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Sample records for stearothermophilus 6-phosphogluconate dehydrogenase

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

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...-phosphogluconate dehydrogenase (6 PGD) in serum and erythrocytes. Measurements of 6-phosphogluconate dehydrogenase are used in the diagnosis and treatment of certain liver diseases (such as hepatitis) and anemias....

  2. Selective inhibition of 6-phosphogluconate dehydrogenase from Trypanosoma brucei

    NASA Astrophysics Data System (ADS)

    Bertelli, Massimo; El-Bastawissy, Eman; Knaggs, Michael H.; Barrett, Michael P.; Hanau, Stefania; Gilbert, Ian H.

    2001-05-01

    A number of triphenylmethane derivatives have been screened against 6-phosphogluconate dehydrogenase from Trypanosoma brucei and sheep liver. Some of these compounds show good inhibition of the enzymes and also selectivity towards the parasite enzyme. Modelling was undertaken to dock the compounds into the active sites of both enzymes. Using a combination of DOCK 3.5 and FLEXIDOCK a correlation was obtained between docking score and both activity for the enzymes and selectivity. Visualisation of the docked structures of the inhibitors in the active sites of the enzymes yielded a possible explanation of the selectivity for the parasite enzyme.

  3. One-step purification of soluble recombinant human 6-phosphogluconate dehydrogenase from Escherichia coli.

    PubMed

    Chan, Barden; Sukhatme, Vikas P

    2013-11-01

    6-Phosphogluconate dehydrogenase (6PGD), the third enzyme in the pentose phosphate pathway, was recently identified as a novel target in human lung cancer. In this report, we present an expression and purification scheme of recombinant human 6PGD from Escherichia coli. Using a DE3 derivative strain expressing tRNAs for seven rare codons in E. coli called Rosetta2 (DE3), a large quantity of soluble human 6PGD can be expressed with an N-terminal histidine tag and purified by a one-step purification procedure to near homogeneity without denaturants or refolding. Three to seven milligrams of purified protein could be obtained from 100 ml of culture. This recombinant human 6PGD follows classic Michaelis-Menton saturation kinetics with respect to both substrates NADP(+) and 6-phosphogluconate. The respective k(cat) and K(m) were comparable to those of 6PGDs purified from mammalian tissues. Using this purified 6PGD enzyme, we devised an endpoint colorimetric assay suitable for high-throughput screening for human 6PGD inhibitors.

  4. Inhibition effects of some metal ions on the rat liver 6-phosphogluconate dehydrogenase

    NASA Astrophysics Data System (ADS)

    Adem, Şevki; Kayhan, Naciye

    2016-04-01

    6-phosphogluconate dehydrogenase is an enzyme in the pentose phosphate path. The main functions of the pathway are the manufacture of the reduced coenzyme NADPH and the formation of ribose 5-phosphate for nucleic acid synthesis and nucleotide. Both NADPH and ribose 5-phosphate involve a critical biochemical process. Metals have been recognized as important toxic agents for living for a long time. It has been considered that they lead to in the emergence of many diseases. To evaluate whether metals is effect towards rat liver 6PGD, we apply various concentrations of metals and enzyme inhibition was analyzed using enzyme activity assays. The IC50 values of Pb+2, Cr+3, Co+2, Ni+2, Cd+2, and Va+2, metals on rat liver 6PGD were calculated as 138,138, 169, 214, 280, and 350 µM, respectively.

  5. Congenital 6-phosphogluconate dehydrogenase (6PGD) deficiency associated with chronic hemolytic anemia in a Spanish family.

    PubMed

    Vives Corrons, J L; Colomer, D; Pujades, A; Rovira, A; Aymerich, M; Merino, A; Aguilar i Bascompte, J L

    1996-12-01

    Clinical and metabolic studies were performed in four members of a Spanish family with partial (50%) 6 phosphogluconate dehydrogenase (6PGD) deficiency. In all cases the activities of 6 phosphogluconolactone (6PGL) and glutathione reductase (GR) were normal, and the molecular characterization performed in the partially purified 6PGD from the propositus showed normal kinetic and electrophoretic patterns. Two females (the propositus and her sister) suffered from a well-compensated chronic nonspherocytic hemolytic anemia (CNSHA) and exhibited decreased RBC glutathione (GSH) stability with increased oxidative susceptibility, defined by enhanced malonyldialdehyde (MDA) generation "in vitro." The other two members of the family (the propositus's mother and brother) were clinically asymptomatic. In the propositus and her sister, RBC metabolism exhibited a markedly abnormal concentration of glycolytic intermediates, mainly characterized by striking increases in fructose 1,6 bisphosphate (50-fold), dihydroxiacetone-phosphate (20-fold) and glyceraldehyde 3-phosphate (tenfold). Although the precise mechanism of the hemolysis in the two patients is unknown, the enhanced oxidative threat observed in their RBCs may interfere in some way with the glycolytic pathway function, leading to a marked increase in certain metabolic intermediates located before the glyceraldehyde 3 phosphate dehydrogenase (GA3PD) step. Since it seems that GA3PD half-life is modulated by fluctuations of the cytosolic redox status, an "in situ" approach was simulated by using permeabilized RBCs. In these conditions, GA3PD activity was significantly lower in the propositus and her sister than in the asymptomatic members of the family and the simultaneous normal control.

  6. 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.

  7. Purification of 6-phosphogluconate dehydrogenase from parsley (Petroselinum hortense) leaves and investigation of some kinetic properties.

    PubMed

    Demir, Hülya; Ciftçi, Mehmet; Küfrevioğlu, O Irfan

    2003-02-01

    In this study, 6-phosphogluconate dehydrogenase (E.C.1.1.44; 6PGD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps that are preparation of homogenate ammonium sulfate fractionation and on DEAE-Sephadex A50 ion exchange. The enzyme was obtained with a yield of 49% and had a specific activity of 18.3 U (mg proteins)(-1) (Lehninger, A.L.; Nelson, D.L.; Cox, M.M. Principles of Biochemistry, 2nd Ed.; Worth Publishers Inc.: N.Y., 2000, 558-560). The overall purification was about 339-fold. A temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method at 340 mn. In order to control the purification of the enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 97.5 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a subunit molecular weight of 24.1 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found as 8.0, 8.0, and 50 degrees C, respectively. In addition, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk plots.

  8. High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+

    PubMed Central

    Huang, Rui; Chen, Hui; Zhong, Chao; Kim, Jae Eung; Zhang, Yi-Heng Percival

    2016-01-01

    Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP+ to NAD+. Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfate (PMS), NAD+, and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP+ to NAD+. This screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT. PMID:27587230

  9. Regulation of Enzyme Activities in Drosophila: Genetic Variation Affecting Induction of Glucose 6-Phosphate and 6-Phosphogluconate Dehydrogenases in Larvae

    PubMed Central

    Cochrane, Bruce J.; Lucchesi, John C.; Laurie-Ahlberg, C. C.

    1983-01-01

    The genetic basis of modulation by dietary sucrose of the enzyme activities glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities in third instar larvae of Drosophila melanogaster was investigated, using isogenic lines derived from wild populations. Considerable genetically determined variation in response was detected among lines that differed only in their third chromosome constitution. Comparison of crossreacting material between a responding and a nonresponding line showed that the G6PD activity variation is due to changes in G6PD protein level. These differences in responses are localized in the fat body, with 300 m m sucrose in the diet resulting in a sixfold stimulation of G6PD activity and a fourfold one of 6PGD in the line showing the strongest response. In this tissue, the responses of the two enzymes are closely correlated with one another. Using recombinant lines, we obtained data that suggested the existence of more than one gene on chromosome III involved in the regulation of G6PD in the fat body, and at least one of these genes affects the level of 6PGD as well. PMID:6416921

  10. Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+ with Its Application to Biobatteries

    PubMed Central

    Chen, Hui; Zhu, Zhiguang; Huang, Rui; Zhang, Yi-Heng Percival

    2016-01-01

    Engineering the coenzyme specificity of redox enzymes plays an important role in metabolic engineering, synthetic biology, and biocatalysis, but it has rarely been applied to bioelectrochemistry. Here we develop a rational design strategy to change the coenzyme specificity of 6-phosphogluconate dehydrogenase (6PGDH) from a hyperthermophilic bacterium Thermotoga maritima from its natural coenzyme NADP+ to NAD+. Through amino acid-sequence alignment of NADP+- and NAD+-preferred 6PGDH enzymes and computer-aided substrate-coenzyme docking, the key amino acid residues responsible for binding the phosphate group of NADP+ were identified. Four mutants were obtained via site-directed mutagenesis. The best mutant N32E/R33I/T34I exhibited a ~6.4 × 104-fold reversal of the coenzyme selectivity from NADP+ to NAD+. The maximum power density and current density of the biobattery catalyzed by the mutant were 0.135 mW cm−2 and 0.255 mA cm−2, ~25% higher than those obtained from the wide-type 6PGDH-based biobattery at the room temperature. By using this 6PGDH mutant, the optimal temperature of running the biobattery was as high as 65 °C, leading to a high power density of 1.75 mW cm−2. This study demonstrates coenzyme engineering of a hyperthermophilic 6PGDH and its application to high-temperature biobatteries. PMID:27805055

  11. Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+ with Its Application to Biobatteries

    NASA Astrophysics Data System (ADS)

    Chen, Hui; Zhu, Zhiguang; Huang, Rui; Zhang, Yi-Heng Percival

    2016-11-01

    Engineering the coenzyme specificity of redox enzymes plays an important role in metabolic engineering, synthetic biology, and biocatalysis, but it has rarely been applied to bioelectrochemistry. Here we develop a rational design strategy to change the coenzyme specificity of 6-phosphogluconate dehydrogenase (6PGDH) from a hyperthermophilic bacterium Thermotoga maritima from its natural coenzyme NADP+ to NAD+. Through amino acid-sequence alignment of NADP+- and NAD+-preferred 6PGDH enzymes and computer-aided substrate-coenzyme docking, the key amino acid residues responsible for binding the phosphate group of NADP+ were identified. Four mutants were obtained via site-directed mutagenesis. The best mutant N32E/R33I/T34I exhibited a ~6.4 × 104-fold reversal of the coenzyme selectivity from NADP+ to NAD+. The maximum power density and current density of the biobattery catalyzed by the mutant were 0.135 mW cm‑2 and 0.255 mA cm‑2, ~25% higher than those obtained from the wide-type 6PGDH-based biobattery at the room temperature. By using this 6PGDH mutant, the optimal temperature of running the biobattery was as high as 65 °C, leading to a high power density of 1.75 mW cm‑2. This study demonstrates coenzyme engineering of a hyperthermophilic 6PGDH and its application to high-temperature biobatteries.

  12. 6-Phosphogluconate dehydrogenase regulates tumor cell migration in vitro by regulating receptor tyrosine kinase c-Met

    SciTech Connect

    Chan, Barden; VanderLaan, Paul A.; Sukhatme, Vikas P.

    2013-09-20

    Highlights: •Expression of 6PGD positively correlates with advancing stage of lung carcinoma. •Knockdown of 6PGD by shRNA potently inhibits c-Met tyrosine phosphorylation. •Exogenous HGF fails to restore c-Met phosphorylation in cells with 6PGD knocked down. •6PGD knockdown results in inhibition of cell migration in vitro. •Constitutively active TPR-cMet significantly restores migration of cells without 6PGD. -- Abstract: 6-Phosphogluconate dehydrogenase (6PGD) is the third enzyme in the oxidative pentose phosphate pathway (PPP). Recently, we reported that knockdown of 6PGD inhibited lung tumor growth in vitro and in a xenograft model in mice. In this study, we continued to examine the functional role of 6PGD in cancer. We show that 6PGD expression positively correlates with advancing stage of lung carcinoma. In search of functional signals related to 6PGD, we discovered that knockdown of 6PGD significantly inhibited phosphorylation of c-Met at tyrosine residues known to be critical for activity. This downregulation of c-Met phosphorylation correlated with inhibition of cell migration in vitro. Overexpression of a constitutively active c-Met specifically rescued the migration but not proliferation phenotype of 6PGD knockdown. Therefore, 6PGD appears to be required for efficient c-Met signaling and migration of tumor cells in vitro.

  13. Autosomal factors with correlated effects on the activities of the glucose 6-phosphate and 6-phosphogluconate dehydrogenases in Drosophila melanogaster.

    PubMed

    Laurie-Ahlberg, C C; Williamson, J H; Cochrane, B J; Wilton, A N; Chasalow, F I

    1981-09-01

    Isogenic lines, in which chromosomes sampled from natural populations of C. melanogaster are substituted into a common genetic background, were used to detect and partially characterize autosomal factors that affect the activities of the two pentose phosphate pathway enzymes, glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD). The chromosome 3 effects on G6PD and 6PGD are clearly correlated; the chromosome 2 effects, which are not so great, also appear to be correlated, but the evidence in this case is not so strong. Examination of activity variation of ten other enzymes revealed that G6PD and 6PGD are not the only pair of enzymes showing a high positive correlation, but it is among the highest in both sets of lines. In addition, there was some evidence that the factor(s) affecting G6PD and 6PGD may also affect two other metabolically related enzymes, transaldolase and phosphoglucose isomerase.--Rocket immunoelectrophoresis was used to estimate specific CRM levels for three of the enzymes studied: G6PD, 6PGD and ME. This experiment shows that a large part of the activity variation is accounted for by variation in CRM level (especially for chromosome 3 lines), but there remains a significant fraction of the genetic component of activity variation that is not explained by CRM level.--These results suggest that the autosomal factors are modifiers involved in regulation of the expression of the X-linked structural genes for G6PD and 6PGD, but a role in determining part of the enzymes' primary structure cannot be excluded with the present evidence.

  14. Autosomal Factors with Correlated Effects on the Activities of the Glucose 6-Phosphate and 6-Phosphogluconate Dehydrogenases in DROSOPHILA MELANOGASTER

    PubMed Central

    Laurie-Ahlberg, C. C.; Williamson, J. H.; Cochrane, B. J.; Wilton, A. N.; Chasalow, F. I.

