molecular genetics of these SDHB-negative tumors requires further study. PMID:23531856

  7. Properties of lactate dehydrogenase in a psychrophilic marine bacterium.

    PubMed Central

    Mitchell, P; Yen, H C; Mathemeier, P F

    1985-01-01

    Lactate dehydrogenase (EC 1.1.1.27) from Vibrio marinus MP-1 was purified 15-fold and ammonium activated. The optimum pH for pyruvate reduction was 7.4. Maximum lactate dehydrogenase activity occurred at 10 to 15 degrees C, and none occurred at 40 degrees C. The crude-extract enzyme was stable between 15 and 20 degrees C and lost 50% of its activity after 60 min at 45 degrees C. The partially purified enzyme was stable between 8 and 15 degrees C and lost 50% of its activity after 60 min at 30 degrees C. The thermal stability of lactate dehydrogenase was increased by mercaptoethanol, with 50% remaining activity at 42 degrees C. Images PMID:4004236

  8. [Features of glutamate dehydrogenase in fetal and adult rumen tissue].

    PubMed

    Kalachniuk, H I; Fomenko, I S; Kalachniuk, L H; Kavai, Sh; Marounek, M; Savka, O H

    2001-01-01

    Glutamate dehydrogenase (GDH) from rumen mucosa of cow fetus, liver and two forms from mucosa (bacterial and tissue) of the adult animal were partly purified and characterized. The activity of the bacterial glutamate dehydrogenase was shown to depend on qualities of a biomass of microbes, adhered on surface of rumen mucosa. All enzymes from tissues (GDHTRF, TRC, TLC), revealed the hypersensibility to increase in the concentration medium of Zn2+, guanosine triphosphate (GTP), acting here in a role of negative modulators, and also adenosine monophosphate (AMP) and leucine, which acted as activators. However, in the same concentrations these effectors do not influence the activity of the bacterial glutamate dehydrogenase. And if all tissues enzymes are highly specific to coenzyme NADH, the bacterial ones almost in 3 times is more active at NADPH use. PMID:11642036

  9. The activity of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) in the sera of patients with brain cancer.

    PubMed

    Jelski, Wojciech; Laniewska-Dunaj, Magdalena; Orywal, Karolina; Kochanowicz, Jan; Rutkowski, Robert; Szmitkowski, Maciej

    2014-12-01

    Human brain tissue contains various alcohol dehydrogenase (ADH) isoenzymes and possess also aldehyde dehydrogenase (ALDH) activity. In our last experiments we have shown that ADH and ALDH are present also in the brain tumour cells. Moreover the activities of total ADH and class I isoenzymes were significantly higher in cancer tissue than healthy cells. It can suggests that these changes may be reflected by enzyme activity in the serum of patients with brain cancer. Serum samples were taken for routine biochemical investigation from 62 patients suffering from brain cancer (36 glioblastoma, 26 meningioma). For the measurement of the activity of class I and II ADH isoenzymes and ALDH activity, the fluorometric methods were used. The total ADH activity and activity of class III and IV isoenzymes were measured by the photometric method. A statistically significant increase of class I alcohol dehydrogenase isoenzymes was found in the sera of patients with brain cancer. The median activity of this class isoenzyme in the patients group increased about 24 % in the comparison to the control level. The total alcohol dehydrogenase activity was also significantly higher (26 %) among patients with brain tumour than healthy ones. The activities of other tested ADH isoenzymes and total ALDH were unchanged. The increase of the activity of total ADH and class I alcohol dehydrogenase isoenzyme in the sera of patients with brain cancer seems to be caused by the release of this isoenzyme from tumour's cells.

  10. Crystal structure of homoisocitrate dehydrogenase from Schizosaccharomyces pombe

    SciTech Connect

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

    2013-09-18

    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 that 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 differences in

  11. Role of lys100 in human dihydroorotate dehydrogenase: mutagenesis studies and chemical rescue by external amines.

    PubMed

    Jiang, W; Locke, G; Harpel, M R; Copeland, R A; Marcinkeviciene, J

    2000-07-11

    Chemical modification, mutagenesis, chemical rescue, and isotope effect studies are used to identify and probe the roles of several conserved amino acid groups in catalysis by human dihydroorotate dehydrogenase. Time- and pH-dependent inactivation of human dihydroorotate dehydrogenase by trinitrobenzenesulfonate implicates at least one critical lysyl residue in catalysis. Of four highly conserved lysines, only the cognate of Lys255 was previously suggested to have catalytic functionality. We now show that replacement of either Lys184 or Lys186 by mutagenesis does not impact, whereas substitution of Lys100 abolishes, enzymatic activity. However, activity is partially restored to K100C (or K100A) by inclusion of exogenous primary amines in reaction mixtures. This rescued activity saturates with respect to numerous amines and exhibits a steric discrimination reflected in K(d,(amine)) values. For all amines, rescued k(cat) values were only approximately 10% of wild type and independent of amine basicity. K(M) values for dihydroorotate and coenzyme Q(0) were similar to wild type. Thus, exogenous amines (as surrogates for Lys100) apparently complement a chemical, not binding, step(s) of catalysis, which does not entail proton transfer. In support of this postulate, solvent kinetic isotope effect analysis indicates that Lys100 stabilizes developing negative charge on the isoalloxazine ring of flavin mononucleotide during hydride transfer, as has been observed for a number of flavoprotein oxidoreductases. Ser215 of human dihydroarotate dehydrogenase (DHODase) was also studied because of its alignment with the putative active-site base Cys130 of Lactococcus lactisDHODase. Substantial retention of activity by S215C, yet complete loss of activity for S215A, is consistent with Ser215 serving as the active-site base in the human enzyme. PMID:10891080

  12. The negative impact of α-ketoglutarate dehydrogenase complex deficiency on matrix substrate-level phosphorylation

    PubMed Central

    Kiss, Gergely; Konrad, Csaba; Doczi, Judit; Starkov, Anatoly A.; Kawamata, Hibiki; Manfredi, Giovanni; Zhang, Steven F.; Gibson, Gary E.; Beal, M. Flint; Adam-Vizi, Vera; Chinopoulos, Christos

    2013-01-01

    A decline in α-ketoglutarate dehydrogenase complex (KGDHC) activity has been associated with neurodegeneration. Provision of succinyl-CoA by KGDHC is essential for generation of matrix ATP (or GTP) by substrate-level phosphorylation catalyzed by succinyl-CoA ligase. Here, we demonstrate ATP consumption in respiration-impaired isolated and in situ neuronal somal mitochondria from transgenic mice with a deficiency of either dihydrolipoyl succinyltransferase (DLST) or dihydrolipoyl dehydrogenase (DLD) that exhibit a 20–48% decrease in KGDHC activity. Import of ATP into the mitochondrial matrix of transgenic mice was attributed to a shift in the reversal potential of the adenine nucleotide translocase toward more negative values due to diminished matrix substrate-level phosphorylation, which causes the translocase to reverse prematurely. Immunoreactivity of all three subunits of succinyl-CoA ligase and maximal enzymatic activity were unaffected in transgenic mice as compared to wild-type littermates. Therefore, decreased matrix substrate-level phosphorylation was due to diminished provision of succinyl-CoA. These results were corroborated further by the finding that mitochondria from wild-type mice respiring on substrates supporting substrate-level phosphorylation exhibited ∼30% higher ADP-ATP exchange rates compared to those obtained from DLST+/− or DLD+/− littermates. We propose that KGDHC-associated pathologies are a consequence of the inability of respiration-impaired mitochondria to rely on “in-house” mitochondrial ATP reserves.—Kiss, G., Konrad, C., Doczi, J., Starkov, A. A., Kawamata, H., Manfredi, G., Zhang, S. F., Gibson, G. E., Beal, M. F., Adam-Vizi, V., Chinopoulos, C. The negative impact of α-ketoglutarate dehydrogenase complex deficiency on matrix substrate-level phosphorylation. PMID:23475850

  13. Cloning, characterization and functional expression of Taenia solium 17 beta-hydroxysteroid dehydrogenase.

    PubMed

    Aceves-Ramos, A; de la Torre, P; Hinojosa, L; Ponce, A; García-Villegas, R; Laclette, J P; Bobes, R J; Romano, M C

    2014-07-01

    The 17β-hydroxysteroid dehydrogenases (17β-HSD) are key enzymes involved in the formation (reduction) and inactivation (oxidation) of sex steroids. Several types have been found in vertebrates including fish, as well as in invertebrates like Caenorhabditis elegans, Ciona intestinalis and Haliotis diversicolor supertexta. To date limited information is available about this enzyme in parasites. We showed previously that Taenia solium cysticerci are able to synthesize sex steroid hormones in vitro when precursors are provided in the culture medium. Here, we identified a T. solium 17β-HSD through in silico blast searches in the T. solium genome database. This coding sequence was amplified by RT-PCR and cloned into the pcDNA 3.1(+) expression vector. The full length cDNA contains 957bp, corresponding to an open reading frame coding for 319 aa. The highest identity (84%) at the protein level was found with the Echinococcus multilocularis 17β-HSD although significant similarities were also found with other invertebrate and vertebrate 17β-HSD sequences. The T. solium Tsol-17βHSD belongs to the short-chain dehydrogenase/reductase (SDR) protein superfamily. HEK293T cells transiently transfected with Tsol17β-HSD induced expression of Tsol17β-HSD that transformed 3H-androstenedione into testosterone. In contrast, 3H-estrone was not significantly transformed into estradiol. In conclusion, T. solium cysticerci express a 17β-HSD that catalyzes the androgen reduction. The enzyme belongs to the short chain dehydrogenases/reductase family and shares motifs and activity with the type 3 enzyme of some other species.

  14. Metabolic characterization of Lactococcus lactis deficient in lactate dehydrogenase using in vivo 13C-NMR.

    PubMed

    Neves, A R; Ramos, A; Shearman, C; Gasson, M J; Almeida, J S; Santos, H

    2000-06-01

    The metabolism of glucose by nongrowing cells of Lactococcus lactis strain FI7851, constructed from the wild-type L. lactis strain MG1363 by disruption of the lactate dehydrogenase (ldh) gene [Gasson, M.J., Benson, K., Swindel, S. & Griffin, H. (1996) Lait 76, 33-40] was studied in a noninvasive manner by 13C-NMR. The kinetics of the build-up and consumption of the pools of intracellular intermediates mannitol 1-phosphate, fructose 1,6-bisphosphate, 3-phosphoglycerate, and phosphoenolpyruvate as well as the utilization of [1-13C]glucose and formation of products (lactate, acetate, mannitol, ethanol, acetoin, 2,3-butanediol) were monitored in vivo with a time resolution of 30 s. The metabolism of glucose by the parental wild-type strain was also examined for comparison. A clear shift from typical homolactic fermentation (parental strain) to a mixed acid fermentation (lactate dehdydrogenase deficient; LDHd strain) was observed. Furthermore, high levels of mannitol were transiently produced and metabolized once glucose was depleted. Mannitol 1-phosphate accumulated intracellularly up to 76 mM concentration. Mannitol was formed from fructose 6-phosphate by the combined action of mannitol-1-phosphate dehydrogenase and phosphatase. The results show that the formation of mannitol 1-phosphate by the LDHd strain during glucose catabolism is a consequence of impairment in NADH oxidation caused by a highly reduced LDH activity, the transient production of mannitol 1-phosphate serving as a regeneration pathway for NAD+ regeneration. Oxygen availability caused a drastic change in the pattern of intermediates and end-products, reinforcing the key-role of the fulfilment of the redox balance. The flux control coefficients for the step catalysed by mannitol-1-phosphate dehydrogenase were calculated and the implications in the design of metabolic engineering strategies are discussed.

  15. [Human semen lactate dehydrogenase isoenzymes in fertility studies (author's transl)].

    PubMed

    Gonzalez Buitrago, J M; García Díez, L C; de Castro, S

    1981-01-01

    The lactate dehydrogenase isoenzyme pattern has been obtained in the semen of 87 males undergoing fertility studies. The proportion of LDH-X, the isoenzyme specific to the spermatozoa, is reduced in proportion to the reduction of the sperm density and motility. LDH-X is the most abundant isoenzyme in the semen of normospermic subjects. As to the other isoenzymes, the most abundant ones are the LDH-2 and the LDH-3. The results obtained lead us to conclude that the measurement of the lactate dehydrogenase isoenzymes may be useful in studies of fertility as an indicative parameter of the quality of the semen.

  16. Malate dehydrogenase isozymes in the longnose dace, Rhinichthys cataractae.

    PubMed

    Starzyk, R M; Merritt, R B

    1980-08-01

    The interspecies homology of dace supernatant (A2,AB,B2) and mitochondrial (C2) malate dehydrogenase isozymes has been established through cell fractionation and tissue distribution studies. Isolated supernatant malate dehydrogenase (s-MDH) isozymes show significant differences in Michaelis constants for oxaloacetate and in pH optima. Shifts in s-MDH isozyme pH optima with temperature may result in immediate compensation for increase in ectotherm body pH with decrease in temperature, but duplicate s-MDH isozymes are probably maintained through selection for tissue specific regulation of metabolism.

  17. Isolation of human lactate dehydrogenase isoenzyme X by affinity chromatography.

    PubMed Central

    Kolk, A H; van Kuyk, L; Boettcher, B

    1978-01-01

    Human isoenzyme LDH-X (lactate dehydrogenase isoenzyme X) was isolated from seminal fluid of frozen semen samples by affinity chromatography by using oxamate-Sepharose and AMP-Sepharose. In the presence of 1.6 mM-NAD+, isoenzyme LDH-X does not bind to AMP-Sepharose, whereas the other lactate dehydrogenase isoenzymes do. This is the crucial point in the isolation of isoenzyme LDH-X from the other isoenzymes. The purified human isoenzyme LDH-X had a specific activity of 146 units/mg of protein. Images Fig. 2. Fig. 3. PMID:213050

  18. Purification and characterization of 3-isopropylmalate dehydrogenase from Thiobacillus thiooxidans.

    PubMed

    Kawaguchi, H; Inagaki, K; Matsunami, H; Nakayama, Y; Tano, T; Tanaka, H

    2000-01-01

    3-Isopropylmalate dehydrogenase was purified to homogeneity from the acidophilic autotroph Thiobacillus thiooxidans. The native enzyme was a dimer of molecular weight 40,000. The apparent K(m) values for 3-isopropylmalate and NAD+ were estimated to be 0.13 mM and 8.7 mM, respectively. The optimum pH for activity was 9.0 and the optimum temperature was 65 degrees C. The properties of the enzyme were similar to those of the Thiobacillus ferrooxidans enzyme, expect for substrate specificity. T. thiooxidans 3-isopropylmalate dehydrogenase could not utilize malate as a substrate.

  19. Reversible inactivation of CO dehydrogenase with thiol compounds

    SciTech Connect

    Kreß, Oliver; Gnida, Manuel; Pelzmann, Astrid M.; Marx, Christian; Meyer-Klaucke, Wolfram; Meyer, Ortwin

    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 proceeds 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 step in

  20. Pyruvate dehydrogenase complex from germinating castor bean endosperm.

    PubMed

    Rapp, B J; Randall, D D

    1980-02-01

    Subcellular organelles from castor bean (Ricinus communis) endosperm were isolated on discontinuous sucrose gradients from germinating seeds which were 1 to 7 days postimbibition. Marker enzyme activities of the organelles were measured (fumarase, catalase, and triose phosphate isomerase) and the homogeneity of the organelle fractions was examined by electron microscopy. Pyruvate dehydrogenase complex activity was measured only in the mitochondrial fraction and attempts to activate or release the enzyme from the proplastid were not successful. A pathway is proposed for the most efficient use of endosperm carbon for de novo fatty acid biosynthesis that does not require the presence of the pyruvate dehydrogenase complex in the proplastid to provide acetyl-coenzymeA.

  1. Complete amino acid sequence and characterization of the reaction mechanism of a glucosamine-induced novel alcohol dehydrogenase from Agrobacterium radiobacter (tumefaciens).

    PubMed

    Iwamoto, Ryoko; Kubota, Humie; Hosoki, Tomoko; Ikehara, Kenji; Tanaka, Mieko

    2002-02-15

    A glucosamine-induced novel alcohol dehydrogenase has been isolated from Agrobacterium radiobacter (tumefaciens) and its fundamental properties have been characterized. The enzyme catalyzes NAD-dependent dehydrogenation of aliphatic alcohols and amino alcohols. In this work, the complete amino acid sequence of the alcohol dehydrogenase was determined by PCR method using genomic DNA of A. radiobacter as template. The enzyme comprises 336 amino acids and has a molecular mass of 36 kDa. The primary structure of the enzyme demonstrates a high homology to structures of alcohol dehydrogenases from Shinorhizobium meliloti (83% identity, 90% positive) and Pseudomonas aeruginosa (65% identity, 76% positive). The two Zn(2+) ion binding sites, both the active site and another site that contributed to stabilization of the enzyme, are conserved in those enzymes. Sequences analysis of the NAD-dependent dehydrogenase family using a hypothetical phylogenetic tree indicates that these three enzymes form a new group distinct from other members of the Zn-containing long-chain alcohol dehydrogenase family. The physicochemical properties of alcohol dehydrogenase from A. radiobacter were characterized as follows. (1) Stereospecificity of the hydride transfer from ethanol to NADH was categorized as pro-R type by NMR spectra of NADH formed in the enzymatic reaction using ethanol-D(6) was used as substrate. (2) Optimal pH for all alcohols with no amino group examined was pH 8.5 (of the C(2)-C(6) alcohols, n-amyl alcohol demonstrated the highest activity). Conversely, glucosaminitol was optimally dehydrogenated at pH 10.0. (3) The rate-determining step of the dehydrogenase for ethanol is deprotonation of the enzyme-NAD-Zn-OHCH(2)CH(3) complex to enzyme-NAD-Zn-O(-)CH(2)CH(3) complex and that for glucosaminitol is H(2)O addition to enzyme-Zn-NADH complex. PMID:11831851

  2. Complete amino acid sequence and characterization of the reaction mechanism of a glucosamine-induced novel alcohol dehydrogenase from Agrobacterium radiobacter (tumefaciens).

    PubMed

    Iwamoto, Ryoko; Kubota, Humie; Hosoki, Tomoko; Ikehara, Kenji; Tanaka, Mieko

    2002-02-15

    A glucosamine-induced novel alcohol dehydrogenase has been isolated from Agrobacterium radiobacter (tumefaciens) and its fundamental properties have been characterized. The enzyme catalyzes NAD-dependent dehydrogenation of aliphatic alcohols and amino alcohols. In this work, the complete amino acid sequence of the alcohol dehydrogenase was determined by PCR method using genomic DNA of A. radiobacter as template. The enzyme comprises 336 amino acids and has a molecular mass of 36 kDa. The primary structure of the enzyme demonstrates a high homology to structures of alcohol dehydrogenases from Shinorhizobium meliloti (83% identity, 90% positive) and Pseudomonas aeruginosa (65% identity, 76% positive). The two Zn(2+) ion binding sites, both the active site and another site that contributed to stabilization of the enzyme, are conserved in those enzymes. Sequences analysis of the NAD-dependent dehydrogenase family using a hypothetical phylogenetic tree indicates that these three enzymes form a new group distinct from other members of the Zn-containing long-chain alcohol dehydrogenase family. The physicochemical properties of alcohol dehydrogenase from A. radiobacter were characterized as follows. (1) Stereospecificity of the hydride transfer from ethanol to NADH was categorized as pro-R type by NMR spectra of NADH formed in the enzymatic reaction using ethanol-D(6) was used as substrate. (2) Optimal pH for all alcohols with no amino group examined was pH 8.5 (of the C(2)-C(6) alcohols, n-amyl alcohol demonstrated the highest activity). Conversely, glucosaminitol was optimally dehydrogenated at pH 10.0. (3) The rate-determining step of the dehydrogenase for ethanol is deprotonation of the enzyme-NAD-Zn-OHCH(2)CH(3) complex to enzyme-NAD-Zn-O(-)CH(2)CH(3) complex and that for glucosaminitol is H(2)O addition to enzyme-Zn-NADH complex.

  3. Amino acid sequence homology among the 2-hydroxy acid dehydrogenases: mitochondrial and cytoplasmic malate dehydrogenases form a homologous system with lactate dehydrogenase.

    PubMed Central

    Birktoft, J J; Fernley, R T; Bradshaw, R A; Banaszak, L J

    1982-01-01

    The amino acid sequence of porcine heart mitochondrial malate dehydrogenase (mMDH; L-malate: NAD+ oxidoreductase, EC 1.1.1.37) has been compared with the sequences of six different lactate dehydrogenases (LDH; L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) and with the "x-ray" sequence of cytoplasmic malate dehydrogenase (sMDH). The main points are that (i) all three enzymes are homologous; (ii) invariant residues in the catalytic center of these enzymes include a histidine and an internally located aspartate that function as a proton relay system; (iii) numerous residues important to coenzyme binding are conserved, including several glycines and charged residues; and (iv) amino acid side chains present in the subunit interface common to the MDHs and LDHs appear to be better conserved than those in the protein interior. It is concluded that LDH, sMDH, and mMDH are derived from a common ancestral gene and probably have similar catalytic mechanisms. PMID:6959107

  4. The conserved Glu-60 residue in Thermoanaerobacter brockii alcohol dehydrogenase is not essential for catalysis

    PubMed Central

    Kleifeld, Oded; Shi, Shu Ping; Zarivach, Raz; Eisenstein, Miriam; Sagi, Irit

    2003-01-01

    Glu-60 of the zinc-dependent Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) is a strictly conserved residue in all members of the alcohol dehydrogenase (ADH) family. Unlike most other ADHs, the crystal structures of TbADH and its analogs, ADH from Clostridium beijerinckii (CbADH), exhibit a unique zinc coordination environment in which this conserved residue is directly coordinated to the catalytic zinc ion in the native form of the enzymes. To explore the role of Glu-60 in TbADH catalysis, we have replaced it by alanine (E60A-TbADH) and aspartate (E60D-TbADH). Steady-state kinetic measurements show that the catalytic efficiency of these mutants is only four- and eightfold, respectively, lower than that of wild-type TbADH. We applied X-ray absorption fine-structure (EXAFS) and near-UV circular dichroism to characterize the local environment around the catalytic zinc ion in the variant enzymes in their native, cofactor-bound, and inhibited forms. We show that the catalytic zinc site in the studied complexes of the variant enzymes exhibits minor changes relative to the analogous complexes of wild-type TbADH. These moderate changes in the kinetic parameters and in the zinc ion environment imply that the Glu-60 in TbADH does not remain bound to the catalytic zinc ion during catalysis. Furthermore, our results suggest that a water molecule replaces this residue during substrate turnover. PMID:12592017

  5. Alcohol and aldehyde dehydrogenases: structures of the human liver enzymes, functional properties and evolutionary aspects.

    PubMed

    Jörnvall, H; Hempel, J; von Bahr-Lindström, H; Höög, J O; Vallee, B L

    1987-01-01

    All three types of subunit of class I human alcohol dehydrogenase have been analyzed both at the protein and cDNA levels, and the structures of alpha, beta 1, beta 2, gamma 1, and gamma 2 subunits are known. The same applies to class II pi subunits. Extensive protein data are also available for class III chi subunits. In the class I human isozymes, amino acid exchanges occur at 35 positions in total, with 21-28 replacements between any pair of the alpha/beta/gamma chains. These values, compared with those from species differences between the corresponding human and horse enzymes, suggest that isozyme developments in the class I enzyme resulted from separate gene duplications after the divergence of the human and equine evolutionary lines. All subunits exhibit some unique properties, with slightly closer similarity between the human gamma and horse enzyme subunits and somewhat greater deviations towards the human alpha subunit. Differences are large also in segments close to the active site zinc ligands and other functionally important positions. Species differences are distributed roughly equally between the two types of domain in the subunit, whereas isozyme differences are considerably more common in the catalytic than in the coenzyme-binding domain. These facts illustrate a functional divergence among the isozymes but otherwise similar changes during evolution. Polymorphic forms of beta and gamma subunits are characterized by single replacements at one and two positions, respectively, explaining known deviating properties. Class II and class III subunits are considerably more divergent. Their homology with class I isozymes exhibits only 60-65% positional identity. Hence, they reflect further steps towards the development of new enzymes, with variations well above the horse/human species levels, in contrast to the class I forms. Again, functionally important residues are affected, and patterns resembling those previously established for the divergently related

  6. NADP+-Preferring D-Lactate Dehydrogenase from Sporolactobacillus inulinus.

    PubMed

    Zhu, Lingfeng; Xu, Xiaoling; Wang, Limin; Dong, Hui; Yu, Bo; Ma, Yanhe

    2015-09-01

    Hydroxy acid dehydrogenases, including l- and d-lactate dehydrogenases (L-LDH and D-LDH), are responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids and extensively used in a wide range of biotechnological applications. A common feature of LDHs is their high specificity for NAD(+) as a cofactor. An LDH that could effectively use NADPH as a coenzyme could be an alternative enzymatic system for regeneration of the oxidized, phosphorylated cofactor. In this study, a d-lactate dehydrogenase from a Sporolactobacillus inulinus strain was found to use both NADH and NADPH with high efficiencies and with a preference for NADPH as its coenzyme, which is different from the coenzyme utilization of all previously reported LDHs. The biochemical properties of the D-LDH enzyme were determined by X-ray crystal structural characterization and in vivo and in vitro enzymatic activity analyses. The residue Asn(174) was demonstrated to be critical for NADPH utilization. Characterization of the biochemical properties of this enzyme will contribute to understanding of the catalytic mechanism and provide referential information for shifting the coenzyme utilization specificity of 2-hydroxyacid dehydrogenases.

  7. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Lactic dehydrogenase immunological test system. 866.5560 Section 866.5560 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... blood cells), myocardial infarction (heart disease), and some forms of leukemia (cancer of the...

  8. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Lactic dehydrogenase immunological test system. 866.5560 Section 866.5560 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... blood cells), myocardial infarction (heart disease), and some forms of leukemia (cancer of the...

  9. A novel small-molecule inhibitor of 3-phosphoglycerate dehydrogenase.

    PubMed

    Mullarky, Edouard; Lairson, Luke L; Cantley, Lewis C; Lyssiotis, Costas A

    2016-07-01

    Serine metabolism is likely to play a critical role in cancer cell growth. A recent study reports the identification of a novel small-molecule inhibitor of serine synthesis that targets 3-phosphoglycerate dehydrogenase (PHGDH), the first enzyme of the serine synthesis pathway, and selectively abrogates the proliferation of PHGDH overexpressing breast cancer cells. PMID:27652319

  10. 21 CFR 862.1565 - 6-Phosphogluconate dehydrogenase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false 6-Phosphogluconate dehydrogenase test system. 862.1565 Section 862.1565 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1565...

  11. Genetics Home Reference: 3-hydroxyacyl-CoA dehydrogenase deficiency

    MedlinePlus

    ... step that metabolizes groups of fats called medium-chain fatty acids and short-chain fatty acids. Mutations in the HADH gene lead ... a shortage of 3-hydroxyacyl-CoA dehydrogenase. Medium-chain and short-chain fatty acids cannot be metabolized ...

  12. Molecular cloning of gluconobacter oxydans DSM 2003 xylitol dehydrogenase gene

    PubMed Central

    Sadeghi, H. Mir Mohammad; Ahmadi, R.; Aghaabdollahian, S.; Mofid, M.R.; Ghaemi, Y.; Abedi, D.

    2011-01-01

    Due to the widespread applications of xylitol dehydrogenase, an enzyme used for the production of xylitol, the present study was designed for the cloning of xylitol dehydrogenase gene from Glcunobacter oxydans DSM 2003. After extraction of genomic DNA from this bacterium, xylitol dehydrogenase gene was replicated using polymerase chain reaction (PCR). The amplified product was entered into pTZ57R cloning vector by T/A cloning method and transformation was performed by heat shocking of the E. coli XL1-blue competent cells. Following plasmid preparation, the cloned gene was digested out and ligated into the expression vector pET-22b(+). Electrophoresis of PCR product showed a 789 bp band. Recombinant plasmid (rpTZ57R) was then constructed. This plasmid was double digested with XhoI and EcoRI resulting in 800 bp and 2900 bp bands. The obtained insert was ligated into pET-22b(+) vector and its orientation was confirmed with XhoI and BamHI restriction enzymes. In conclusion, in the present study the recombinant expression vector containing xylitol dehydrogenase gene has been constructed and can be used for the production of this enzyme in high quantities. PMID:22110522

  13. Effects of aerobic training on pyruvate dehydrogenase and pyruvate dehydrogenase kinase in human skeletal muscle.

    PubMed

    LeBlanc, Paul J; Peters, Sandra J; Tunstall, Rebecca J; Cameron-Smith, David; Heigenhauser, George J F

    2004-06-01

    This study examined the effects of short- and long-term aerobic training on the stable up-regulation of pyruvate dehydrogenase (PDH) and PDH kinase (PDK) in human skeletal muscle. We hypothesized that 8 weeks, but not 1 week, of aerobic training would increase total PDH (PDHt) and PDK activities compared to pretraining, and this would be detectable at the level of gene transcription (mRNA) and/or gene translation (protein). Resting muscle biopsies were taken before and after 1 and 8 weeks of aerobic cycle exercise training. PDHt and PDK activities, and their respective protein and mRNA expression, did not differ after 1 week of aerobic training. PDHt activity increased 31% after 8 weeks and this may be partially due to a 1.3-fold increase in PDH-E(1)alpha protein expression. PDK activity approximately doubled after 8 weeks of aerobic training and this was attributed to a 1.3-fold increase in PDK2 isoform protein expression. Similar to 1 week, no changes were observed at the mRNA level after 8 weeks of training. These findings suggest that aerobically trained human skeletal muscle has an increased maximal capacity to utilize carbohydrates, evident by increased PDHt, but increased metabolic control sensitivity to pyruvate through increased contribution of PDK2 to total PDK activity. PMID:15020699

  14. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme...