    1981-01-01

    Isogenic lines, in which chromosomes sampled from natural populations of D. melanogaster are substituted into a common genetic background, were used to detect and partially characterize autosomal factors that affect the activities of the two pentose phosphate pathway enzymes, glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD). The chromosome 3 effects on G6PD and 6PGD are clearly correlated; the chromosome 2 effects, which are not so great, also appear to be correlated, but the evidence in this case is not so strong. Examination of activity variation of ten other enzymes revealed that G6PD and 6PGD are not the only pair of enzymes showing a high positive correlation, but it is among the highest in both sets of lines. In addition, there was some evidence that the factor(s) affecting G6PD and 6PGD may also affect two other metabolically related enzymes, transaldolase and phosphoglucose isomerase.—Rocket immunoelectrophoresis was used to estimate specific CRM levels for three of the enzymes studied: G6PD, 6PGD and ME. This experiment shows that a large part of the activity variation is accounted for by variation in CRM level (especially for chromosome 3 lines), but there remains a significant fraction of the genetic component of activity variation that is not explained by CRM level.—These results suggest that the autosomal factors are modifiers involved in regulation of the expression of the X-linked structural genes for G6PD and 6PGD, but a role in determining part of the enzymes' primary structure cannot be excluded with the present evidence. PMID:6804300

  15. Defects in Peroxisomal 6-Phosphogluconate Dehydrogenase Isoform PGD2 Prevent Gametophytic Interaction in Arabidopsis thaliana1[OPEN

    PubMed Central

    Hölscher, Christian; Meyer, Tanja; Fischer, Kerstin

    2016-01-01

    We studied the localization of 6-phosphogluconate dehydrogenase (PGD) isoforms of Arabidopsis (Arabidopsis thaliana). Similar polypeptide lengths of PGD1, PGD2, and PGD3 obscured which isoform may represent the cytosolic and/or plastidic enzyme plus whether PGD2 with a peroxisomal targeting motif also might target plastids. Reporter-fusion analyses in protoplasts revealed that, with a free N terminus, PGD1 and PGD3 accumulate in the cytosol and chloroplasts, whereas PGD2 remains in the cytosol. Mutagenesis of a conserved second ATG enhanced the plastidic localization of PGD1 and PGD3 but not PGD2. Amino-terminal deletions of PGD2 fusions with a free C terminus resulted in peroxisomal import after dimerization, and PGD2 could be immunodetected in purified peroxisomes. Repeated selfing of pgd2 transfer (T-)DNA alleles yielded no homozygous mutants, although siliques and seeds of heterozygous plants developed normally. Detailed analyses of the C-terminally truncated PGD2-1 protein showed that peroxisomal import and catalytic activity are abolished. Reciprocal backcrosses of pgd2-1 suggested that missing PGD activity in peroxisomes primarily affects the male gametophyte. Tetrad analyses in the quartet1-2 background revealed that pgd2-1 pollen is vital and in vitro germination normal, but pollen tube growth inside stylar tissues appeared less directed. Mutual gametophytic sterility was overcome by complementation with a genomic construct but not with a version lacking the first ATG. These analyses showed that peroxisomal PGD2 activity is required for guided growth of the male gametophytes and pollen tube-ovule interaction. Our report finally demonstrates an essential role of oxidative pentose-phosphate pathway reactions in peroxisomes, likely needed to sustain critical levels of nitric oxide and/or jasmonic acid, whose biosynthesis both depend on NADPH provision. PMID:26941195

  16. Increased activity of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in purified cell suspensions and single cells from the uterine cervix in cervical intraepithelial neoplasia.

    PubMed Central

    Jonas, S. K.; Benedetto, C.; Flatman, A.; Hammond, R. H.; Micheletti, L.; Riley, C.; Riley, P. A.; Spargo, D. J.; Zonca, M.; Slater, T. F.

    1992-01-01

    The activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase have been measured in squamous epithelial cells of the uterine cervix from normal patients and cases of cervical intraepithelial neoplasia (CIN). A biochemical cycling method, which uses only simple equipment and is suited to routine use and to automation, was applied to cells separated by gradient centrifugation. In addition, cells were examined cytochemically, and the intensity of staining in the cytoplasm of single whole cells was measured using computerised microcytospectrophotometry. Twenty per cent of cells in samples from normal patients (n=61) showed staining intensities above an extinction of 0.15 at 540 nm, compared to 71% of cases of CIN 1 (n=14), 91% of cases of CIN 2 (n=11) and 67% of cases of CIN 3 (n=15). The cytochemical data do not allow definitive distinctions to be made between different grades of CIN whereas the biochemical assay applied to cell lysates shows convincing differences between normal samples and cases of CIN. There are no false negatives for CIN 3 (n=14) and CIN 2 (n=10) and 11% false negatives for CIN 1 (n=9) and 14% of false positives for normal cases (n=21). The results of this preliminary study with reference to automation are discussed [corrected]. Images Figure 1 PMID:1637668

  17. Key Enzymes of the Semiphosphorylative Entner-Doudoroff Pathway in the Haloarchaeon Haloferax volcanii: Characterization of Glucose Dehydrogenase, Gluconate Dehydratase, and 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase

    PubMed Central

    Sutter, Jan-Moritz; Tästensen, Julia-Beate; Johnsen, Ulrike; Soppa, Jörg

    2016-01-01

    ABSTRACT The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. So far, the key enzymes of this pathway, glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (KDPGA), have not been characterized, and their functional involvement in glucose degradation has not been demonstrated. Here we report that the genes HVO_1083 and HVO_0950 encode GDH and KDPGA, respectively. The recombinant enzymes show high specificity for glucose and KDPG and did not convert the corresponding C4 epimers galactose and 2-keto-3-deoxy-6-phosphogalactonate at significant rates. Growth studies of knockout mutants indicate the functional involvement of both GDH and KDPGA in glucose degradation. GAD was purified from H. volcanii, and the encoding gene, gad, was identified as HVO_1488. GAD catalyzed the specific dehydration of gluconate and did not utilize galactonate at significant rates. A knockout mutant of GAD lost the ability to grow on glucose, indicating the essential involvement of GAD in glucose degradation. However, following a prolonged incubation period, growth of the Δgad mutant on glucose was recovered. Evidence is presented that under these conditions, GAD was functionally replaced by xylonate dehydratase (XAD), which uses both xylonate and gluconate as substrates. Together, the characterization of key enzymes and analyses of the respective knockout mutants present conclusive evidence for the in vivo operation of the spED pathway for glucose degradation in H. volcanii. IMPORTANCE The work presented here describes the identification and characterization of the key enzymes glucose dehydrogenase, gluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase and their encoding genes of the proposed semiphosphorylative Entner-Doudoroff pathway in the haloarchaeon Haloferax volcanii. The functional involvement of the three enzymes was

  18. A survey for isoenzymes of glucosephosphate isomerase, phosphoglucomutase, glucose-6-phosphate dehydrogenase and 6-Phosphogluconate dehydrogenase in C3-, C 4-and crassulacean-acid-metabolism plants, and green algae.

    PubMed

    Herbert, M; Burkhard, C; Schnarrenberger, C

    1979-01-01

    Two isoenzymes each of glucosephosphate isomerase (EC 5.3.1.9), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.43) were separated by (NH4)2SO4 gradient solubilization and DEAE-cellulose ion-exchange chromatography from green leaves of the C3-plants spinach (Spinacia oleracea L.), tobacco (Nicotiana tabacum L.) and wheat (Triticum aestivum L.), of the Crassulacean-acid-metabolism plants Crassula lycopodioides Lam., Bryophyllum calycinum Salisb. and Sedum rubrotinctum R.T. Clausen, and from the green algae Chlorella vulgaris and Chlamydomonas reinhardii. After isolation of cell organelles from spinach leaves by isopyenic centrifugation in sucrose gradients one of two isoenzymes of each of the four enzymes was found to be associated with whole chloroplasts while the other was restricted to the soluble cell fraction, implying the same intracellular distribution of these isoenzymes also in the other species.Among C4-plants, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were found in only one form in corn (Zea mays L.), sugar cane (Saccharum officinarum L.) and Coix lacrymajobi L., but as two isoenzymes in Atriplex spongiosa L. and Portulaca oleracea L. In corn, the two dehydrogenases were mainly associated with isolated mesophyll protoplasts while in Atriplex spongiosa they were of similar specific activity in both mesophyll protoplasts and bundle-sheath strands. In all five C4-plants three isoenzymes of glucosephosphate isomerase and phosphoglucomutase were found. In corn two were localized in the bundle-sheath strands and the third one in the mesophyll protoplasts. The amount of activity of the enzymes was similar in each of the two cell fractions. Apparently, C4 plants have isoenzymes not only in two cell compartments, but also in physiologically closely linked cell types such as mesophyll and bundle-sheath cells.

  19. Purification of rat kidney glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase enzymes using 2',5'-ADP Sepharose 4B affinity in a single chromatography step.

    PubMed

    Adem, Sevki; Ciftci, Mehmet

    2012-01-01

    The enzymes of glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), and glutathione reductase (GR) were purified from rat kidney in one chromatographic step consisting of the use of the 2',5'-ADP Sepharose 4B by using different elution buffers. This purification procedure was accomplished with the preparation of the homogenate and affinity chromatography on 2',5'-ADP Sepharose 4B. The purity and subunit molecular weights of the enzymes were checked on SDS-PAGE and purified enzymes showed a single band on the gel. The native molecular weights of the enzymes were found with Sephadex G-150 gel filtration chromatography. Using this procedure, G6PG, having the specific activity of 32 EU/mg protein, was purified 531-fold with a yield of 88%; 6PGD, having the specific activity of 25 EU/mg protein, was purified 494-fold with a yield of 73%; and GR, having the specific activity of 33 EU/mg protein, was purified 477-fold with a yield of 76%. Their native molecular masses were estimated to be 144 kDa for G6PD, 110 kDa for 6PGD, and 121 kDa for GR and the subunit molecular weights were found to be 68, 56, and 61 kDa, respectively. A new modified method to purify G6PD, 6PGD, and GR, namely one chromatographic step using the 2',5'-ADP Sepharose 4B, is described for the first time in this study. This procedure has several advantages for purification of enzymes, such as, rapid purification, produces high yield, and uses less chemical materials.

  20. Stability engineering of the Geobacillus stearothermophilus alcohol dehydrogenase and application for the synthesis of a polyamide 12 precursor.

    PubMed

    Kirmair, Ludwig; Seiler, Daniel Leonard; Skerra, Arne

    2015-12-01

    The thermostable NAD(+)-dependent alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH) was exploited with regard to the biocatalytic synthesis of ω-oxo lauric acid methyl ester (OLAMe), a key intermediate for biobased polyamide 12 production, from the corresponding long-chain alcohol. Recombinant BsADH was produced in Escherichia coli as a homogeneous tetrameric enzyme and showed high activity towards the industrially relevant substrate ω-hydroxy lauric acid methyl ester (HLAMe) with K M = 86 μM and 44 U mg(-1). The equilibrium constant for HLAMe oxidation to the aldehyde (OLAMe) with NAD(+) was determined as 2.16 × 10(-3) from the kinetic parameters of the BsADH-catalyzed forward and reverse reactions. Since BsADH displayed limited stability under oxidizing conditions, the predominant oxidation-prone residue Cys257 was mutated to Leu based on sequence homology with related enzymes and computational simulation. This substitution resulted in an improved BsADH variant exhibiting prolonged stability and an elevated inactivation temperature. Semi-preparative biocatalysis at 60 °C using the stabilized enzyme, employing butyraldehyde for in situ cofactor regeneration with only catalytic amounts of NAD(+), yielded up to 23 % conversion of HLAMe to OLAMe after 30 min. In contrast to other oxidoreductases, no overoxidation to the dodecanoic diacid monomethyl ester was detected. Thus, the mutated BsADH offers a promising biocatalyst for the selective oxidation of fatty alcohols to yield intermediates for industrial polymer production.

  1. Structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus at 1.8 A resolution.

    PubMed

    Skarzyński, T; Moody, P C; Wonacott, A J

    1987-01-05

    The structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus has been crystallographically refined at 1.8 A resolution using restrained least-squares refinement methods. The final crystallographic R-factor for 93,120 reflexions with F greater than 3 sigma (F) is 0.177. The asymmetric unit of the crystal contains a complete tetramer, the final model of which incorporates a total of 10,272 unique protein and coenzyme atoms together with 677 bound solvent molecules. The structure has been analysed with respect to molecular symmetry, intersubunit contacts, coenzyme binding and active site geometry. The refined model shows the four independent subunits to be remarkable similar apart from local deviations due to intermolecular contacts within the crystal lattice. A number of features are revealed that had previously been misinterpreted from an earlier 2.7 A electron density map. Arginine at position 195 (previously thought to be a glycine) contributes to the formation of the anion binding sites in the active site pocket, which are involved in binding of the substrate and inorganic phosphates during catalysis. This residue seems to be structurally equivalent to the conserved Arg194 in the enzyme from other sources. In the crystal both of the anion binding sites are occupied by sulphate ions. The ND atom of the catalytically important His176 is hydrogen-bonded to the main-chain carbonyl oxygen of Ser177, thus fixing the plane of the histidine imidazole ring and preventing rotation. The analysis has revealed the presence of several internal salt-bridges stabilizing the tertiary and quaternary structure. A significant number of buried water molecules have been found that play an important role in the structural integrity of the molecule.

  2. Redesign of the coenzyme specificity in L-lactate dehydrogenase from bacillus stearothermophilus using site-directed mutagenesis and media engineering.

    PubMed

    Holmberg, N; Ryde, U; Bülow, L

    1999-10-01

    L-lactate dehydrogenase (LDH) from Bacillus stearothermophilus is a redox enzyme which has a strong preference for NADH over NADPH as coenzyme. To exclude NADPH from the coenzyme-binding pocket, LDH contains a conserved aspartate residue at position 52. However, this residue is probably not solely responsible for the NADH specificity. In this report we examine the possibilities of altering the coenzyme specificity of LDH by introducing a range of different point mutations in the coenzyme-binding domain. Furthermore, after choosing the mutant with the highest selectivity for NADPH, we also investigated the possibility of further altering the coenzyme specificity by adding an organic solvent to the reaction mixture. The LDH mutant, I51K:D52S, exhibited a 56-fold increased specificity to NADPH over the wild-type LDH in a reaction mixture containing 15% methanol. Furthermore, the NADPH turnover number of this mutant was increased almost fourfold as compared with wild-type LDH. To explain the altered coenzyme specificity exhibited by the D52SI51K double mutant, molecular dynamics simulations were performed.

  3. Use of differential dye-ligand chromatography with affinity elution for enzyme purification: 2-keto-3-deoxy-6-phosphogluconate aldolase from Zymomonas mobilis.