  15. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme...

  16. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme...

  17. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme...

  18. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme...

  19. 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.

  20. Glutamate dehydrogenases: the why and how of coenzyme specificity.

    PubMed

    Engel, Paul C

    2014-01-01

    NAD(+) and NADP(+), chemically similar and with almost identical standard oxidation-reduction potentials, nevertheless have distinct roles, NAD(+) serving catabolism and ATP generation whereas NADPH is the biosynthetic reductant. Separating these roles requires strict specificity for one or the other coenzyme for most dehydrogenases. In many organisms this holds also for glutamate dehydrogenases (GDH), NAD(+)-dependent for glutamate oxidation, NADP(+)-dependent for fixing ammonia. In higher animals, however, GDH has dual specificity. It has been suggested that GDH in mitochondria reacts only with NADP(H), the NAD(+) reaction being an in vitro artefact. However, contrary evidence suggests mitochondrial GDH not only reacts with NAD(+) but maintains equilibrium using the same pool as accessed by β-hydroxybutyrate dehydrogenase. Another complication is the presence of an energy-linked dehydrogenase driving NADP(+) reduction by NADH, maintaining the coenzyme pools at different oxidation-reduction potentials. Its coexistence with GDH makes possible a futile cycle, control of which is not yet properly explained. Structural studies show NAD(+)-dependent, NADP(+)-dependent and dual-specificity GDHs are closely related and a few site-directed mutations can reverse specificity. Specificity for NAD(+) or for NADP(+) has probably emerged repeatedly during evolution, using different structural solutions on different occasions. In various GDHs the P7 position in the coenzyme-binding domain plays a key role. However, whereas in other dehydrogenases an acidic P7 residue usually hydrogen bonds to the 2'- and 3'-hydroxyls, dictating NAD(+) specificity, among GDHs, depending on detailed conformation of surrounding residues, an acidic P7 may permit binding of NAD(+) only, NADP(+) only, or in higher animals both.

  1. Marked reduction of alcohol dehydrogenase in keratoconus corneal fibroblasts

    PubMed Central

    Kanoff, J.M.; Shankardas, J.; Dimitrijevich, S.

    2009-01-01

    Purpose To identify differentially expressed genes in keratoconus (KC) corneal fibroblasts. Methods Stromal keratocytes (having a fibroblast morphology) from KC keratoplasty specimens and eye bank donor corneas were isolated and expanded using a serum containing medium. RNA was isolated from three KC fibroblast cultures and five eye bank donor cornea fibroblast cultures. The targets from the cultured fibroblasts were hybridized to the Affymetrix U133 Plus 2.0 microarrays. Western blot analyses of cell lysates were performed to examine protein levels of interest in the two groups. Protein levels of select differentially expressed genes were further examined by immunohistochemistry. Keratocyte staining of archived KC keratoplasty specimens were graded using a 0 to 3+ scale and compared to five archived whole globes having normal corneas as well as to 10 Fuchs’ dystrophy keratoplasty specimens. Results Microarray analysis revealed up to a 212 fold reduction in the mRNA levels of alcohol dehydrogenase (class 1) beta polypeptide (ADH1B) in KC fibroblasts (p=0.04). Decreased alcohol dehydrogenase in KC fibroblasts was confirmed by western blot analysis of early passage primary keratocyte cell lysates. Immunohistochemistry using a monoclonal mouse immunoglobulin G (IgG) against human liver alcohol dehydrogenase revealed a dramatic difference in protein staining in the keratocytes of the KC group compared to the normal cornea group. Immunohistochemistry also showed decreased immunostaining against alcohol dehydrogenase in the KC stromal sections compared to those obtained from Fuchs’ endothelial corneal dystrophy samples. Conclusions Decreased alcohol dehydrogenase in KC corneal fibroblasts represents a strong marker and possible mediator of keratoconus. PMID:19365573

  2. Short Chain Dehydrogenase/Reductase Rdhe2 Is a Novel Retinol Dehydrogenase Essential for Frog Embryonic Development*

    PubMed Central

    Belyaeva, Olga V.; Lee, Seung-Ah; Adams, Mark K.; Chang, Chenbei; Kedishvili, Natalia Y.

    2012-01-01

    The enzymes responsible for the rate-limiting step in retinoic acid biosynthesis, the oxidation of retinol to retinaldehyde, during embryogenesis and in adulthood have not been fully defined. Here, we report that a novel member of the short chain dehydrogenase/reductase superfamily, frog sdr16c5, acts as a highly active retinol dehydrogenase (rdhe2) that promotes retinoic acid biosynthesis when expressed in mammalian cells. In vivo assays of rdhe2 function show that overexpression of rdhe2 in frog embryos leads to posteriorization and induction of defects resembling those caused by retinoic acid toxicity. Conversely, antisense morpholino-mediated knockdown of endogenous rdhe2 results in phenotypes consistent with retinoic acid deficiency, such as defects in anterior neural tube closure, microcephaly with small eye formation, disruption of somitogenesis, and curved body axis with bent tail. Higher doses of morpholino induce embryonic lethality. Analyses of retinoic acid levels using either endogenous retinoic acid-sensitive gene hoxd4 or retinoic acid reporter cell line both show that the levels of retinoic acid are significantly decreased in rdhe2 morphants. Taken together, these results provide strong evidence that Xenopus rdhe2 functions as a retinol dehydrogenase essential for frog embryonic development in vivo. Importantly, the retinol oxidizing activity of frog rdhe2 is conserved in its mouse homologs, suggesting that rdhe2-related enzymes may represent the previously unrecognized physiologically relevant retinol dehydrogenases that contribute to retinoic acid biosynthesis in higher vertebrates. PMID:22291023

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

    PubMed

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

    2012-06-01

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

  4. Designing a highly active soluble PQQ-glucose dehydrogenase for efficient glucose biosensors and biofuel cells

    SciTech Connect

    Durand, Fabien; Stines-Chaumeil, Claire; Flexer, Victoria; Andre, Isabelle; Mano, Nicolas

    2010-11-26

    Research highlights: {yields} A new mutant of PQQ-GDH designed for glucose biosensors application. {yields} First mutant of PQQ-GDH with higher activity for D-glucose than the Wild type. {yields} Position N428 is a key point to increase the enzyme activity. {yields} Molecular modeling shows that the N428 C mutant displays a better interaction for PQQ than the WT. -- Abstract: We report for the first time a soluble PQQ-glucose dehydrogenase that is twice more active than the wild type for glucose oxidation and was obtained by combining site directed mutagenesis, modelling and steady-state kinetics. The observed enhancement is attributed to a better interaction between the cofactor and the enzyme leading to a better electron transfer. Electrochemical experiments also demonstrate the superiority of the new mutant for glucose oxidation and make it a promising enzyme for the development of high-performance glucose biosensors and biofuel cells.

  5. Ozone inhalation in rats: effects on alkaline phosphatase and lactic dehydrogenase isoenzymes in lavage and plasma

    SciTech Connect

    Nachtman, J.P.; Moon, H.L.; Miles, R.C.

    1988-10-01

    Ozone is found in urban and rural atmospheres and is produced from a variety of natural and man-made sources. Animal studies conducted at typical ambient levels result in reproducible morphological, biochemical and functional effects. Ozone damages type I epithelial cells, induces proliferation of type II cells and produces inflammation of the terminal bronchiolar-alveolar duct region. Ozone increases lung oxygen utilization and increases glutathione metabolism. Ozone increases airway resistance. The authors measured lactic dehydrogenase (LD) isoenzymes to ascertain the tissue giving rise to the increased LD activity in lavage. They also assayed acid phosphatase, alkaline phosphatase, creatine kinase activities, and protein levels since these parameters were increased in rat lung lavage after particulate exposure. They determined white cell differential and red cell morphology parameters because previous investigators reported that ozone increased neutrophil/lymphocyte ratio.

  6. Catalytic reaction of cytokinin dehydrogenase: preference for quinones as electron acceptors.

    PubMed Central

    Frébortová, Jitka; Fraaije, Marco W; Galuszka, Petr; Sebela, Marek; Pec, Pavel; Hrbác, Jan; Novák, Ondrej; Bilyeu, Kristin D; English, James T; Frébort, Ivo

    2004-01-01

    The catalytic reaction of cytokinin oxidase/dehydrogenase (EC 1.5.99.12) was studied in detail using the recombinant flavoenzyme from maize. Determination of the redox potential of the covalently linked flavin cofactor revealed a relatively high potential dictating the type of electron acceptor that can be used by the enzyme. Using 2,6-dichlorophenol indophenol, 2,3-dimethoxy-5-methyl-1,4-benzoquinone or 1,4-naphthoquinone as electron acceptor, turnover rates with N6-(2-isopentenyl)adenine of approx. 150 s(-1) could be obtained. This suggests that the natural electron acceptor of the enzyme is quite probably a p-quinone or similar compound. By using the stopped-flow technique, it was found that the enzyme is rapidly reduced by N6-(2-isopentenyl)adenine (k(red)=950 s(-1)). Re-oxidation of the reduced enzyme by molecular oxygen is too slow to be of physiological relevance, confirming its classification as a dehydrogenase. Furthermore, it was established for the first time that the enzyme is capable of degrading aromatic cytokinins, although at low reaction rates. As a result, the enzyme displays a dual catalytic mode for oxidative degradation of cytokinins: a low-rate and low-substrate specificity reaction with oxygen as the electron acceptor, and high activity and strict specificity for isopentenyladenine and analogous cytokinins with some specific electron acceptors. PMID:14965342

  7. Succinate dehydrogenase mutant of Listonella anguillarum protects rainbow trout against vibriosis.

    PubMed

    Altinok, Ilhan; Capkin, Erol; Karsi, Attila

    2015-10-13

    Listonella anguillarum is a Gram-negative facultative anaerobic rod causing hemorrhagic septicemia in marine and rarely in freshwater fish. Succinate dehydrogenase (SDH) plays an important role in the tricarboxylic acid (TCA) cycle by oxidizing succinate to fumarate while reducing ubiquinone to ubiquinol. Recent studies indicate that central metabolic pathways, including the TCA cycle, contribute to bacterial virulence. However, the role of SDH in L. anguillarum virulence has not been studied. Here, we report in-frame deletion of the succinate dehydrogenase iron-sulfur protein (SDHB) and its role in L. anguillarum virulence in rainbow trout. To accomplish this goal, upstream and downstream regions of the L. anguillarum sdhB gene were amplified in-frame and cloned into a suicide plasmid. The chromosomal sdhB gene of L. anguillarum was deleted by homologous recombination. Virulence and immunogenicity of the L. anguillarum ΔsdhB mutant (LaΔsdhB) were determined in rainbow trout. Results show that LaΔsdhB was highly attenuated in rainbow trout, and fish immunized with LaΔsdhB displayed high relative survival rate after exposure to wild type L. anguillarum. These findings indicate SDH is important in L. anguillarum virulence in rainbow trout, and LaΔsdhB could be used as an immersion, oral, or injection vaccine to protect rainbow trout against vibriosis.

  8. Deletion of the Bacillus subtilis isocitrate dehydrogenase gene causes a block at stage I of sporulation.

    PubMed Central

    Jin, S; Levin, P A; Matsuno, K; Grossman, A D; Sonenshein, A L

    1997-01-01

    A Bacillus subtilis mutant with a deletion of citC, the gene encoding isocitrate dehydrogenase, the third enzyme of the tricarboxylic acid branch of the Krebs cycle, had a greatly reduced ability to sporulate. Analysis of expression of lacZ fusions to various sporulation gene promoters revealed that in the citC mutant development is probably blocked between stage 0 and stage II. That is, genes expressed very early in sporulation, under the direct control of the Spo0A transcription factor, were induced normally in the citC mutant. However, genes expressed after asymmetric septation (stage II) in wild-type cells were not induced in the citC mutant. Analysis of cell morphology by thin-section electron microscopy and immunofluorescence microscopy showed that the mutant formed axial chromosomal filaments and accumulated rings of FtsZ protein at potential polar division sites but failed to form asymmetric division septa, indicating that sporulation is blocked at stage I. The growth and sporulation defects of the B. subtilis citC mutant were fully overcome by introduction and expression of the Escherichia coli icd gene, encoding an isocitrate dehydrogenase similar to the enzyme from B. subtilis. PMID:9244258

  9. Mechanistic studies of the coenzyme F420 reducing formate dehydrogenase from Methanobacterium formicicum.

    PubMed

    Schauer, N L; Ferry, J G; Honek, J F; Orme-Johnson, W H; Walsh, C

    1986-11-01

    Mechanistic studies have been undertaken on the coenzyme F420 dependent formate dehydrogenase from Methanobacterium formicicum. The enzyme was specific for the si face hydride transfer to C5 of F420 and joins three other F420-recognizing methanogen enzymes in this stereospecificity, consistent perhaps with a common type of binding site for this 8-hydroxy-5-deazariboflavin. While catalysis probably occurs by hydride transfer from formate to the enzyme to generate an EH2 species and then by hydride transfer back out to F420, the formate-derived hydrogen exchanged with solvent protons before transfer back out to F420. The kinetics of hydride transfer from formate revealed that this step is not rate determining, which suggests that the rate-determining step is an internal electron transfer. The deflavo formate dehydrogenase was amenable to reconstitution with flavin analogues. The enzyme was sensitive to alterations in FAD structure in the 6-, 7-, and 8-loci of the benzenoid moiety in the isoalloxazine ring.

  10. CYTOKININ OXIDASE/DEHYDROGENASE3 Maintains Cytokinin Homeostasis during Root and Nodule Development in Lotus japonicus.

    PubMed

    Reid, Dugald E; Heckmann, Anne B; Novák, Ondřej; Kelly, Simon; Stougaard, Jens

    2016-02-01

    Cytokinins are required for symbiotic nodule development in legumes, and cytokinin signaling responses occur locally in nodule primordia and in developing nodules. Here, we show that the Lotus japonicus Ckx3 cytokinin oxidase/dehydrogenase gene is induced by Nod factor during the early phase of nodule initiation. At the cellular level, pCkx3::YFP reporter-gene studies revealed that the Ckx3 promoter is active during the first cortical cell divisions of the nodule primordium and in growing nodules. Cytokinin measurements in ckx3 mutants confirmed that CKX3 activity negatively regulates root cytokinin levels. Particularly, tZ and DHZ type cytokinins in both inoculated and uninoculated roots were elevated in ckx3 mutants, suggesting that these are targets for degradation by the CKX3 cytokinin oxidase/dehydrogenase. The effect of CKX3 on the positive and negative roles of cytokinin in nodule development, infection and regulation was further clarified using ckx3 insertion mutants. Phenotypic analysis indicated that ckx3 mutants have reduced nodulation, infection thread formation and root growth. We also identify a role for cytokinin in regulating nodulation and nitrogen fixation in response to nitrate as ckx3 phenotypes are exaggerated at increased nitrate levels. Together, these findings show that cytokinin accumulation is tightly regulated during nodulation in order to balance the requirement for cell divisions with negative regulatory effects of cytokinin on infection events and root development. PMID:26644503

  11. A cysteine endopeptidase isolated from castor bean endosperm microbodies processes the glyoxysomal malate dehydrogenase precursor protein.

    PubMed

    Gietl, C; Wimmer, B; Adamec, J; Kalousek, F

    1997-03-01

    A plant cysteine endopeptidase with a molecular mass of 35 kD was purified from microbodies of germinating castor bean (Ricinus communis) endosperm by virtue of its capacity to specifically process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro. Processing of the glyoxysomal malate dehydrogenase precursor occurs sequentially in three steps, the first intermediate resulting from cleavage after arginine-13 within the presequence and the second from cleavage after arginine-33. The endopeptidase is unable to remove the presequences of prethiolases from rape (Brassica napus) glyoxysomes and rat peroxisomes at the expected cleavage site. Protein sequence analysis of N-terminal and internal peptides revealed high identity to the mature papain-type cysteine endopeptidases from cotyledons of germinating mung bean (Vigna mungo) and French bean (Phaseolus vulgaris) seeds. These endopeptidases are synthesized with an extended pre-/prosequence at the N terminus and have been considered to be processed in the endoplasmic reticulum and targeted to protein-storing vacuoles.

  12. Deletion of lactate dehydrogenase in Enterobacter aerogenes to enhance 2,3-butanediol production.

    PubMed

    Jung, Moo-Young; Ng, Chiam Yu; Song, Hyohak; Lee, Jinwon; Oh, Min-Kyu

    2012-07-01

    2,3-Butanediol is an important bio-based chemical product, because it can be converted into several C4 industrial chemicals. In this study, a lactate dehydrogenase-deleted mutant was constructed to improve 2,3-butanediol productivity in Enterobacter aerogenes. To delete the gene encoding lactate dehydrogenase, λ Red recombination method was successfully adapted for E. aerogenes. The resulting strain produced a very small amount of lactate and 16.7% more 2,3-butanediol than that of the wild-type strain in batch fermentation. The mutant and its parental strain were then cultured with six different carbon sources, and the mutant showed higher carbon source consumption and microbial growth rates in all media. The 2,3-butanediol titer reached 69.5 g/l in 54 h during fed-batch fermentation with the mutant,which was 27.4% higher than that with the parental strain.With further optimization of the medium and aeration conditions,118.05 g/l 2,3-butanediol was produced in 54 h during fed-batch fermentation with the mutant. This is by far the highest titer of 2,3-butanediol with E. aerogenes achieved by metabolic pathway engineering.

  13. Role of a putative tungsten-dependent formylmethanofuran dehydrogenase in Methanosarcina acetivorans.

    PubMed

    Matschiavelli, Nicole; Rother, Michael

    2015-04-01

    Methanogenesis, the biological production of methane, is the sole means for energy conservation for methanogenic archaea. Among the few methanogens shown to grow on carbon monoxide (CO) is Methanosarcina acetivorans, which produces, beside methane, acetate and formate in the process. Since CO-dependent methanogenesis proceeds via formation of formylmethanofuran from CO2 and methanofuran, catalyzed by formylmethanofuran dehydrogenase, we were interested whether this activity could participate in the formate formation from CO. The genome of M. acetivorans encodes four putative formylmethanofuran dehydrogenases, two annotated as molybdenum-dependent and the remaining two as tungsten-dependent enzymes. A mutant lacking one of the putative tungsten enzymes grew very slowly on CO and only after a prolonged adaptation period, which suggests an important role for this isoform during growth on CO. Methanol- and CO-dependent growth of the mutant required the presence of molybdenum indicating an indispensable function of this metal in the remaining isoforms. CO-dependent formate formation could not be observed in the mutant indicating involvement of the respective isoform in the process. However, addition of formaldehyde, which spontaneously reacts with tetrahydrosarcinapterin (H4SPT) to methenyl-H4SPT, led to near-wild-type formate production rates, which argues for an alternative route of formate formation in this organism.

  14. Analyzing Xanthine Dehydrogenase Iron-Sulfur Clusters Using Electron Paramagnetic Resonance Spectroscopy

    SciTech Connect

    Hodson, R.

    2004-02-05

    Xanthine dehydrogenase is a metalloenzyme that is present in a variety of eukaryotic and prokaryotic organisms. The oxidation of the xanthine occurs at the molybdenum site, and the catalytic cycle is completed by electron transfer to the iron-sulfur (Fe/S) clusters and finally the flavin, where they are accepted by nicotinamide adenine dinucleotide (NAD). Since the site giving rise to the Fe/S I electron paramagnetic resonance (EPR) signal is thought to be the initial recipient of the electrons from the Mo, we wish to understand which EPR signal is associated with which Fe/S cluster in the structure in order to develop an understanding of the electron flow within the molecule. Samples of xanthine dehydrogenase wild-type and mutant forms were analyzed with EPR spectroscopy techniques at low and high temperatures. The results showed an altered Fe/S I signal along with an unaltered Fe/S II signal. The converted Cysteine, in the mutant, did affect the Fe/S cluster immediately adjacent to it. Therefore, the Fe/S I signal arises from the Fe/S cluster closest to the Mo and immediately adjacent to the mutated amino acid, and the Fe/S II signal must arise from the more distant Fe/S cluster.

  15. Succinate dehydrogenase mutation underlies global epigenomic divergence in gastrointestinal stromal tumor.

    PubMed

    Killian, J Keith; Kim, Su Young; Miettinen, Markku; Smith, Carly; Merino, Maria; Tsokos, Maria; Quezado, Martha; Smith, William I; Jahromi, Mona S; Xekouki, Paraskevi; Szarek, Eva; Walker, Robert L; Lasota, Jerzy; Raffeld, Mark; Klotzle, Brandy; Wang, Zengfeng; Jones, Laura; Zhu, Yuelin; Wang, Yonghong; Waterfall, Joshua J; O'Sullivan, Maureen J; Bibikova, Marina; Pacak, Karel; Stratakis, Constantine; Janeway, Katherine A; Schiffman, Joshua D; Fan, Jian-Bing; Helman, Lee; Meltzer, Paul S

    2013-06-01

    Gastrointestinal stromal tumors (GIST) harbor driver mutations of signal transduction kinases such as KIT, or, alternatively, manifest loss-of-function defects in the mitochondrial succinate dehydrogenase (SDH) complex, a component of the Krebs cycle and electron transport chain. We have uncovered a striking divergence between the DNA methylation profiles of SDH-deficient GIST (n = 24) versus KIT tyrosine kinase pathway-mutated GIST (n = 39). Infinium 450K methylation array analysis of formalin-fixed paraffin-embedded tissues disclosed an order of magnitude greater genomic hypermethylation relative to SDH-deficient GIST versus the KIT-mutant group (84.9 K vs. 8.4 K targets). Epigenomic divergence was further found among SDH-mutant paraganglioma/pheochromocytoma (n = 29), a developmentally distinct SDH-deficient tumor system. Comparison of SDH-mutant GIST with isocitrate dehydrogenase-mutant glioma, another Krebs cycle-defective tumor type, revealed comparable measures of global hypo- and hypermethylation. These data expose a vital connection between succinate metabolism and genomic DNA methylation during tumorigenesis, and generally implicate the mitochondrial Krebs cycle in nuclear epigenomic maintenance. PMID:23550148

  16. Succinate dehydrogenase mutation underlies global epigenomic divergence in gastrointestinal stromal tumor.

    PubMed

    Killian, J Keith; Kim, Su Young; Miettinen, Markku; Smith, Carly; Merino, Maria; Tsokos, Maria; Quezado, Martha; Smith, William I; Jahromi, Mona S; Xekouki, Paraskevi; Szarek, Eva; Walker, Robert L; Lasota, Jerzy; Raffeld, Mark; Klotzle, Brandy; Wang, Zengfeng; Jones, Laura; Zhu, Yuelin; Wang, Yonghong; Waterfall, Joshua J; O'Sullivan, Maureen J; Bibikova, Marina; Pacak, Karel; Stratakis, Constantine; Janeway, Katherine A; Schiffman, Joshua D; Fan, Jian-Bing; Helman, Lee; Meltzer, Paul S

    2013-06-01

    Gastrointestinal stromal tumors (GIST) harbor driver mutations of signal transduction kinases such as KIT, or, alternatively, manifest loss-of-function defects in the mitochondrial succinate dehydrogenase (SDH) complex, a component of the Krebs cycle and electron transport chain. We have uncovered a striking divergence between the DNA methylation profiles of SDH-deficient GIST (n = 24) versus KIT tyrosine kinase pathway-mutated GIST (n = 39). Infinium 450K methylation array analysis of formalin-fixed paraffin-embedded tissues disclosed an order of magnitude greater genomic hypermethylation relative to SDH-deficient GIST versus the KIT-mutant group (84.9 K vs. 8.4 K targets). Epigenomic divergence was further found among SDH-mutant paraganglioma/pheochromocytoma (n = 29), a developmentally distinct SDH-deficient tumor system. Comparison of SDH-mutant GIST with isocitrate dehydrogenase-mutant glioma, another Krebs cycle-defective tumor type, revealed comparable measures of global hypo- and hypermethylation. These data expose a vital connection between succinate metabolism and genomic DNA methylation during tumorigenesis, and generally implicate the mitochondrial Krebs cycle in nuclear epigenomic maintenance.

  17. Cardiac Hypertrophy in Mice with Long-Chain Acyl-CoA Dehydrogenase (LCAD) or Very Long-Chain Acyl-CoA Dehydrogenase (VLCAD) Deficiency

    PubMed Central

    Cox, Keith B.; Liu, Jian; Tian, Liqun; Barnes, Stephen; Yang, Qinglin; Wood, Philip A.

    2009-01-01

    Cardiac hypertrophy is a common finding in human patients with inborn errors of long-chain fatty acid oxidation. Mice with either very long-chain acyl-CoA dehydrogenase deficiency (VLCAD−/−) or long-chain acyl-CoA dehydrogenase deficiency (LCAD−/−) develop cardiac hypertrophy. Cardiac hypertrophy, initially measured using heart/body weight ratios, was manifested most severely in LCAD−/− male mice. VLCAD−/− mice, as a group, showed a mild increase in normalized cardiac mass (8.8% hypertrophy compared to all wild-type [WT] mice). In contrast, LCAD−/− mice as a group showed more severe cardiac hypertrophy (32.2% increase compared to all WT mice). Based on a clear male predilection, we investigated the role of dietary plant estrogenic compounds commonly found in mouse diets due to soy or alfalfa components providing natural phytoestrogens or isoflavones in cardioprotection of LCAD−/− mice. Male LCAD−/− mice fed an isoflavone-free test diet had more severe cardiac hypertrophy (58.1% hypertrophy compared to WT mice fed the same diet. There were no significant differences in the female groups fed any of the diets. Echocardiography measurement performed on male LCAD deficient mice fed a standard diet at ~3 months of age confirmed the substantial cardiac hypertrophy in these mice compared with WT controls. Left ventricular wall thickness of interventricular septum and posterior wall was remarkably increased in LCAD−/− mice compared with that of WT controls. Accordingly, the calculated LV mass after normalization to body weight was increased about 40% in the LCAD−/− mice compared with WT mice. In summary, we found that metabolic cardiomyopathy, expressed as hypertrophy, developed in mice due to either VLCAD deficiency or LCAD deficiency; however, LCAD deficiency was the most profound and appeared to be attenuated either by endogenous estrogen in females or phytoestrogens in the diet as isoflavones in males. PMID:19736549

  18. Anomalous behaviour of yeast isocitrate dehydrogenase during isoelectric focusing

    PubMed Central

    Illingworth, John A.

    1972-01-01

    Isoelectric focusing of yeast isocitrate dehydrogenase apparently reveals a number of `isoenzymes'. These have isoelectric points near pH5.5 in crude material, but during purification the mean isoelectric point progressively rises to pH7.0 and the band pattern changes. The shift in isoelectric point during purification is apparently genuine, since it is also manifested in the electrophoretic and chromatographic properties of the enzyme. The multiple forms, however, are an artifact, generated by exposure of the enzyme to Ampholine, since their activities vary with the protein/Ampholine ratio and they cannot be observed in any system from which Ampholine is excluded. There are no detectable isoenzymes of yeast isocitrate dehydrogenase. PMID:4571177

  19. Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation

    NASA Astrophysics Data System (ADS)

    Tan, Tien-Chye; Kracher, Daniel; Gandini, Rosaria; Sygmund, Christoph; Kittl, Roman; Haltrich, Dietmar; Hällberg, B. Martin; Ludwig, Roland; Divne, Christina

    2015-07-01

    A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization.

  20. Theoretical analysis of the glutamate dehydrogenase kinetics under physiological conditions.

    PubMed

    Popova, S V; Reich, J G

    1983-01-01

    A kinetic model of the glutamate dehydrogenase reaction has been formulated for the reversible reaction including all seven reactants (substrates and cofactors NAD(H) and NADP(H)). The model parameters have been evaluated from published initial-rate data. Analysis of the model at cofactor concentration near to that in the intact mitochondrion has shown that the competition for active sites between cofactors and substrates simultaneously present in mitochondria diminishes the steady-state rate of the reaction by a factor of 10 to 100 as compared to the maximal reaction rate. The model predicts near-equilibrium of the reaction substrates with NAD+/NADH cofactor pair and off-equilibrium with NADP+/NADPH. Substrate cycling with futile transfer of hydrogen from NADP+-system to NAD+-system has been found to account under in vivo conditions for no more than 2% of the maximal glutamate dehydrogenase activity in the mitochondria.

  1. Ribitol dehydrogenase from Klebsiella aerogenes. Purification and subunit structure

    PubMed Central

    Taylor, Susan S.; Rigby, Peter W. J.; Hartley, Brian S.

    1974-01-01

    Ribitol dehydrogenase has been purified to homogeneity from several strains of Klebsiella aerogenes. One strain yields 3–6g of pure enzyme from 1kg of cells. The enzyme is a tetramer of four subunits, mol.wt. 27000. Preliminary studies of the activity of the enzyme are reported. Peptide `maps' together with the amino acid composition indicate that the subunits are identical. ImagesPLATE 2PLATE 1 PMID:4618776

  2. Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria.

    PubMed

    Watanabe, Seiya; Sueda, Rui; Fukumori, Fumiyasu; Watanabe, Yasuo

    2015-01-01

    Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with Agrobacterium species, and are synthesized by well-studied NAD(P)H-dependent dehydrogenases (synthases), which catalyze the reductive condensation of α-ketoglutarate or pyruvate with L-arginine. The corresponding genes are transferred into plant cells via a tumor-inducing (Ti) plasmid. In addition to the reverse oxidative reaction(s), the genes noxB-noxA and ooxB-ooxA are considered to be involved in opine catabolism as (membrane-associated) oxidases; however, their properties have not yet been elucidated in detail due to the difficulties associated with purification (and preservation). We herein successfully expressed Nox/Oox-like genes from Pseudomonas putida in P. putida cells. The purified protein consisted of different α-, β-, and γ-subunits encoded by the OdhA, OdhB, and OdhC genes, which were arranged in tandem on the chromosome (OdhB-C-A), and exhibited dehydrogenase (but not oxidase) activity toward nopaline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol. The enzyme contained FAD, FMN, and [2Fe-2S]-iron sulfur as prosthetic groups. On the other hand, the gene cluster from Bradyrhizobium japonicum consisted of OdhB1-C-A-B2, from which two proteins, OdhAB1C and OdhAB2C, appeared through the assembly of each β-subunit together with common α- and γ-subunits. A poor phylogenetic relationship was detected between OdhB1 and OdhB2 in spite of them both functioning as octopine dehydrogenases, which provided clear evidence for the acquisition of novel functions by "subunit-exchange". To the best of our knowledge, this is the first study to have examined flavin-containing opine dehydrogenase. PMID:26382958

  3. Hydroxysteroid dehydrogenases (HSDs) in bacteria: a bioinformatic perspective.

    PubMed

    Kisiela, Michael; Skarka, Adam; Ebert, Bettina; Maser, Edmund

    2012-03-01

    Steroidal compounds including cholesterol, bile acids and steroid hormones play a central role in various physiological processes such as cell signaling, growth, reproduction, and energy homeostasis. Hydroxysteroid dehydrogenases (HSDs), which belong to the superfamily of short-chain dehydrogenases/reductases (SDR) or aldo-keto reductases (AKR), are important enzymes involved in the steroid hormone metabolism. HSDs function as an enzymatic switch that controls the access of receptor-active steroids to nuclear hormone receptors and thereby mediate a fine-tuning of the steroid response. The aim of this study was the identification of classified functional HSDs and the bioinformatic annotation of these proteins in all complete sequenced bacterial genomes followed by a phylogenetic analysis. For the bioinformatic annotation we constructed specific hidden Markov models in an iterative approach to provide a reliable identification for the specific catalytic groups of HSDs. Here, we show a detailed phylogenetic analysis of 3α-, 7α-, 12α-HSDs and two further functional related enzymes (3-ketosteroid-Δ(1)-dehydrogenase, 3-ketosteroid-Δ(4)(5α)-dehydrogenase) from the superfamily of SDRs. For some bacteria that have been previously reported to posses a specific HSD activity, we could annotate the corresponding HSD protein. The dominating phyla that were identified to express HSDs were that of Actinobacteria, Proteobacteria, and Firmicutes. Moreover, some evolutionarily more ancient microorganisms (e.g., Cyanobacteria and Euryachaeota) were found as well. A large number of HSD-expressing bacteria constitute the normal human gastro-intestinal flora. Another group of bacteria were originally isolated from natural habitats like seawater, soil, marine and permafrost sediments. These bacteria include polycyclic aromatic hydrocarbons-degrading species such as Pseudomonas, Burkholderia and Rhodococcus. In conclusion, HSDs are found in a wide variety of microorganisms including

  4. R-lipoic acid inhibits mammalian pyruvate dehydrogenase kinase.

    PubMed

    Korotchkina, Lioubov G; Sidhu, Sukhdeep; Patel, Mulchand S

    2004-10-01

    The four pyruvate dehydrogenase kinase (PDK) and two pyruvate dehydrogenase phosphatase (PDP) isoenzymes that are present in mammalian tissues regulate activity of the pyruvate dehydrogenase complex (PDC) by phosphorylation/dephosphorylation of its pyruvate dehydrogenase (E1) component. The effect of lipoic acids on the activity of PDKs and PDPs was investigated in purified proteins system. R-lipoic acid, S-lipoic acid and R-dihydrolipoic acid did not significantly affect activities of PDPs and at the same time inhibited PDKs to different extents (PDK1>PDK4 approximately PDK2>PDK3 for R-LA). Since lipoic acids inhibited PDKs activity both when reconstituted in PDC and in the presence of E1 alone, dissociation of PDK from the lipoyl domains of dihydrolipoamide acetyltransferase in the presence of lipoic acids is not a likely explanation for inhibition. The activity of PDK1 towards phosphorylation sites 1, 2 and 3 of E1 was decreased to the same extent in the presence of R-lipoic acid, thus excluding protection of the E1 active site by lipoic acid from phosphorylation. R-lipoic acid inhibited autophosphorylation of PDK2 indicating that it exerted its effect on PDKs directly. Inhibition of PDK1 by R-lipoic acid was not altered by ADP but was decreased in the presence of pyruvate which itself inhibits PDKs. An inhibitory effect of lipoic acid on PDKs would result in less phosphorylation of E1 and hence increased PDC activity. This finding provides a possible mechanism for a glucose (and lactate) lowering effect of R-lipoic acid in diabetic subjects. PMID:15512796

  5. Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria

    PubMed Central

    Watanabe, Seiya; Sueda, Rui; Fukumori, Fumiyasu; Watanabe, Yasuo

    2015-01-01

    Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with Agrobacterium species, and are synthesized by well-studied NAD(P)H-dependent dehydrogenases (synthases), which catalyze the reductive condensation of α-ketoglutarate or pyruvate with L-arginine. The corresponding genes are transferred into plant cells via a tumor-inducing (Ti) plasmid. In addition to the reverse oxidative reaction(s), the genes noxB-noxA and ooxB-ooxA are considered to be involved in opine catabolism as (membrane-associated) oxidases; however, their properties have not yet been elucidated in detail due to the difficulties associated with purification (and preservation). We herein successfully expressed Nox/Oox-like genes from Pseudomonas putida in P. putida cells. The purified protein consisted of different α-, β-, and γ-subunits encoded by the OdhA, OdhB, and OdhC genes, which were arranged in tandem on the chromosome (OdhB-C-A), and exhibited dehydrogenase (but not oxidase) activity toward nopaline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol. The enzyme contained FAD, FMN, and [2Fe-2S]-iron sulfur as prosthetic groups. On the other hand, the gene cluster from Bradyrhizobium japonicum consisted of OdhB1-C-A-B2, from which two proteins, OdhAB1C and OdhAB2C, appeared through the assembly of each β-subunit together with common α- and γ-subunits. A poor phylogenetic relationship was detected between OdhB1 and OdhB2 in spite of them both functioning as octopine dehydrogenases, which provided clear evidence for the acquisition of novel functions by “subunit-exchange”. To the best of our knowledge, this is the first study to have examined flavin-containing opine dehydrogenase. PMID:26382958

  6. Delineation of an in vivo inhibitor for Aspergillus glutamate dehydrogenase.

    PubMed

    Choudhury, Rajarshi; Noor, Shahid; Varadarajalu, Lakshmi Prabha; Punekar, Narayan S

    2008-01-01

    NADP-glutamate dehydrogenase (NADP-GDH) along with glutamine synthetase plays a pivotal role in ammonium assimilation. Specific inhibitors were valuable in defining the importance of glutamine synthetase in nitrogen metabolism. Selective in vivo inhibition of NADP-GDH has so far been an elusive desideratum. Isophthalate, a potent in vitro inhibitor of Aspergillus niger NADP-GDH [Noor S, Punekar NS. Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon-nitrogen interface. Microbiology 2005;151:1409-19], was evaluated for its efficacy in vivo. Dimethyl ester of isophthalate (DMIP), but not isophthalate, inhibited A. niger growth on agar as well as in liquid culture. This was ascribed to the inability of isophthalate to enter fungal mycelia. Subsequent to DMIP addition however, intracellular isophthalate could be demonstrated. Apart from NAD-GDH, no other enzyme including NAD-glutamate synthase was inhibited by isophthalate. A cross-over at NADP-GDH step of metabolism was observed as a direct consequence of isophthalate (formed in vivo from DMIP) inhibiting this enzyme. Addition of ammonium to DMIP-treated A. niger mycelia resulted in intensive vacuolation, retraction of cytoplasm and autolysis. Taken together, these results implicate glutamate dehydrogenase and NADP-GDH in particular, as a key target of in vivo isophthalate inhibition during ammonium assimilation. PMID:22578865

  7. Cloning, purification and crystallization of Thermus thermophilus proline dehydrogenase

    SciTech Connect

    White, Tommi A.; Tanner, John J.

    2005-08-01

    Cloning, purification and crystallization of T. thermophilus proline dehydrogenase is reported. The detergent n-octyl β-d-glucopyranoside was used to reduce polydispersity, which enabled crystallization. Nature recycles l-proline by converting it to l-glutamate. This four-electron oxidation process is catalyzed by the two enzymes: proline dehydrogenase (PRODH) and Δ{sup 1}-pyrroline-5-carboxylate dehydrogenase. This note reports the cloning, purification and crystallization of Thermus thermophilus PRODH, which is the prototype of a newly discovered superfamily of bacterial monofunctional PRODHs. The results presented here include production of a monodisperse protein solution through use of the detergent n-octyl β-d-glucopyranoside and the growth of native crystals that diffracted to 2.3 Å resolution at Advanced Light Source beamline 4.2.2. The space group is P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 82.2, b = 89.6, c = 94.3 Å. The asymmetric unit is predicted to contain two protein molecules and 46% solvent. Molecular-replacement trials using a fragment of the PRODH domain of the multifunctional Escherichia coli PutA protein as the search model (24% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of T. thermophilus PRODH will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

  8. Making biochemistry count: life among the amino acid dehydrogenases.

    PubMed

    Engel, Paul C

    2011-04-01

    The guiding principle of the IAS Medal Lecture and of the research it covered was that searching mathematical analysis, depending on good measurements, must underpin sound biochemical conclusions. This was illustrated through various experiences with the amino acid dehydrogenases. Topics covered in the present article include: (i) the place of kinetic measurement in assessing the metabolic role of GDH (glutamate dehydrogenase); (ii) the discovery of complex regulatory behaviour in mammalian GDH, involving negative co-operativity in coenzyme binding; (iii) an X-ray structure solution for a bacterial GDH providing insight into catalysis; (iv) almost total positive co-operativity in glutamate binding to clostridial GDH; (v) unexpected outcomes with mutations at the catalytic aspartate site in GDH; (vi) reactive cysteine as a counting tool in the construction of hybrid oligomers to probe the basis of allosteric interaction; (vii) tryptophan-to-phenylalanine mutations in analysis of allosteric conformational change; (viii) site-directed mutagenesis to alter substrate specificity in GDH and PheDH (phenylalanine dehydrogenase); and (ix) varying strengths of binding of the 'wrong' enantiomer in engineered mutant enzymes and implications for resolution of racemates.

  9. Characterization of a cellobiose dehydrogenase from Humicola insolens.

    PubMed Central

    Schou, C; Christensen, M H; Schülein, M

    1998-01-01

    The major cellobiose dehydrogenase (oxidase) (CBDH) secreted by the soft-rot thermophilic fungus Humicola insolens during growth on cellulose has been isolated and purified. It was shown to be a haemoflavoprotein with a molecular weight of 92 kDa and a pI of 4.0, capable of oxidizing the anomeric carbon of cellobiose, soluble cellooligosaccharides, lactose, xylobiose and maltose. Possible electron acceptors are 2,6-dichlorophenol-indophenol (DCPIP), Methylene Blue, 3,5-di-t-butyl-1,2-benzoquinone, potassium ferricyanide, cytochrome c and molecular oxygen. The oxidation of the prosthetic groups by oxygen was monitored at 449 nm for the flavin group and at 562 nm for the haem group. The curves were very similar to those of the cellobiose dehydrogenase from Phanerochaete chrysosporium, suggesting a similar mechanism. The pH-optima for the oxidation varied remarkably depending on the electron acceptor. For the organic electron acceptors, the pH-optima ranged from pH 4 for Methylene Blue to pH 7 for DCPIP and the benzoquinone. In the case of the FeIII-containing electron acceptors, the enzyme displayed alkaline pH-optima, in contrast to the properties of cellobiose dehydrogenases from Phanerochaete chrysosporium and Myceliophthora (Sporotrichum) thermophila. The enzyme has optimal activity at 65 degrees C. PMID:9461557

  10. Functional Analysis of a Mosquito Short Chain Dehydrogenase Cluster

    PubMed Central

    Mayoral, Jaime G.; Leonard, Kate T.; Defelipe, Lucas A.; Turjansksi, Adrian G.; Nouzova, Marcela; Noriegal, Fernando G.

    2013-01-01

    The short chain dehydrogenases (SDR) constitute one the oldest and largest families of enzymes with over 46,000 members in sequence databases. About 25% of all known dehydrogenases belong to the SDR family. SDR enzymes have critical roles in lipid, amino acid, carbohydrate, hormone and xenobiotic metabolism as well as in redox sensor mechanisms. This family is present in archaea, bacteria, and eukaryota, emphasizing their versatility and fundamental importance for metabolic processes. We identified a cluster of eight SDRs in the mosquito Aedes aegypti (AaSDRs). Members of the cluster differ in tissue specificity and developmental expression. Heterologous expression produced recombinant proteins that had diverse substrate specificities, but distinct from the conventional insect alcohol (ethanol) dehydrogenases. They are all NADP+-dependent and they have S-enantioselectivity and preference for secondary alcohols with 8–15 carbons. Homology modeling was used to build the structure of AaSDR1 and two additional cluster members. The computational study helped explain the selectivity towards the (10S)-isomers as well as the reduced activity of AaSDR4 and AaSDR9 for longer isoprenoid substrates. Similar clusters of SDRs are present in other species of insects, suggesting similar selection mechanisms causing duplication and diversification of this family of enzymes. PMID:23238893

  11. 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.

  12. Structural insights on mouse L-threonine dehydrogenase: A regulatory role of Arg180 in catalysis.

    PubMed

    He, Chao; Huang, Xianyu; Liu, Yanhong; Li, Fudong; Yang, Yang; Tao, Hongru; Han, Chuanchun; Zhao, Chen; Xiao, Yazhong; Shi, Yunyu

    2015-12-01

    Mouse L-threonine dehydrogenase (mTDH), which belongs to the short-chain dehydrogenase/reductase (SDR) superfamily and mediates threonine catabolism, plays pivotal roles in both powerful biosynthesis and signaling in mouse stem cells and has a regulatory residue Arg180. Here we determined three crystal structures of mTDH: wild-type (WT) in the apo form; in complex with NAD(+) and a substrate analog, glycerol, or with only NAD(+); as well as the R180K variant with NAD(+). This is the first description of a structure for mammalian SDR-type TDH. Structural comparison revealed the structural basis for SDR-type TDH catalysis remains strictly conserved in bacteria and mammals. Kinetic enzyme assays, and isothermal titration calorimetry (ITC) measurements indicated the R180K mutation has little effect on NAD(+) binding affinity, whereas affects the substrate's affinity for the enzyme. The crystal structure of R180K with NAD(+), biochemical and spectroscopic studies suggested that the R180K mutant should bind NAD(+) in a similar way and have a similar folding to the WT. However, the R180K variant may have difficulty adopting the closed form due to reduced interaction of residue 180 with a loop which connects a key position for mTDH switching between the closed and open forms in mTDH catalysis, and thereby exhibited a significantly decreased kcat/Km value toward the substrate, L-Thr. In sum, our results suggest that activity of GalE-like TDH can be regulated by remote interaction, such as hydrogen bonding and hydrophobic interaction around the Arg180 of mTDH. PMID:26492815

  13. Mechanism of protection against alcoholism by an alcohol dehydrogenase polymorphism: development of an animal model

    PubMed Central

    Rivera-Meza, Mario; Quintanilla, María Elena; Tampier, Lutske; Mura, Casilda V.; Sapag, Amalia; Israel, Yedy

    2010-01-01

    Humans who carry a point mutation in the gene coding for alcohol dehydrogenase-1B (ADH1B*2; Arg47His) are markedly protected against alcoholism. Although this mutation results in a 100-fold increase in enzyme activity, it has not been reported to cause higher levels of acetaldehyde, a metabolite of ethanol known to deter alcohol intake. Hence, the mechanism by which this mutation confers protection against alcoholism is unknown. To study this protective effect, the wild-type rat cDNA encoding rADH-47Arg was mutated to encode rADH-47His, mimicking the human mutation. The mutated cDNA was incorporated into an adenoviral vector and administered to genetically selected alcohol-preferring rats. The Vmax of rADH-47His was 6-fold higher (P<0.001) than that of the wild-type rADH-47Arg. Animals transduced with rAdh-47His showed a 90% (P<0.01) increase in liver ADH activity and a 50% reduction (P<0.001) in voluntary ethanol intake. In animals transduced with rAdh-47His, administration of ethanol (1g/kg) produced a short-lived increase of arterial blood acetaldehyde concentration to levels that were 3.5- to 5-fold greater than those in animals transduced with the wild-type rAdh-47Arg vector or with a noncoding vector. This brief increase (burst) in arterial acetaldehyde concentration after ethanol ingestion may constitute the mechanism by which humans carrying the ADH1B*2 allele are protected against alcoholism.—Rivera-Meza, M., Quintanilla, M. E., Tampier, L., Mura, C. V., Sapag, A., Israel, Y. Mechanism of protection against alcoholism by an alcohol dehydrogenase polymorphism: development of an animal model. PMID:19710201

  14. Structural insights on mouse L-threonine dehydrogenase: A regulatory role of Arg180 in catalysis.

    PubMed

    He, Chao; Huang, Xianyu; Liu, Yanhong; Li, Fudong; Yang, Yang; Tao, Hongru; Han, Chuanchun; Zhao, Chen; Xiao, Yazhong; Shi, Yunyu

    2015-12-01

    Mouse L-threonine dehydrogenase (mTDH), which belongs to the short-chain dehydrogenase/reductase (SDR) superfamily and mediates threonine catabolism, plays pivotal roles in both powerful biosynthesis and signaling in mouse stem cells and has a regulatory residue Arg180. Here we determined three crystal structures of mTDH: wild-type (WT) in the apo form; in complex with NAD(+) and a substrate analog, glycerol, or with only NAD(+); as well as the R180K variant with NAD(+). This is the first description of a structure for mammalian SDR-type TDH. Structural comparison revealed the structural basis for SDR-type TDH catalysis remains strictly conserved in bacteria and mammals. Kinetic enzyme assays, and isothermal titration calorimetry (ITC) measurements indicated the R180K mutation has little effect on NAD(+) binding affinity, whereas affects the substrate's affinity for the enzyme. The crystal structure of R180K with NAD(+), biochemical and spectroscopic studies suggested that the R180K mutant should bind NAD(+) in a similar way and have a similar folding to the WT. However, the R180K variant may have difficulty adopting the closed form due to reduced interaction of residue 180 with a loop which connects a key position for mTDH switching between the closed and open forms in mTDH catalysis, and thereby exhibited a significantly decreased kcat/Km value toward the substrate, L-Thr. In sum, our results suggest that activity of GalE-like TDH can be regulated by remote interaction, such as hydrogen bonding and hydrophobic interaction around the Arg180 of mTDH.

  15. Nicotinamide adenine dinucleotide-dependent and nicotinamide adenine dinucleotide-independent lactate dehydrogenases in homofermentative and heterofermentative lactic acid bacteria.

    PubMed

    Doelle, H W

    1971-12-01

    Three homofermentative (Lactobacillus plantarum B38, L. plantarum B33, Pediococcus pentosaceus B30) and three heterofermentative (Leuconostoc mesenteroides 39, L. oenos B70, Lactobacillus brevis) lactic acid bacteria were examined for the presence or absence of nicotinamide adenine dinucleotide (NAD)-dependent and NAD-independent d- and l-lactate dehydrogenases. Two of the six strains investigated, P. pentosaceus and L. oenos, did not exhibit an NAD-independent enzyme activity capable of reducing dichlorophenol indophenol. The pH optima of the lactic dehydrogenases were determined. The NAD-dependent enzymes from homofermentative strains exhibited optima at pH 7.8 to 8.8, whereas values from 9.0 to 10.0 were noted for these enzymes from heterofermentative organisms. The optima for the NAD-independent enzymes were between 5.8 and 6.6. The apparent Michaelis-Menten constants determined for both NAD and the substrates demonstrated the existence of a greater affinity for d- than l-lactic acid. A comparison of the specific NAD-dependent and NAD-independent lactate dehydrogenase activities revealed a direct correlation of the d/l ratios of these activities with the type of lactic acid produced during the growth of the organism.

  16. The E1 beta-subunit of pyruvate dehydrogenase is surface-expressed in Lactobacillus plantarum and binds fibronectin.

    PubMed

    Vastano, Valeria; Salzillo, Marzia; Siciliano, Rosa A; Muscariello, Lidia; Sacco, Margherita; Marasco, Rosangela

    2014-01-01

    Lactobacillus plantarum is among the species with a probiotic activity. Adhesion of probiotic bacteria to host tissues is an important principle for strain selection, because it represents a crucial step in the colonization process of either pathogens or commensals. Most bacterial adhesins are proteins, and a major target for them is fibronectin, an extracellular matrix glycoprotein. In this study we demonstrate that PDHB, a component of the pyruvate dehydrogenase complex, is a factor contributing to fibronectin-binding in L. plantarum LM3. By means of fibronectin overlay immunoblotting assay, we identified a L. plantarum LM3 surface protein with apparent molecular mass of 35 kDa. Mass spectrometric analysis shows that this protein is the pyruvate dehydrogenase E1 beta-subunit (PDHB). The corresponding pdhB gene is located in a 4-gene cluster encoding pyruvate dehydrogenase. In LM3-B1, carrying a null mutation in pdhB, the 35 kDa adhesin was not anymore detectable by immunoblotting assay. Nevertheless, the pdhB null mutation did not abolish pdhA, pdhC, and pdhD transcription in LM3-B1. By adhesion assays, we show that LM3-B1 cells bind to immobilized fibronectin less efficiently than wild type cells. Moreover, we show that pdhB expression is negatively regulated by the CcpA protein and is induced by bile.

  17. Establishment of permanent chimerism in a lactate dehydrogenase-deficient mouse mutant with hemolytic anemia

    SciTech Connect

    Datta, T.; Doermer, P.

    1987-12-01

    Pluripotent hemopoietic stem cell function was investigated in the homozygous muscle type lactate dehydrogenase (LDH-A) mutant mouse using bone marrow transplantation experiments. Hemopoietic tissues of LDH-A mutants showed a marked decreased in enzyme activity that was associated with severe hemolytic anemia. This condition proved to be transplantable into wild type mice (+/+) through total body irradiation (TBI) at a lethal dose of 8.0 Gy followed by engraftment of mutant bone marrow cells. Since the mutants are extremely radiosensitive (lethal dose50/30 4.4 Gy vs 7.3 Gy in +/+ mice), 8.0-Gy TBI followed by injection of even high numbers of normal bone marrow cells did not prevent death within 5-6 days. After a nonlethal dose of 4.0 Gy and grafting of normal bone marrow cells, a transient chimerism showing peripheral blood characteristics of the wild type was produced that returned to the mutant condition within 12 weeks. The transfusion of wild type red blood cells prior to and following 8.0-Gy TBI and reconstitution with wild type bone marrow cells prevented the early death of the mutants and permanent chimerism was achieved. The chimeras showed all hematological parameters of wild type mice, and radiosensitivity returned to normal. It is concluded that the mutant pluripotent stem cells are functionally comparable to normal stem cells, emphasizing the significance of this mouse model for studies of stem cell regulation.

  18. Characterization of Arabidopsis lines deficient in GAPC-1, a cytosolic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Rius, Sebastián P; Casati, Paula; Iglesias, Alberto A; Gomez-Casati, Diego F

    2008-11-01

    Phosphorylating glyceraldehyde-3-P dehydrogenase (GAPC-1) is a highly conserved cytosolic enzyme that catalyzes the conversion of glyceraldehyde-3-P to 1,3-bis-phosphoglycerate; besides its participation in glycolysis, it is thought to be involved in additional cellular functions. To reach an integrative view on the many roles played by this enzyme, we characterized a homozygous gapc-1 null mutant and an as-GAPC1 line of Arabidopsis (Arabidopsis thaliana). Both mutant plant lines show a delay in growth, morphological alterations in siliques, and low seed number. Embryo development was altered, showing abortions and empty embryonic sacs in basal and apical siliques, respectively. The gapc-1 line shows a decrease in ATP levels and reduced respiratory rate. Furthermore, both lines exhibit a decrease in the expression and activity of aconitase and succinate dehydrogenase and reduced levels of pyruvate and several Krebs cycle intermediates, as well as increased reactive oxygen species levels. Transcriptome analysis of the gapc-1 mutants unveils a differential accumulation of transcripts encoding for enzymes involved in carbon partitioning. According to these studies, some enzymes involved in carbon flux decreased (phosphoenolpyruvate carboxylase, NAD-malic enzyme, glucose-6-P dehydrogenase) or increased (NAD-malate dehydrogenase) their activities compared to the wild-type line. Taken together, our data indicate that a deficiency in the cytosolic GAPC activity results in modifications of carbon flux and mitochondrial dysfunction, leading to an alteration of plant and embryo development with decreased number of seeds, indicating that GAPC-1 is essential for normal fertility in Arabidopsis plants. PMID:18820081

  19. 2-Oxoglutarate dehydrogenase and pyruvate dehydrogenase activities in plant mitochondria: interaction via a common coenzyme a pool.

    PubMed

    Dry, I B; Wiskich, J T

    1987-08-15

    2-Oxoglutarate (2-OG)-dependent O2 uptake by washed or purified turnip (Brassica rapa L.) and pea (Pisum sativum L. cv. Massey Gem) leaf mitochondria, in the presence of malonate, was inhibited between 65 and 90% by micromolar levels of pyruvate. The inhibition was not observed in the absence of malonate and was reversed by alpha-cyano-4-hydroxycinnamic acid. The inhibition was also reversed by oxaloacetate or by malate, but not by any other tricarboxylic acid cycle intermediates. The stimulation of O2 uptake by oxaloacetate was half maximal at 8-9 microM and was transient, indicating its action was not mediated through the complete metabolic removal of pyruvate. Pyruvate had not effect on 2-OG oxidation under conditions in which pyruvate dehydrogenase was not active, indicating that pyruvate metabolism, rather than pyruvate itself, was responsible for producing the inhibition of 2-OG oxidation. Similar results were obtained with detergent-treated mitochondrial extracts with the exception that the inhibition of 2-OG oxidation by pyruvate could also be reversed by coenzyme A. The results suggest that pyruvate inhibits 2-oxoglutarate oxidation, in intact plant mitochondria, by sequestering intramitochondrial CoA as acetyl-CoA and, in the absence of citrate synthase activity, reduces the amount of free coenzyme A available for 2-oxoglutarate dehydrogenase. These results indicate that pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase share a common CoA pool within plant mitochondria and that the turnover of the acyl-CoA product of one enzyme will dramatically influence the activity of the other.