    PubMed

    Scopes, R K

    1984-02-01

    2-Keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) has been isolated from extracts of Zymomonas mobilis using differential dye-ligand chromatography and affinity elution with product/product analog. The one-step procedure gives an enzyme with specific activity 600 units mg-1. Only 1 out of 47 dyes, Procion Yellow MX-GR, bound the enzyme completely in 20 mM phosphate buffer, pH 6.5. A column of Navy HE-R adsorbent was used first to remove most of the potentially adsorbing proteins.

  4. The bacterial Entner-Doudoroff pathway does not replace glycolysis in Saccharomyces cerevisiae due to the lack of activity of iron-sulfur cluster enzyme 6-phosphogluconate dehydratase.

    PubMed

    Benisch, Feline; Boles, Eckhard

    2014-02-10

    Replacement of the glycolytic pathway of Saccharomyces cerevisiae by a bacterial Entner-Doudoroff pathway (EDP) would result in lower ATP production and therefore a lower biomass yield is expected that would further allow higher products yields in the fermentation of sugars. To establish catabolism of glucose via the EDP in S. cerevisiae requires expression of only two additional enzyme activities, 6-phosphogluconate dehydratase (PGDH) and KDPG aldolase. In this work, KDPG aldolase from Escherichia coli could be successfully expressed in the yeast cytosol with very high enzyme activity. Nevertheless, simultaneous expression of KDPG aldolase and a codon optimized PGDH gene of E. coli could not replace glycolysis or the pentose phosphate pathway in growth experiments. It could be shown that this was due to the very low enzyme activity of PGDH. This bacterial enzyme is a [4Fe-4S] iron-sulfur cluster protein. Several attempts to improve the availability of iron-sulfur clusters or iron in the yeast cells, to attract the iron-sulfur cluster assembly machinery to Leu1-PGDH fusion proteins or to localize the PGDH in the mitochondria did not result in improved enzyme activities. From our results we conclude that establishing functional expression of iron-sulfur cluster enzymes will be a major task for the integration of the EDP and other biochemical pathways in yeast.

  5. Quantitative Analysis of X Chromosome Effects on the Activities of the Glucose 6-Phosphate and 6-Phosphogluconate Dehydrogenases of DROSOPHILA MELANOGASTER

    PubMed Central

    Miyashita, Naohiko; Laurie-Ahlberg, Cathy C.; Wilton, Alan N.; Emigh, Ted H.

    1986-01-01

    By combining 20 X chromosomes with five autosomal backgrounds, the relative importance of these factors with respect to the activity variations of G6PD and 6PGD in Drosophila melanogaster were investigated. Analysis of variance revealed that there exist significant X chromosome, autosomal background and genetic interaction effects. The effect of the X chromosome was due mainly to the two allozymic forms of each enzyme, but some within-allozyme effects were also detected. From the estimated variance components, it was concluded that the variation attributed to the autosomal background is much larger than the variation attributed to the X chromosome, even when the effect of the allozymes is included. The segregation of the allozymes seems to account for about 10% of the total activity variation of each enzyme. The variation due to the interaction between the X chromosome and the autosomal background is much smaller than variations attributed either to the X chromosome or to the autosomal background. The interaction effect is indicated by the change of the ranking of the X chromosomes for different autosomal backgrounds. Highly significant and positive correlation between G6PD and 6PGD activities was detected. Again, the contribution of the autosomal background to the correlation was much larger than that attributed to the X chromosome. PMID:3087815

  6. Control of glycolytic flux in Zymomonas mobilis by glucose 6-phosphate dehydrogenase activity

    SciTech Connect

    Snoep, J.L. |; Arfman, N.; Yomano, L.P.; Ingram, L.O.; Westerhoff, H.V.; Conway, T.

    1996-07-20

    Alycolytic genes in Zymomonas mobilis are highly expressed and constitute half of the cytoplasmic protein. The first four genes (glf, zwf, edd, glk) in this pathway form an operon encoding a glucose permease, glucose 6-phosphate dehydrogenase (G6-P dehydrogenase), 6-phosphogluconate dehydratase, and glucokinase, respectively. Each gene was overexpressed from a tac promoter to investigate the control of glycolysis during the early stages of batch fermentation when flux (qCO{sub 2}) is highest. Almost half of flux control appears to reside with G6-P dehydrogenase (C{sub G6-P dehydrogenase}{sup J} = 0.4). Although Z. mobilis exhibits one of the highest rates of glycolysis known, recombinants with elevated G6-P dehydrogenase had a 10% to 13% higher glycolytic flux than the native organism. A small increase in flux was also observed for recombinants expressing glf. Results obtained did not allow a critical evaluation of glucokinase and this enzyme may also represent an important control point. 6-Phosphogluconate dehydratase appears to be saturating at native levels. With constructs containing the full operon, growth rate and flux were both reduced, complicating interpretations. However, results obtained were also consistent with G6-P dehydrogenase as a primary site of control. Flux was 17% higher in operon constructs which exhibited a 17% increase in G6-P dehydrogenase specific activity, relative to the average of other operon constructs which contain a frameshift mutation in zwf.

  7. Cloning, expression, and isolation of the mannitol transport protein from the thermophilic bacterium Bacillus stearothermophilus.

    PubMed Central

    Henstra, S A; Tolner, B; ten Hoeve Duurkens, R H; Konings, W N; Robillard, G T

    1996-01-01

    A mannitol phosphotransferase system (PTS) was identified in Bacillus stearothermophilus by in vitro complementation with Escherichia coli EI, HPr, and IIA(Mtl). Degenerate primers based on regions of high amino acid similarity in the E. coli and Staphylococcus carnosus EII(Mt1) were used to develop a digoxigenin-labeled probe by PCR. Using this probe, we isolated three overlapping DNA fragments totaling 7.2 kb which contain the genes mtlA, mtlR, mtlF, and mtlD, encoding the mannitol IICB,a regulator, IIA, and a mannitol-1-phosphate dehydrogenase, respectively. The mtl4 gene consists of 1,413 bp coding for a 471-amino-acid protein with a calculated mass of 50.1 kDa. The amino acid sequence shows high similarity with the sequence of IICB(Mtl) of S. carnosus and the IICB part of the IICBA(Mtl)s of E. coli and B. subtilis. The enzyme could be functionally expressed in E. coli by placing it behind the strong tac promoter. The rate of thermal inactivation at 60 degrees C of B. stearothermophilus HCB(Mt1) expressed in E. coli was two times lower than that of E. coli IICB(Mtl). IICB(Mtl) in B. stearothermophilus is maximally active at 85 degrees C and thus very thermostable. The enzyme was purified on Ni-nitrilotriacetic acid resin to greater than 95% purity after six histidines were fused to the C-terminal part of the transporter. PMID:8824601

  8. A dehydrogenase-mediated recycling system of NADPH in plant peroxisomes.

    PubMed Central

    Corpas, F J; Barroso, J B; Sandalio, L M; Distefano, S; Palma, J M; Lupiáñez, J A; Del Río, L A

    1998-01-01

    The presence of the two NADP-dependent dehydrogenases of the pentose phosphate pathway has been investigated in plant peroxisomes from pea (Pisum sativum L.) leaves. Both enzymes, glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44), were present in the matrix of leaf peroxisomes, and their kinetic properties were studied. G6PDH and 6PGDH showed a typical Michaelis-Menten kinetic saturation curve, and had specific activities of 12.4 and 29.6 mU/mg protein, respectively. The Km values of G6PDH and 6PGDH for glucose 6-phosphate and for 6-phosphogluconate were 107.3 and 10.2 microM, respectively. Dithiothreitol did not inhibit G6PDH activity. By isoelectric focusing of peroxisomal matrices, the G6PDH activity was resolved into three isoforms with isoelectric points of 5.55, 5.30 and 4.85. The isoelectric point of peroxisomal 6PGDH was 5.10. Immunoblot analyses of peroxisomal matrix with an antibody against yeast G6PDH revealed a single cross-reactive band of 56 kDa. Post-embedment, EM immunogold labelling of G6PDH confirmed that this enzyme was localized in the peroxisomal matrices, the thylakoid membrane and matrix of chloroplasts, and the cytosol. The presence of the two oxidative enzymes of the pentose phosphate pathway in plant peroxisomes implies that these organelles have the capacity to reduce NADP+ to NADPH for its re-utilization in the peroxisomal metabolism. NADPH is particularly required for the ascorbate-glutathione cycle, which has been recently demonstrated in plant peroxisomes [Jiménez, Hernández, del Río and Sevilla (1997) Plant Physiol. 114, 275-284] and represents an important antioxidant protection system against H2O2 generated in peroxisomes. PMID:9480890

  9. Crystal structure of novel NADP-dependent 3-hydroxyisobutyrate dehydrogenase from Thermus thermophilus HB8.

    PubMed

    Lokanath, Neratur K; Ohshima, Noriyasu; Takio, Koji; Shiromizu, Ikuya; Kuroishi, Chizu; Okazaki, Nobuo; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Miyano, Masashi; Kunishima, Naoki

    2005-09-30

    3-Hydroxyisobutyrate, a central metabolite in the valine catabolic pathway, is reversibly oxidized to methylmalonate semialdehyde by a specific dehydrogenase belonging to the 3-hydroxyacid dehydrogenase family. To gain insight into the function of this enzyme at the atomic level, we have determined the first crystal structures of the 3-hydroxyisobutyrate dehydrogenase from Thermus thermophilus HB8: holo enzyme and sulfate ion complex. The crystal structures reveal a unique tetrameric oligomerization and a bound cofactor NADP+. This bacterial enzyme may adopt a novel cofactor-dependence on NADP, whereas NAD is preferred in eukaryotic enzymes. The protomer folds into two distinct domains with open/closed interdomain conformations. The cofactor NADP+ with syn nicotinamide and the sulfate ion are bound to distinct sites located at the interdomain cleft of the protomer through an induced-fit domain closure upon cofactor binding. From the structural comparison with the crystal structure of 6-phosphogluconate dehydrogenase, another member of the 3-hydroxyacid dehydrogenase family, it is suggested that the observed sulfate ion and the substrate 3-hydroxyisobutyrate share the same binding pocket. The observed oligomeric state might be important for the catalytic function through forming the active site involving two adjacent subunits, which seems to be conserved in the 3-hydroxyacid dehydrogenases. A kinetic study confirms that this enzyme has strict substrate specificity for 3-hydroxyisobutyrate and serine, but it cannot distinguish the chirality of the substrates. Lys165 is likely the catalytic residue of the enzyme.

  10. 21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the culture filtrate that results from a pure culture fermentation of a nonpathogenic and nontoxicogenic strain of Bacillus stearothermophilus....

  11. 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

  12. Genetics of thermophilic bacteria. [Bacillus stearothermophilus:a2

    SciTech Connect

    Welker, N.E.

    1991-01-01

    Organisms adapted to high temperature have evolved a variety of unique solutions to the biochemical problems imposed by this environment. Adaptation is commonly used to describe the biochemical properties of organisms which have become adapted to their environment (genetic adaptation). It can also mean the direct response-at the cellular level-of an organism to changes in temperature (physiological adaptation). Thermophilic bacilli (strains of Bacillus stearothermophilus) can exhibit a variety of biochemical adaptations in response to changes in temperature. These include changes in the composition and stability of the membrane, metabolic potential, the transport of amino acids, regulatory mechanisms, ribose methylation of tRNA, protein thermostability, and nutritional requirements. The objectives of the research were to develop efficient and reliable genetic systems to analyze and manipulate B. Stearothermophilus, and to use these systems initiate a biochemical, molecular, and genetic investigations of genes that are required for growth at high temperature.

  13. DECONTAMINATION ASSESSMENT OF BACILLUS ANTHRACIS, BACILLUS SUBTILIS, AND GEOBACILLUS STEAROTHERMOPHILUS SPORES ON INDOOR SURFACTS USING A HYDROGEN PERIOXIDE GAS GENERATOR

    EPA Science Inventory

    Aims: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using hydrogen peroxide gas. Methods and Results: B. anthracis, B. subtilis, and G. Stearothermophilus spores were dried on seven...

  14. 21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true α-Amylase enzyme preparation from Bacillus... preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the culture... Bacillus stearothermophilus. Its characterizing enzyme activity is α-amylase (1,4 α-D...

  15. Metabolism of phenol and cresols by Bacillus stearothermophilus.

    PubMed Central

    Buswell, J A

    1975-01-01

    An obligate thermophilic strain of Bacillus stearothermophilus, strain PH24, isolated from industrial sediment by elective culture, grew readily at 55 C on phenol or on one of the isomers of cresol as the major carbon source. Intact cells grown in the presence of phenol, o-cresol, m-cresol, or p-cresol were induced to oxidize, without lag, these substrates together with catechol, 3-methylcatechol, and 4-methylcatechol. Cell extracts prepared from B. stearothermophilus PH24 after growth in the presence of phenol converted phenol to catechol with a concomitant uptake of 1 mol of oxygen per mol of substrate in reaction mixtures supplemented with reduced nicotinamide adenine dinucleotide. These preparations also catalyzed the oxidation of o-cresol to 3-methylcatechol and of m-cresol and p-cresol to 4-methylcatechol. Enzyme activity was inhibited by 1 mM p-chloromercuribenzoate and by 0.1 mM 0-phenanthroline. Catechol and the corresponding methylcatechol intermediates were further dissimilated by cell extracts of phenol-grown cells via the meta-cleavage route to yield 2-hydroxymuconic semialdehyde and the respective methylated derivatives. PMID:1194230

  16. FORMALDEHYDE GAS INACTIVATION OF BACILLUS ANTHRACIS, BACILLUS SUBTILIS AND GEOBACILLUS STEAROTHERMOPHILUS SPORES ON INDOOR SURFACE MATERIALS.

    EPA Science Inventory

    Research evaluated the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface material using formaldehyde gas. Spores were dried on seven types of indoor surfaces and exposed to 1100 ppm formaldehyde gas for 10 hr. Fo...

  17. Structure of the Apo Form of Bacillus stearothermophilus Phosphofructokinase

    SciTech Connect

    Mosser, Rockann; Reddy, Manchi C.M.; Bruning, John B.; Sacchettini, James C.; Reinhart, Gregory D.

    2012-02-08

    The crystal structure of the unliganded form of Bacillus stearothermophilus phosphofructokinase (BsPFK) was determined using molecular replacement to 2.8 {angstrom} resolution (Protein Data Bank entry 3U39). The apo BsPFK structure serves as the basis for the interpretation of any structural changes seen in the binary or ternary complexes. When the apo BsPFK structure is compared with the previously published liganded structures of BsPFK, the structural impact that the binding of the ligands produces is revealed. This comparison shows that the apo form of BsPFK resembles the substrate-bound form of BsPFK, a finding that differs from previous predictions.