  20. Evidence for distinct dehydrogenase and isomerase sites within a single 3. beta. -hydroxysteroid dehydrogenase/5-ene-4-ene isomerase protein

    SciTech Connect

    Luu-The, V.; Takahashi, Masakazu; de Launoit, Y.; Dumont, M.; Lachance, Y.; Labrie, F. )

    1991-09-10

    Complementary DNA encoding human 3{beta}-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3-{beta}-HSD) has been expressed in transfected GH{sub 4}C{sub 1} with use of the cytomegalovirus promoter. The activity of the expressed protein clearly shows that both dehydrogenase and isomerase enzymatic activities are present within a single protein. However, such findings do not indicate whether the two activities reside within one or two closely related catalytic sites. With use of ({sup 3}H)-5-androstenedione, the intermediate compound in dehydroepiandrosterone (DHEA) transformation into 4-androstenedione by 3{beta}-HSD, the present study shows that 4MA (N,N-diethyl-4-methyl-3-oxo-4-aza-5{alpha}-androstane-17{beta}-carboxamide) and its analogues of 5-androstenedione to 4-androstenedione with an approximately 1,000-fold higher K{sub i} value. The present results thus strongly suggest that dehydrogenase and isomerase activities are present at separate sites on the 3-{beta}-HSD protein. Such data suggest that the irreversible step in the transformation of DHEA to 4-androstenedione is due to a separate site possessing isomerase activity that converts the 5-ene-3-keto to a much more stable 4-ene-3-keto configuration.

  1. Succinate dehydrogenase in Arabidopsis thaliana is regulated by light via phytochrome A.

    PubMed

    Popov, Vasily N; Eprintsev, Alexander T; Fedorin, Dmitry N; Igamberdiev, Abir U

    2010-01-01

    The effect of light on succinate dehydrogenase (SDH) activity and mRNA content was studied in Arabidopsis thaliana plants. The transition from darkness to light caused a short transient increase in the SDH activity followed by a decrease to a half of the original activity. The white or red light were found to be down-regulating factors for the mRNA content of the sdh1-2 and sdh2-3 genes and SDH catalytic activity both in A. thaliana wild-type plants and in the mutant deficient in the phytochrome B gene, but not in the mutant deficient in the phytochrome A gene, while the far-red light of 730 nm reversed the red light effect. It is concluded that phytochrome A participates in the regulation of mitochondrial respiration through effect on SDH expression.

  2. Dye-linked D-amino acid dehydrogenases: biochemical characteristics and applications in biotechnology.

    PubMed

    Satomura, Takenori; Sakuraba, Haruhiko; Suye, Shin-Ichiro; Ohshima, Toshihisa

    2015-11-01

    Dye-linked D-amino acid dehydrogenases (Dye-DADHs) catalyze the dehydrogenation of free D-amino acids in the presence of an artificial electron acceptor. Although Dye-DADHs functioning in catabolism of L-alanine and as primary enzymes in electron transport chains are widely distributed in mesophilic Gram-negative bacteria, biochemical and biotechnological information on these enzymes remains scanty. This is in large part due to their instability after isolation. On the other hand, in the last decade, several novel types of Dye-DADH have been found in thermophilic bacteria and hyperthermophilic archaea, where they contribute not only to L-alanine catabolism but also to the catabolism of other amino acids, including D-arginine and L-hydroxyproline. In this minireview, we summarize recent developments in our understanding of the biochemical characteristics of Dye-DADHs and their specific application to electrochemical biosensors. PMID:26362681

  3. Escherichia coli derivatives lacking both alcohol dehydrogenase and phosphotransacetylase grow anaerobically by lactate fermentation

    SciTech Connect

    Gupta, S.; Clark, D.P. )

    1989-07-01

    Escherichia coli mutants lacking alcohol dehydrogenase (adh mutants) cannot synthesize the fermentation product ethanol and are unable to grow anaerobically on glucose and other hexoses. Similarly, phosphotransacetylase-negative mutants (pta mutants) neither excrete acetate nor grow anaerobically. However, when a strain carrying an adh deletion was selected for anaerobic growth on glucose, spontaneous pta mutants were isolated. Strains carrying both adh and pta mutations were observed by in vivo nuclear magnetic resonance and shown to produce lactic acid as the major fermentation product. Various combinations of adh pta double mutants regained the ability to grow anaerobically on hexoses, by what amounts to a homolactic fermentation. Unlike wild-type strains, such adh pta double mutants were unable to grow anaerobically on sorbitol or on glucuronic acid. The growth properties of strains carrying various mutations affecting the enzymes of fermentation are discussed terms of redox balance.

  4. Dye-linked D-amino acid dehydrogenases: biochemical characteristics and applications in biotechnology.

    PubMed

    Satomura, Takenori; Sakuraba, Haruhiko; Suye, Shin-Ichiro; Ohshima, Toshihisa

    2015-11-01

    Dye-linked D-amino acid dehydrogenases (Dye-DADHs) catalyze the dehydrogenation of free D-amino acids in the presence of an artificial electron acceptor. Although Dye-DADHs functioning in catabolism of L-alanine and as primary enzymes in electron transport chains are widely distributed in mesophilic Gram-negative bacteria, biochemical and biotechnological information on these enzymes remains scanty. This is in large part due to their instability after isolation. On the other hand, in the last decade, several novel types of Dye-DADH have been found in thermophilic bacteria and hyperthermophilic archaea, where they contribute not only to L-alanine catabolism but also to the catabolism of other amino acids, including D-arginine and L-hydroxyproline. In this minireview, we summarize recent developments in our understanding of the biochemical characteristics of Dye-DADHs and their specific application to electrochemical biosensors.

  5. Increased riboflavin production by manipulation of inosine 5'-monophosphate dehydrogenase in Ashbya gossypii.

    PubMed

    Buey, Rubén M; Ledesma-Amaro, Rodrigo; Balsera, Mónica; de Pereda, José María; Revuelta, José Luis

    2015-11-01

    Guanine nucleotides are the precursors of essential biomolecules including nucleic acids and vitamins such as riboflavin. The enzyme inosine-5'-monophosphate dehydrogenase (IMPDH) catalyzes the ratelimiting step in the guanine nucleotide de novo biosynthetic pathway and plays a key role in controlling the cellular nucleotide pools. Thus, IMPDH is an important metabolic bottleneck in the guanine nucleotide synthesis, susceptible of manipulation by means of metabolic engineering approaches. Herein, we report the functional and structural characterization of the IMPDH enzyme from the industrial fungus Ashbya gossypii. Our data show that the overexpression of the IMPDH gene increases the metabolic flux through the guanine pathway and ultimately enhances 40 % riboflavin production with respect to the wild type. Also, IMPDH disruption results in a 100-fold increase of inosine excretion to the culture media. Our results contribute to the developing metabolic engineering toolbox aiming at improving the production of metabolites with biotechnological interest in A. gossypii. PMID:26150243

  6. In crystallo posttranslational modification within a MauG/pre-methylamine dehydrogenase complex.

    SciTech Connect

    Jensen, L. M. R.; Sanishvili, R.; Davidson, V. L.; Wilmot, C. M.; Biosciences Division; Univ. of Minnesota; Univ. of Mississippi

    2010-03-12

    MauG is a diheme enzyme responsible for the posttranslational modification of two tryptophan residues to form the tryptophan tryptophylquinone (TTQ) cofactor of methylamine dehydrogenase (MADH). MauG converts preMADH, containing monohydroxylated {beta}Trp{sup 57}, to fully functional MADH by catalyzing the insertion of a second oxygen atom into the indole ring and covalently linking {beta}Trp{sup 57} to {beta}Trp{sup 108}. We have solved the x-ray crystal structure of MauG complexed with preMADH to 2.1 angstroms. The c-type heme irons and the nascent TTQ site are separated by long distances over which electron transfer must occur to achieve catalysis. In addition, one of the hemes has an atypical His-Tyr axial ligation. The crystalline protein complex is catalytically competent; upon addition of hydrogen peroxide, MauG-dependent TTQ synthesis occurs.

  7. The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates.

    PubMed Central

    Zammit, V A; Newsholme, E A

    1976-01-01

    Comparison of the activities of hexokinase, phosphorylase and phosphofructokinase in muscles from marine invertebrates indicates that they can be divided into three groups. First, the activities of the three enzymes are low in coelenterate muscles, catch muscles of molluscs and muscles of echinoderms; this indicates a low rate of carbohydrate (and energy) utilization by these muscles. Secondly, high activities of phosphorylase and phosphofructokinase relative to those of hexokinase are found in, for example, lobster abdominal and scallop snap muscles; this indicates that these muscles depend largely on anaerobic degradation of glycogen for energy production. Thirdly, high activities of hexokinase are found in the radular muscles of prosobranch molluscs and the fin muscles of squids; this indicates a high capacity for glucose utilization, which is consistent with the high activities of enzymes of the tricarboxylic acid cycle in these muscles [Alp, Newsholme & Zammit (1976) Biochem. J. 154, 689-700]. 2. The activities of lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase and glutamate-oxaloacetate transaminase were measured in order to provide a qualitative indication of the importance of different processes for oxidation of glycolytically formed NADH. The muscles are divided into four groups: those that have a high activity of lactate dehydrogenase relative to the activities of phosphofructokinase (e.g. crustacean muscles); those that have high activities of octopine dehydrogenase but low activities of lactate dehydrogenase (e.g. scallop snap muscle); those that have moderate activities of both lactate dehydrogenase and octopine dehydrogenase (radular muscles of prosobranchs), and those that have low activities of both lactate dehydrogenase and octopine dehydrogenase, but which possess activities of phosphoenolpyruvate carboxykinase (oyster adductor muscles). It is

  8. Developmentally Regulated RNA Transcripts Coding for Alcohol Dehydrogenase in DROSOPHILA AFFINIDISJUNCTA

    PubMed Central

    Rowan, Robert G.; Brennan, Mark D.; Dickinson, W. J.

    1986-01-01

    The organization of the gene coding for alcohol dehydrogenase (Adh) in Drosophila affinidisjuncta has been determined by physically mapping Adh RNA transcripts to cloned genomic DNA. Two distinct transcript types accumulate with developmental specificity. Because only a single genomic Adh locus is detected in D. affinidisjuncta , and since all Adh transcripts appear to be identical except at their termini, the two Adh RNA types are products of the same gene. One type of transcript, abundant in adults, contains a small 5' terminal exon that is completely lacking in the other type of transcript, which accumulates in larvae. This 5' end difference suggests that the D. affinidisjuncta Adh gene, like the homologous gene from the distantly related species D. melanogaster, is expressed from two promoters. According to the transcription map, these D. affinidisjuncta promoters are separated by approximately 560 base pairs of genomic DNA sequence. D. affinidisjuncta Adh transcripts also resemble D. melanogaster Adh transcripts in both their overall organization and their developmental distribution. Multiple 3' ends are responsible for the size heterogeneity of both types of D. affinidisjuncta Adh RNA, and some of these also appear with stage specificity. PMID:2429897

  9. Evidence for involvement of medium chain acyl-CoA dehydrogenase in the metabolism of phenylbutyrate

    PubMed Central

    Kormanik, Kaitlyn; Kang, Heejung; Cuebas, Dean; Vockley, Jerry; Mohsen, Al-Walid

    2012-01-01

    Sodium phenylbutyrate is used for treating urea cycle disorders, providing an alternative for ammonia excretion. Following conversion to its CoA ester, phenylbutyryl-CoA is postulated to undergo one round of β-oxidation to phenylacetyl-CoA, the active metabolite. Molecular modeling suggests that medium chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3), a key enzyme in straight chain fatty acid β-oxidation, could utilize phenylbutyryl-CoA as substrate. Moreover, phenylpropionyl-CoA has been shown to be a substrate for MCAD and its intermediates accumulate in patients with MCAD deficiency. We have examined the involvement of MCAD and other acyl-CoA dehydrogenases (ACADs) in the metabolism of phenylbutyryl-CoA. Anaerobic titration of purified recombinant human MCAD with phenylbutyryl-CoA caused changes in the MCAD spectrum that are similar to those induced by octanoyl-CoA, its bona fide substrate, and unique to the development of the charge transfer ternary complex. The calculated apparent dissociation constant (KD app) for these substrates was 2.16 μM and 0.12 μM, respectively. The MCAD reductive and oxidative half reactions were monitored using the electron transfer flavoprotein (ETF) fluorescence reduction assay. The catalytic efficiency and the Km for phenylbutyryl-CoA were 0.2 mM−1· sec−1 and 5.3 μM compared to 4.0 mM−1· sec−1 and 2.8 μM for octanoyl-CoA. Extracts of wild type and MCAD-deficient lymphoblast cells were tested for the ability to reduce ETF using phenylbutyryl-CoA as substrate. While ETF reduction activity was detected in extracts of wild type cells, it was undetectable in extracts of cells deficient in MCAD. The results are consistent with MCAD playing a key role in phenylbutyrate metabolism. PMID:23141465

  10. Site-directed mutagenesis of aldehyde dehydrogenase-2 suggests three distinct pathways of nitroglycerin biotransformation.

    PubMed

    Wenzl, M Verena; Beretta, Matteo; Griesberger, Martina; Russwurm, Michael; Koesling, Doris; Schmidt, Kurt; Mayer, Bernd; Gorren, Antonius C F

    2011-08-01

    To elucidate the mechanism underlying reduction of nitroglycerin (GTN) to nitric oxide (NO) by mitochondrial aldehyde dehydrogenase (ALDH2), we generated mutants of the enzyme lacking the cysteines adjacent to reactive Cys302 (C301S and C303S), the glutamate that participates as a general base in aldehyde oxidation (E268Q) or combinations of these residues. The mutants were characterized regarding acetaldehyde dehydrogenation, GTN-triggered enzyme inactivation, GTN denitration, NO formation, and soluble guanylate cyclase activation. Lack of the cysteines did not affect dehydrogenase activity but impeded GTN denitration, aggravated GTN-induced enzyme inactivation, and increased NO formation. A triple mutant lacking the cysteines and Glu268 catalyzed sustained formation of superstoichiometric amounts of NO and exhibited slower rates of inactivation. These results suggest three alternative pathways for the reaction of ALDH2 with GTN, all involving formation of a thionitrate/sulfenyl nitrite intermediate at Cys302 as the initial step. In the first pathway, which predominates in the wild-type enzyme and reflects clearance-based GTN denitration, the thionitrate apparently reacts with one of the adjacent cysteine residues to yield nitrite and a protein disulfide. The predominant reaction catalyzed by the single and double cysteine mutants requires Glu268 and results in irreversible enzyme inactivation. Finally, combined lack of the cysteines and Glu268 shifts the reaction toward formation of the free NO radical, presumably through homolytic cleavage of the sulfenyl nitrite intermediate. Although the latter reaction accounts for less than 10% of total turnover of GTN metabolism catalyzed by wild-type ALDH2, it is most likely essential for vascular GTN bioactivation.

  11. Application of NAD-dependent polyol dehydrogenases for enzymatic mannitol/sorbitol production with coenzyme regeneration.

    PubMed

    Parmentier, S; Arnaut, F; Soetaert, W; Vandamme, E J

    2003-01-01

    D-Mannitol and D-sorbitol were produced enzymatically from D-fructose using NAD-dependent polyol dehydrogenases. For the production of D-mannitol the Leuconostoc mesenteroides mannitol dehydrogenase could be used. Gluconobacter oxydans cell extract contained however both mannitol and sorbitol dehydrogenase. When this cell extract was used, the reduction of D-fructose resulted in a mixture of D-sorbitol and D-mannitol. To determine the optimal bioconversion conditions the polyol dehydrogenases were characterized towards pH- and temperature-optimum and -stability. As a compromise between enzyme activity and stability, the bioconversion reactions were performed at pH 6.5 and 25 degrees C. Since the polyol dehydrogenases are NADH-dependent, an efficient coenzyme regeneration was needed. Regeneration of NADH was accomplished by formate dehydrogenase-mediated oxidation of formate into CO2.

  12. 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

  13. Isolation, sequence, and characterization of the Cercospora nicotianae phytoene dehydrogenase gene.

    PubMed

    Ehrenshaft, M; Daub, M E

    1994-08-01

    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.

  14. Characterization of testis-specific isoenzyme of human pyruvate dehydrogenase.

    PubMed

    Korotchkina, Lioubov G; Sidhu, Sukhdeep; Patel, Mulchand S

    2006-04-01

    Pyruvate dehydrogenase (PDH), the first component of the human pyruvate dehydrogenase complex, has two isoenzymes, somatic cell-specific PDH1 and testis-specific PDH2 with 87% sequence identity in the alpha subunit of alpha(2) beta(2) PDH. The presence of functional testis-specific PDH2 is important for sperm cells generating nearly all their energy from carbohydrates via pyruvate oxidation. Kinetic and regulatory properties of recombinant human PDH2 and PDH1 were compared in this study. Site-specific phosphorylation/dephosphorylation of the three phosphorylation sites by four PDH kinases (PDK1-4) and two PDH phosphatases (PDP1-2) were investigated by substituting serines with alanine or glutamate in PDHs. PDH2 was found to be very similar to PDH1 as follows: (i) in specific activities and kinetic parameters as determined by the pyruvate dehydrogenase complex assay; (ii) in thermostability at 37 degrees C; (iii) in the mechanism of inactivation by phosphorylation of three sites; and (iv) in the phosphorylation of sites 1 and 2 by PDK3. In contrast, the differences for PDH2 were indicated as follows: (i) by a 2.4-fold increase in binding affinity for the PDH-binding domain of dihydrolipoamide acetyltransferase as measured by surface plasmon resonance; (ii) by possible involvement of Ser-264 (site 1) of PDH2 in catalysis as evident by its kinetic behavior; and (iii) by the lower activities of PDK1, PDK2, and PDK4 as well as PDP1 and PDP2 toward PDH2. These differences between PDH2 and PDH1 are less than expected from substitution of 47 amino acids in each PDH2 alpha subunit. The multiple substitutions may have compensated for any drastic alterations in PDH2 structure thereby preserving its kinetic and regulatory characteristics largely similar to that of PDH1. PMID:16436377

  15. 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

  16. Glucose metabolism in perfused skeletal muscle. Pyruvate dehydrogenase activity in starvation, diabetes and exercise.

    PubMed Central

    Hagg, S A; Taylor, S I; Ruberman, N B

    1976-01-01

    1. The interconversion of pyruvate dehydrogenase between its inactive phosphorylated and active dephosphorylated forms was studied in skeletal muscle. 2. Exercise, induced by electrical stimulation of the sciatic nerve (5/s), increased the measured activity of (active) pyruvate dehydrogenase threefold in intact anaesthetized rated within 2 min. No further increase was seen after 15 min of stimulation. 3. In the perfused rat hindquarter, (active) pyruvate dehydrogenase activity was decreased by 50% in muscle of starved and diabetic rats. Exercise produced a twofold increase in its activity in all groups; however, the relative differences between fed, starved and diabetic groups persisted. 4. Perfusion of muslce with acetoacetate (2 mM) decreased (active) pyruvate dehydrogenase activity by 50% at rest but not during exercise. 5. Whole-tissue concentrations of pyruvate and citrate, inhibitors of (active) pyruvate dehydrogenase kinase and (inactive) pyruvate dehydrogenase phosphate phosphatase respectively, were not altered by excerise. A decrease in the ATP/ADP ratio was observed, but did not appear to be sufficient to account for the increase in (active) pyruvate dehydrogenase activity. 6. The results suggest that interconversion of the phosphorylated and dephosphorylated forms of pyruvate dehydrogenase plays a major role in the regulation of pyruvate oxidation by eomparison of enzyme activity with measurements of lactate oxidation in the perfused hindquarter [see the preceding paper, Berger et al. (1976)] suggest that pyruvate oxidation is also modulated by the concentrations of substrates, cofactors and inhibitors of (active) pyruvate dehydrogenase activity. PMID:825112

  17. Structural studies of MFE-1: the 1.9 A crystal structure of the dehydrogenase part of rat peroxisomal MFE-1.

    PubMed

    Taskinen, Jukka P; Kiema, Tiila R; Hiltunen, J Kalervo; Wierenga, Rik K

    2006-01-27

    The 1.9 A structure of the C-terminal dehydrogenase part of the rat peroxisomal monomeric multifunctional enzyme type 1 (MFE-1) has been determined. In this construct (residues 260-722 and referred to as MFE1-DH) the N-terminal hydratase part of MFE-1 has been deleted. The structure of MFE1-DH shows that it consists of an N-terminal helix, followed by a Rossmann-fold domain (domain C), followed by two tightly associated helical domains (domains D and E), which have similar topology. The structure of MFE1-DH is compared with the two known homologous structures: human mitochondrial 3-hydroxyacyl-CoA dehydrogenase (HAD; sequence identity is 33%) (which is dimeric and monofunctional) and with the dimeric multifunctional alpha-chain (alphaFOM; sequence identity is 28%) of the bacterial fatty acid beta-oxidation alpha2beta2-multienzyme complex. Like MFE-1, alphaFOM has an N-terminal hydratase part and a C-terminal dehydrogenase part, and the structure comparisons show that the N-terminal helix of MFE1-DH corresponds to the alphaFOM linker helix, located between its hydratase and dehydrogenase part. It is also shown that this helix corresponds to the C-terminal helix-10 of the hydratase/isomerase superfamily, suggesting that functionally it belongs to the N-terminal hydratase part of MFE-1.

  18. Methylotrophic Bacillus methanolicus Encodes Two Chromosomal and One Plasmid Born NAD+ Dependent Methanol Dehydrogenase Paralogs with Different Catalytic and Biochemical Properties

    PubMed Central

    Müller, Jonas E. N.; Kupper, Christiane E.; Schneider, Olha; Vorholt, Julia A.; Ellingsen, Trond E.; Brautaset, Trygve

    2013-01-01

    Bacillus methanolicus can utilize methanol as the sole carbon source for growth and it encodes an NAD+-dependent methanol dehydrogenase (Mdh), catalyzing the oxidation of methanol to formaldehyde. Recently, the genomes of the B. methanolicus strains MGA3 (ATCC53907) and PB1 (NCIMB13113) were sequenced and found to harbor three different putative Mdh encoding genes, each belonging to the type III Fe-NAD+-dependent alcohol dehydrogenases. In each strain, two of these genes are encoded on the chromosome and one on a plasmid; only one chromosomal act gene encoding the previously described activator protein ACT was found. The six Mdhs and the ACT proteins were produced recombinantly in Escherichia coli, purified, and characterized. All Mdhs required NAD+ as cosubstrate, were catalytically stimulated by ACT, exhibited a broad and different substrate specificity range and displayed both dehydrogenase and reductase activities. All Mdhs catalyzed the oxidation of methanol; however the catalytic activity for methanol was considerably lower than for most other alcohols tested, suggesting that these enzymes represent a novel class of alcohol dehydrogenases. The kinetic constants for the Mdhs were comparable when acting as pure enzymes, but together with ACT the differences were more pronounced. Quantitative PCR experiments revealed major differences with respect to transcriptional regulation of the paralogous genes. Taken together our data indicate that the repertoire of methanol oxidizing enzymes in thermotolerant bacilli is larger than expected with complex mechanisms involved in their regulation. PMID:23527128

  19. In vitro hydrogen production by glucose dehydrogenase and hydrogenase

    SciTech Connect

    Woodward, J.; Mattingly, S.M.; Danson, M.

    1996-07-01

    A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. The reaction is based on the oxidation of glucose by Thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of NADPH by Pyrococcus furiosus hydrogenase. Stoichiometric yields of hydrogen were produced from glucose with the continuous recycling of cofactor. This simple system may provide a method for the biological production of hydrogen from renewable sources. In addition, the other product of this reaction, gluconic acid, is a high-value chemical commodity. 23 refs., 5 figs.

  20. Lactate Dehydrogenase B Controls Lysosome Activity and Autophagy in Cancer.

    PubMed

    Brisson, Lucie; Bański, Piotr; Sboarina, Martina; Dethier, Coralie; Danhier, Pierre; Fontenille, Marie-Joséphine; Van Hée, Vincent F; Vazeille, Thibaut; Tardy, Morgane; Falces, Jorge; Bouzin, Caroline; Porporato, Paolo E; Frédérick, Raphaël; Michiels, Carine; Copetti, Tamara; Sonveaux, Pierre

    2016-09-12

    Metabolic adaptability is essential for tumor progression and includes cooperation between cancer cells with different metabolic phenotypes. Optimal glucose supply to glycolytic cancer cells occurs when oxidative cancer cells use lactate preferentially to glucose. However, using lactate instead of glucose mimics glucose deprivation, and glucose starvation induces autophagy. We report that lactate sustains autophagy in cancer. In cancer cells preferentially to normal cells, lactate dehydrogenase B (LDHB), catalyzing the conversion of lactate and NAD(+) to pyruvate, NADH and H(+), controls lysosomal acidification, vesicle maturation, and intracellular proteolysis. LDHB activity is necessary for basal autophagy and cancer cell proliferation not only in oxidative cancer cells but also in glycolytic cancer cells. PMID:27622334

  1. Synthesis of brequinar analogue inhibitors of malaria parasite dihydroorotate dehydrogenase.

    PubMed

    Boa, Andrew N; Canavan, Shane P; Hirst, Paul R; Ramsey, Christopher; Stead, Andrew M W; McConkey, Glenn A

    2005-03-15

    A series of 2-phenyl quinoline-4-carboxylic acid derivatives related to brequinar, an inhibitor of human dihydroorotate dehydrogenase (DHODH), has been prepared and evaluated as inhibitors of DHODH from the malaria parasite Plasmodium falciparum. Brequinar was essentially inactive against PfDHODH (IC(50) 880 microM) whereas several members of the series inhibited PfDHODH. Unexpectedly, replacement of the carboxylic acid required for brequinar to inhibit hDHODH was not essential in the diisopropylamides that inhibited PfDHODH.

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

    PubMed Central

    Hart, G J; Dickinson, F M

    1977-01-01

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

  3. Direct Observation of Correlated Interdomain Motion in Alcohol Dehydrogenase

    SciTech Connect

    Biehl, Ralf; Monkenbusch, Michael; Richter, Dieter; Hoffmann, Bernd; Merkel, Rudolf; Falus, Peter; Preost, Sylvain

    2008-09-26

    Interdomain motions in proteins are essential to enable or promote biochemical function. Neutron spin-echo spectroscopy is used to directly observe the domain dynamics of the protein alcohol dehydrogenase. The collective motion of domains as revealed by their coherent form factor relates to the cleft opening dynamics between the binding and the catalytic domains enabling binding and release of the functional important cofactor. The cleft opening mode hardens as a result of an overall stiffening of the domain complex due to the binding of the cofactor.

  4. In vitro hydrogen production by glucose dehydrogenase and hydrogenase

    SciTech Connect

    Woodward, J.

    1996-10-01

    A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. The reaction is based upon the oxidation of glucose by Thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of NADPH by Pyrococcus furiosus hydrogenase. Stoichiometric yields of hydrogen were produced from glucose with continuous cofactor recycle. This simple system may provide a method for the biological production of hydrogen from renewable sources. In addition, the other product of this reaction, gluconic acid, is a high-value commodity chemical.

  5. Lactate dehydrogenase in two digenetic trematodes and their host.

    PubMed

    Haque, M; Siddiqi, A H; Siddiqui, J

    1990-12-01

    Polyacrylamide gel electrophoresis of the two digenetic trematodes, Gigantocotyle explanatum from the liver and Gastrothylax crumenifer from the rumen of the water buffalo, Bubalus bubalis revealed the presence of at least six and seven isoenzymes of lactate dehydrogenase (LDH), respectively in a partially purified enzyme preparation. The respective host tissues showed five isoenzymes of LDH, which are characteristic to the vertebrates. Both parachloromercuribenzoate and iodoacetate affected the LDH activity of the parasites and host tissues differently. Spectrophotometric analysis also showed different specific activity and susceptibility to the action of thiol inhibitors. The host LDH was quite stable at 57 degrees C for 30 min, but that of the parasites was less stable.

  6. [Polymorphism of the gene for subunit 6 of the NADh dehydrogenase complex (ND6) in ethnic russian population in Russia].

    PubMed

    Kornienko, I V; Vodolazhskiĭ, D I; Mikhalkovich, L S; Pavlichenko, G N; Ivanov, P L

    2003-01-01

    A sample of ethnic Russians of Russia was tested for polymorphism of the NADH dehydrogenase subunit 6 (ND6) gene mapping to the mtDNA region 14,170-14,569. Genetic diversity of ND6 haplotypes was estimated at 0.406, and probability of haplotype random match, at 0.598. Combined with typing the mtDNA control region, analysis of the ND6 gene polymorphism was assumed to improve the reliability of forensic identification. Several point substitutions in the ND6 gene region proved to be associated with particular transitions in the mtDNA control region; the association was characterized with the phi coefficient.

  7. Methylmalonic semialdehyde dehydrogenase deficiency: demonstration of defective valine and beta-alanine metabolism and reduced malonic semialdehyde dehydrogenase activity in cultured fibroblasts

    SciTech Connect

    Gray, R.G.; Pollitt, R.J.; Webley, J.

    1987-08-01

    Intact cultured fibroblasts from a child with a new metabolic disorder, thought to be due to a deficiency of methylmalonic semialdehyde dehydrogenase, produced labeled CO/sub 2/ normally from (1-/sup 14/C)valine but not from (2-/sup 14/C)valine. CO/sub 2/ production from labeled beta-alanine was also much reduced, confirming the suspicion that malonic semialdehyde dehydrogenase is also deficient in this condition. An assay for malonic semialdehyde dehydrogenase in cell homogenates showed low activity but it was impossible to assess the degree of reduction.