  18. Purification and characterization of alkaline phosphatase from Bacillus stearothermophilus.

    PubMed

    Mori, S; Okamoto, M; Nishibori, M; Ichimura, M; Sakiyama, J; Endo, H

    1999-06-01

    Soluble alkaline phosphatase from the thermophilic bacterium Bacillus stearothermophilus was purified by a combination of chromatographic methods, and its properties were examined. The purified enzyme had specific activity of 4.43 micromol p-nitrophenol/min per mg of protein and seemed to be a single band on SDS/PAGE with a molecular mass of 32 kDa. Its apparent Km for p-nitrophenyl phosphate was 1.114 mM. The enzyme exhibited an optimal pH of approx. 9.0 and exhibited its highest activity at 60-70 degrees C. It also showed a bivalent cation requirement for activity, with maximal enhancement in the presence of Mg2+. In addition, significant thermal stability was observed in comparison with counterparts from mesophiles. Its partial N-terminal sequence was T1FSIVAFDPATGELGIAVQ19 as estimated by automated Edman degradation method. A search on the SwissProt database did not reveal any similar protein sequences from other sources.

  19. Reproduction of Bacillus stearothermophilus as a Function of Temperature and Pressure

    PubMed Central

    Yayanos, A. Aristides; Van Boxtel, R.; Dietz, Allan S.

    1983-01-01

    The colony-forming ability and the rate of reproduction of Bacillus stearothermophilus were determined as a function of temperature and pressure. Colonies were formed between 39 and 70°C at atmospheric pressure and between 54 and 67°C at 45 MPa. Colonies did not form at 55.9 MPa. The rate of reproduction in broth cultures decreased with increasing pressure at all temperatures. The rate of reproduction diminished rapidly with pressure above 10.4 MPa. Therefore, increased hydrostatic pressure was not sufficient to enable B. stearothermophilus to function beyond the temperature limiting growth and reproduction at atmospheric pressure, and B. stearothermophilus should grow in naturally or artificially warmed regions of the deep sea, where the pressure is less than approximately 50 MPa, although growth rates would be low above 10 MPa. PMID:16346444

  20. Analysis of the tryptophanase expression in Symbiobacterium thermophilum in a coculture with Geobacillus stearothermophilus.

    PubMed

    Watsuji, Tomo-O; Takano, Hideaki; Yamabe, Tomoya; Tamazawa, Satoshi; Ikemura, Hiroka; Ohishi, Takanori; Matsuda, Tohyo; Shiratori-Takano, Hatsumi; Beppu, Teruhiko; Ueda, Kenji

    2014-12-01

    The tryptophanase-positive Symbiobacterium thermophilum is a free-living syntrophic bacterium that grows effectively in a coculture with Geobacillus stearothermophilus. Our studies have shown that S. thermophilum growth depends on the high CO2 and low O2 condition established by the precedent growth of G. stearothermophilus. The use of an anoxic atmosphere containing high CO2 allows S. thermophilum to grow independently of G. stearothermophilus, but the cellular yield is ten times lower than that achieved in the coculture. In this study, we characterized the coculture-dependent expression and activity of tryptophanase in S. thermophilum. S. thermophilum cells accumulated a marked amount of indole in a coculture with G. stearothermophilus, but not in the bacterium's pure culture irrespective of the addition of tryptophan. S. thermophilum cells accumulated indole in its pure culture consisting of conditioned medium (medium supplied with culture supernatant of G. stearothermophilus). Proteomic analysis identified the protein specifically produced in the S. thermophilum cells grown in conditioned medium, which was a tryptophanase encoded by tna2 (STH439). An attempt to isolate the tryptophanase-inducing component from the culture supernatant of G. stearothermophilus was unsuccessful, but we did discover that the indole accumulation occurs when 10 mM bicarbonate is added to the medium. RT-PCR analysis showed that the addition of bicarbonate stimulated transcription of tna2. The transcriptional start site, identified within the tna2 promoter, was preceded by the -24 and -12 consensus sequences specified by an alternative sigma factor, σ(54). The evidence suggests that the transcription of some genes involved in amino acid metabolism is σ(54)-dependent, and that a bacterial enhancer-binding protein containing a PAS domain controls the transcription under the presence of high levels of bicarbonate.

  1. Development and application of Geobacillus stearothermophilus growth model for predicting spoilage of evaporated milk.

    PubMed

    Kakagianni, Myrsini; Gougouli, Maria; Koutsoumanis, Konstantinos P

    2016-08-01

    The presence of Geobacillus stearothermophilus spores in evaporated milk constitutes an important quality problem for the milk industry. This study was undertaken to provide an approach in modelling the effect of temperature on G. stearothermophilus ATCC 7953 growth and in predicting spoilage of evaporated milk. The growth of G. stearothermophilus was monitored in tryptone soy broth at isothermal conditions (35-67 °C). The data derived were used to model the effect of temperature on G. stearothermophilus growth with a cardinal type model. The cardinal values of the model for the maximum specific growth rate were Tmin = 33.76 °C, Tmax = 68.14 °C, Topt = 61.82 °C and μopt = 2.068/h. The growth of G. stearothermophilus was assessed in evaporated milk at Topt in order to adjust the model to milk. The efficiency of the model in predicting G. stearothermophilus growth at non-isothermal conditions was evaluated by comparing predictions with observed growth under dynamic conditions and the results showed a good performance of the model. The model was further used to predict the time-to-spoilage (tts) of evaporated milk. The spoilage of this product caused by acid coagulation when the pH approached a level around 5.2, eight generations after G. stearothermophilus reached the maximum population density (Nmax). Based on the above, the tts was predicted from the growth model as the sum of the time required for the microorganism to multiply from the initial to the maximum level ( [Formula: see text] ), plus the time required after the [Formula: see text] to complete eight generations. The observed tts was very close to the predicted one indicating that the model is able to describe satisfactorily the growth of G. stearothermophilus and to provide realistic predictions for evaporated milk spoilage.

  2. Differential response of NADP-dehydrogenases and carbon metabolism in leaves and roots of two durum wheat (Triticum durum Desf.) cultivars (Karim and Azizi) with different sensitivities to salt stress.

    PubMed

    Bouthour, Donia; Kalai, Tawba; Chaffei, Haouari C; Gouia, Houda; Corpas, Francisco J

    2015-05-01

    Wheat (Triticum durum Desf.) is a common Mediterranean species of considerable agronomic importance. Salinity is one of the major threats to sustainable agricultural production mainly because it limits plant productivity. After exposing the Karim and Azizi durum wheat cultivars, which are of agronomic significance in Tunisia, to 100mM NaCl salinity, growth parameters (dry weight and length), proline content and chlorophylls were evaluated in their leaves and roots. In addition, we analyzed glutathione content and key enzymatic activities, including phosphoenolpyruvate carboxylase (PEPC), NADP-isocitrate dehydrogenase (NADP-ICDH), NADP-malic enzyme (NADP-ME), glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), involved in the carbon metabolism and NADPH-generating system. The sensitivity index indicates that cv Karim was more tolerant to salinity than cv Azizi. This higher tolerance was corroborated at the biochemical level, as cv Karim showed a greater capacity to accumulate proline, especially in leaves, while the enzyme activities studied were differentially regulated in both organs, with NADP-ICDH being the only activity to be unaffected in all organs. In summary, the data indicate that higher levels of proline accumulation and the differential responses of some key enzymes involved in the carbon metabolism and NADPH regeneration contribute to the salinity tolerance mechanism and lead to increased biomass accumulation in cv Karim.

  3. 21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false α-Amylase enzyme preparation from Bacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1012 α-Amylase enzyme preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the...

  4. 21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false α-Amylase enzyme preparation from Bacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1012 α-Amylase enzyme preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the...

  5. 21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false α-Amylase enzyme preparation from Bacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1012 α-Amylase enzyme preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the...

  6. Substrate-Ligand Interactions in Geobacillus Stearothermophilus Nitric Oxide Synthase†

    PubMed Central

    Kabir, Mariam; Sudhamsu, Jawahar; Crane, Brian R.; Yeh, Syun-Ru; Rousseau, Denis L.

    2012-01-01

    Ntric oxide synthase (NOS) generates NO via a sequential two-step reaction, L-arginine (L-Arg) → N-hydroxy-L-arginine (NOHA) → L-citrulline + NO. Each step of the reaction follows a distinct mechanism defined by the chemical environment introduced by each substrate bound to the heme active site. The dioxygen complex of the NOS enzyme from a thermophilic bacterium, Geobacillus stearothermophilus (gsNOS), is unusually stable; hence it provides a unique model for the studies of the mechanistic differences between the two steps of the NOS reaction. By using CO as a structural probe, it was found that gsNOS exhibits two conformations in the absence of substrate, as indicated by the presence of two sets of the νFe-CO/νC-O modes in the resonance Raman spectra. In the νFe-CO versus νC-O inverse correlation plot, one set of the data falls on the correlation line characterized by mammalian NOSs (mNOS), whereas the other set of the data lies on a new correlation line defined by a bacterial NOS from Bacillus subtilis (bsNOS), reflecting a difference in the proximal Fe-Cys bond strength in the two conformers of gsNOS. The addition of L-Arg stabilizes the conformer associated with the mNOS correlation line, whereas NOHA stabilizes the conformer associated with the bsNOS correlation line, although both substrates introduce a positive electrostatic potential to the distal heme pocket. To assess how substrate-binding affects the Fe-Cys bond strength, the frequency of the Fe-Cys stretching mode of gsNOS was monitored by resonance Raman spectroscopy with 363.8 nm excitation. In the substrate-free form, the Fe-Cys stretching mode was detected at 342.5 cm−1 similar to that of bsNOS. The binding of L-Arg and NOHA brings about a small decrease and increase in the Fe-Cys stretching frequency, respectively. The implication of these unique structural features on the oxygen chemistry of NOS is discussed. PMID:18956884

  7. Substrate-ligand interactions in Geobacillus stearothermophilus nitric oxide synthase.

    PubMed

    Kabir, Mariam; Sudhamsu, Jawahar; Crane, Brian R; Yeh, Syun-Ru; Rousseau, Denis L

    2008-11-25

    Nitric oxide synthase (NOS) generates NO via a sequential two-step reaction [l-arginine (l-Arg) --> N-hydroxy-l-arginine (NOHA) --> l-citrulline + NO]. Each step of the reaction follows a distinct mechanism defined by the chemical environment introduced by each substrate bound to the heme active site. The dioxygen complex of the NOS enzyme from a thermophilic bacterium, Geobacillus stearothermophilus (gsNOS), is unusually stable; hence, it provides a unique model for the studies of the mechanistic differences between the two steps of the NOS reaction. By using CO as a structural probe, we found that gsNOS exhibits two conformations in the absence of substrate, as indicated by the presence of two sets of nu(Fe-CO)/nu(C-O) modes in the resonance Raman spectra. In the nu(Fe-CO) versus nu(C-O) inverse correlation plot, one set of data falls on the correlation line characterized by mammalian NOSs (mNOS), whereas the other set of data lies on a new correlation line defined by a bacterial NOS from Bacillus subtilis (bsNOS), reflecting a difference in the proximal Fe-Cys bond strength in the two conformers of gsNOS. The addition of l-Arg stabilizes the conformer associated with the mNOS correlation line, whereas NOHA stabilizes the conformer associated with the bsNOS correlation line, although both substrates introduce a positive electrostatic potential into the distal heme pocket. To assess how substrate binding affects Fe-Cys bond strength, the frequency of the Fe-Cys stretching mode of gsNOS was monitored by resonance Raman spectroscopy with 363.8 nm excitation. In the substrate-free form, the Fe-Cys stretching mode was detected at 342.5 cm(-1), similar to that of bsNOS. The binding of l-Arg and NOHA brings about a small decrease and increase in the Fe-Cys stretching frequency, respectively. The implication of these unique structural features with respect to the oxygen chemistry of NOS is discussed.

  8. Solution structure of the HU protein from Bacillus stearothermophilus.

    PubMed

    Vis, H; Mariani, M; Vorgias, C E; Wilson, K S; Kaptein, R; Boelens, R

    1995-12-08

    The histone-like protein HU from Bacillus stearothermophilus is a dimer with a molecular mass of 19.5 kDa that is capable of bending DNA. An X-ray structure has been determined, but no structure could be established for a large part of the supposed DNA-binding beta-arms. Using distance and dihedral constraints derived from triple-resonance NMR data of a 13C/15N doubly-labelled HU protein 49 distance geometry structures were calculated, which were refined by means of restrained Molecular Dynamics. From this set a total of 25 refined structures were selected having low constraint energy and few constraint violations. The ensemble of 25 structures display a root-mea-square co-ordinate deviation of 0.36 A with respect to the average structure, calculated over the backbone heavy atoms of residues 2 to 54 and 75 to 90 (and residues 2' to 54' and 75' to 90' of the second monomer). The structure of the core is very similar to that observed in the X-ray structure, with a pairwise r.m.s.d. of 1.06 A. The structure of the beta-hairpin arm contains a double flip-over at the prolines in the two strands of the beta-arm. Strong 15N-NH heteronuclear nuclear Overhauser effects indicate that the beta-arm and especially the tip is flexible. This explains the disorder observed in the solution and X-ray structures of the beta-arm, in respect of the core of the protein. Overlayed onto itself the beta-arm is better defined, with an r.m.s.d. of 1.0 A calculated over the backbone heavy atoms of residues 54 to 59 and 69 to 74. The tip of the arm adopts a well-defined 4:6 beta-hairpin conformation similar to the iron co-ordinating beta-arms of rubredoxin.

  9. 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

  10. Folding domains and intramolecular ionic interactions of lysine residues in glyceraldehyde 3-phosphate dehydrogenase.

    PubMed Central

    Lambert, J M; Perham, R N

    1977-01-01

    1. Treatment with methyl acetimidate was used to probe the topography of several tetrameric glyceraldehyde 3-phosphate dehydrogenases, in particular the holoenzymes from rabbit muscle and Bacillus stearothermophilus. During the course of the reaction with the rabbit muscle enzyme, the number of amino groups fell rapidly from the starting value of 27 per subunit to a value of approx. five per subunit. This number could be lowered further to values between one and two per subunit by a second treatment with methyl acetimidate. The enzyme remained tetrameric throughout and retained 50% of its initial catalytic activity at the end of the experiment. 2. Use of methyl [1-14C]acetimidate and small-scale methods of protein chemistry showed that only one amino group per subunit, that of lysine-306, was completely unavailable for reaction with imido ester in the native enzyme. This results is consistent with the structure of the highly homologous glyceraldehyde 3-phosphate dehydrogenase of lobster muscle deduced from X-ray-crystallographic analysis, since lysine-306 can be seen to form an intrachain ion-pair with aspartic acid-241 in the hydrophobic environment of a subunit-subunit interface. 3. Several other amino groups in the rabbit muscle enzyme that reacted only slowly with the reagent were also identified chemically. These were found to be located entirely in the C-terminal half of the polypeptides chain, which comprises a folding domain associated with catalytic activity and subunit contact in the three-dimensional structure. Slow reaction of these 'surface' amino groups with methyl acetimidate is attributed to intramolecular ionic interactions of the amino groups with neighbouring side-chain carboxyl groups, a conclusion that is compatible with the reported three-dimensional structure and with the dependence of the reaction of ionic stength. 4. Very similar results were obtained with the enzymes from B. stearothermophilus and from ox muscle and ox liver, supporting

  11. Production and characterization of a mesophilic lipase isolated from Bacillus stearothermophilus AB-1.

    PubMed

    Abada, Emad Abd El-Moniem

    2008-04-15

    Using Bacillus stearothermophilus AB-1 isolated from air, the production of lipase was attempted along with its purification and characterization studies. When different carbon and nitrogen sources were supplemented in the culture medium, xylose, tryptophan, alanine, phenylalanine and potassium nitrate were found to be the best. During cultivation, the strain secreted most of its lipase content after 48 h. In particular, the lipase produced in the culture broth showed 300 U mL(-1) when cultivated at optimal temperature and pH of 35 degrees C and 7.5, respectively. The enzyme was purified using 60% ammonium sulfate precipitation and sephadex G200 column chromatography. The enzyme was stable up to 40 degrees C and in the range of pH 7-8. This research reports for the first time the characterization of mesophilic lipase from Bacillus stearothermophilus AB-1 isolated from air.

  12. Thermal Adaptation of Dihydrofolate Reductase from the Moderate Thermophile Geobacillus stearothermophilus

    PubMed Central

    2014-01-01

    The thermal melting temperature of dihydrofolate reductase from Geobacillus stearothermophilus (BsDHFR) is ∼30 °C higher than that of its homologue from the psychrophile Moritella profunda. Additional proline residues in the loop regions of BsDHFR have been proposed to enhance the thermostability of BsDHFR, but site-directed mutagenesis studies reveal that these proline residues contribute only minimally. Instead, the high thermal stability of BsDHFR is partly due to removal of water-accessible thermolabile residues such as glutamine and methionine, which are prone to hydrolysis or oxidation at high temperatures. The extra thermostability of BsDHFR can be obtained by ligand binding, or in the presence of salts or cosolvents such as glycerol and sucrose. The sum of all these incremental factors allows BsDHFR to function efficiently in the natural habitat of G. stearothermophilus, which is characterized by temperatures that can reach 75 °C. PMID:24730604

  13. Extraction of Copper from Malanjkhand Low-Grade Ore by Bacillus stearothermophilus.

    PubMed

    Singh, Sradhanjali; Sukla, Lala Behari; Mishra, Baroda Kanta

    2011-10-01

    Thermophilic bacteria are actively prevalent in hot water springs. Their potential to grow and sustain at higher temperatures makes them exceptional compare to other microorganism. The present study was initiated to isolate, identify and determine the feasibility of extraction of copper using thermophilic heterotrophic bacterial strain. Bacillus stearothermophilus is a thermophilic heterotrophic bacterium isolated from hot water spring, Atri, Orissa, India. This bacterium was adapted to low-grade chalcopyrite ore and its efficiency to solubilize copper from Malanjkhand low-grade ore was determined. The low-grade copper ore contains 0.27% Cu, in which the major copper-bearing mineral is chalcopyrite associated with other minerals present as minor phase. Variation in parameters such as pulp-density and temperatures were studied. After 30 days of incubation, it was found that Bacillus stearothermophilus solubilize copper up to 81.25% at pH 6.8 at 60°C.

  14. Effect of Hyperbaric Carbon Dioxide on Spores and Vegetative Cells of Bacillus stearothermophilus

    DTIC Science & Technology

    1994-05-01

    SUBJECT TERMS BACILLUS STEAROTHERMOPHILUS THERM0PHILIC BACTERIA THERM0PHILIC SPOILAGE 15. NUMBER OF PAGES 39 16 PRICE CODE 17. SECURITY...both vegetative and sporeforming bacteria in model food systems and ration items, (3) select optimum atmosphere/pressures as a result of time...atmospheres and stopped at 25 atmospheres of pure C02 (10). Doyle revealed that at atmospheric pressure 100% C02 delayed toxin production by C

  15. Isolation of Glucocardiolipins from Geobacillus stearothermophilus NRS 2004/3a

    PubMed Central

    Schäffer, Christina; Beckedorf, Anke I.; Scheberl, Andrea; Zayni, Sonja; Peter-Katalinić, Jasna; Messner, Paul

    2002-01-01

    Glucose-substituted cardiolipins account for about 4 mol% of total phospholipid extracted from exponentially grown cells of Geobacillus stearothermophilus NRS 2004/3a. Individual glucocardiolipin species exhibited differences in fatty acid substitution, with iso-C15:0 and anteiso-C17:0 prevailing. The compounds were purified to homogeneity by a novel protocol and precharacterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. PMID:12426359

  16. A thermostable, sequence-specific restriction endonuclease from Bacillus stearothermophilus: BstPI.

    PubMed Central

    Pugatsch, T; Weber, H

    1979-01-01

    A restriction endonuclease, BstPI, was purified from a strain of B. stearothermophilus, and its cleavage specificity was determined. The enzyme cleaves at palindromic sites of the general structure: (Formula: see text) where N.N' can be any base pair. It produces phosphorylated 5'-termini which are single stranded over a length of 5 nucleotides. Ends generated by cleavage with BstPI can be rejoined by DNA ligase. Images PMID:503858

  17. Gene cloning, sequence analysis, purification, and characterization of a thermostable aminoacylase from Bacillus stearothermophilus.

    PubMed Central

    Sakanyan, V; Desmarez, L; Legrain, C; Charlier, D; Mett, I; Kochikyan, A; Savchenko, A; Boyen, A; Falmagne, P; Pierard, A

    1993-01-01

    A genomic DNA fragment encoding aminoacylase activity of the eubacterium Bacillus stearothermophilus was cloned into Escherichia coli. Transformants expressing aminoacylase activity were selected by their ability to complement E. coli mutants defective in acetylornithine deacetylase activity, the enzyme that converts N-acetylornithine to ornithine in the arginine biosynthetic pathway. The 2.3-kb cloned fragment has been entirely sequenced. Analysis of the sequence revealed two open reading frames, one of which encoded the aminoacylase. B. stearothermophilus aminoacylase, produced in E. coli, was purified to near homogeneity in three steps, one of which took advantage of the intrinsic thermostability of the enzyme. The enzyme exists as homotetramer of 43-kDa subunits as shown by cross-linking experiments. The deacetylating capacity of purified aminoacylase varies considerably depending on the nature of the amino acid residue in the substrate. The enzyme hydrolyzes N-acyl derivatives of aromatic amino acids most efficiently. Comparison of the predicted amino acid sequence of B. stearothermophilus aminoacylase with those of eubacterial acetylornithine deacylase, succinyldiaminopimelate desuccinylase, carboxypeptidase G2, and eukaryotic aminoacylase I suggests a common origin for these enzymes. Images PMID:8285691

  18. Lactate dehydrogenase test

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003471.htm Lactate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Lactate dehydrogenase (LDH) is a protein that helps produce energy ...

  19. Expression and Characterization of Geobacillus stearothermophilus SR74 Recombinant α-Amylase in Pichia pastoris

    PubMed Central

    Gandhi, Sivasangkary; Salleh, Abu Bakar; Rahman, Raja Noor Zaliha Raja Abd; Chor Leow, Thean; Oslan, Siti Nurbaya

    2015-01-01

    Geobacillus stearothermophilus SR74 is a locally isolated thermophilic bacteria producing thermostable and thermoactive α-amylase. Increased production and commercialization of thermostable α-amylase strongly warrant the need of a suitable expression system. In this study, the gene encoding the thermostable α-amylase in G. stearothermophilus SR74 was amplified, sequenced, and subcloned into P. pastoris GS115 strain under the control of a methanol inducible promoter, alcohol oxidase (AOX). Methanol induced recombinant expression and secretion of the protein resulted in high levels of extracellular amylase production. YPTM medium supplemented with methanol (1% v/v) was the best medium and once optimized, the maximum recombinant α-amylase SR74 achieved in shake flask was 28.6 U mL−1 at 120 h after induction. The recombinant 59 kDa α-amylase SR74 was purified 1.9-fold using affinity chromatography with a product yield of 52.6% and a specific activity of 151.8 U mg−1. The optimum pH of α-amylase SR74 was 7.0 and the enzyme was stable between pH 6.0–8.0. The purified enzyme was thermostable and thermoactive, exhibiting maximum activity at 65°C with a half-life (t1/2) of 88 min at 60°C. In conclusion, thermostable α-amylase SR74 from G. stearothermophilus SR74 would be beneficial for industrial applications, especially in liquefying saccrification. PMID:26090417

  20. Expression and Characterization of Geobacillus stearothermophilus SR74 Recombinant α-Amylase in Pichia pastoris.

    PubMed

    Gandhi, Sivasangkary; Salleh, Abu Bakar; Rahman, Raja Noor Zaliha Raja Abd; Chor Leow, Thean; Oslan, Siti Nurbaya

    2015-01-01

    Geobacillus stearothermophilus SR74 is a locally isolated thermophilic bacteria producing thermostable and thermoactive α-amylase. Increased production and commercialization of thermostable α-amylase strongly warrant the need of a suitable expression system. In this study, the gene encoding the thermostable α-amylase in G. stearothermophilus SR74 was amplified, sequenced, and subcloned into P. pastoris GS115 strain under the control of a methanol inducible promoter, alcohol oxidase (AOX). Methanol induced recombinant expression and secretion of the protein resulted in high levels of extracellular amylase production. YPTM medium supplemented with methanol (1% v/v) was the best medium and once optimized, the maximum recombinant α-amylase SR74 achieved in shake flask was 28.6 U mL(-1) at 120 h after induction. The recombinant 59 kDa α-amylase SR74 was purified 1.9-fold using affinity chromatography with a product yield of 52.6% and a specific activity of 151.8 U mg(-1). The optimum pH of α-amylase SR74 was 7.0 and the enzyme was stable between pH 6.0-8.0. The purified enzyme was thermostable and thermoactive, exhibiting maximum activity at 65°C with a half-life (t₁/₂) of 88 min at 60°C. In conclusion, thermostable α-amylase SR74 from G. stearothermophilus SR74 would be beneficial for industrial applications, especially in liquefying saccrification.

  1. ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method.

    PubMed

    Yang, Mu; Wang, Ganggang

    2016-09-15

    The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication. In this work, a non-radioactive method for measuring ATPase activity of DnaBBst helicase was described. The working parameters and conditions were optimized. Furthermore, this method was applied to investigate effects of DnaG primase, ssDNA and helicase loader protein (DnaI) on ATPase activity of DnaBBst. Our results showed this method was sensitive and efficient. Moreover, it is suitable for the investigation of functional interaction between DnaB and related factors.

  2. Biotransformation of 2,6-diaminopurine nucleosides by immobilized Geobacillus stearothermophilus.

    PubMed

    De Benedetti, Eliana C; Rivero, Cintia W; Britos, Claudia N; Lozano, Mario E; Trelles, Jorge A

    2012-01-01

    An efficient and green bioprocess to obtain 2,6-diaminopurine nucleosides using thermophilic bacteria is herein reported. Geobacillus stearothermophilus CECT 43 showed a conversion rate of 90 and 83% at 2 h to obtain 2,6-diaminopurine-2'-deoxyriboside and 2,6-diaminopurine riboside, respectively. The selected biocatalyst was successfully stabilized in an agarose matrix and used to produce up to 23.4 g of 2,6-diaminopurine-2'-deoxyriboside in 240 h of process. These nucleoside analogues can be used as prodrug precursors or in antisense oligonucleotide synthesis.

  3. Die another day: Fate of heat-treated Geobacillus stearothermophilus ATCC 12980 spores during storage under growth-preventing conditions.

    PubMed

    Mtimet, Narjes; Trunet, Clément; Mathot, Anne-Gabrielle; Venaille, Laurent; Leguérinel, Ivan; Coroller, Louis; Couvert, Olivier

    2016-06-01

    Geobacillus stearothermophilus spores are recognized as one of the most wet-heat resistant among aerobic spore-forming bacteria and are responsible for 35% of canned food spoilage after incubation at 55 °C. The purpose of this study was to investigate and model the fate of heat-treated survivor spores of G. stearothermophilus ATCC 12980 in growth-preventing environment. G. stearothermophilus spores were heat-treated at four different conditions to reach one or two decimal reductions. Heat-treated spores were stored in nutrient broth at different temperatures and pH under growth-preventing conditions. Spore survival during storage was evaluated by count plating over a period of months. Results reveal that G. stearothermophilus spores surviving heat treatment lose their viability during storage under growth-preventing conditions. Two different subpopulations were observed during non-thermal inactivation. They differed according to the level of their resistance to storage stress, and the proportion of each subpopulation can be modulated by heat treatment conditions. Finally, tolerance to storage stress under growth-preventing conditions increases at refrigerated temperature and neutral pH regardless of heat treatment conditions. Such results suggest that spore inactivation due to heat treatment could be completed by storage under growth-preventing conditions.

  4. A group IIC-type intron interrupts the rRNA methylase gene of Geobacillus stearothermophilus strain 10.

    PubMed

    Moretz, Samuel E; Lampson, Bert C

    2010-10-01

    Group IIC introns insert next to the stem-loop structure of rho-independent transcription terminators, thus avoiding intact genes. The insertion sites of 17 copies of the G.st.I1 intron from Geobacillus stearothermophilus were compared. One copy of the intron was found to interrupt an open reading frame (ORF) encoding an rRNA methylase.