  8. 4-dihydrotrisporin-dehydrogenase, an enzyme of the sex hormone pathway of Mucor mucedo: purification, cloning of the corresponding gene, and developmental expression.

    PubMed

    Wetzel, Jana; Scheibner, Olaf; Burmester, Anke; Schimek, Christine; Wöstemeyer, Johannes

    2009-01-01

    The NADP-dependent 4-dihydrotrisporin-dehydrogenase is a (-) mating-type-specific enzyme in the pathway from beta-carotene to trisporic acid. This substance and its isomers and derivatives represent the general system of sexual communication in zygomycetes. The (-) mating type of Mucor mucedo was stimulated by trisporic acid and the enzyme was purified by ion exchange and affinity chromatography. Several peptides of the 26-kDa protein, digested with trypsin, were sequenced by mass spectrometry. Oligonucleotides based on protein sequence data were used for PCR amplification of genomic DNA. The primary PCR fragment was sequenced and the complete gene, TSP2, was isolated. A labeled TSP2 hybridization probe detects a single-copy gene in the genome of M. mucedo. Northern blot analysis with RNAs from different growth stages reveals that the expression of the gene depends on the developmental stage of the mycelium in both mating types of M. mucedo. At the enzyme level, activity is found exclusively in the (-) mating type. However, renaturation of proteins in sodium dodecyl sulfate-containing gels revealed the TSP2 gene product in both mating types. Analyzing the protein sequence places the enzyme in the short chain dehydrogenase superfamily. Thus, it has an evolutionary origin distinct from that of the previously isolated 4-dihydromethyltrisporate dehydrogenase, which belongs to the aldo/keto reductase superfamily. Apart from the TSP2 genes in the three sequenced zygomycetous genomes (Phycomyces blakesleeanus, Rhizopus oryzae, and Mucor circinelloides), the closest relative is the Myxococcus xanthus CsgA gene product, which is also a short chain dehydrogenase, involved in C signaling and fruiting body formation.

  9. 4-Dihydrotrisporin-Dehydrogenase, an Enzyme of the Sex Hormone Pathway of Mucor mucedo: Purification, Cloning of the Corresponding Gene, and Developmental Expression▿

    PubMed Central

    Wetzel, Jana; Scheibner, Olaf; Burmester, Anke; Schimek, Christine; Wöstemeyer, Johannes

    2009-01-01

    The NADP-dependent 4-dihydrotrisporin-dehydrogenase is a (−) mating-type-specific enzyme in the pathway from β-carotene to trisporic acid. This substance and its isomers and derivatives represent the general system of sexual communication in zygomycetes. The (−) mating type of Mucor mucedo was stimulated by trisporic acid and the enzyme was purified by ion exchange and affinity chromatography. Several peptides of the 26-kDa protein, digested with trypsin, were sequenced by mass spectrometry. Oligonucleotides based on protein sequence data were used for PCR amplification of genomic DNA. The primary PCR fragment was sequenced and the complete gene, TSP2, was isolated. A labeled TSP2 hybridization probe detects a single-copy gene in the genome of M. mucedo. Northern blot analysis with RNAs from different growth stages reveals that the expression of the gene depends on the developmental stage of the mycelium in both mating types of M. mucedo. At the enzyme level, activity is found exclusively in the (−) mating type. However, renaturation of proteins in sodium dodecyl sulfate-containing gels revealed the TSP2 gene product in both mating types. Analyzing the protein sequence places the enzyme in the short chain dehydrogenase superfamily. Thus, it has an evolutionary origin distinct from that of the previously isolated 4-dihydromethyltrisporate dehydrogenase, which belongs to the aldo/keto reductase superfamily. Apart from the TSP2 genes in the three sequenced zygomycetous genomes (Phycomyces blakesleeanus, Rhizopus oryzae, and Mucor circinelloides), the closest relative is the Myxococcus xanthus CsgA gene product, which is also a short chain dehydrogenase, involved in C signaling and fruiting body formation. PMID:18931040

  10. Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose.

    PubMed

    Wang, Qingzhao; Ingram, Lonnie O; Shanmugam, K T

    2011-11-22

    Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(-)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L(-1) of optically pure D(-)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 μmoles min(-1) (mg protein)(-1). By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50 °C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(-) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates. PMID:22065761

  11. Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation

    PubMed Central

    Tan, Tien-Chye; Kracher, Daniel; Gandini, Rosaria; Sygmund, Christoph; Kittl, Roman; Haltrich, Dietmar; Hällberg, B. Martin; Ludwig, Roland; Divne, Christina

    2015-01-01

    A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization. PMID:26151670

  12. Structural analysis of fungus-derived FAD glucose dehydrogenase

    PubMed Central

    Yoshida, Hiromi; Sakai, Genki; Mori, Kazushige; Kojima, Katsuhiro; Kamitori, Shigehiro; Sode, Koji

    2015-01-01

    We report the first three-dimensional structure of fungus-derived glucose dehydrogenase using flavin adenine dinucleotide (FAD) as the cofactor. This is currently the most advanced and popular enzyme used in glucose sensor strips manufactured for glycemic control by diabetic patients. We prepared recombinant nonglycosylated FAD-dependent glucose dehydrogenase (FADGDH) derived from Aspergillus flavus (AfGDH) and obtained the X-ray structures of the binary complex of enzyme and reduced FAD at a resolution of 1.78 Å and the ternary complex with reduced FAD and D-glucono-1,5-lactone (LGC) at a resolution of 1.57 Å. The overall structure is similar to that of fungal glucose oxidases (GOxs) reported till date. The ternary complex with reduced FAD and LGC revealed the residues recognizing the substrate. His505 and His548 were subjected for site-directed mutagenesis studies, and these two residues were revealed to form the catalytic pair, as those conserved in GOxs. The absence of residues that recognize the sixth hydroxyl group of the glucose of AfGDH, and the presence of significant cavity around the active site may account for this enzyme activity toward xylose. The structural information will contribute to the further engineering of FADGDH for use in more reliable and economical biosensing technology for diabetes management. PMID:26311535

  13. Kinetic properties of aldehyde dehydrogenase from sheep liver mitochondria.

    PubMed Central

    Hart, G J; Dickinson, F M

    1978-01-01

    The kinetics of the NAD+-dependent oxidation of aldehydes, catalysed by aldehyde dehydrogenase purified from sheep liver mitochondria, were studied in detail. Lag phases were observed in the assays, the length of which were dependent on the enzyme concentration. The measured rates after the lag phase was over were directly proportional to the enzyme concentration. If enzyme was preincubated with NAD+, the lag phase was eliminated. Double-reciprocal plots with aldehyde as the variable substrate were non-linear, showing marked substrate activation. With NAD+ as the variable substrate, double-reciprocal plots were linear, and apparently parallel. Double-reciprocal plots with enzyme modified with disulfiram (tetraethylthiuram disulphide) or iodoacetamide, such that at pH 8.0 the activity was decreased to 50% of the control value, showed no substrate activation, and the plots were linear. At pH 7.0, the kinetic parameters Vmax. and Km NAD+- for the oxidation of acetaldehyde and butyraldehyde by the native enzyme are almost identical. Formaldehyde and propionaldehyde show the same apparent maximum rate. Aldehyde dehydrogenase is able to catalyse the hydrolysis of p-nitrophenyl esters. This esterase activity was stimulated by both NAD+ and NADH, the maximum rate for the NAD+ stimulated esterase reaction being roughly equal to the maximum rate for the oxidation of aldehydes. The mechanistic implications of the above behaviour are discussed. PMID:217355

  14. Glutamate dehydrogenase from pumpkin cotyledons: characterization and isoenzymes.

    PubMed

    Chou, K H; Splittstoesser, W E

    1972-04-01

    Glutamate dehydrogenase from pumpkin (Cucurbita moschata Pior. cultivar Dickinson Field) cotyledons was found in both soluble and particulate fractions with the bulk of the activity in the soluble fraction. Both enzymes used NAD(H) and NADP(H) but NAD(H) was favored. The enzymes were classified as glutamate-NAD oxidoreductase, deaminating (EC 1.4.1.3). Both enzymes were heat stable, had a pH optimum for reductive amination of 8.0, and were inhibited by high concentrations of NH(4) (+) or alpha-ketoglutarate. The soluble enzyme was more sensitive to NH(4) (+) inhibition and was activated by metal ions after ammonium sulfate fractionation while the solubilized particulate enzyme was not. Inhibition by ethylenediaminetetraacetate was restored by several divalent ions and inhibition by p-hydroxymercuribenzoate was reversed by glutathione. Particulate glutamate dehydrogenase showed a greater activity with NADP. The molecular weights of the enzymes are 250,000. Separation of the enzymes by disc gel electrophoresis showed that during germination the soluble isoenzymes increased from 1 to 7 in number, while only one particulate isoenzyme was found at any time. This particulate isoenzyme was identical with one of the soluble isoenzymes. A number of methods indicated that the soluble isoenzymes were not simply removed from the particulate fraction and that true isoenzymes were found.

  15. High-pressure-induced water penetration into 3-isopropylmalate dehydrogenase

    SciTech Connect

    Nagae, Takayuki; Kawamura, Takashi; Chavas, Leonard M. G.; Niwa, Ken; Hasegawa, Masashi; Kato, Chiaki; Watanabe, Nobuhisa

    2012-03-01

    Structures of 3-isopropylmalate dehydrogenase were determined at pressures ranging from 0.1 to 650 MPa. Comparison of these structures gives a detailed picture of the swelling of a cavity at the dimer interface and the generation of a new cleft on the molecular surface, which are accompanied by water penetration. Hydrostatic pressure induces structural changes in proteins, including denaturation, the mechanism of which has been attributed to water penetration into the protein interior. In this study, structures of 3-isopropylmalate dehydrogenase (IPMDH) from Shewanella oneidensis MR-1 were determined at about 2 Å resolution under pressures ranging from 0.1 to 650 MPa using a diamond anvil cell (DAC). Although most of the protein cavities are monotonically compressed as the pressure increases, the volume of one particular cavity at the dimer interface increases at pressures over 340 MPa. In parallel with this volume increase, water penetration into the cavity could be observed at pressures over 410 MPa. In addition, the generation of a new cleft on the molecular surface accompanied by water penetration could also be observed at pressures over 580 MPa. These water-penetration phenomena are considered to be initial steps in the pressure-denaturation process of IPMDH.

  16. Crystal structure of a chimaeric bacterial glutamate dehydrogenase.

    PubMed

    Oliveira, Tânia; Sharkey, Michael A; Engel, Paul C; Khan, Amir R

    2016-06-01

    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(+) versus NADP(+), but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies, as shown by the apo structure of glutamate dehydrogenase from Clostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia coli enzyme 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 parent E. coli domain II, although there are subtle differences in catalytic activity. PMID:27303899

  17. Retinol dehydrogenases: membrane-bound enzymes for the visual function.

    PubMed

    Lhor, Mustapha; Salesse, Christian

    2014-12-01

    Retinoid metabolism is important for many physiological functions, such as differenciation, growth, and vision. In the visual context, after the absorption of light in rod photoreceptors by the visual pigment rhodopsin, 11-cis retinal is isomerized to all-trans retinal. This retinoid subsequently undergoes a series of modifications during the visual cycle through a cascade of reactions occurring in photoreceptors and in the retinal pigment epithelium. Retinol dehydrogenases (RDHs) are enzymes responsible for crucial steps of this visual cycle. They belong to a large family of proteins designated as short-chain dehydrogenases/reductases. The structure of these RDHs has been predicted using modern bioinformatics tools, which allowed to propose models with similar structures including a common Rossman fold. These enzymes undergo oxidoreduction reactions, whose direction is dictated by the preference and concentration of their individual cofactor (NAD(H)/NADP(H)). This review presents the current state of knowledge on functional and structural features of RDHs involved in the visual cycle as well as knockout models. RDHs are described as integral or peripheral enzymes. A topology model of the membrane binding of these RDHs via their N- and (or) C-terminal domain has been proposed on the basis of their individual properties. Membrane binding is a crucial issue for these enzymes because of the high hydrophobicity of their retinoid substrates.

  18. Human mutations in glucose 6-phosphate dehydrogenase reflect evolutionary history.

    PubMed

    Notaro, R; Afolayan, A; Luzzatto, L

    2000-03-01

    Glucose 6-phosphate dehydrogenase (G6PD) is a cytosolic enzyme encoded by a housekeeping X-linked gene whose main function is to produce NADPH, a key electron donor in the defense against oxidizing agents and in reductive biosynthetic reactions. Inherited G6PD deficiency is associated with either episodic hemolytic anemia (triggered by fava beans or other agents) or life-long hemolytic anemia. We show here that an evolutionary analysis is a key to understanding the biology of a housekeeping gene. From the alignment of the amino acid (aa) sequence of 52 glucose 6-phosphate dehydrogenase (G6PD) species from 42 different organisms, we found a striking correlation between the aa replacements that cause G6PD deficiency in humans and the sequence conservation of G6PD: two-thirds of such replacements are in highly and moderately conserved (50-99%) aa; relatively few are in fully conserved aa (where they might be lethal) or in poorly conserved aa, where presumably they simply would not cause G6PD deficiency. This is consistent with the notion that all human mutants have residual enzyme activity and that null mutations are lethal at some stage of development. Comparing the distribution of mutations in a human housekeeping gene with evolutionary conservation is a useful tool for pinpointing amino acid residues important for the stability or the function of the corresponding protein. In view of the current explosive increase in full genome sequencing projects, this tool will become rapidly available for numerous other genes.

  19. New model for polymerization of oligomeric alcohol dehydrogenases into nanoaggregates.

    PubMed

    Barzegar, Abolfazl; Moosavi-Movahedi, Ali A; Kyani, Anahita; Goliaei, Bahram; Ahmadian, Shahin; Sheibani, Nader

    2010-02-01

    Polymerization and self-assembly of proteins into nanoaggregates of different sizes and morphologies (nanoensembles or nanofilaments) is a phenomenon that involved problems in various neurodegenerative diseases (medicine) and enzyme instability/inactivity (biotechnology). Thermal polymerization of horse liver alcohol dehydrogenase (dimeric) and yeast alcohol dehydrogenase (tetrameric), as biotechnological ADH representative enzymes, was evaluated for the development of a rational strategy to control aggregation. Constructed ADH nuclei, which grew to larger amorphous nanoaggregates, were prevented via high repulsion strain of the net charge values. Good correlation between the variation in scattering and lambda(-2) was related to the amorphousness of the nanoaggregated ADHs, shown by electron microscopic images. Scattering corrections revealed that ADH polymerization was related to the quaternary structural changes, including delocalization of subunits without unfolding, i.e. lacking the 3D conformational and/or secondary-ordered structural changes. The results demonstrated that electrostatic repulsion was not only responsible for disaggregation but also caused a delay in the onset of aggregation temperature, decreasing maximum values of aggregation and amounts of precipitation. Together, our results demonstrate and propose a new model of self-assembly for ADH enzymes based on the construction of nuclei, which grow to formless nanoaggregates with minimal changes in the tertiary and secondary conformations. PMID:19444390

  20. A Formate Dehydrogenase Confers Tolerance to Aluminum and Low pH1[OPEN

    PubMed Central

    Gong, Yu Long; Fan, Wei; Xu, Jia Meng; Liu, Yu; Cao, Meng Jie; Wang, Ming-Hu

    2016-01-01

    Formate dehydrogenase (FDH) is involved in various higher plant abiotic stress responses. Here, we investigated the role of rice bean (Vigna umbellata) VuFDH in Al and low pH (H+) tolerance. Screening of various potential substrates for the VuFDH protein demonstrated that it functions as a formate dehydrogenase. Quantitative reverse transcription-PCR and histochemical analysis showed that the expression of VuFDH is induced in rice bean root tips by Al or H+ stresses. Fluorescence microscopic observation of VuFDH-GFP in transgenic Arabidopsis plants indicated that VuFDH is localized in the mitochondria. Accumulation of formate is induced by Al and H+ stress in rice bean root tips, and exogenous application of formate increases internal formate content that results in the inhibition of root elongation and induction of VuFDH expression, suggesting that formate accumulation is involved in both H+- and Al-induced root growth inhibition. Over-expression of VuFDH in tobacco (Nicotiana tabacum) results in decreased sensitivity to Al and H+ stress due to less production of formate in the transgenic tobacco lines under Al and H+ stresses. Moreover, NtMATE and NtALS3 expression showed no changes versus wild type in these over-expression lines, suggesting that herein known Al-resistant mechanisms are not involved. Thus, the increased Al tolerance of VuFDH over-expression lines is likely attributable to their decreased Al-induced formate production. Taken together, our findings advance understanding of higher plant Al toxicity mechanisms, and suggest a possible new route toward the improvement of plant performance in acidic soils, where Al toxicity and H+ stress coexist. PMID:27021188

  1. Molecular and biochemical characterization of mannitol-1-phosphate dehydrogenase from the model brown alga Ectocarpus sp.

    PubMed

    Bonin, Patricia; Groisillier, Agnès; Raimbault, Alice; Guibert, Anaïs; Boyen, Catherine; Tonon, Thierry

    2015-09-01

    The sugar alcohol mannitol is important in the food, pharmaceutical, medical and chemical industries. It is one of the most commonly occurring polyols in nature, with the exception of Archaea and animals. It has a range of physiological roles, including as carbon storage, compatible solute, and osmolyte. Mannitol is present in large amounts in brown algae, where its synthesis involved two steps: a mannitol-1-phosphate dehydrogenase (M1PDH) catalyzes a reversible reaction between fructose-6-phosphate (F6P) and mannitol-1-phosphate (M1P) (EC 1.1.1.17), and a mannitol-1-phosphatase hydrolyzes M1P to mannitol (EC 3.1.3.22). Analysis of the model brown alga Ectocarpus sp. genome provided three candidate genes for M1PDH activities. We report here the sequence analysis of Ectocarpus M1PDHs (EsM1PDHs), and the biochemical characterization of the recombinant catalytic domain of EsM1PDH1 (EsM1PDH1cat). Ectocarpus M1PDHs are representatives of a new type of modular M1PDHs among the polyol-specific long-chain dehydrogenases/reductases (PSLDRs). The N-terminal domain of EsM1PDH1 was not necessary for enzymatic activity. Determination of kinetic parameters indicated that EsM1PDH1cat displayed higher catalytic efficiency for F6P reduction compared to M1P oxidation. Both activities were influenced by NaCl concentration and inhibited by the thioreactive compound pHMB. These observations were completed by measurement of endogenous M1PDH activity and of EsM1PDH gene expression during one diurnal cycle. No significant changes in enzyme activity were monitored between day and night, although transcription of two out of three genes was altered, suggesting different levels of regulation for this key metabolic pathway in brown algal physiology. PMID:26232554

  2. A Formate Dehydrogenase Confers Tolerance to Aluminum and Low pH.

    PubMed

    Lou, He Qiang; Gong, Yu Long; Fan, Wei; Xu, Jia Meng; Liu, Yu; Cao, Meng Jie; Wang, Ming-Hu; Yang, Jian Li; Zheng, Shao Jian

    2016-05-01

    Formate dehydrogenase (FDH) is involved in various higher plant abiotic stress responses. Here, we investigated the role of rice bean (Vigna umbellata) VuFDH in Al and low pH (H(+)) tolerance. Screening of various potential substrates for the VuFDH protein demonstrated that it functions as a formate dehydrogenase. Quantitative reverse transcription-PCR and histochemical analysis showed that the expression of VuFDH is induced in rice bean root tips by Al or H(+) stresses. Fluorescence microscopic observation of VuFDH-GFP in transgenic Arabidopsis plants indicated that VuFDH is localized in the mitochondria. Accumulation of formate is induced by Al and H(+) stress in rice bean root tips, and exogenous application of formate increases internal formate content that results in the inhibition of root elongation and induction of VuFDH expression, suggesting that formate accumulation is involved in both H(+)- and Al-induced root growth inhibition. Over-expression of VuFDH in tobacco (Nicotiana tabacum) results in decreased sensitivity to Al and H(+) stress due to less production of formate in the transgenic tobacco lines under Al and H(+) stresses. Moreover, NtMATE and NtALS3 expression showed no changes versus wild type in these over-expression lines, suggesting that herein known Al-resistant mechanisms are not involved. Thus, the increased Al tolerance of VuFDH over-expression lines is likely attributable to their decreased Al-induced formate production. Taken together, our findings advance understanding of higher plant Al toxicity mechanisms, and suggest a possible new route toward the improvement of plant performance in acidic soils, where Al toxicity and H(+) stress coexist. PMID:27021188

  3. Dichloroacetate, the Pyruvate Dehydrogenase Complex and the Modulation of mESC Pluripotency

    PubMed Central

    Rodrigues, Ana Sofia; Correia, Marcelo; Gomes, Andreia; Pereira, Sandro L.; Perestrelo, Tânia; Sousa, Maria Inês; Ramalho-Santos, João

    2015-01-01

    Introduction The pyruvate dehydrogenase (PDH) complex is localized in the mitochondrial matrix catalyzing the irreversible decarboxylation of pyruvate to acetyl-CoA and NADH. For proper complex regulation the E1-α subunit functions as an on/off switch regulated by phosphorylation/dephosphorylation. In different cell types one of the four-pyruvate dehydrogenase kinase isoforms (PDHK1-4) can phosphorylate this subunit leading to PDH inactivation. Our previous results with human Embryonic Stem Cells (hESC), suggested that PDHK could be a key regulator in the metabolic profile of pluripotent cells, as it is upregulated in pluripotent stem cells. Therefore, we wondered if metabolic modulation, via inexpensive pharmacological inhibition of PDHK, could impact metabolism and pluripotency. Methods/Results In order to assess the importance of the PDH cycle in mouse Embryonic Stem Cells (mESC), we incubated cells with the PDHK inhibitor dichloroacetate (DCA) and observed that in its presence ESC started to differentiate. Changes in mitochondrial function and proliferation potential were also found and protein levels for PDH (both phosphorylated and non-phosphorylated) and PDHK1 were monitored. Interestingly, we were also able to describe a possible pathway that involves Hif-1α and p53 during DCA-induced loss of pluripotency. Results with ESCs treated with DCA were comparable to those obtained for cells grown without Leukemia Inhibitor Factor (LIF), used in this case as a positive control for differentiation. Conclusions DCA negatively affects ESC pluripotency by changing cell metabolism and elements related to the PDH cycle, suggesting that PDHK could function as a possible metabolic gatekeeper in ESC, and may be a good target to modulate metabolism and differentiation. Although further molecular biology-based experiments are required, our data suggests that inactive PDH favors pluripotency and that ESC have similar strategies as cancer cells to maintain a glycolytic

  4. Characterization of homogeneous recombinant rat ovarian 20alpha-hydroxysteroid dehydrogenase: fluorescent properties and inhibition profile.

    PubMed Central

    Ma, H; Penning, T M

    1999-01-01

    In rat ovary, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), a member of the aldo-keto reductase (AKR) superfamily, converts progesterone into the inactive progestin 20alpha-hydroxyprogesterone and has been implicated in the termination of pregnancy. Here we report a convenient overexpression system that permits the purification of milligram quantities of homogeneous recombinant 20alpha-HSD with wild-type enzyme activity. The availability of this enzyme has permitted detailed kinetic, inhibition and fluorescence analyses. The enzyme exhibited narrow steroid specificity, catalysing reactions only at C-20; it reduced progesterone and 17alpha-hydroxyprogesterone and oxidized 20alpha-hydroxypregnanes. It also turned over common AKR substrates, such as 9, 10-phenanthrenequinone and 4-nitrobenzaldehyde. The intrinsic fluorescence spectrum of 20alpha-HSD was characterized and was quenched on the binding of NADP(H), yielding a KNADPd of 0.36 microM and a KNADPHd of 0.64 microM. NADP(H) binding generated an energy transfer band that could not be quenched by steroids. Inhibition studies conducted with non-steroidal and steroidal anti-inflammatory drugs and synthetic oestrogens indicated that even though rat ovarian 20alpha-HSD and rat liver 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) share more than 67% amino acid identity, their inhibition profiles are markedly different. Unlike 3alpha-HSD, most of these compounds did not inhibit 20alpha-HSD. Only meclofenamic acid and hexoestrol were potent competitive inhibitors for 20alpha-HSD, yielding K(i) values of 18.9 and 14.3 microM respectively. These studies suggest that selective non-steroidal AKR inhibitors could be developed for 20alpha-HSD that might be useful in maintaining pregnancy and that specific inhibitors might be developed from either N-phenylanthranilates or biphenols. PMID:10417353

  5. Characterization of homogeneous recombinant rat ovarian 20alpha-hydroxysteroid dehydrogenase: fluorescent properties and inhibition profile.

    PubMed

    Ma, H; Penning, T M

    1999-08-01

    In rat ovary, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), a member of the aldo-keto reductase (AKR) superfamily, converts progesterone into the inactive progestin 20alpha-hydroxyprogesterone and has been implicated in the termination of pregnancy. Here we report a convenient overexpression system that permits the purification of milligram quantities of homogeneous recombinant 20alpha-HSD with wild-type enzyme activity. The availability of this enzyme has permitted detailed kinetic, inhibition and fluorescence analyses. The enzyme exhibited narrow steroid specificity, catalysing reactions only at C-20; it reduced progesterone and 17alpha-hydroxyprogesterone and oxidized 20alpha-hydroxypregnanes. It also turned over common AKR substrates, such as 9, 10-phenanthrenequinone and 4-nitrobenzaldehyde. The intrinsic fluorescence spectrum of 20alpha-HSD was characterized and was quenched on the binding of NADP(H), yielding a KNADPd of 0.36 microM and a KNADPHd of 0.64 microM. NADP(H) binding generated an energy transfer band that could not be quenched by steroids. Inhibition studies conducted with non-steroidal and steroidal anti-inflammatory drugs and synthetic oestrogens indicated that even though rat ovarian 20alpha-HSD and rat liver 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) share more than 67% amino acid identity, their inhibition profiles are markedly different. Unlike 3alpha-HSD, most of these compounds did not inhibit 20alpha-HSD. Only meclofenamic acid and hexoestrol were potent competitive inhibitors for 20alpha-HSD, yielding K(i) values of 18.9 and 14.3 microM respectively. These studies suggest that selective non-steroidal AKR inhibitors could be developed for 20alpha-HSD that might be useful in maintaining pregnancy and that specific inhibitors might be developed from either N-phenylanthranilates or biphenols. PMID:10417353

  6. 11β-Hydroxysteroid Dehydrogenases: Intracellular Gate-Keepers of Tissue Glucocorticoid Action

    PubMed Central

    Chapman, Karen; Holmes, Megan

    2013-01-01

    Glucocorticoid action on target tissues is determined by the density of “nuclear” receptors and intracellular metabolism by the two isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD) which catalyze interconversion of active cortisol and corticosterone with inert cortisone and 11-dehydrocorticosterone. 11β-HSD type 1, a predominant reductase in most intact cells, catalyzes the regeneration of active glucocorticoids, thus amplifying cellular action. 11β-HSD1 is widely expressed in liver, adipose tissue, muscle, pancreatic islets, adult brain, inflammatory cells, and gonads. 11β-HSD1 is selectively elevated in adipose tissue in obesity where it contributes to metabolic complications. Similarly, 11β-HSD1 is elevated in the ageing brain where it exacerbates glucocorticoid-associated cognitive decline. Deficiency or selective inhibition of 11β-HSD1 improves multiple metabolic syndrome parameters in rodent models and human clinical trials and similarly improves cognitive function with ageing. The efficacy of inhibitors in human therapy remains unclear. 11β-HSD2 is a high-affinity dehydrogenase that inactivates glucocorticoids. In the distal nephron, 11β-HSD2 ensures that only aldosterone is an agonist at mineralocorticoid receptors (MR). 11β-HSD2 inhibition or genetic deficiency causes apparent mineralocorticoid excess and hypertension due to inappropriate glucocorticoid activation of renal MR. The placenta and fetus also highly express 11β-HSD2 which, by inactivating glucocorticoids, prevents premature maturation of fetal tissues and consequent developmental “programming.” The role of 11β-HSD2 as a marker of programming is being explored. The 11β-HSDs thus illuminate the emerging biology of intracrine control, afford important insights into human pathogenesis, and offer new tissue-restricted therapeutic avenues. PMID:23899562

  7. The Role of Ala198 in the Stability and Coenzyme Specificity of Bacterial Formate Dehydrogenases

    PubMed Central

    Alekseeva, A. A.; Fedorchuk, V. V.; Zarubina, S. A.; Sadykhov, E. G.; Matorin, A. D.; Savin, S. S.; Tishkov, V. I.