  5. Bacterial metabolites from intra- and inter-species influencing thermotolerance: the case of Bacillus cereus and Geobacillus stearothermophilus.

    PubMed

    Gómez-Govea, Mayra Alejandra; García, Santos; Heredia, Norma

    2016-11-28

    Bacterial metabolites with communicative functions could provide protection against stress conditions to members of the same species. Yet, information remains limited about protection provided by metabolites in Bacillus cereus and inter-species. This study investigated the effect of extracellular compounds derived from heat shocked (HS) and non-HS cultures of B. cereus and Geobacillus stearothermophilus on the thermotolerance of non-HS vegetative and sporulating B. cereus. Cultures of B. cereus and G. stearothermophilus were subjected to HS (42 or 65 °C respectively for 30 min) or non-HS treatments. Cells and supernatants were separated, mixed in a combined array, and then exposed to 50 °C for 60 min and viable cells determined. For spores, D values (85 and 95 °C) were evaluated after 120 h. In most cases, supernatants from HS B. cereus cultures added to non-HS B. cereus cells caused their thermotolerance to increase (D 50 12.2-51.9) when compared to supernatants from non-HS cultures (D 50 7.4-21.7). While the addition of supernatants from HS and non-HS G. stearothermophilus cultures caused the thermotolerance of non-HS cells from B. cereus to decrease initially (D 50 3.7-7.1), a subsequent increase was detected in most cases (D 50 18-97.7). In most cases, supernatants from sporulating G. stearothermophilus added to sporulating cells of B. cereus caused the thermotolerance of B. cereus 4810 spores to decline, whereas that of B. cereus 14579 increased. This study clearly shows that metabolites in supernatants from either the same or different species (such as G. stearothermophilus) influence the thermotolerance of B. cereus.

  6. 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.

  7. Occurrence of cold-labile NAD-specific glutamate dehydrogenase in Bacillus species.

    PubMed

    Jahns, T

    1992-09-15

    A nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (NAD-GluDH; EC 1.4.1.3) inactivated by incubation at low temperatures was detected in several species of the genus Bacillus, including strains of B. cereus, B. laterosporus, B. lentus, B. panthotenicus, B. pasteurii, B. sphaericus, B. stearothermophilus, B. subtilis and B. thuringiensis. Incubation of cell-free extracts of these strains at 0 degrees C resulted in an 80-100% inactivation of NAD-GluDH activity within 120 min. The addition of 20% glycerol protected the enzyme from this inactivation in the cold. Strains of B. fastidiosus, B. licheniformis, B. macerans, B. megaterium and B. pumilus were found to lack NAD-GluDH activity.

  8. Relief of Casein Inhibition of Bacillus stearothermophilus by Iron, Calcium, and Magnesium1

    PubMed Central

    Ashton, D. H.; Busta, F. F.; Warren, J. A.

    1968-01-01

    Growth of Bacillus stearothermophilus strain NCA 1518 Smooth in Dextrose Tryptone Agar (DTA) was inhibited by sodium caseinate. Binding studies indicated that sodium caseinate, when present in DTA, had the capacity to effect an iron deficiency which could cause inhibition of growth. Additions of essential cations, iron (1 mM), calcium (5 mM), magnesium (10 mM), or hydrogen ion (pH 5.7), relieved inhibition. Responses to and interactions among these relief factors were analyzed statistically. Equations were fitted to the data and were used to estimate responses to all treatment combinations within the ranges tested. Results from these studies indicated that calcium, magnesium, and hydrogen ion acted by decreasing the binding capacity of the protein for iron, rendering this metal available for metabolic needs. Evidence was obtained that ferrous rather than ferric iron was the limiting factor in DTA containing sodium caseinate. PMID:5694503

  9. Investigation of Sterilization Mechanism for Geobacillus stearothermophilus Spores with Plasma-Excited Neutral Gas

    NASA Astrophysics Data System (ADS)

    Matsui, Kei; Ikenaga, Noriaki; Sakudo, Noriyuki

    2015-09-01

    We investigate the mechanism of the sterilization with plasma-excited neutral gas that uniformly sterilizes both the space and inner wall of the reactor chamber at atmospheric pressure. Only reactive neutral species such as plasma-excited gas molecules and radicals are separated from the plasma and sent to the reactor chamber for chemical sterilization. The plasma source gas uses humidified mixture of nitrogen and oxygen. Geobacillus stearothermophilus spores and tyrosine which is amino acid are treated by the plasma-excited neutral gas. Shape change of the treated spore is observed by SEM, and chemical modification of the treated tyrosine is analyzed by HPLC. As a result, the surface of the treated spore shows depression. Hydroxylation and nitration of tyrosine are shown after the treatment. For these reasons, we believe that the sterilization with plasma-excited neutral gas results from the deformation of spore structure due to the chemical modification of amino acid.

  10. Keratinous waste decomposition and peptide production by keratinase from Geobacillus stearothermophilus AD-11.

    PubMed

    Gegeckas, Audrius; Gudiukaitė, Renata; Debski, Janusz; Citavicius, Donaldas

    2015-04-01

    A keratinolytic proteinase was cloned from thermophilic bacterium Geobacillus stearothermophilus AD-11 and was expressed in Escherichia coli BL21(DE3). Recombinant keratinolytic proteinase (RecGEOker) with an estimated molecular weight of 57 kDa was purified and keratinase activity was measured. RecGEOker showed optimal activity at pH 9 and 60 °C. Recombinant keratinolytic proteinase showed the highest substrate specificity toward keratin from wool > collagen > sodium caseinate > gelatin > and BSA in descending order. RecGEOker is applicable for efficient keratin waste biodegradation and can replace conventional non-biological hydrolysis processes. High-value small peptides obtained from enzymatic biodegradation by RecGEOker are suitable for industrial application in white and/or green biotechnology for use as major additives in various products.

  11. Bacillus stearothermophilus Neopullulanase Selective Hydrolysis of Amylose to Maltose in the Presence of Amylopectin

    PubMed Central

    Kamasaka, Hiroshi; Sugimoto, Kazuhisa; Takata, Hiroki; Nishimura, Takahisa; Kuriki, Takashi

    2002-01-01

    The specificity of Bacillus stearothermophilus TRS40 neopullulanase toward amylose and amylopectin was analyzed. Although this neopullulanase completely hydrolyzed amylose to produce maltose as the main product, it scarcely hydrolyzed amylopectin. The molecular mass of amylopectin was decreased by only one order of magnitude, from approximately 108 to 107 Da. Furthermore, this neopullulanase selectively hydrolyzed amylose when starch was used as a substrate. This phenomenon, efficient hydrolysis of amylose but not amylopectin, was also observed with cyclomaltodextrinase from alkaliphilic Bacillus sp. strain A2-5a and maltogenic amylase from Bacillus licheniformis ATCC 27811. These three enzymes hydrolyzed cyclomaltodextrins and amylose much faster than pullulan. Other amylolytic enzymes, such as bacterial saccharifying α-amylase, bacterial liquefying α-amylase, β-amylase, and neopullulanase from Bacillus megaterium, did not exhibit this distinct substrate specificity at all, i.e., the preference of amylose to amylopectin. PMID:11916682

  12. Characteristic views of E. coli and B. stearothermophilus 30S ribosomal subunits in the electron microscope.

    PubMed Central

    van Heel, M; Stöffler-Meilicke, M

    1985-01-01

    Large sets of electron microscopic images of the 30S ribosomal subunits of Bacillus stearothermophilus (914 molecules) and Escherichia coli (422 molecules) were analysed with image processing techniques. Using computer alignment and a new multivariate statistical classification scheme, three predominant views of the subunit were found for both species. These views, which together account for approximately 90% of the population of images, were determined to a reproducible resolution of up to 1.7 nm, thus elucidating many new structural details. The angular spread of the molecular orientations around the three main stable positions is remarkably small (less than 8 degrees). Some of the current models for the small ribosomal subunit are incompatible with our new results. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3908096

  13. The abp gene in Geobacillus stearothermophilus T-6 encodes a GH27 β-L-arabinopyranosidase.

    PubMed

    Salama, Rachel; Alalouf, Onit; Tabachnikov, Orly; Zolotnitsky, Gennady; Shoham, Gil; Shoham, Yuval

    2012-07-30

    In this study we demonstrate that the abp gene in Geobacillus stearothermophilus T-6 encodes a family 27 glycoside hydrolase β-L-arabinopyranosidase. The catalytic constants towards the chromogenic substrate pNP-β-L-arabinopyranoside were 0.8±0.1 mM, 6.6±0.3 s(-1), and 8.2±0.3 s(-1) mM(-1) for K(m), k(cat) and k(cat)/K(m), respectively. (13)C NMR spectroscopy unequivocally showed that Abp is capable of removing β-L-arabinopyranose residues from the natural arabino-polysaccharide, larch arabinogalactan. Most family 27 enzymes are active on galactose and contain a conserved Asp residue, whereas in Abp this residue is Ile67, which shifts the specificity of the enzyme towards arabinopyranoside.

  14. Bleach-boosting effect of crude xylanase from Bacillus stearothermophilus SDX on wheat straw pulp.

    PubMed

    Garg, Gaurav; Dhiman, Saurabh Sudha; Mahajan, Ritu; Kaur, Amanjot; Sharma, Jitender

    2011-01-31

    Pretreatment of wheat straw pulp using cellulase-free xylanase produced from Bacillus stearothermophilus SDX at 60°C for 120min resulted in 4.75% and 22.31% increase in brightness and whiteness, respectively. Enzyme dose of 10U/g of oven dried pulp at pH 9 decreased the kappa number and permanganate number by 7.14% and 5.31%, respectively. Further chlorine dioxide and alkaline bleaching sequences (CDED(1)D(2)) resulted in 1.76% and 3.63% increase in brightness and whiteness, respectively. Enzymatic prebleaching of pulp decreased 20% of chlorine consumption without any decrease in brightness. Improvement in various pulp properties like viscosity, burst factor, burstness, breaking length, double fold, gurley porosity, tear factor, and tearness were also observed after bleaching of xylanase treated wheat straw pulp.

  15. Improving thermal and detergent stability of Bacillus stearothermophilus neopullulanase by rational enzyme design.

    PubMed

    Ece, Selin; Evran, Serap; Janda, Jan-Oliver; Merkl, Rainer; Sterner, Reinhard

    2015-06-01

    Neopullulanase, a glycosyl hydrolase from Bacillus stearothermophilus (bsNpl), is a potentially valuable enzyme for starch and detergent industries. However, as the protein is not active at elevated temperatures and high surfactant concentrations, we aimed to increase its stability by rational enzyme design. Nine potentially destabilizing cavities were identified in the crystal structure of the enzyme. Based on computational predictions, these cavities were filled by residues with bulkier side chains. The five Asp46Glu, Val239Leu, Val404Leu, Ser407Thr and Ala566Leu exchanges resulted in a drastic stabilization of bsNpl against inactivation by heat and detergents. The catalytic activity of the variants was identical to the wild-type enzyme.

  16. Purification and characterization of cloned alkaline protease gene of Geobacillus stearothermophilus.

    PubMed

    Iqbal, Irfana; Aftab, Muhammad Nauman; Afzal, Mohammed; Ur-Rehman, Asad; Aftab, Saima; Zafar, Asma; Ud-Din, Zia; Khuharo, Ateeque Rahman; Iqbal, Jawad; Ul-Haq, Ikram

    2015-02-01

    Thermostable alkaline serine protease gene of Geobacillus stearothermophilus B-1172 was cloned and expressed in Escherichia coli BL21 (DE3) using pET-22b(+), as an expression vector. The growth conditions were optimized for maximal production of the protease using variable fermentation parameters, i.e., pH, temperature, and addition of an inducer. Protease, thus produced, was purified by ammonium sulfate precipitation followed by ion exchange chromatography with 13.7-fold purification, with specific activity of 97.5 U mg(-1) , and a recovery of 23.6%. Molecular weight of the purified protease, 39 kDa, was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 90 °C at pH 9. The enzyme activity was steady in the presence of EDTA indicating that the protease was not a metalloprotease. No significant change in the activity of protease after addition of various metal ions further strengthened this fact. However, an addition of 1% Triton X-100 or SDS surfactants constrained the enzyme specific activity to 34 and 19%, respectively. Among organic solvents, an addition of 1-butanol (20%) augmented the enzyme activity by 29% of the original activity. With casein as a substrate, the enzyme activity under optimized conditions was found to be 73.8 U mg(-1) . The effect of protease expression on the host cells growth was also studied and found to negatively affect E. coli cells to certain extent. Catalytic domains of serine proteases from eight important thermostable organisms were analyzed through WebLogo and found to be conserved in all serine protease sequences suggesting that protease of G. stearothermophilus could be beneficially used as a biocontrol agent and in many industries including detergent industry.