    2015-01-01

    It has been shown by an X-ray structural analysis that the amino acid residues Ala198, which are located in the coenzyme-binding domain of NAD+-dependent formate dehydrogenases (EC 1.2.1.2., FDH) from bacteria Pseudomonas sp.101 and Moraxella sp. C-1 (PseFDH and MorFDH, respectively), have non-optimal values of the angles ψ and φ. These residues were replaced with Gly by site-directed mutagenesis. The mutants PseFDH A198G and MorFDH A198G were expressed in E.coli cells and obtained in active and soluble forms with more than 95% purity. The study of thermal inactivation kinetics showed that the mutation A198G results in a 2.5- fold increase in stability compared to one for the wild-type enzymes. Kinetic experiments indicate that A198G replacement reduces the KMNAD+ value from 60 to 35 and from 80 to 45 μM for PseFDH and MorFDH, respectively, while the KMHCOO- value remains practically unchanged. Amino acid replacement A198G was also added to the mutant PseFDH D221S with the coenzyme specificity changed from NAD+ to NADP+. In this case, an increase in thermal stability was also observed, but the influence of the mutation on the kinetic parameters was opposite: KM increased from 190 to 280 μM and from 43 to 89 mM for NADP+ and formate, respectively. According to the data obtained, inference could be drawn that earlier formate dehydrogenase from bacterium Pseudomonas sp. 101 was specific to NADP+, but not to NAD+. PMID:25927002

  8. Structural Determinants of Oligomerization of !1-Pyrroline-5-Carboxylate Dehydrogenase: Identification of a Hexamerization Hot Spot

    PubMed Central

    Luo, Min; Singh, Ranjan K.; Tanner, John J.

    2013-01-01

    The aldehyde dehydrogenase (ALDH) superfamily member !1-pyrroline-5-carboxylate dehydrogenase (P5CDH) catalyzes the NAD+-dependent oxidation of glutamate semialdehyde to glutamate, which is the final step of proline catabolism. Defects in P5CDH activity lead to the metabolic disorder type II hyperprolinemia, P5CDH is essential for virulence of the fungal pathogen Cryptococcus neoformans, and bacterial P5CDHs have been targeted for vaccine development. Although the enzyme oligomeric state is known to be important for ALDH function, the oligomerization of P5CDH has remained relatively unstudied. Here we determine the oligomeric states and quaternary structures of four bacterial P5CDHs using a combination of small-angle X-ray scattering, X-ray crystallography, and dynamic light scattering. The P5CDHs from Thermus thermophilus and Deinococcus radiodurans form trimer-of-dimers hexamers in solution, which is the first observation of a hexameric ALDH in solution. In contrast, two Bacillus P5CDHs form dimers in solution but do not assemble into a higher order oligomer. Site-directed mutagenesis was used to identify a hexamerization hot spot that is centered on an arginine residue in the NAD+-binding domain. Mutation of this critical Arg residue to Ala in either of the hexameric enzymes prevents hexamer formation in solution. Paradoxically, the dimeric Arg-to-Ala T. thermophilus mutant enzyme packs as a hexamer in the crystal state, which illustrates the challenges associated with predicting the biological assembly in solution from crystal structures. The observation of different oligomeric states among P5CDHs suggests potential differences in cooperativity and protein-protein interactions. PMID:23747974

  9. Crystal structure studies of NADP{sup +} dependent isocitrate dehydrogenase from Thermus thermophilus exhibiting a novel terminal domain

    SciTech Connect

    Kumar, S.M.; Pampa, K.J.; Manjula, M.; Abdoh, M.M.M.; Kunishima, Naoki; Lokanath, N.K.

    2014-06-20

    Highlights: • We determined the structure of isocitrate dehydrogenase with citrate and cofactor. • The structure reveals a unique novel terminal domain involved in dimerization. • Clasp domain shows significant difference, and catalytic residues are conserved. • Oligomerization of the enzyme is quantized with subunit-subunit interactions. • Novel domain of this enzyme is classified as subfamily of the type IV. - Abstract: NADP{sup +} dependent isocitrate dehydrogenase (IDH) is an enzyme catalyzing oxidative decarboxylation of isocitrate into oxalosuccinate (intermediate) and finally the product α-ketoglutarate. The crystal structure of Thermus thermophilus isocitrate dehydrogenase (TtIDH) ternary complex with citrate and cofactor NADP{sup +} was determined using X-ray diffraction method to a resolution of 1.80 Å. The overall fold of this protein was resolved into large domain, small domain and a clasp domain. The monomeric structure reveals a novel terminal domain involved in dimerization, very unique and novel domain when compared to other IDH’s. And, small domain and clasp domain showing significant differences when compared to other IDH’s of the same sub-family. The structure of TtIDH reveals the absence of helix at the clasp domain, which is mainly involved in oligomerization in other IDH’s. Also, helices/beta sheets are absent in the small domain, when compared to other IDH’s of the same sub family. The overall TtIDH structure exhibits closed conformation with catalytic triad residues, Tyr144-Asp248-Lys191 are conserved. Oligomerization of the protein is quantized using interface area and subunit–subunit interactions between protomers. Overall, the TtIDH structure with novel terminal domain may be categorized as a first structure of subfamily of type IV.

  10. Converting NAD-specific inositol dehydrogenase to an efficient NADP-selective catalyst, with a surprising twist.

    PubMed

    Zheng, Hongyan; Bertwistle, Drew; Sanders, David A R; Palmer, David R J

    2013-08-27

    myo-Inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis converts myo-inositol to scyllo-inosose and is strictly dependent on NAD for activity. We sought to alter the coenzyme specificity to generate an NADP-dependent enzyme in order to enhance our understanding of coenzyme selectivity and to create an enzyme capable of recycling NADP in biocatalytic processes. Examination of available structural information related to the GFO/MocA/IDH family of dehydrogenases and precedents for altering coenzyme selectivity allowed us to select residues for substitution, and nine single, double, and triple mutants were constructed. Mutagenesis experiments with B. subtilis IDH proved extremely successful; the double mutant D35S/V36R preferred NADP to NAD by a factor of 5. This mutant is an excellent catalyst with a second-order rate constant with respect to NADP of 370 000 s⁻¹ M⁻¹, and the triple mutant A12K/D35S/V36R had a value of 570 000 s⁻¹ M⁻¹, higher than that of the wild-type IDH with NAD. The high-resolution X-ray crystal structure of the double mutant A12K/D35S was solved in complex with NADP. Surprisingly, the binding of the coenzyme is altered such that although the nicotinamide ring maintains the required position for catalysis, the coenzyme has twisted by nearly 90°, so the adenine moiety no longer binds to a hydrophobic cleft in the Rossmann fold as in the wild-type enzyme. This change in binding conformation has not previously been observed in mutated dehydrogenases.

  11. Creation of a thermostable NADP⁺-dependent D-amino acid dehydrogenase from Ureibacillus thermosphaericus strain A1 meso-diaminopimelate dehydrogenase by site-directed mutagenesis.

    PubMed

    Akita, Hironaga; Doi, Katsumi; Kawarabayasi, Yutaka; Ohshima, Toshihisa

    2012-09-01

    A thermostable, NADP(+)-dependent D: -amino acid dehydrogenase (DAADH) was created from the meso-diaminopimelate dehydrogenase of Ureibacillus thermosphaericus strain A1 by introducing five point mutations into amino acid residues located in the active site. The recombinant protein, expressed in Escherichia coli, was purified to homogeneity using a two-step separation procedure and then characterized. In the presence of NADP(+), the protein catalyzed the oxidative deamination of several D: -amino acids, including D: -cyclohexylalanine, D: -isoleucine and D: -2-aminooctanoate, but not meso-diaminopimelate, confirming the creation of a NADP(+)-dependent DAADH. For the reverse reaction, the corresponding 2-oxo acids were aminated in the presence of NADPH and ammonia. In addition, the D: -amino acid dehydrogenase showed no loss of activity at 65 °C, indicating the mutant enzyme was more thermostable than its parental meso-diaminopimelate dehydrogenase.

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

    PubMed Central

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

    2010-01-01

    In approximately one billion people, a point mutation inactivates a key detoxifying enzyme, aldehyde dehydrogenase (ALDH2). This mitochondrial enzyme metabolizes toxic biogenic and environmental aldehydes, including the endogenously produced 4-hydroxynonenal (4HNE) and the environmental pollutant, acrolein. ALDH2 also bioactivates nitroglycerin, but it is best known for its role in ethanol metabolism. The accumulation of acetaldehyde following the consumption of even a single alcoholic beverage leads to the Asian Alcohol-induced Flushing Syndrome in ALDH2*2 homozygotes. The ALDH2*2 allele is semi-dominant and heterozygotic individuals exhibit a similar, but not as severe phenotype. We recently identified a small molecule, Alda-1, which activates wild-type ALDH2 and restores near wild-type activity to ALDH2*2. The structures of Alda-1 bound to ALDH2 and ALDH2*2 reveal how Alda-1 activates the wild-type enzyme and how it restores the activity of ALDH2*2 by acting as a structural chaperone. PMID:20062057

  13. A model system for QTL analysis: Effects of alcohol dehydrogenase genotype on alcohol pharmacokinetics

    SciTech Connect

    Martin, N.G.; Nightingale, B.; Whitfield, J.B.

    1994-09-01

    There is much interest in the detection of quantitative trait loci (QTL) - major genes which affect quantitative phenotypes. The relationship of polymorphism at known alcohol metabolizing enzyme loci to alcohol pharmacokinetics is a good model system. The three class I alcohol dehydrogenase genes are clustered on chromosome 4 and protein electrophoresis has revealed polymorphisms at the ADH2 and ADH3 loci. While different activities of the isozymes have been demonstrated in vitro, little work has been done in trying to relate ADH polymorphism to variation in ethanol metabolism in vivo. We previously measured ethanol metabolism and psychomotor reactivity in 206 twin pairs and demonstrated that most of the repeatable variation was genetic. We have now recontacted the twins to obtain DNA samples and used PCR with allele specific primers to type the ADH2 and ADH3 polymorphisms in 337 individual twins. FISHER has been used to estimate fixed effects of typed polymorphisms simultaneously with remaining linked and unlinked genetic variance. The ADH2*1-2 genotypes metabolize ethanol faster and attain a lower peak blood alcohol concentration than the more common ADH2*1-1 genotypes, although less than 3% of the variance is accounted for. There is no effect of ADH3 genotype. However, sib-pair linkage analysis suggests that there is a linked polymorphism which has a much greater effect on alcohol metabolism that those typed here.

  14. Directed evolution of leucine dehydrogenase for improved efficiency of L-tert-leucine synthesis.

    PubMed

    Zhu, Lin; Wu, Zhe; Jin, Jian-Ming; Tang, Shuang-Yan

    2016-07-01

    L-tert-Leucine and its derivatives are used as synthetic building blocks for pharmaceutical active ingredients, chiral auxiliaries, and ligands. Leucine dehydrogenase (LeuDH) is frequently used to prepare L-tert-leucine from the α-keto acid precursor trimethylpyruvate (TMP). In this study, a high-throughput screening method for the L-tert-leucine synthesis reaction based on a spectrophotometric approach was developed. Directed evolution strategy was applied to engineer LeuDH from Lysinibacillus sphaericus for improved efficiency of L-tert-leucine synthesis. After two rounds of random mutagenesis, the specific activity of LeuDH on the substrate TMP was enhanced by more than two-fold, compared with that of the wild-type enzyme, while the activity towards its natural substrate, leucine, decreased. The catalytic efficiencies (k cat/K m) of the best mutant enzyme, H6, on substrates TMP and NADH were all enhanced by more than five-fold as compared with that of the wild-type enzyme. The efficiency of L-tert-leucine synthesis by mutant H6 was significantly improved. A productivity of 1170 g/l/day was achieved for the mutant enzyme H6, compared with 666 g/l/day for the wild-type enzyme.

  15. Ethanol-Induced Alcohol Dehydrogenase E (AdhE) Potentiates Pneumolysin in Streptococcus pneumoniae

    PubMed Central

    Luong, Truc Thanh; Kim, Eun-Hye; Bak, Jong Phil; Nguyen, Cuong Thach; Choi, Sangdun; Briles, David E.; Pyo, Suhkneung

    2014-01-01

    Alcohol impairs the host immune system, rendering the host more vulnerable to infection. Therefore, alcoholics are at increased risk of acquiring serious bacterial infections caused by Streptococcus pneumoniae, including pneumonia. Nevertheless, how alcohol affects pneumococcal virulence remains unclear. Here, we showed that the S. pneumoniae type 2 D39 strain is ethanol tolerant and that alcohol upregulates alcohol dehydrogenase E (AdhE) and potentiates pneumolysin (Ply). Hemolytic activity, colonization, and virulence of S. pneumoniae, as well as host cell myeloperoxidase activity, proinflammatory cytokine secretion, and inflammation, were significantly attenuated in adhE mutant bacteria (ΔadhE strain) compared to D39 wild-type bacteria. Therefore, AdhE might act as a pneumococcal virulence factor. Moreover, in the presence of ethanol, S. pneumoniae AdhE produced acetaldehyde and NADH, which subsequently led Rex (redox-sensing transcriptional repressor) to dissociate from the adhE promoter. An increase in AdhE level under the ethanol condition conferred an increase in Ply and H2O2 levels. Consistently, S. pneumoniae D39 caused higher cytotoxicity to RAW 264.7 cells than the ΔadhE strain under the ethanol stress condition, and ethanol-fed mice (alcoholic mice) were more susceptible to infection with the D39 wild-type bacteria than with the ΔadhE strain. Taken together, these data indicate that AdhE increases Ply under the ethanol stress condition, thus potentiating pneumococcal virulence. PMID:25312953

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

    SciTech Connect

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

    2010-04-19

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

  17. Ethanol-induced alcohol dehydrogenase E (AdhE) potentiates pneumolysin in Streptococcus pneumoniae.

    PubMed

    Luong, Truc Thanh; Kim, Eun-Hye; Bak, Jong Phil; Nguyen, Cuong Thach; Choi, Sangdun; Briles, David E; Pyo, Suhkneung; Rhee, Dong-Kwon

    2015-01-01

    Alcohol impairs the host immune system, rendering the host more vulnerable to infection. Therefore, alcoholics are at increased risk of acquiring serious bacterial infections caused by Streptococcus pneumoniae, including pneumonia. Nevertheless, how alcohol affects pneumococcal virulence remains unclear. Here, we showed that the S. pneumoniae type 2 D39 strain is ethanol tolerant and that alcohol upregulates alcohol dehydrogenase E (AdhE) and potentiates pneumolysin (Ply). Hemolytic activity, colonization, and virulence of S. pneumoniae, as well as host cell myeloperoxidase activity, proinflammatory cytokine secretion, and inflammation, were significantly attenuated in adhE mutant bacteria (ΔadhE strain) compared to D39 wild-type bacteria. Therefore, AdhE might act as a pneumococcal virulence factor. Moreover, in the presence of ethanol, S. pneumoniae AdhE produced acetaldehyde and NADH, which subsequently led Rex (redox-sensing transcriptional repressor) to dissociate from the adhE promoter. An increase in AdhE level under the ethanol condition conferred an increase in Ply and H2O2 levels. Consistently, S. pneumoniae D39 caused higher cytotoxicity to RAW 264.7 cells than the ΔadhE strain under the ethanol stress condition, and ethanol-fed mice (alcoholic mice) were more susceptible to infection with the D39 wild-type bacteria than with the ΔadhE strain. Taken together, these data indicate that AdhE increases Ply under the ethanol stress condition, thus potentiating pneumococcal virulence.

  18. 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.

  19. Electrochemical conversion of carbon dioxide to methanol with the assistance of formate dehydrogenase and methanol dehydrogenase as biocatalysts

    SciTech Connect

    Kuwabata, Susumu; Tsuda, Ryo; Yoneyama, Hiroshi )

    1994-06-15

    Electrolysis at potentials between -0.7 and -0.9 V vs SCE of carbon dioxide-saturated phosphate buffer solutions (pH7) containing formate dehydrogenase (FDH) and either methyl viologen (MV[sup 2+]) or pyrroloquinolinequinone (PQQ) as an electron mediator yielded formate with current efficiencies as high as 90%. The enzyme was durable as long as the electrolysis was carried out in the dark. Electrolysis of phosphate buffer solutions containing sodium formate in the presence of methanol dehydrogenase (MDH) and MV[sup 2+] at -0.7 V vs SCE yielded formaldehyde if the concentration of the enzyme used was low, whereas both formaldehyde and methanol were produced for relatively high concentrations of the enzyme where the methanol production began to occur when the formaldehyde produced accumulated. The use of PQQ in place of MV[sup 2+] as the electron mediator exclusively produced methanol alone after some induction period in the electrolysis. On the basis of these results, successful attempts have been made to reduce carbon dioxide to methanol with cooperative assistance of FDH and MDH in the presence of PQQ as the electron mediator. The role of enzyme and mediator in these reduction processes is discussed in detail. 34 refs., 10 figs., 2 tabs.

  20. 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

    2015-01-01

    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.

  1. In vivo regulation of alcohol dehydrogenase and lactate dehydrogenase in Rhizopus oryzae to improve L-lactic acid fermentation.

    PubMed

    Thitiprasert, Sitanan; Sooksai, Sarintip; Thongchul, Nuttha

    2011-08-01

    Rhizopus oryzae is becoming more important due to its ability to produce an optically pure L: -lactic acid. However, fermentation by Rhizopus usually suffers from low yield because of production of ethanol as a byproduct. Limiting ethanol production in living immobilized R. oryzae by inhibition of alcohol dehydrogenase (ADH) was observed in shake flask fermentation. The effects of ADH inhibitors added into the medium on the regulation of ADH and lactate dehydrogenase (LDH) as well as the production of cell biomass, lactic acid, and ethanol were elucidated. 1,2-diazole and 2,2,2-trifluroethanol were found to be the effective inhibitors used in this study. The highest lactic acid yield of 0.47 g/g glucose was obtained when 0.01 mM 2,2,2-trifluoroethanol was present during the production phase of the pregrown R. oryzae. This represents about 38% increase in yield as compared with that from the simple glucose fermentation. Fungal metabolism was suppressed when iodoacetic acid, N-ethylmaleimide, 4,4'-dithiodipyridine, or 4-hydroxymercury benzoic acid were present. Dramatic increase in ADH and LDH activities but slight change in product yields might be explained by the inhibitors controlling enzyme activities at the pyruvate branch point. This showed that in living R. oryzae, the inhibitors regulated the flux through the related pathways. PMID:21416338

  2. The diagnostic value of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) measurement in the sera of gastric cancer patients.

    PubMed

    Jelski, Wojciech; Orywal, Karolina; Laniewska, Magdalena; Szmitkowski, Maciej

    2010-12-01

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are present in gastric cancer cells (GC). Moreover, the activity of total ADH and class IV isoenzymes is significantly higher in cancer tissue than in healthy mucosa. The activity of these enzymes in cancer cells is probably reflected in the sera and could thus be helpful for diagnostics of gastric cancer. The aim of this study was to investigate a potential role of ADH and ALDH as tumor markers for gastric cancer. We defined diagnostic sensitivity, specificity, predictive value for positive and negative results, and receiver-operating characteristics (ROC) curve for tested enzymes. Serum samples were taken from 168 patients with gastric cancer before treatment and from 168 control subjects. Total ADH activity and class III and IV isoenzymes were measured by photometric but ALDH activity and ADH I and II by the fluorometric method, with class-specific fluorogenic substrates. There was significant increase in the activity of ADH IV isoenzyme and ADH total in the sera of gastric cancer patients compared to the control. The diagnostic sensitivity for ADH IV was 73%, specificity 79%, positive and negative predictive values were 81 and 72% respectively. Area under ROC curve for ADH IV was 0.67. The results suggest a potential role for ADH IV as marker of gastric cancer.

  3. In vivo regulation of alcohol dehydrogenase and lactate dehydrogenase in Rhizopus oryzae to improve L-lactic acid fermentation.

    PubMed

    Thitiprasert, Sitanan; Sooksai, Sarintip; Thongchul, Nuttha

    2011-08-01

    Rhizopus oryzae is becoming more important due to its ability to produce an optically pure L: -lactic acid. However, fermentation by Rhizopus usually suffers from low yield because of production of ethanol as a byproduct. Limiting ethanol production in living immobilized R. oryzae by inhibition of alcohol dehydrogenase (ADH) was observed in shake flask fermentation. The effects of ADH inhibitors added into the medium on the regulation of ADH and lactate dehydrogenase (LDH) as well as the production of cell biomass, lactic acid, and ethanol were elucidated. 1,2-diazole and 2,2,2-trifluroethanol were found to be the effective inhibitors used in this study. The highest lactic acid yield of 0.47 g/g glucose was obtained when 0.01 mM 2,2,2-trifluoroethanol was present during the production phase of the pregrown R. oryzae. This represents about 38% increase in yield as compared with that from the simple glucose fermentation. Fungal metabolism was suppressed when iodoacetic acid, N-ethylmaleimide, 4,4'-dithiodipyridine, or 4-hydroxymercury benzoic acid were present. Dramatic increase in ADH and LDH activities but slight change in product yields might be explained by the inhibitors controlling enzyme activities at the pyruvate branch point. This showed that in living R. oryzae, the inhibitors regulated the flux through the related pathways.

  4. [Activity of NADP-dependent glycerol-3-phosphate dehydrogenase in skeletal muscles of animals].

    PubMed

    Epifanova, Iu E; Glushankov, E P; Kolotilova, A I

    1978-01-01

    The NADP-dependent glycerol-3-phosphate dehydrogenase activity was studied in sketetal muscles of the rat, rabbit and frog. The dehydrogenase activity in the skeletal muscles of the rat and rabbit was higher than that of the frog. The enzyme activity was found to depend upon the buffer, being higher in tris-HCl buffer than in triethanolamine buffer.

  5. Analysis of rat cytosolic 9-cis-retinol dehydrogenase activity and enzymatic characterization of rat ADHII.

    PubMed

    Popescu, G; Napoli, J L

    2000-01-01

    We report the characterization of two enzymes that catalyze NAD(+)-dependent 9-cis-retinol dehydrogenase activity in rat liver cystol. Alcohol dehydrogenase class I (ADHI) contributes > 80% of the NA D+-dependent 9-cis-retinol dehydrogenase activity recovered, whereas alcohol dehydrogenase class II (ADHII), not identified previously at the protein level, nor characterized enzymatically in rat, accounts for approximately 2% of the activity. Rat ADHII exhibits properties different from those described for human ADHII. Moreover, rat ADHII-catalyzed rates of ethanol dehydrogenation are markedly lower than octanol or retinoid dehydrogenation rates. Neither ethanol nor 4-methylpyrazole inhibits the 9-cis-retinol dehydrogenase activity of rat ADHII. We propose that ADHII represents the previously observed additional retinoid oxidation activity of rat liver cytosol which occurred in the presence of either ethanol or 4-methylpyrazole. We also show that human and rat ADHII differ considerably in enzymatic properties. PMID:10606766

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

    SciTech Connect

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

    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 are consistent with an association between increased IMP dehydrogenase expression and either enhanced cell proliferation or malignant transformation.

  7. Isolation, characterization and evaluation of the Pichia pastoris sorbitol dehydrogenase promoter for expression of heterologous proteins.

    PubMed

    Periyasamy, Sankar; Govindappa, Nagaraj; Sreenivas, Suma; Sastry, Kedarnath

    2013-11-01

    Sorbitol is used as a non-repressive carbon source to develop fermentation process for Mut(s) recombinant clones obtained using the AOX1 promoter in Pichia pastoris. Sorbitol dehydrogenase is an enzyme in the carbohydrate metabolism that catalyzes reduction of D-fructose into D-sorbitol in the presence of NADH. The small stretch of 211bps upstream region of sorbitol dehydrogenase coding gene has all the promoter elements like CAAT box, GC box, etc. It is able to promote protein production under repressive as well as non-repressive carbon sources. In this study, the strength of the sorbitol dehydrogenase promoter was evaluated by expression of two heterologous proteins: human serum albumin and erythrina trypsin inhibitor. Sorbitol dehydrogenase promoter allowed constitutive expression of recombinant proteins in all carbon sources that were tested to grow P. pastoris and showed activity similar to GAP promoter. The sorbitol dehydrogenase promoter was active in all the growth phases of the P. pastoris.

  8. Changing kinetic properties of glucose-6-phosphate dehydrogenase from pea chloroplasts during photosynthetic induction

    SciTech Connect

    Yuan, X.; Anderson, L.E.

    1987-04-01

    The first enzyme of the oxidative pentose phosphate pathway, glucose-6-P dehydrogenase (EC 1.1.1.49), is inactivated when pea chloroplasts are irradiated. They have examined the kinetics of light inactivation of glucose-6-P dehydrogenase in intact chloroplasts during photosynthetic induction and the kinetic parameters of the active (dark) and less active (light) form of the dehydrogenase. Light inactivation of the dehydrogenase is rapid and occurs before photosynthetic O/sub 2/ evolution is measureable in intact chloroplasts. Likewise dark activation is quite rapid. The major change in the kinetic parameters of glucose-6-phosphate dehydrogenase is in maximal velocity. This light inactivation probably prevents operation of a futile cycle involving glucose-6-P, NADPH and oxidative and reductive pentose phosphate pathway enzymes.

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

    PubMed

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

    1998-01-01

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

  10. Characterization of betaine aldehyde dehydrogenase (BetB) as an essential virulence factor of Brucella abortus.

    PubMed

    Lee, Jin Ju; Kim, Jae Hong; Kim, Dae Geun; Kim, Dong Hyeok; Simborio, Hannah Leah; Min, Won Gi; Rhee, Man Hee; Lim, Jong Hwan; Chang, Hong Hee; Kim, Suk

    2014-01-10

    The pathogenic mechanisms of Brucellosis used to adapt to the harsh intracellular environment of the host cell are not fully understood. The present study investigated the in vitro and in vivo characteristics of B. abortus betaine aldehyde dehydrogenase (BetB) (Gene Bank ID: 006932) using a betB deletion mutant constructed from virulent B. abortus 544. In test under stress conditions, including osmotic- and acid stress-resistance, the betB mutant had a lower osmotic-resistance than B. abortus wild-type. In addition, the betB mutant showed higher internalization rates compared to the wild-type strain; however, it also displayed replication failures in HeLa cells and RAW 264.7 macrophages. During internalization, compared to the wild-type strain, the betB mutant was more adherent to the host surface and showed enhanced phosphorylation of protein kinases, two processes that promote phagocytic activity, in host cells. During intracellular trafficking, colocalization of B. abortus-containing phagosomes with LAMP-1 was elevated in betB mutant-infected cells compared to the wild-type cells. In mice, the betB mutant was predominantly cleared from spleens compared to the wild-type strain after 2 weeks post-infection, and the vaccination test with the live betB mutant showed effective protection against challenge infection with the virulent wild-type strain. These findings suggested that the B. abortus betB gene substantially affects the phagocytic pathway in human phagocytes and in host cells in mice. Furthermore, this study highlights the potential use of the B. abortus betB mutant as a live vaccine for the control of brucellosis.

  11. Identification and regional localization of a human IMP dehydrogenase-like locus (IMPHDL1) at 16p13. 13

    SciTech Connect

    Doggett, N.A.; Tesmer, J.G.; Duesing, L.A. ); Callen, D.F.; Chen, Z.L.; Moore, S. ); Stallings, R.L. )

    1993-12-01

    Sequence-tagged sites (STS)s are versatile chromosomal markers for a variety of genome mapping efforts. In this report, the authors describe a randomly generated STS (323F4) from human chromosome 16 genomic DNA that has 90.0% sequence identity to the type I human inosine-5[prime]-monophosphate dehydrogenase (IMPDH1) gene and 72% identity to the type II human inosine-5[prime]-monophosphate dehydrogenase (IMPDH2) gene. Additional sequencing by primer walking has provided a total of 1380 bp of the human chromosome 16 sequence. The IMPDH-like sequence 323F4 was regionally localized by PCR analysis of a panel of somatic cell hybrids containing different portions of human chromosome 16 to 16p13.3-13.12, between the breakpoints found in hybrids CY196/CY197 and CY198. This regional mapping assignment was further refined to subband 16p13.3 by high-resolution fluorescence in situ hybridization using cosmid 323F4 as a probe. The authors conclude that a third, previously undescribed IMPDH locus, termed IMPDHL1, exists at human chromosome 16p13.13. 11 refs., 2 figs.