  17. Toxicological effects of thiomersal and ethylmercury: Inhibition of the thioredoxin system and NADP{sup +}-dependent dehydrogenases of the pentose phosphate pathway

    SciTech Connect

    Rodrigues, Juan; Branco, Vasco; Lu, Jun; Holmgren, Arne; Carvalho, Cristina

    2015-08-01

    Mercury (Hg) is a strong toxicant affecting mainly the central nervous, renal, cardiovascular and immune systems. Thiomersal (TM) is still in use in medical practice as a topical antiseptic and as a preservative in multiple dose vaccines, routinely given to young children in some developing countries, while other forms of mercury such as methylmercury represent an environmental and food hazard. The aim of the present study was to determine the effects of thiomersal (TM) and its breakdown product ethylmercury (EtHg) on the thioredoxin system and NADP{sup +}-dependent dehydrogenases of the pentose phosphate pathway. Results show that TM and EtHg inhibited the thioredoxin system enzymes in purified suspensions, being EtHg comparable to methylmercury (MeHg). Also, treatment of neuroblastoma and liver cells with TM or EtHg decreased cell viability (GI{sub 50}: 1.5 to 20 μM) and caused a significant (p < 0.05) decrease in the overall activities of thioredoxin (Trx) and thioredoxin reductase (TrxR) in a concentration- and time-dependent manner in cell lysates. Compared to control, the activities of Trx and TrxR in neuroblastoma cells after EtHg incubation were reduced up to 60% and 80% respectively, whereas in hepatoma cells the reduction was almost 100%. In addition, the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were also significantly inhibited by all mercurials, with inhibition intensity of Hg{sup 2+} > MeHg ≈ EtHg > TM (p < 0.05). Cell incubation with sodium selenite alleviated the inhibitory effects on TrxR and glucose-6-phosphate dehydrogenase. Thus, the molecular mechanism of toxicity of TM and especially of its metabolite EtHg encompasses the blockage of the electrons from NADPH via the thioredoxin system. - Highlights: • TM and EtHg inhibit Trx and TrxR both in purified suspensions and cell lysates. • TM and EtHg also inhibit the activities of G6PDH and 6PGDH in cell lysates, • Co-exposure to selenite alleviates

  18. Direct fermentation of potato starch and potato residues to lactic acid by Geobacillus stearothermophilus under non-sterile conditions

    PubMed Central

    Smerilli, Marina; Neureiter, Markus; Wurz, Stefan; Haas, Cornelia; Frühauf, Sabine; Fuchs, Werner

    2015-01-01

    BACKGROUND Lactic acid is an important biorefinery platform chemical. The use of thermophilic amylolytic microorganisms to produce lactic acid by fermentation constitutes an efficient strategy to reduce operating costs, including raw materials and sterilization costs. RESULTS A process for the thermophilic production of lactic acid by Geobacillus stearothermophilus directly from potato starch was characterized and optimized. Geobacillus stearothermophilus DSM 494 was selected out of 12 strains screened for amylolytic activity and the ability to form lactic acid as the major product of the anaerobic metabolism. In total more than 30 batches at 3–l scale were run at 60 °C under non-sterile conditions. The process developed produced 37 g L−1 optically pure (98%) L-lactic acid in 20 h from 50 g L−1 raw potato starch. As co-metabolites smaller amounts (<7% w/v) of acetate, formate and ethanol were formed. Yields of lactic acid increased from 66% to 81% when potato residues from food processing were used as a starchy substrate in place of raw potato starch. CONCLUSIONS Potato starch and residues were successfully converted to lactic acid by G. stearothermophilus. The process described in this study provides major benefits in industrial applications and for the valorization of starch-rich waste streams. © 2015 The Authors.Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. PMID:25937690

  19. Glucose-6-phosphate dehydrogenase

    MedlinePlus

    ... Elsevier Saunders; 2012:chap 42. Read More Enzyme Glucose-6-phosphate dehydrogenase deficiency Hemoglobin Review Date 2/11/2016 Updated by: ... A.M. Editorial team. Related MedlinePlus Health Topics G6PD Deficiency Browse the Encyclopedia A.D.A.M., Inc. ...

  20. HIV-1 Gag p17 presented as virus-like particles on the E2 scaffold from Geobacillus stearothermophilus induces sustained humoral and cellular immune responses in the absence of IFNγ production by CD4+ T cells

    PubMed Central

    Caivano, Antonella; Doria-Rose, Nicole A.; Buelow, Benjamin; Sartorius, Rossella; Trovato, Maria; D’Apice, Luciana; Domingo, Gonzalo J.; Sutton, William F.; Haigwood, Nancy L.; De Berardinis, Piergiuseppe

    2012-01-01

    We have constructed stable virus-like particles displaying the HIV-1 Gag(p17) protein as an N-terminal fusion with an engineered protein domain from the Geobacillus stearothermophilus pyruvate dehydrogenase subunit E2. Mice immunized with the Gag(p17)-E2 60-mer scaffold particles mounted a strong and sustained antibody response. Antibodies directed to Gag(p17) were boosted significantly with additional immunizations, while anti-E2 responses reached a plateau. The isotype of the induced antibodies was biased towards IgG1, and the E2-primed CD4+ T cells did not secrete IFNγ. Using transgenic mouse model systems, we demonstrated that CD8+ T cells primed with E2 particles were able to exert lytic activity and produce IFNγ. These results show that the E2 scaffold represents a powerful vaccine delivery system for whole antigenic proteins or polyepitope engineered proteins, evoking antibody production and antigen specific CTL activity even in the absence of IFNγ-producing CD4+ T cells. PMID:20850858

  1. HIV-1 Gag p17 presented as virus-like particles on the E2 scaffold from Geobacillus stearothermophilus induces sustained humoral and cellular immune responses in the absence of IFN{gamma} production by CD4+ T cells

    SciTech Connect

    Caivano, Antonella; Doria-Rose, Nicole A.; Buelow, Benjamin; Sartorius, Rossella; Trovato, Maria; D'Apice, Luciana; Domingo, Gonzalo J.; Sutton, William F.; Haigwood, Nancy L.; De Berardinis, Piergiuseppe

    2010-11-25

    We have constructed stable virus-like particles displaying the HIV-1 Gag(p17) protein as an N-terminal fusion with an engineered protein domain from the Geobacillus stearothermophilus pyruvate dehydrogenase subunit E2. Mice immunized with the Gag(p17)-E2 60-mer scaffold particles mounted a strong and sustained antibody response. Antibodies directed to Gag(p17) were boosted significantly with additional immunizations, while anti-E2 responses reached a plateau. The isotype of the induced antibodies was biased towards IgG1, and the E2-primed CD4+ T cells did not secrete IFN{gamma}. Using transgenic mouse model systems, we demonstrated that CD8+ T cells primed with E2 particles were able to exert lytic activity and produce IFN{gamma}. These results show that the E2 scaffold represents a powerful vaccine delivery system for whole antigenic proteins or polyepitope engineered proteins, evoking antibody production and antigen specific CTL activity even in the absence of IFN{gamma}-producing CD4+ T cells.

  2. Electron transfer kinetics of caa3 oxidase from Bacillus stearothermophilus: a hypothesis for thermophilicity.

    PubMed Central

    Giuffrè, A; Watmough, N J; Giannini, S; Brunori, M; Konings, W N; Greenwood, C

    1999-01-01

    The O2 reaction and the reverse electron transfer of the thermophilic caa3 terminal oxidase of Bacillus stearothermophilus have been studied by laser flash-photolysis. The results show that both reactions, although studied at a temperature of 20 degreesC, far from the optimal temperature of > 60 degreesC for caa3, follow a kinetic behavior essentially identical to that observed with the electrostatic complex between mammalian cyt c and cyt c oxidase. In the O2 reaction cyt a and cyt a3 are very quickly oxidized; cyt a is then re-reduced via CuA, whereas cyt c oxidation is apparently rate-limited by the oxidation of CuA. Upon photodissociation of the mixed valence-CO caa3, reverse electron transfer from the binuclear center to cyt a3+ (tau1 = 3 micros) and CuA2+ (tau2 = 64 micros) is observed, while cyt c is not reduced by any detectable level. These results seem to rule out accounting for enzymatic thermophilicity by altered kinetics of intramolecular electron transfer involving the cyt center in the reduced configuration, which is very fast. On the basis of these results and previous data, we propose that thermophilicity involves an increased activation barrier for the reduction of cyt a3-CuB in the configuration typical of the oxidized site. PMID:9876155

  3. The HPr Proteins from the Thermophile Bacillus stearothermophilus Can Form Domain-swapped Dimers

    SciTech Connect

    Sridharan, Sudharsan; Razvi, Abbas; Scholtz, J. Martin; Sacchettini, James C.

    2010-07-20

    The study of proteins from extremophilic organisms continues to generate interest in the field of protein folding because paradigms explaining the enhanced stability of these proteins still elude us and such studies have the potential to further our knowledge of the forces stabilizing proteins. We have undertaken such a study with our model protein HPr from a mesophile, Bacillus subtilis, and a thermophile, Bacillus stearothermophilus. We report here the high-resolution structures of the wild-type HPr protein from the thermophile and a variant, F29W. The variant proved to crystallize in two forms: a monomeric form with a structure very similar to the wild-type protein as well as a domain-swapped dimer. Interestingly, the structure of the domain-swapped dimer for HPr is very different from that observed for a homologous protein, Crh, from B. subtilis. The existence of a domain-swapped dimer has implications for amyloid formation and is consistent with recent results showing that the HPr proteins can form amyloid fibrils. We also characterized the conformational stability of the thermophilic HPr proteins using thermal and solvent denaturation methods and have used the high-resolution structures in an attempt to explain the differences in stability between the different HPr proteins. Finally, we present a detailed analysis of the solution properties of the HPr proteins using a variety of biochemical and biophysical methods.

  4. Crystal Structure of the Geobacillus stearothermophilus Carboxylesterase Est55 and Its Activation of Prodrug CPT-11

    PubMed Central

    Liu, Ping; Ewis, Hosam E.; Tai, Phang C.; Lu, Chung-Dar; Weber, Irene T.

    2007-01-01

    Several mammalian carboxylesterases were shown to activate the prodrug irinotecan (CPT-11) to produce SN-38, a topoisomerase inhibitor used in cancer therapy. However, the potential use of bacterial carboxylesterases, which have the advantage of high stability, has not been explored. We present the crystal structure of the carboxyesterase Est55 from Geobacillus stearothermophilus and evaluation of its enzyme activity on CPT-11. Crystal structures were determined at pH 6.2 and 6.8 and resolution of 2.0 and 1.58 Å, respectively. Est55 folds into three domains, a catalytic domain, an α/β domain and a regulatory domain. The structure is in an inactive form; the side chain of His409, one of the catalytic triad residues, is directed away from the other catalytic residues Ser194 and Glu310. Moreover, the adjacent Cys408 is triply oxidized and lies in the oxyanion hole, which would block the binding of substrate, suggesting a regulatory role. However, Cys408 is not essential for enzyme activity. Mutation of Cys408 showed that hydrophobic side chains were favorable, while polar serine was unfavorable for enzyme activity. Est55 was shown to hydrolyze CPT-11 into the active form SN-38. The mutant C408V provided a more stable enzyme for activation of CPT-11. Therefore, engineered thermostable Est55 is a candidate for use with irinotecan in enzyme-prodrug cancer therapy. PMID:17239398

  5. Racemization of alanine by the alanine racemases from Salmonella typhimurium and Bacillus stearothermophilus: energetic reaction profiles

    SciTech Connect

    Faraci, W.S.; Walsh, C.T.

    1988-05-03

    Alanine racemases are bacterial pyridoxal 5'-phosphate (PLP) dependent enzymes providing D-alanine as an essential building block for biosynthesis of the peptidoglycan layer of the cell wall. Two isozymic alanine racemases, encoded by the dadB gene and the alr gene, from the Gram-negative mesophilic Salmonella typhimurium and one from the Gram-positive thermophilic Bacillus stearothermophilus have been examined for the racemization mechanism. Substrate deuterium isotope effects and solvent deuterium isotope effects have been measured in both L ..-->.. D and D..-->.. L directions for all three enzymes to assess the degree to which abstraction of the ..cap alpha..-proton or protonation of substrate PLP carbanion is limiting in catalysis. Additionally, experiments measuring internal return of ..cap alpha..-/sup 3/H from substrate to product and solvent exchange/substrate conversion experiments in /sup 3/H/sub 2/O have been used with each enzyme to examine the partitioning of substrate PLP carbanion intermediates and to obtain the relative heights of kinetically significant energy barriers in alanine racemase catalysis.

  6. Enhanced maltose production through mutagenesis of acceptor binding subsite +2 in Bacillus stearothermophilus maltogenic amylase.

    PubMed

    Sun, Yecheng; Duan, Xuguo; Wang, Lei; Wu, Jing

    2016-01-10

    Maltogenic amylases are used to decrease the maltotriose content of high maltose syrups. However, due to the interplay between the hydrolysis and transglycosylation activities of maltogenic amylases, the maltotriose contents of these syrups are still greater than that necessary for pure maltose preparation. In this study, the maltogenic amylase from Bacillus stearothermophilus was engineered to decrease its transglycosylation activity with the expectation that this would enhance maltose production. Site-directed mutagenesis was used to generate Trp 177 variants W177F, W177Y, W177L, W177N, and W177S. The transglycosylation activities of the mutant enzymes decreased as the hydrophilicity of the residue at position 177 increased. The mutant enzymes exhibited notable enhancements in maltose production, with a minimum of maltotriose contents of 0.2%, compared with 3.2% for the wild-type enzyme. Detailed characterization of the mutant enzymes suggests that the best of them, W177S, will deliver performance superior to that of the wild-type under industrial conditions.

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

    PubMed Central

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

    2003-01-01

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

  8. Effect on pH heating medium on the thermal resistance of Bacillus stearothermophilus spores.

    PubMed

    López, M; González, I; Condón, S; Bernardo, A

    1996-01-01

    The influence of the pH of heating medium on heat resistance of Bacillus stearothermophilus spores (ATCC 7953, 12980, 15951 and 15952) were studied. The pH values tested were: 4.0, 5.0, 6.0 and 7.0 at temperatures of 115, 120, 125, 130 and 135 degrees C. It was found that at low treatment temperatures (115 degrees C) D-values decreased between 7- and 10-fold with 7953, 12980 and 15951 strains and about 23-fold with 15952 strain when pH dropped from 7.0 to 4.0. At highest treatment temperatures (135 degrees C) D-values obtained with pH 6.0 and 7.0 did not show any significant statistical differences (p > 0.05). z-Values appeared to be higher when the medium was acidified, ranging from 7.58 to 8.20 and 9.43 10.0 for spores suspended in McIlvaine buffer pH 7.0 and pH 4.0, respectively, although the difference was not statistically significant. Heat resistance of strain ATCC 7953 at 120, 128, and 135 degrees C in asparagus purée and tomato purée at pH 5.0 under continuous monitoring of pH was also determined. D-values obtained in asparagus purée were similar to those obtained in buffer at the same pH, whereas those observed in tomato purée were found to be lower.

  9. Structural insights into methanol-stable variants of lipase T6 from Geobacillus stearothermophilus.

    PubMed

    Dror, Adi; Kanteev, Margarita; Kagan, Irit; Gihaz, Shalev; Shahar, Anat; Fishman, Ayelet

    2015-11-01

    Enzymatic production of biodiesel by transesterification of triglycerides and alcohol, catalyzed by lipases, offers an environmentally friendly and efficient alternative to the chemically catalyzed process while using low-grade feedstocks. Methanol is utilized frequently as the alcohol in the reaction due to its reactivity and low cost. However, one of the major drawbacks of the enzymatic system is the presence of high methanol concentrations which leads to methanol-induced unfolding and inactivation of the biocatalyst. Therefore, a methanol-stable lipase is of great interest for the biodiesel industry. In this study, protein engineering was applied to substitute charged surface residues with hydrophobic ones to enhance the stability in methanol of a lipase from Geobacillus stearothermophilus T6. We identified a methanol-stable variant, R374W, and combined it with a variant found previously, H86Y/A269T. The triple mutant, H86Y/A269T/R374W, had a half-life value at 70 % methanol of 324 min which reflects an 87-fold enhanced stability compared to the wild type together with elevated thermostability in buffer and in 50 % methanol. This variant also exhibited an improved biodiesel yield from waste chicken oil compared to commercial Lipolase 100L® and Novozyme® CALB. Crystal structures of the wild type and the methanol-stable variants provided insights regarding structure-stability correlations. The most prominent features were the extensive formation of new hydrogen bonds between surface residues directly or mediated by structural water molecules and the stabilization of Zn and Ca binding sites. Mutation sites were also characterized by lower B-factor values calculated from the X-ray structures indicating improved rigidity.