  12. Glyceraldehyde 3-phosphate dehydrogenase is bound to the fibrous sheath of mammalian spermatozoa.

    PubMed

    Westhoff, D; Kamp, G

    1997-08-01

    Evidence is provided that the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase is covalently linked to the fibrous sheath. The fibrous sheath is a typical structure of mammalian spermatozoa surrounding the axoneme in the principal piece of the flagellum. More than 90% of boar sperm glyceraldehyde 3-phosphate dehydrogenase activity is sedimented after cell disintegration by centrifugation. Detergents, different salt concentrations or short term incubation with chymotrypsin do not solubilize the enzyme, whereas digestion with trypsin or elastase does. Short term incubation with trypsin (15 minutes) even resulted in an activation of glyceraldehyde 3-phosphate dehydrogenase. Purification on phenyl-Sepharose yielded a homogeneous glyceraldehyde 3-phosphate dehydrogenase as judged from gel electrophoresis SDS-PAGE and native gradient PAGE. The molecular masses are 41.5 and 238 kDa, respectively, suggesting native glyceraldehyde 3-phosphate dehydrogenase to be a hexamer. Rabbit polyclonal antibodies raised to purified glyceraldehyde 3-phosphate dehydrogenase show a high specificity for mammalian spermatozoal glyceraldehyde 3-phosphate dehydrogenase, while other proteins of boar spermatozoa or the muscle glyceraldehyde 3-phosphate dehydrogenase are not labelled. Immunogold staining performed in a post-embedding procedure reveals the localization of glyceraldehyde 3-phosphate dehydrogenase along the fibrous sheath in spermatozoa of boar, bull, rat, stallion and man. Other structures such as the cell membrane, dense fibres, the axoneme or the mitochondria are free of label. During the process of sperm maturation, most of the cytoplasm of the sperm midpiece is removed as droplets during the passage through the epididymis. The labelling of this cytoplasm, in immature boar spermatozoa and in the droplets, indicates that glyceraldehyde 3-phosphate dehydrogenase is completely removed from the midpiece during sperm maturation in the epididymis. The inverse

  13. Affinity chromatography of the Neurospora NADP-specific glutamate dehydrogenase, its mutational variants and hybrid hexamers.

    PubMed Central

    Watson, D H; Wootton, J C

    1977-01-01

    The synthesis of an affinity adsorbent, 8-(6-aminohexyl)aminoadenosine 2'-phosphate-Sepharose 4B, is described. The assembly of the 2'-AMP ligand and the hexanediamide spacer arm was synthesized in free solution before its attachment to the Sepharose matrix. This adsorbent retarded the hexameric NADP-specific glutamate dehydrogenase of Neurospora crassa, showing a capacity for this enzyme similar to that of comparable coenzyme-analogue adsorbents for other dehydrogenases. The enzyme was eluted either at pH 6.8 in a concentration gradient of NADP+, or at pH 8.5 in the presence of NADP+ in concentration gradients of either dicarboxylates or NaCl. Anomalous effects of dicarboxylates in facilitating elution are discussed. 2'-AMP and its derivatives, 8-bromoadenosine 2'-phosphate and 8-(l-aminohexyl)aminoadenosine 2'-phosphate, which were used in the synthesis of the adsorbent, all acted as enzyme inhibitors competitive with NADP+. The chromatographic properties of the wild-type enzyme were compared with those of mutationally modified variants containing defined amino acid substitutions. This approach was used to assess the biospecificity of adsorption and elution and the contribution of non-specific binding. The adsorbent showed a low capacity for the enzyme from mutant am1 (Ser-336 replaced by Phe), a variant that has a localized defect in NADP binding, but an otherwise almost normal conformation, suggesting that non-specific interactions are at most weak. The enzyme from mutant am3, a variant modified in a conformational equilibrium, was fully retarded by the adsorbent, but showed a significantly earlier elution position than the wild-type enzyme. This is consistent with measurements in free solution that showed the am3 enzyme to have a higher Ki for 2'-AMP than the wild-type enzyme. The enzyme from mutant am19 was eluted as two distinct peaks at both pH 6.8 and 8.5. The adsorbent was used to separate hybrid hexamers constructed in vitro by a freeze-thaw procedure

  14. Context of action of Proline Dehydrogenase (ProDH) in the Hypersensitive Response of Arabidopsis

    PubMed Central

    2014-01-01

    Background Proline (Pro) dehydrogenase (ProDH) potentiates the oxidative burst and cell death of the plant Hypersensitive Response (HR) by mechanisms not yet elucidated. ProDH converts Pro into ∆1 pyrroline-5-carboxylate (P5C) and can act together with P5C dehydrogenase (P5CDH) to produce Glu, or with P5C reductase (P5CR) to regenerate Pro and thus stimulate the Pro/P5C cycle. To better understand the effects of ProDH in HR, we studied the enzyme at three stages of the defense response differing in their ROS and cell death levels. In addition, we tested if ProDH requires P5CDH to potentiate HR. Results Control and infected leaves of wild type and p5cdh plants were used to monitor ProDH activity, in vivo Pro catabolism, amino acid content, and gene expression. Wild type plants activated ProDH at all HR stages. They did not consume Pro during maximal ROS accumulation, and maintained almost basal P5C levels at all conditions. p5cdh mutants activated ProDH as wild type plants. They achieved maximum oxidative burst and cell death levels producing normal HR lesions, but evidenced premature defense activation. Conclusion ProDH activation has different effects on HR. Before the oxidative burst it leads to Pro consumption involving the action of P5CDH. During the oxidative burst, ProDH becomes functionally uncoupled to P5CDH and apparently works with P5CR. The absence of P5CDH does not reduce ROS, cell death, or pathogen resistance, indicating this enzyme is not accompanying ProDH in the potentiation of these defense responses. In contrast, p5cdh infected plants displayed increased ROS burst and earlier initiation of HR cell death. In turn, our results suggest that ProDH may sustain HR by participating in the Pro/P5C cycle, whose action on HR must be formally evaluated in a future. PMID:24410747

  15. Protein-mediated assembly of succinate dehydrogenase and its cofactors.

    PubMed

    Van Vranken, Jonathan G; Na, Un; Winge, Dennis R; Rutter, Jared

    2015-01-01

    Succinate dehydrogenase (or complex II; SDH) is a heterotetrameric protein complex that links the tribarboxylic acid cycle with the electron transport chain. SDH is composed of four nuclear-encoded subunits that must translocate independently to the mitochondria and assemble into a mature protein complex embedded in the inner mitochondrial membrane. Recently, it has become clear that failure to assemble functional SDH complexes can result in cancer and neurodegenerative syndromes. The effort to thoroughly elucidate the SDH assembly pathway has resulted in the discovery of four subunit-specific assembly factors that aid in the maturation of individual subunits and support the assembly of the intact complex. This review will focus on these assembly factors and assess the contribution of each factor to the assembly of SDH. Finally, we propose a model of the SDH assembly pathway that incorporates all extant data.

  16. The reaction of choline dehydrogenase with some electron acceptors.

    PubMed Central

    Barrett, M C; Dawson, A P

    1975-01-01

    1. The choline dehydrogenase (EC 1.1.99.1) WAS SOLUBILIZED FROM ACETONE-DRIED POWDERS OF RAT LIVER MITOCHONDRIA BY TREATMENT WITH Naja naja venom. 2. The kinetics of the reaction of enzyme with phenazine methosulphate and ubiquinone-2 as electron acceptors were investigated. 3. With both electron acceptors the reaction mechanism appears to involve a free, modified-enzyme intermediate. 4. With some electron acceptors the maximum velocity of the reaction is independent of the nature of the acceptor. With phenazine methosulphate and ubiquinone-2 as acceptors the Km value for choline is also independent of the nature of the acceptor molecule. 5. The mechanism of the Triton X-100-solubilized enzyme is apparently the smae as that for the snake venom solubilized enzyme. PMID:1218095

  17. The reaction of choline dehydrogenase with some electron acceptors.

    PubMed

    Barrett, M C; Dawson, A P

    1975-12-01

    1. The choline dehydrogenase (EC 1.1.99.1) WAS SOLUBILIZED FROM ACETONE-DRIED POWDERS OF RAT LIVER MITOCHONDRIA BY TREATMENT WITH Naja naja venom. 2. The kinetics of the reaction of enzyme with phenazine methosulphate and ubiquinone-2 as electron acceptors were investigated. 3. With both electron acceptors the reaction mechanism appears to involve a free, modified-enzyme intermediate. 4. With some electron acceptors the maximum velocity of the reaction is independent of the nature of the acceptor. With phenazine methosulphate and ubiquinone-2 as acceptors the Km value for choline is also independent of the nature of the acceptor molecule. 5. The mechanism of the Triton X-100-solubilized enzyme is apparently the smae as that for the snake venom solubilized enzyme.

  18. The antibiotic potential of prokaryotic IMP dehydrogenase inhibitors

    PubMed Central

    Hedstrom, Lizbeth; Liechti, George; Goldberg, Joanna B.; Gollapalli, Deviprasad R.

    2016-01-01

    Inosine 5′-monophosphate dehydrogenase (IMPDH) catalyzes the first committed step of guanosine 5′-monophosphate (GMP) biosynthesis, and thus regulates the guanine nucleotide pool, which in turn governs proliferation. Human IMPDHs are validated targets for immunosuppressive, antiviral and anticancer drugs, but as yet microbial IMPDHs have not been exploited in antimicrobial chemotherapy. Selective inhibitors of IMPDH from Cryptosporidium parvum have recently been discovered that display anti-parasitic activity in cell culture models of infection. X-ray crystal structure and mutagenesis experiments identified the structural features that determine inhibitor susceptibility. These features are found in IMPDHs from a wide variety of pathogenic bacteria, including select agents and multiply drug resistant strains. A second generation inhibitor displays antibacterial activity against Helicobacter pylori, demonstrating the antibiotic potential of IMPDH inhibitors. PMID:21517780

  19. [Sorbitol-6-Phosphate Dehydrogenase Gene Polymorhism in Malus Mill. (Rosaceae)].

    PubMed

    Boris, K V; Kudryavtsev, A M; Kochieva, E Z

    2015-11-01

    The sorbitol-6-phosphate dehydrogenase gene (S6PDH) sequences of six representatives of the genus Malus, which belong to five different taxonomic sections, were examined for the first time. The exon-intron structure and polymorphism of the nucleotide and amino acid sequences of these genes was characterized. The intraspecific polymorphism of the S6PDH gene was assessed for the first time in 40 Russian and foreign apple (Malus domestica) cultivars. It was demonstrated that the interspecific polymorphism level of the S6PDH coding sequences in the studied. representatives of the genus Malus was 4%, and the intraspecific polymorphism level of M. domestica cultivars was very low, constituting 0.96%. PMID:26845854

  20. Engineered PQQ-Glucose Dehydrogenase as a Universal Biosensor Platform.

    PubMed

    Guo, Zhong; Murphy, Lindy; Stein, Viktor; Johnston, Wayne A; Alcala-Perez, Siro; Alexandrov, Kirill

    2016-08-17

    Biosensors with direct electron output hold promise for nearly seamless integration with portable electronic devices. However, so far, they have been based on naturally occurring enzymes that significantly limit the spectrum of detectable analytes. Here, we present a novel biosensor architecture based on analyte-driven intermolecular recombination and activity reconstitution of a re-engineered component of glucometers: PQQ-glucose dehydrogenase. We demonstrate that this sensor architecture can be rapidly adopted for the detection of immunosuppressant drugs, α-amylase protein, or protease activity of thrombin and Factor Xa. The biosensors could be stored in dried form without appreciable loss of activity. We further show that ligand-induced activity of the developed biosensors could be directly monitored by chronoamperometry, enabling construction of disposable sensory electrodes. We expect that this architecture could be expanded to the detection of other biochemical activities, post-translational modifications, nucleic acids, and inorganic molecules.

  1. Fabricating polystyrene fiber-dehydrogenase assemble as a functional biocatalyst.

    PubMed

    An, Hongjie; Jin, Bo; Dai, Sheng

    2015-01-01

    Immobilization of the enzymes on nano-structured materials is a promising approach to enhance enzyme stabilization, activation and reusability. This study aimed to develop polystyrene fiber-enzyme assembles to catalyze model formaldehyde to methanol dehydrogenation reaction, which is an essential step for bioconversion of CO2 to a renewable bioenergy. We fabricated and modified electrospun polystyrene fibers, which showed high capability to immobilize dehydrogenase for the fiber-enzyme assembles. Results from evaluation of biochemical activities of the fiber-enzyme assemble showed that nitriation with the nitric/sulfuric acid ratio (v/v, 10:1) and silanization treatment delivered desirable enzyme activity and long-term storage stability, showing great promising toward future large-scale applications. PMID:25435501

  2. IMP Dehydrogenase: Structural Schizophrenia and an Unusual Base

    SciTech Connect

    Hedstrom,L.; Gan, L.

    2006-01-01

    Textbooks describe enzymes as relatively rigid templates for the transition state of a chemical reaction, and indeed an enzyme such as chymotrypsin, which catalyzes a relatively simple hydrolysis reaction, is reasonably well described by this model. Inosine monophosphate dehydrogenase (IMPDH) undergoes a remarkable array of conformational transitions in the course of a complicated catalytic cycle, offering a dramatic counterexample to this view. IMPDH displays several other unusual mechanistic features, including an Arg residue that may act as a general base catalyst and a dynamic monovalent cation site. Further, IMPDH appears to be involved in 'moon-lighting' functions that may require additional conformational states. How the balance between conformational states is maintained and how the various conformational states interconvert is only beginning to be understood.

  3. Method To Identify Specific Inhibiutors Of Imp Dehydrogenase

    DOEpatents

    Collart, Frank R.; Huberman, Eliezer

    2000-11-28

    This invention relates to methods to identify specific inhibitors of the purine nucleotide synthesis enzyme, IMP dehydrogenase (IMPDH). IMPDH is an essential enzyme found in all free-living organisms from humans to bacteria and is an important therapeutic target. The invention allows the identification of specific inhibitors of any IMPDH enzyme which can be expressed in a functional form in a recombinant host cell. A variety of eukaryotic or prokaryotic host systems commonly used for the expression of recombinant proteins are suitable for the practice of the invention. The methods are amenable to high throughput systems for the screening of inhibitors generated by combinatorial chemistry or other methods such as antisense molecule production. Utilization of exogenous guanosine as a control component of the methods allows for the identification of inhibitors specific for IMPDH rather than other causes of decreased cell proliferation.

  4. The role of Pyruvate Dehydrogenase Complex in cardiovascular diseases.

    PubMed

    Sun, Wanqing; Liu, Quan; Leng, Jiyan; Zheng, Yang; Li, Ji

    2015-01-15

    The regulation of mammalian myocardial carbohydrate metabolism is complex; many factors such as arterial substrate and hormone levels, coronary flow, inotropic state and the nutritional status of the tissue play a role in regulating mammalian myocardial carbohydrate metabolism. The Pyruvate Dehydrogenase Complex (PDHc), a mitochondrial matrix multienzyme complex, plays an important role in energy homeostasis in the heart by providing the link between glycolysis and the tricarboxylic acid (TCA) cycle. In TCA cycle, PDHc catalyzes the conversion of pyruvate into acetyl-CoA. This review determines that there is altered cardiac glucose in various pathophysiological states consequently causing PDC to be altered. This review further summarizes evidence for the metabolism mechanism of the heart under normal and pathological conditions including ischemia, diabetes, hypertrophy and heart failure.

  5. [Sorbitol-6-Phosphate Dehydrogenase Gene Polymorhism in Malus Mill. (Rosaceae)].

    PubMed

    Boris, K V; Kudryavtsev, A M; Kochieva, E Z

    2015-11-01

    The sorbitol-6-phosphate dehydrogenase gene (S6PDH) sequences of six representatives of the genus Malus, which belong to five different taxonomic sections, were examined for the first time. The exon-intron structure and polymorphism of the nucleotide and amino acid sequences of these genes was characterized. The intraspecific polymorphism of the S6PDH gene was assessed for the first time in 40 Russian and foreign apple (Malus domestica) cultivars. It was demonstrated that the interspecific polymorphism level of the S6PDH coding sequences in the studied. representatives of the genus Malus was 4%, and the intraspecific polymorphism level of M. domestica cultivars was very low, constituting 0.96%.

  6. Microbial metabolic activity in soil as measured by dehydrogenase determinations

    NASA Technical Reports Server (NTRS)

    Casida, L. E., Jr.

    1977-01-01

    The dehydrogenase technique for measuring the metabolic activity of microorganisms in soil was modified to use a 6-h, 37 C incubation with either glucose or yeast extract as the electron-donating substrate. The rate of formazan production remained constant during this time interval, and cellular multiplication apparently did not occur. The technique was used to follow changes in the overall metabolic activities of microorganisms in soil undergoing incubation with a limiting concentration of added nutrient. The sequence of events was similar to that obtained by using the Warburg respirometer to measure O2 consumption. However, the major peaks of activity occurred earlier with the respirometer. This possibly is due to the lack of atmospheric CO2 during the O2 consumption measurements.

  7. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Alzheimer's disease.

    PubMed

    El Kadmiri, N; Slassi, I; El Moutawakil, B; Nadifi, S; Tadevosyan, A; Hachem, A; Soukri, A

    2014-12-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous enzyme that catalyzes the sixth step of glycolysis and thus, serves to break down glucose for energy production. Beyond the traditional aerobic metabolism of glucose, recent studies have highlighted additional roles played by GAPDH in non-metabolic processes, such as control of gene expression and redox post-translational modifications. Neuroproteomics have revealed high affinity interactions between GAPDH and Alzheimer's disease-associated proteins, including the β-amyloid, β-amyloid precursor protein and tau. This neuronal protein interaction may lead to impairment of the GAPDH glycolytic function in Alzheimer's disease and may be a forerunner of its participation in apoptosis. The present review examines the crucial implication of GAPDH in neurodegenerative processes and clarifies its role in apoptotic cell death.

  8. Engineered PQQ-Glucose Dehydrogenase as a Universal Biosensor Platform.

    PubMed

    Guo, Zhong; Murphy, Lindy; Stein, Viktor; Johnston, Wayne A; Alcala-Perez, Siro; Alexandrov, Kirill

    2016-08-17

    Biosensors with direct electron output hold promise for nearly seamless integration with portable electronic devices. However, so far, they have been based on naturally occurring enzymes that significantly limit the spectrum of detectable analytes. Here, we present a novel biosensor architecture based on analyte-driven intermolecular recombination and activity reconstitution of a re-engineered component of glucometers: PQQ-glucose dehydrogenase. We demonstrate that this sensor architecture can be rapidly adopted for the detection of immunosuppressant drugs, α-amylase protein, or protease activity of thrombin and Factor Xa. The biosensors could be stored in dried form without appreciable loss of activity. We further show that ligand-induced activity of the developed biosensors could be directly monitored by chronoamperometry, enabling construction of disposable sensory electrodes. We expect that this architecture could be expanded to the detection of other biochemical activities, post-translational modifications, nucleic acids, and inorganic molecules. PMID:27463000

  9. Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Hong, Y. S.; Ciszak, Ewa; Patel, Mulchand

    2000-01-01

    Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For

  10. Xanthine Dehydrogenase Is Transported to the Drosophila Eye

    PubMed Central

    Reaume, A. G.; Clark, S. H.; Chovnick, A.

    1989-01-01

    The rosy (ry) locus in Drosophila melanogaster codes for the enzyme xanthine dehydrogenase. Mutants that have no enzyme activity are characterized by a brownish eye color phenotype reflecting a deficiency in the red eye pigment. This report demonstrates that enzyme which is synthesized in some tissue other than the eye is transported and sequestered at the eye. Previous studies find that no leader sequence is associated with this molecule but a peroxisomal targeting sequence has been noted, and the enzyme has been localized to peroxisomes. This represents a rare example of an enzyme involved in intermediary metabolism being transported from one tissue to another and may also be the first example of a peroxisomal protein being secreted from a cell. PMID:2513252

  11. Purification and characterization of aldehyde dehydrogenase from rat liver mitochondria.

    PubMed

    Senior, D J; Tsai, C S

    1988-04-01

    Nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-dependent dehydrogenase activities from rat liver mitochondria have been copurified to homogeneity using combined DEAE, Sepharose, and affinity chromatographic procedures. The enzyme has a native molecular weight of 240,000 and subunit molecular weight of 60,000. The enzyme is tetrameric consisting of four identical subunits as revealed by electrophoresis and terminal analyses. A partial summary of physical properties is provided. The amino acid composition by acid hydrolysis is reported. Specific activities for various NAD(P)+ analogs and alkanal substrates were compared. The action of the effectors chloral hydrate, disulfiram, diethylstilbestrol, and Mg2+ and K+ ions were also investigated. PMID:3355167

  12. Involvement of snapdragon benzaldehyde dehydrogenase in benzoic acid biosynthesis.

    PubMed

    Long, Michael C; Nagegowda, Dinesh A; Kaminaga, Yasuhisa; Ho, Kwok Ki; Kish, Christine M; Schnepp, Jennifer; Sherman, Debra; Weiner, Henry; Rhodes, David; Dudareva, Natalia

    2009-07-01

    Benzoic acid (BA) is an important building block in a wide spectrum of compounds varying from primary metabolites to secondary products. Benzoic acid biosynthesis from L-phenylalanine requires shortening of the propyl side chain by two carbons, which can occur via a beta-oxidative pathway or a non-beta-oxidative pathway, with benzaldehyde as a key intermediate. The non-beta-oxidative route requires benzaldehyde dehydrogenase (BALDH) to convert benzaldehyde to BA. Using a functional genomic approach, we identified an Antirrhinum majus (snapdragon) BALDH, which exhibits 40% identity to bacterial BALDH. Transcript profiling, biochemical characterization of the purified recombinant protein, molecular homology modeling, in vivo stable isotope labeling, and transient expression in petunia flowers reveal that BALDH is capable of oxidizing benzaldehyde to BA in vivo. GFP localization and immunogold labeling studies show that this biochemical step occurs in the mitochondria, raising a question about the role of subcellular compartmentalization in BA biosynthesis.

  13. Benzaldehyde dehydrogenase from chitosan-treated Sorbus aucuparia cell cultures.

    PubMed

    Gaid, Mariam M; Sircar, Debabrata; Beuerle, Till; Mitra, Adinpunya; Beerhues, Ludger

    2009-09-01

    Cell cultures of Sorbus aucuparia respond to the addition of chitosan with the accumulation of the biphenyl phytoalexin aucuparin. The carbon skeleton of this inducible defense compound is formed by biphenyl synthase (BIS) from benzoyl-CoA and three molecules of malonyl-CoA. The formation of benzoyl-CoA proceeds via benzaldehyde as an intermediate. Benzaldehyde dehydrogenase (BD), which converts benzaldehyde into benzoic acid, was detected in cell-free extracts from S. aucuparia cell cultures. BD and BIS were induced by chitosan treatment. The preferred substrate for BD was benzaldehyde (K(m)=49 microM). Cinnamaldehyde and various hydroxybenzaldehydes were relatively poor substrates. BD activity was strictly dependent on the presence of NAD(+) as a cofactor (K(m)=67 microM).

  14. [Effect Of Polyelectrolytes on Catalytic Activity of Alcohol Dehydrogenase].

    PubMed

    Dubrovsky, A V; Musina, E V; Kim, A L; Tikhonenko, S A

    2016-01-01

    Fluorescent and optical spectroscopy were used to study the interaction of alcohol dehydrogenase (ADH) with negatively charged polystyrene sulfonate (PSS) and dextran sulfate (DS), as well as positively charged poly(diallyldimethylammonium) (PDADMA). As found, DS and PDADMA did not affect the structural and catalytic enzyme properties. In contrast, PSS slightly decreased the protein self-fluorescence over 1 h of incubation, which is associated with partial destruction of its quaternary (globular) structure. Investigation of the ADH activity with and without PSS showed its dependency on the incubation time and the PSS presence. Sodium chloride (2.0 M and 0.2 M) or ammonium sulfate (0.1 M) added to the reaction mixture did not completely protect the enzyme quaternary structure from the PSS action. However ammonium sulfate or 0.2 M sodium chloride stabilized the enzyme and partially inhibited the negative PSS effect. PMID:27266256

  15. Mechanistic enzymology of CO dehydrogenase from Clostridium thermoaceticum

    SciTech Connect

    Ragsdale, S.W.

    1992-01-01

    The final steps in acetyl-CoA biosynthesis by anaerobic bacteria are performed by carbon monoxide dehydrogenase (CODH), a nickel/iron-sulfur protein. An important achievement was to establish conditions under which acetyl-CoA synthesis by purified enzymes equals the in vivo rate of acetate synthesis. Under these optimized conditions we established that the rate limiting step in the synthesis of acetyl-CoA from methyl-H[sub 4]folate, CO and CoA is likely to be the methylation of CODH by the methylated corrinoid/iron-sulfur protein. We then focused on stopped flow studies of this rate limiting transmethylation reaction and established its mechanism. We have studied the carbonylation of CODH by infrared and resonance Raman spectroscopy and determined that the [Ni-Fe[sup 3-4]S[sub 4

  16. Inhibitor-bound structures of human pyruvate dehydrogenase kinase 4.

    PubMed

    Kukimoto-Niino, Mutsuko; Tokmakov, Alexander; Terada, Takaho; Ohbayashi, Naomi; Fujimoto, Takako; Gomi, Sumiko; Shiromizu, Ikuya; Kawamoto, Masaki; Matsusue, Tomokazu; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2011-09-01

    The mitochondrial pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. PDC activity is tightly regulated by four members of a family of pyruvate dehydrogenase kinase isoforms (PDK1-4), which phosphorylate and inactivate PDC. Recently, the development of specific inhibitors of PDK4 has become an especially important focus for the pharmaceutical management of diabetes and obesity. In this study, crystal structures of human PDK4 complexed with either AMPPNP, ADP or the inhibitor M77976 were determined. ADP-bound PDK4 has a slightly wider active-site cleft and a more disordered ATP lid compared with AMPPNP-bound PDK4, although both forms of PDK4 assume open conformations with a wider active-site cleft than that in the closed conformation of the previously reported ADP-bound PDK2 structure. M77976 binds to the ATP-binding pocket of PDK4 and causes local conformational changes with complete disordering of the ATP lid. M77976 binding also leads to a large domain rearrangement that further expands the active-site cleft of PDK4 compared with the ADP- and AMPPNP-bound forms. Biochemical analyses revealed that M77976 inhibits PDK4 with increased potency compared with the previously characterized PDK inhibitor radicicol. Thus, the present structures demonstrate for the first time the flexible and dynamic aspects of PDK4 in the open conformation and provide a basis for the development of novel inhibitors targeting the nucleotide-binding pocket of PDK4. PMID:21904029

  17. Recommended nomenclature for the vertebrate alcohol dehydrogenase gene family.

    PubMed

    Duester, G; Farrés, J; Felder, M R; Holmes, R S; Höög, J O; Parés, X; Plapp, B V; Yin, S J; Jörnvall, H

    1999-08-01

    The alcohol dehydrogenase (ADH) gene family encodes enzymes that metabolize a wide variety of substrates, including ethanol, retinol, other aliphatic alcohols, hydroxysteroids, and lipid peroxidation products. Studies on 19 vertebrate animals have identified ADH orthologs across several species, and this has now led to questions of how best to name ADH proteins and genes. Seven distinct classes of vertebrate ADH encoded by non-orthologous genes have been defined based upon sequence homology as well as unique catalytic properties or gene expression patterns. Each class of vertebrate ADH shares <70% sequence identity with other classes of ADH in the same species. Classes may be further divided into multiple closely related isoenzymes sharing >80% sequence identity such as the case for class I ADH where humans have three class I ADH genes, horses have two, and mice have only one. Presented here is a nomenclature that uses the widely accepted vertebrate ADH class system as its basis. It follows the guidelines of human and mouse gene nomenclature committees, which recommend coordinating names across species boundaries and eliminating Roman numerals and Greek symbols. We recommend that enzyme subunits be referred to by the symbol "ADH" (alcohol dehydrogenase) followed by an Arabic number denoting the class; i.e. ADH1 for class I ADH. For genes we recommend the italicized root symbol "ADH" for human and "Adh" for mouse, followed by the appropriate Arabic number for the class; i.e. ADH1 or Adh1 for class I ADH genes. For organisms where multiple species-specific isoenzymes exist within a class, we recommend adding a capital letter after the Arabic number; i.e. ADH1A, ADH1B, and ADH1C for human alpha, beta, and gamma class I ADHs, respectively. This nomenclature will accommodate newly discovered members of the vertebrate ADH family, and will facilitate functional and evolutionary studies. PMID:10424757

  18. STRUCTURE AND KINETICS OF MONOFUNCTIONAL PROLINE DEHYDROGENASE FROM THERMUS THERMOPHILUS

    PubMed Central

    White, Tommi A.; Krishnan, Navasona; Becker, Donald F.; Tanner, John J.

    2009-01-01

    Proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) catalyze the two-step oxidation of proline to glutamate. They are distinct monofunctional enzymes in all eukaryotes and some bacteria, but are fused into bifunctional enzymes known as Proline utilization A (PutA) in other bacteria. Here we report the first structure and biochemical data for a monofunctional PRODH. The 2.0 Å resolution structure of Thermus thermophilus PRODH reveals a distorted (βα)8 barrel catalytic core domain and a hydrophobic α-helical domain located above the carboxyl terminal ends of the strands of the barrel. Although the catalytic core is similar to that of the PutA PRODH domain, the FAD conformation of T. thermophilus PRODH is remarkably different and likely reflects unique requirements for membrane association and communication with P5CDH. Also, the FAD of T. thermophilus PRODH is highly solvent exposed compared to PutA due to a 4-Å shift of helix 8. Structure-based sequence analysis of the PutA/PRODH family led us to identify 9 conserved motifs involved in cofactor and substrate recognition. Biochemical studies show that the midpoint potential of the FAD is −75 mV and the kinetic parameters for proline are Km=27 mM and kcat=13 s−1. 3,4-dehydro-L-proline was found to be an efficient substrate and L-tetrahydro-2-furoic acid is a competitive inhibitor (KI=1.0 mM). Finally, we demonstrate that T. thermophilus PRODH reacts with O2 producing superoxide. This is significant because superoxide production underlies the role of human PRODH in p53-mediated apoptosis, implying commonalities between eukaryotic and bacterial monofunctional PRODHs. PMID:17344208

  19. Lactate dehydrogenase concentration in nasal wash fluid indicates severity of rhinovirus-induced wheezy bronchitis in preschool children.

    PubMed

    Cangiano, Giulia; Proietti, Elena; Kronig, Marie Noelle; Kieninger, Elisabeth; Sadeghi, Christine D; Gorgievski, Meri; Barbani, Maria Teresa; Midulla, Fabio; Tapparel, Caroline; Kaiser, Laurent; Alves, Marco P; Regamey, Nicolas

    2014-12-01

    The clinical course of rhinovirus (RV)-associated wheezing illnesses is difficult to predict. We measured lactate dehydrogenase concentrations, RV load, antiviral and proinflammatory cytokines in nasal washes obtained from 126 preschool children with RV wheezy bronchitis. lactate dehydrogenase values were inversely associated with subsequent need for oxygen therapy. lactate dehydrogenase may be a useful biomarker predicting disease severity in RV wheezy bronchitis.

  20. Determining structure and function of steroid dehydrogenase enzymes by sequence analysis, homology modeling, and rational mutational analysis.

    PubMed

    Duax, William L; Thomas, James; Pletnev, Vladimir; Addlagatta, Anthony; Huether, Robert; Habegger, Lukas; Weeks, Charles M

    2005-12-01

    The short-chain oxidoreductase (SCOR) family of enzymes includes over 6,000 members identified in sequenced genomes. Of these enzymes, approximately 300 have been characterized functionally, and the three-dimensional crystal structures of approximately 40 have been reported. Since some SCOR enzymes are steroid dehydrogenases involved in hypertension, diabetes, breast cancer, and polycystic kidney disease, it is important to characterize the other members of the family for which the biological functions are currently unknown and to determine their three-dimensional structure and mechanism of action. Although the SCOR family appears to have only a single fully conserved residue, it was possible, using bioinformatics methods, to determine characteristic fingerprints composed of 30-40 residues that are conserved at the 70% or greater level in SCOR subgroups. These fingerprints permit reliable prediction of several important structure-function features including cofactor preference, catalytic residues, and substrate specificity. Human type 1 3beta-hydroxysteroid dehydrogenase isomerase (3beta-HSDI) has 30% sequence identity with a human UDP galactose 4-epimerase (UDPGE), a SCOR family enzyme for which an X-ray structure has been reported. Both UDPGE and 3-HSDI appear to trace their origins back to bacterial 3alpha,20beta-HSD. Combining three-dimensional structural information and sequence data on the 3alpha,20beta-HSD, UDPGE, and 3beta-HSDI subfamilies with mutational analysis, we were able to identify the residues critical to the dehydrogenase function of 3-HSDI. We also identified the residues most probably responsible for the isomerase activity of 3beta-HSDI. We test our predictions by specific mutations based on sequence analysis and our structure-based model.

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

    PubMed Central

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

    2016-01-01

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

  2. Determining Structure and Function of Steroid Dehydrogenase Enzymes by Sequence Analysis, Homology Modeling, and Rational Mutational Analysis

    PubMed Central

    DUAX, WILLIAM L.; THOMAS, JAMES; PLETNEV, VLADIMIR; ADDLAGATTA, ANTHONY; HUETHER, ROBERT; HABEGGER, LUKAS; WEEKS, CHARLES M.

    2006-01-01

    The short-chain oxidoreductase (SCOR) family of enzymes includes over 6,000 members identified in sequenced genomes. Of these enzymes, ~300 have been characterized functionally, and the three-dimensional crystal structures of ~40 have been reported. Since some SCOR enzymes are steroid dehydrogenases involved in hypertension, diabetes, breast cancer, and polycystic kidney disease, it is important to characterize the other members of the family for which the biological functions are currently unknown and to determine their three-dimensional structure and mechanism of action. Although the SCOR family appears to have only a single fully conserved residue, it was possible, using bioinformatics methods, to determine characteristic fingerprints composed of 30–40 residues that are conserved at the 70% or greater level in SCOR subgroups. These fingerprints permit reliable prediction of several important structure-function features including cofactor preference, catalytic residues, and substrate specificity. Human type 1 3β-hydroxysteroid dehydrogenase isomerase (3β-HSDI) has 30% sequence identity with a human UDP galactose 4-epimerase (UDPGE), a SCOR family enzyme for which an X-ray structure has been reported. Both UDPGE and 3-HSDI appear to trace their origins back to bacterial 3α,20β-HSD. Combining three-dimensional structural information and sequence data on the 3α,20β-HSD, UDPGE, and 3β-HSDI subfamilies with mutational analysis, we were able to identify the residues critical to the dehydrogenase function of 3-HSDI. We also identified the residues most probably responsible for the isomerase activity of 3β-HSDI. We test our predictions by specific mutations based on sequence analysis and our structure-based model. PMID:16467263

  3. pH-dependent electron transfer reaction and direct bioelectrocatalysis of the quinohemoprotein pyranose dehydrogenase.

    PubMed

    Takeda, Kouta; Matsumura, Hirotoshi; Ishida, Takuya; Yoshida, Makoto; Igarashi, Kiyohiko; Samejima, Masahiro; Ohno, Hiroyuki; Nakamura, Nobuhumi

    2016-08-26

    A pyranose dehydrogenase from Coprinopsis cinerea (CcPDH) is an extracellular quinohemoeprotein, which consists a b-type cytochrome domain, a pyrroloquinoline-quinone (PQQ) domain, and a family 1-type carbohydrate-binding module. The electron transfer reaction of CcPDH was studied using some electron acceptors and a carbon electrode at various pH levels. Phenazine methosulfate (PMS) reacted directly at the PQQ domain, whereas cytochrome c (cyt c) reacted via the cytochrome domain of intact CcPDH. Thus, electrons are transferred from reduced PQQ in the catalytic domain of CcPDH to heme b in the N-terminal cytochrome domain, which acts as a built-in mediator and transfers electron to a heterogenous electron transfer protein. The optimal pH values of the PMS reduction (pH 6.5) and the cyt c reduction (pH 8.5) differ. The catalytic currents for the oxidation of l-fucose were observed within a range of pH 4.5 to 11. Bioelectrocatalysis of CcPDH based on direct electron transfer demonstrated that the pH profile of the biocatalytic current was similar to the reduction activity of cyt c characters.

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

    SciTech Connect

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

    1985-12-01

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

  5. Biochemical Characterization and Complete Conversion of Coenzyme Specificity of Isocitrate Dehydrogenase from Bifidobacterium longum

    PubMed Central

    Huang, Shi-Ping; Cheng, Hong-Mei; Wang, Peng; Zhu, Guo-Ping

    2016-01-01

    Bifidobacterium longum is a very important gram-positive non-pathogenic bacterium in the human gastrointestinal tract for keeping the digestive and immune system healthy. Isocitrate dehydrogenase (IDH) from B. longum (BlIDH), a novel member in Type II subfamily, was overexpressed, purified and biochemically characterized in detail. The active form of BlIDH was an 83-kDa homodimer. Kinetic analysis showed BlIDH was a NADP+-dependent IDH (NADP-IDH), with a 567- and 193-fold preference for NADP+ over NAD+ in the presence of Mg2+ and Mn2+, respectively. The maximal activity for BlIDH occurred at 60 °C (with Mn2+) and 65 °C (with Mg2+), and pH 7.5 (with Mn2+) and pH 8.0 (with Mg2+). Heat-inactivation profiles revealed that BlIDH retained 50% of maximal activity after incubation at 45 °C for 20 min with either Mn2+ or Mg2+. Furthermore, the coenzyme specificity of BlIDH can be completely reversed from NADP+ to NAD+ by a factor of 2387 by replacing six residues. This current work, the first report on the coenzyme specificity conversion of Type II NADP-IDHs, would provide better insight into the evolution of NADP+ use by the IDH family. PMID:26927087

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

    PubMed Central

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

    2014-01-01

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

  7. Biochemical Characterization and Complete Conversion of Coenzyme Specificity of Isocitrate Dehydrogenase from Bifidobacterium longum.

    PubMed

    Huang, Shi-Ping; Cheng, Hong-Mei; Wang, Peng; Zhu, Guo-Ping

    2016-02-26

    Bifidobacterium longum is a very important gram-positive non-pathogenic bacterium in the human gastrointestinal tract for keeping the digestive and immune system healthy. Isocitrate dehydrogenase (IDH) from B. longum (BlIDH), a novel member in Type II subfamily, was overexpressed, purified and biochemically characterized in detail. The active form of BlIDH was an 83-kDa homodimer. Kinetic analysis showed BlIDH was a NADP⁺-dependent IDH (NADP-IDH), with a 567- and 193-fold preference for NADP⁺ over NAD⁺ in the presence of Mg(2+) and Mn(2+), respectively. The maximal activity for BlIDH occurred at 60 °C (with Mn(2+)) and 65 °C (with Mg(2+)), and pH 7.5 (with Mn(2+)) and pH 8.0 (with Mg(2+)). Heat-inactivation profiles revealed that BlIDH retained 50% of maximal activity after incubation at 45 °C for 20 min with either Mn(2+) or Mg(2+). Furthermore, the coenzyme specificity of BlIDH can be completely reversed from NADP⁺ to NAD⁺ by a factor of 2387 by replacing six residues. This current work, the first report on the coenzyme specificity conversion of Type II NADP-IDHs, would provide better insight into the evolution of NADP⁺ use by the IDH family.

  8. Substitution of arginine for histidine-47 in the coenzyme binding site of yeast alcohol dehydrogenase I

    SciTech Connect

    Gould, R.M.; Plapp, B.V. )

    1990-06-12

    Molecular modeling of alcohol dehydrogenases suggests that His-47 in the yeast enzyme (His-44 in the protein sequence, corresponding to Arg-47 in the horse liver enzyme) binds the pyrophosphate of the NAD coenzyme. His-47 in the Saccharomyces cerevisiae isoenzyme I was substituted with an arginine by a directed mutation. Steady-state kinetic results at pH 7.3 and 30{degree}C of the mutant and wild-type enzymes were consistent with an ordered Bi-Bi mechanism. The substitution decreased dissociation constants by 4-fold for NAD{sup +} and 2-fold for NADH while turnover numbers were decreased by 4-fold for ethanol oxidation and 6-fold for acetaldehyde reduction. The magnitudes of these effects are smaller than those found for the same mutation in the human liver {beta} enzyme, suggesting that other amino acid residues in the active site modulate the effects of the substitution. The pH dependencies of dissociation constants and other kinetic constants were similar in the two yeast enzymes. Thus, it appears that His-47 is not solely responsible for a pK value near 7 that controls activity and coenzyme binding rates in the wild-type enzyme. The small substrate deuterium isotope effect above pH 7 and the single exponential phase of NADH production during the transient oxidation of ethanol by the Arg-47 enzyme suggest that the mutation makes an isomerization of the enzyme-NAD{sup +} complex limiting for turnover with ethanol.

  9. Surface modification of silicon dioxide, silicon nitride and titanium oxynitride for lactate dehydrogenase immobilization.

    PubMed

    Saengdee, Pawasuth; Chaisriratanakul, Woraphan; Bunjongpru, Win; Sripumkhai, Witsaroot; Srisuwan, Awirut; Jeamsaksiri, Wutthinan; Hruanun, Charndet; Poyai, Amporn; Promptmas, Chamras

    2015-05-15

    Three different types of surface, silicon dioxide (SiO2), silicon nitride (Si3N4), and titanium oxynitride (TiON) were modified for lactate dehydrogenase (LDH) immobilization using (3-aminopropyl)triethoxysilane (APTES) to obtain an amino layer on each surface. The APTES modified surfaces can directly react with LDH via physical attachment. LDH can be chemically immobilized on those surfaces after incorporation with glutaraldehyde (GA) to obtain aldehyde layers of APTES-GA modified surfaces. The wetting properties, chemical bonding composition, and morphology of the modified surface were determined by contact angle (CA) measurement, Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM), respectively. In this experiment, the immobilized protein content and LDH activity on each modified surface was used as an indicator of surface modification achievement. The results revealed that both the APTES and APTES-GA treatments successfully link the LDH molecule to those surfaces while retaining its activity. All types of tested surfaces modified with APTES-GA gave better LDH immobilizing efficiency than APTES, especially the SiO2 surface. In addition, the SiO2 surface offered the highest LDH immobilization among tested surfaces, with both APTES and APTES-GA modification. However, TiON and Si3N4 surfaces could be used as alternative candidate materials in the preparation of ion-sensitive field-effect transistor (ISFET) based biosensors, including lactate sensors using immobilized LDH on the ISFET surface.

  10. A Putative Hydroxysteroid Dehydrogenase Involved in Regulating Plant Growth and Development1

    PubMed Central

    Li, Fengling; Asami, Tadao; Wu, Xianzhong; Tsang, Edward W.T.; Cutler, Adrian J.

    2007-01-01

    We have functionally characterized an Arabidopsis (Arabidopsis thaliana) gene AtHSD1 (At5g50600) that encodes a protein with homology to animal 11-β-hydroxysteroid dehydrogenase (HSD). Transgenic Arabidopsis plants overexpressing AtHSD1 (designated AOHSD plants) under the control of the cauliflower mosaic virus 35S promoter showed increased growth and seed yield as well as increased tolerance of saline stress and reduced seed dormancy. In canola (Brassica napus), transgenic plants overexpressing AtHSD1 also outgrew wild-type plants. AOHSD phenotypes were similar to those of plants that overproduced brassinosteroids (BRs) or overexpressed the BR receptor gene BRI1. A loss-of-function hsd mutant produced by RNA interference displayed a semidwarfed phenotype with reduced sensitivity to BRs. In contrast, AOHSD plants were hypersensitive to BRs and exhibited increased catabolism of abscisic acid (ABA). Germination of AOHSD seeds was less sensitive to ABA, while hsd seed was more sensitive to ABA during germination. AtHSD transcription was rapidly induced by BR treatment in wild type and was expressed widely in aerial plant parts, especially vascular tissues. This study demonstrates that AtHSD1 is involved in regulating growth and development in plants and is likely to promote or mediate BR effects. The gene has significant potential for improving growth and yield of canola and other agricultural crops. PMID:17616511

  11. pH-dependent electron transfer reaction and direct bioelectrocatalysis of the quinohemoprotein pyranose dehydrogenase.

    PubMed

    Takeda, Kouta; Matsumura, Hirotoshi; Ishida, Takuya; Yoshida, Makoto; Igarashi, Kiyohiko; Samejima, Masahiro; Ohno, Hiroyuki; Nakamura, Nobuhumi

    2016-08-26

    A pyranose dehydrogenase from Coprinopsis cinerea (CcPDH) is an extracellular quinohemoeprotein, which consists a b-type cytochrome domain, a pyrroloquinoline-quinone (PQQ) domain, and a family 1-type carbohydrate-binding module. The electron transfer reaction of CcPDH was studied using some electron acceptors and a carbon electrode at various pH levels. Phenazine methosulfate (PMS) reacted directly at the PQQ domain, whereas cytochrome c (cyt c) reacted via the cytochrome domain of intact CcPDH. Thus, electrons are transferred from reduced PQQ in the catalytic domain of CcPDH to heme b in the N-terminal cytochrome domain, which acts as a built-in mediator and transfers electron to a heterogenous electron transfer protein. The optimal pH values of the PMS reduction (pH 6.5) and the cyt c reduction (pH 8.5) differ. The catalytic currents for the oxidation of l-fucose were observed within a range of pH 4.5 to 11. Bioelectrocatalysis of CcPDH based on direct electron transfer demonstrated that the pH profile of the biocatalytic current was similar to the reduction activity of cyt c characters. PMID:27338639

  12. Sorbitol dehydrogenase is a cytosolic protein required for sorbitol metabolism in Arabidopsis thaliana.

    PubMed

    Aguayo, María Francisca; Ampuero, Diego; Mandujano, Patricio; Parada, Roberto; Muñoz, Rodrigo; Gallart, Marta; Altabella, Teresa; Cabrera, Ricardo; Stange, Claudia; Handford, Michael

    2013-05-01

    Sorbitol is converted to fructose in Rosaceae species by SORBITOL DEHYDROGENASE (SDH, EC 1.1.1.14), especially in sink organs. SDH has also been found in non-Rosaceae species and here we show that the protein encoded by At5g51970 in Arabidopsis thaliana (L.) Heynh. possesses the molecular characteristics of an SDH. Using a green fluorescent protein-tagged version and anti-SDH antisera, we determined that SDH is cytosolically localized, consistent with bioinformatic predictions. We also show that SDH is widely expressed, and that SDH protein accumulates in both source and sink organs. In the presence of NAD+, recombinant SDH exhibited greatest oxidative activity with sorbitol, ribitol and xylitol as substrates; other sugar alcohols were oxidized to a lesser extent. Under standard growth conditions, three independent sdh- mutants developed as wild-type. Nevertheless, all three exhibited reduced dry weight and primary root length compared to wild-type when grown in the presence of sorbitol. Additionally, under short-day conditions, the mutants were more resistant to dehydration stress, as shown by a reduced loss of leaf water content when watering was withheld, and a greater survival rate on re-watering. This evidence suggests that limitations in the metabolism of sugar alcohols alter the growth of Arabidopsis and its response to drought.

  13. Baseline sensitivity and resistance risk assessmemt of Rhizoctonia cerealis to thifluzamide, a succinate dehydrogenase inhibitor.

    PubMed

    Zhang, Yu; Lu, Jingle; Wang, Jianxin; Zhou, MingGuo; Chen, Changjun

    2015-10-01

    During 2010-2012, a total of 120 isolates of Rhizoctonia cerealis were collected from wheat with symptoms of sharp eyespot in four provinces (Henan, Shandong, Anhui and Jiangsu) in China. All the isolates were determined for baseline sensitivity to thifluzamide, a succinate dehydrogenase inhibitor (SDHI) with strong antifungal activity. The sampled pathogenic populations, never exposed to SDHIs, had similar sensitivity to trifluzamide (0.025-0.359 µg/ml) in the four regions and over the two years. The baseline sensitivity was distributed as a skewed unimodal curve with a mean EC50 value (effective concentrations for 50% inhibiting mycelial growth) of 0.064 ± 0.013 µg/ml. The resistance risk of R. cerealis to thifluzamide was further evaluated in vitro. Two thifluzamide-resistant mutants of R. cerealis were obtained by culturing on thifluzamide-amended plates. The resistance factors (RF = EC50 value of a mutant/EC50 value of the wild type progenitor of the mutant) were 120 and 40 for two R. cerealis mutants, respectively. All the mutants exhibited similar fitness after 10 successive transfers when compared to their wild-type parents in mycelial growth, sclerotia production, and virulence. However, the two thifluzamide-resistant mutants differed significantly in sensitivity to boscalid and flutolanil. Therefore, a low-to-moderate risk of resistance development was recommended for thifluzamide. PMID:26453237

  14. Testis-specific expression of a functional retroposon encoding glucose-6-phosphate dehydrogenase in the mouse

    SciTech Connect

    Hendriksen, P.J.M. |; Hoogerbrugge, J.W.; Baarends, W.M.

    1997-05-01

    The X-chromosomal gene glucose-6-phosphate dehydrogenase (G6pd) is known to be expressed in most cell types of mammalian species. In the mouse, we have detected a novel gene, designated G6pd-2, encoding a G6PD isoenzyme. G6pd-2 does not contain introns and appears to represent a retroposed gene. This gene is uniquely transcribed in postmeiotic spermatogenic cells in which the X-encoded G6pd gene is not transcribed. Expression of the G6pd-2 sequence in a bacterial system showed that the encoded product is an active enzyme. Zymogramic analysis demonstrated that recombinant G6PD-2, but not recombinant G6PD-1 (the X-chromosome-encoded G6PD), formed tetramers under reducing conditions. Under the same conditions, G6PD tetramers were also found in extracts of spermatids and spermatozoa, indicating the presence of G6pd-2-encoded isoenzyme in these cell types. G6pd-2 is one of the very few known expressed retroposons encoding a functional protein, and the presence of this gene is probably related to X chromosome inactivation during spermatogenesis. 62 refs., 7 figs.

  15. Biochemical Characterization and Complete Conversion of Coenzyme Specificity of Isocitrate Dehydrogenase from Bifidobacterium longum.

    PubMed

    Huang, Shi-Ping; Cheng, Hong-Mei; Wang, Peng; Zhu, Guo-Ping

    2016-01-01

    Bifidobacterium longum is a very important gram-positive non-pathogenic bacterium in the human gastrointestinal tract for keeping the digestive and immune system healthy. Isocitrate dehydrogenase (IDH) from B. longum (BlIDH), a novel member in Type II subfamily, was overexpressed, purified and biochemically characterized in detail. The active form of BlIDH was an 83-kDa homodimer. Kinetic analysis showed BlIDH was a NADP⁺-dependent IDH (NADP-IDH), with a 567- and 193-fold preference for NADP⁺ over NAD⁺ in the presence of Mg(2+) and Mn(2+), respectively. The maximal activity for BlIDH occurred at 60 °C (with Mn(2+)) and 65 °C (with Mg(2+)), and pH 7.5 (with Mn(2+)) and pH 8.0 (with Mg(2+)). Heat-inactivation profiles revealed that BlIDH retained 50% of maximal activity after incubation at 45 °C for 20 min with either Mn(2+) or Mg(2+). Furthermore, the coenzyme specificity of BlIDH can be completely reversed from NADP⁺ to NAD⁺ by a factor of 2387 by replacing six residues. This current work, the first report on the coenzyme specificity conversion of Type II NADP-IDHs, would provide better insight into the evolution of NADP⁺ use by the IDH family. PMID:26927087

  16. Improved poly(3-hydroxybutyrate) production in Escherichia coli by inactivation of cytochrome bd-II oxidase or/and NDH-II dehydrogenase in low efficient respiratory chains.

    PubMed

    Liu, Qiaojie; Lin, Zhenquan; Zhang, Yan; Li, Yifan; Wang, Zhiwen; Chen, Tao

    2014-12-20

    In order to redirect more carbon flux from TCA cycle into poly(3-hydroxybutyrate) (PHB) biosynthesis pathway via increasing respiratory efficiency, appB and ndh genes encoding cytochrome bd-II oxidase and NDH-II dehydrogenase were inactivated in Escherichia coli JM109/pBHR68. All appB or/and ndh knockout strains exhibited significantly increased PHB accumulation accompanying with increased NAD(P)H/NAD(P)(+) ratio and intracellular acetyl-CoA pool. Among them, the Δndh strain could accumulate up to 6.16g/L PHB from 20g/L glucose and 3.5g/L PHB from 20g/L xylose, respectively, a 1.76-fold and 3.43-fold increase compared to the wild-type control. The PHB production of this strain reached 28.23g/L in a 5-L fermentor study, which was 2.70-fold as much as that of the wild-type control. These results indicated that inactivating the cytochrome bd-II oxidase or/and NDH-II dehydrogenase of the aerobic respiratory chain is a simple and effective strategy to improve PHB biosynthesis in E. coli. To date, this is the first time to improve PHB production by inactivation of cytochrome bd-II oxidase or/and NDH-II dehydrogenase in low efficient respiratory chains.

  17. Intracellular glycerol influences resistance to freeze stress in Saccharomyces cerevisiae: analysis of a quadruple mutant in glycerol dehydrogenase genes and glycerol-enriched cells.

    PubMed

    Izawa, Shingo; Sato, Machiko; Yokoigawa, Kumio; Inoue, Yoshiharu

    2004-11-01

    Glycerol is well known as a cryoprotectant similar to trehalose. However, there is little information about the effects of intracellular glycerol on the freeze-thaw stress tolerance of yeast. Through analysis of a quadruple-knockout mutant of glycerol dehydrogenase genes (ara1 Delta gcy1 Delta gre3 Delta ypr1 Delta) in Saccharomyces cerevisiae, we revealed that the decrease in glycerol dehydrogenase activity led to increased levels of intracellular glycerol. We also found that this mutant showed higher tolerance to freeze stress than wild type strain W303-1A. Furthermore, we demonstrated that intracellular-glycerol-enriched cells cultured in glycerol medium acquire tolerance to freeze stress and retain high leavening ability in dough even after frozen storage for 7 days. These results suggest the possibility of using intracellular-glycerol-enriched cells to develop better frozen dough.

  18. Intracellular glycerol influences resistance to freeze stress in Saccharomyces cerevisiae: analysis of a quadruple mutant in glycerol dehydrogenase genes and glycerol-enriched cells.

    PubMed

    Izawa, Shingo; Sato, Machiko; Yokoigawa, Kumio; Inoue, Yoshiharu

    2004-11-01

    Glycerol is well known as a cryoprotectant similar to trehalose. However, there is little information about the effects of intracellular glycerol on the freeze-thaw stress tolerance of yeast. Through analysis of a quadruple-knockout mutant of glycerol dehydrogenase genes (ara1 Delta gcy1 Delta gre3 Delta ypr1 Delta) in Saccharomyces cerevisiae, we revealed that the decrease in glycerol dehydrogenase activity led to increased levels of intracellular glycerol. We also found that this mutant showed higher tolerance to freeze stress than wild type strain W303-1A. Furthermore, we demonstrated that intracellular-glycerol-enriched cells cultured in glycerol medium acquire tolerance to freeze stress and retain high leavening ability in dough even after frozen storage for 7 days. These results suggest the possibility of using intracellular-glycerol-enriched cells to develop better frozen dough. PMID:15127164

  19. Periplasmic nitrate reductase and formate dehydrogenase: similar molecular architectures with very different enzymatic activities.

    PubMed

    Cerqueira, Nuno M F S A; Gonzalez, Pablo J; Fernandes, Pedro A; Moura, José J G; Ramos, Maria João

    2015-11-17

    It is remarkable how nature has been able to construct enzymes that, despite sharing many similarities, have simple but key differences that tune them for completely different functions in living cells. Periplasmic nitrate reductase (Nap) and formate dehydrogenase (Fdh) from the DMSOr family are representative examples of this. Both enzymes share almost identical three-dimensional protein foldings and active sites, in terms of coordination number, geometry and nature of the ligands. The substrates of both enzymes (nitrate and formate) are polyatomic anions that also share similar charge and stereochemistry. In terms of the catalytic mechanism, both enzymes have a common activation mechanism (the sulfur-shift mechanism) that ensures a constant coordination number around the metal ion during the catalytic cycle. In spite of these similarities, they catalyze very different reactions: Nap abstracts an oxygen atom from nitrate releasing nitrite, whereas FdH catalyzes a hydrogen atom transfer from formate and releases carbon dioxide. In this Account, a critical analysis of structure, function, and catalytic mechanism of the molybdenum enzymes periplasmic nitrate reductase (Nap) and formate dehydrogenase (Fdh) is presented. We conclude that the main structural driving force that dictates the type of reaction, catalyzed by each enzyme, is a key difference on one active site residue that is located in the top region of the active sites of both enzymes. In both enzymes, the active site is centered on the metal ion of the cofactor (Mo in Nap and Mo or W in Fdh) that is coordinated by four sulfur atoms from two pyranopterin guanosine dinucleotide (PGD) molecules and by a sulfido. However, while in Nap there is a Cys directly coordinated to the Mo ion, in FdH there is a SeCys instead. In Fdh there is also an important His that interacts very closely with the SeCys, whereas in Nap the same position is occupied by a Met. The role of Cys in Nap and SeCys in FdH is similar in both

  20. Biophysical and mutagenic analysis of Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase activity and specificity.

    PubMed Central

    Burdette, D S; Secundo, F; Phillips, R S; Dong, J; Scott, R A; Zeikus, J G

    1997-01-01

    The Thermoanaerobacter ethanolicus 39E adhB gene encoding the secondary-alcohol dehydrogenase (secondary ADH) was overexpressed in Escherichia coli at more than 10% of total protein. The recombinant enzyme was purified in high yield (67%) by heat-treatment at 85 degrees C and (NH4)2SO4 precipitation. Site-directed mutants (C37S, H59N, D150N, D150Eand D150C were analysed to test the peptide sequence comparison-based predictions of amino acids responsible for putative catalytic Zn binding. X-ray absorption spectroscopy confirmed the presence of a protein-bound Zn atom with ZnS1(imid)1(N,O)3 co-ordination sphere. Inductively coupled plasma atomic emission spectrometry measured 0.48 Zn atoms per wild-type secondary ADH subunit. The C37S, H59N and D150N mutant enzymes bound only 0.11, 0.13 and 0.33 Zn per subunit respectively,suggesting that these residues are involved in Zn liganding. The D150E and D150C mutants retained 0.47 and 1.2 Zn atoms per subun