  10. Thermostable, alkaline and detergent-tolerant lipase from a newly isolated thermophilic Bacillus stearothermophilus.

    PubMed

    Ben Bacha, Abir; Moubayed, Nadine M S; Abid, Islam

    2015-04-01

    Lipases are the enzymes of choice for laundry detergent industries, owing to their triglyceride removing ability from the soiled fabric, which eventually reduces the usage of phosphate-based chemical cleansers in the detergent formulation. In this study, a novel thermo-alkaline lipase-producing strain identified as Bacillus stearothermophilus was isolated from the soil samples of olive oil mill. Enhanced lipase production was observed at 55 degrees C, pH 11 and after 48 h of incubation. Among the substrates tested, xylose (a carbon source), peptone (a nitrogen source) and olive oil at a concentration of 1% were suitable substrates for enhancing lipase production. MgSO4 and Tween-80 were suitable substrates for maximizing lipase production. The enzyme was purified to homogeneity by a single CM-Sephadex column chromatography and revealed molecular mass of 67 kDa. The enzyme (BL1) was active over a wide range of pH from 9.0 to 13.0, with an optimum at pH 11.0, exhibited maximal activity at 55 degreesC and retained more than 70% of its activity after incubation at 70 degrees C or pH 13 for 0.5 h or 24 h, respectively. The enzyme hydrolyzed both short and long-chain triacylglycerols at comparable rates. BL1 was studied in a preliminary evaluation for use in detergent formulation solutions. This novel lipase showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40 degrees C, and good stability towards oxidizing agents. Additionally, the enzyme showed excellent stability and compatibility with various commercial detergents, suggesting its potential as an additive in detergent formulations.

  11. Purification and characterization of alpha-L-arabinofuranosidase from Bacillus stearothermophilus T-6.

    PubMed Central

    Gilead, S; Shoham, Y

    1995-01-01

    Bacillus stearothermophilus T-6 produced an alpha-L-arabinofuranosidase when grown in the presence of L-arabinose, sugar beet arabinan, or oat spelt xylan. At the end of a fermentation, about 40% of the activity was extracellular, and enzyme activity in the cell-free supernatant could reach 25 U/ml. The enzymatic activity in the supernatant was concentrated against polyethylene glycol 20000, and the enzyme was purified eightfold by anion-exchange and hydrophobic interaction chromatographies. The molecular weight of T-6 alpha-L-arabinofuranosidase was 256,000, and it consisted of four identical subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The native enzyme had a pI of 6.5 and was most active at 70 degrees C and at pH 5.5 to 6.0. Its thermostability at pH 7.0 was characterized by half-lives of 53, 15, and 1 h at 60, 65, and 70 degrees C, respectively. Kinetic experiments at 60 degrees C with p-nitrophenyl alpha-L-arabinofuranoside as a substrate gave a Vmax, a Km, and an activation energy of 749 U/mg, 0.42 mM, and 16.6 kcal/mol, (ca. 69.5 kJ/mol), respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by 1 mM Hg2+. T-6 alpha-L-arabinofuranosidase released L-arabinose from arabinan and had low activity on oat spelt xylan. The enzyme acted cooperatively with T-6 xylanase in hydrolyzing oat spelt xylan, and L-arabinose, xylose, and xylobiose were detected as the end reaction products.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7887599

  12. Inactivation of Geobacillus stearothermophilus spores by alkaline hydrolysis applied to medical waste treatment.

    PubMed

    Pinho, Sílvia C; Nunes, Olga C; Lobo-da-Cunha, Alexandre; Almeida, Manuel F

    2015-09-15

    Although alkaline hydrolysis treatment emerges as an alternative disinfection/sterilization method for medical waste, information on its effects on the inactivation of biological indicators is scarce. The effects of alkaline treatment on the resistance of Geobacillus stearothermophilus spores were investigated and the influence of temperature (80 °C, 100 °C and 110 °C) and NaOH concentration was evaluated. In addition, spore inactivation in the presence of animal tissues and discarded medical components, used as surrogate of medical waste, was also assessed. The effectiveness of the alkaline treatment was carried out by determination of survival curves and D-values. No significant differences were seen in D-values obtained at 80 °C and 100 °C for NaOH concentrations of 0.5 M and 0.75 M. The D-values obtained at 110 °C (2.3-0.5 min) were approximately 3 times lower than those at 100 °C (8.8-1.6 min). Independent of the presence of animal tissues and discarded medical components, 6 log10 reduction times varied between 66 and 5 min at 100 °C-0.1 M NaOH and 110 °C-1 M NaOH, respectively. The alkaline treatment may be used in future as a disinfection or sterilization alternative method for contaminated waste.

  13. Molecular cloning and characterization of an enzyme hydrolyzing p-nitrophenyl alpha-D-glucoside from Bacillus stearothermophilus SA0301.

    PubMed

    Kobayashi, Atsushi; Tonozuka, Takashi; Sato, Kimihiko; Suyama, Mikita; Sasaki, Jun; Nyamdawaa, Batbold; Sakaguchi, Masayoshi; Sakano, Yoshiyuki

    2006-02-01

    Bacillus stearothermophilus SA0301 produces an extracellular oligo-1,6-glucosidase (bsO16G) that also hydrolyzes p-nitrophenyl alpha-D-glucoside (Tonozuka et al., J. Appl. Glycosci., 45, 397-400 (1998)). We cloned a gene for an enzyme hydrolyzing p-nitrophenyl alpha-D-glucoside, which was different from the one mentioned above, from B. stearothermophilus SA0301. The k(0)/K(m) values of bsO16G for isomaltotriose and isomaltose were 13.2 and 1.39 s(-1).mM(-1) respectively, while the newly cloned enzyme did not hydrolyze isomaltotriose, and the k(0)/K(m) value for isomaltose was 0.81 s(-1).mM(-1). The primary structure of the cloned enzyme more closely resembled those of trehalose-6-phosphate hydrolases than those of oligo-1,6-glucosidases, and the cloned enzyme hydrolyzed trehalose 6-phosphate. An open reading frame encoding a protein homologous to the trehalose-specific IIBC component of the phopshotransferase system was also found upstream of the gene for this enzyme.

  14. Walking dead: Permeabilization of heat-treated Geobacillus stearothermophilus ATCC 12980 spores under growth-preventing conditions.

    PubMed

    Mtimet, Narjes; Trunet, Clément; Mathot, Anne-Gabrielle; Venaille, Laurent; Leguérinel, Ivan; Coroller, Louis; Couvert, Olivier

    2017-06-01

    Although heat treatment is probably the oldest and the most common method used to inactivate spores in food processes, the specific mechanism of heat killing of spores is still not fully understood. The purpose of this study is to investigate the evolution of the permeabilization and the viability of heat-treated spores during storage under growth-preventing conditions. Geobacillus stearothermophilus spores were heat-treated under various conditions of temperature and pH, and then stored under conditions of temperature and pH that prevent growth. Spore survival was evaluated by count plating immediately after heat treatment, and then during storage over a period of months. Flow cytometry analyses were performed to investigate the Syto 9 permeability of heat-treated spores. Sub-lethally heat-treated spores of G. stearothermophilus were physically committed to permeabilization after heat treatment. However, prolonged heat treatment may abolish the spore permeabilization and block heat-treated spores in the refractive state. However, viability loss and permeabilization during heat treatment seem to be two different mechanisms that occur independently, and the loss of permeabilization properties takes place at a much slower rate than spore killing. Under growth-preventing conditions, viable heat-treated spores presumably lose their viability due to the permeabilization phenomena, which makes them more susceptible to the action of adverse conditions precluding growth.

  15. Quantitative assessment of the risk of microbial spoilage in foods. Prediction of non-stability at 55 °C caused by Geobacillus stearothermophilus in canned green beans.

    PubMed

    Rigaux, Clémence; André, Stéphane; Albert, Isabelle; Carlin, Frédéric

    2014-02-03

    Microbial spoilage of canned foods by thermophilic and highly heat-resistant spore-forming bacteria, such as Geobacillus stearothermophilus, is a persistent problem in the food industry. An incubation test at 55 °C for 7 days, then validation of biological stability, is used as an indicator of compliance with good manufacturing practices. We propose a microbial risk assessment model predicting the percentage of non-stability due to G. stearothermophilus in canned green beans manufactured by a French company. The model accounts for initial microbial contaminations of fresh unprocessed green beans with G. stearothermophilus, cross-contaminations in the processing chain, inactivation processes and probability of survival and growth. The sterilization process is modeled by an equivalent heating time depending on sterilization value F₀ and on G. stearothermophilus resistance parameter z(T). Following the recommendations of international organizations, second order Monte-Carlo simulations are used, separately propagating uncertainty and variability on parameters. As a result of the model, the mean predicted non-stability rate is of 0.5%, with a 95% uncertainty interval of [0.1%; 1.2%], which is highly similar to data communicated by the French industry. A sensitivity analysis based on Sobol indices and some scenario tests underline the importance of cross-contamination at the blanching step, in addition to inactivation due to the sterilization process.

  16. LACTIC DEHYDROGENASES OF PSEUDOMONAS NATRIEGENS.

    PubMed

    WALKER, H; EAGON, R G

    1964-07-01

    Walker, Hazel (University of Georgia, Athens), and R. G. Eagon. Lactic dehydrogenases of Pseudomonas natriegens. J. Bacteriol. 88:25-30. 1964.-Lactic dehydrogenases specific for d- and l-lactate were demonstrated in Pseudomonas natriegens. The l-lactic dehydrogenase showed considerable heat stability, and 40% of the activity remained in extracts after heating at 60 C for 10 min. An essential thiol group for enzyme activity was noted. The results of these experiments were consistent with the view that lactate was dehydrogenated initially by a flavin cofactor and that electrons were transported through a complete terminal oxidase system to oxygen. The intracellular site of these lactic dehydrogenases was shown to be the cell membrane. It was suggested that the main physiological role of these lactic dehydrogenases is that of lactate utilization.

  17. The Glucuronic Acid Utilization Gene Cluster from Bacillus stearothermophilus T-6

    PubMed Central

    Shulami, Smadar; Gat, Orit; Sonenshein, Abraham L.; Shoham, Yuval

    1999-01-01

    A λ-EMBL3 genomic library of Bacillus stearothermophilus T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting β-xylosidase activity were isolated. The clones overlap each other and together represent a 23.5-kb chromosomal segment. The segment contains a cluster of xylan utilization genes, which are organized in at least three transcriptional units. These include the gene for the extracellular xylanase, xylanase T-6; part of an operon coding for an intracellular xylanase and a β-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 genes involved in the utilization of α-d-glucuronic acid (GlcUA). The first four genes in the potential GlcUA operon (orf1, -2, -3, and -4) code for a putative sugar transport system with characteristic components of the binding-protein-dependent transport systems. The most likely natural substrate for this transport system is aldotetraouronic acid [2-O-α-(4-O-methyl-α-d-glucuronosyl)-xylotriose] (MeGlcUAXyl3). The following two genes code for an intracellular α-glucuronidase (aguA) and a β-xylosidase (xynB). Five more genes (kdgK, kdgA, uxaC, uxuA, and uxuB) encode proteins that are homologous to enzymes involved in galacturonate and glucuronate catabolism. The gene cluster also includes a potential regulatory gene, uxuR, the product of which resembles repressors of the GntR family. The apparent transcriptional start point of the cluster was determined by primer extension analysis and is located 349 bp from the initial ATG codon. The potential operator site is a perfect 12-bp inverted repeat located downstream from the promoter between nucleotides +170 and +181. Gel retardation assays indicated that UxuR binds specifically to this sequence and that this binding is efficiently prevented in vitro by MeGlcUAXyl3, the most likely molecular inducer. PMID:10368143

  18. Selective oxidation of glycerol to 1,3-dihydroxyacetone by covalently immobilized glycerol dehydrogenases with higher stability and lower product inhibition.

    PubMed

    Rocha-Martin, Javier; Acosta, Andreína; Berenguer, Jose; Guisan, Jose M; Lopez-Gallego, Fernando

    2014-10-01

    Glycerol dehydrogenase (GlyDH) catalyzes the regioselective oxidation of glycerol to yield 1,3-dihydroxyacetone (DHA); an important building block in chemical industry. Three recombinant GlyDHs from Geobacillus stearothermophilus, from Citrobacter braakii and from Cellulomonas sp. were stabilized by covalent immobilization. The highest activity recoveries (40-50%) of the insoluble preparations were obtained by immobilizing these enzymes in presence of polyethylene glycol (PEG). Noteworthy, these immobilized preparations were more stable and less inhibited by DHA than their soluble counterparts. In particular, GlyDH from G.stearothermophilus immobilized on agarose activated with both amine and glyoxyl groups and crosslinked with dextran aldehyde was 3.7-fold less inhibited by DHA than its soluble form and retained 100% of its initial activity after 18h of incubation at 65°C and pH 7. This is one of the few examples where the same immobilization protocol has minimized enzyme product inhibition and maximized thermal stability.

  19. Lactate dehydrogenase-elevating virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This book chapter describes the taxonomic classification of Lactate dehydrogenase-elevating virus (LDV). Included are: host, genome, classification, morphology, physicochemical and physical properties, nucleic acid, proteins, lipids, carbohydrates, geographic range, phylogenetic properties, biologic...

  20. Crystallization and preliminary crystallographic studies of the recombinant l-N-carbamoylase from Geobacillus stearothermophilus CECT43

    PubMed Central

    Martínez-Rodríguez, Sergio; García-Pino, Abel; Las Heras-Vázquez, Francisco Javier; Clemente-Jiménez, Josefa María; Rodríguez-Vico, Felipe; Loris, Remy; García-Ruiz, Juan Ma.; Gavira, Jose Antonio

    2008-01-01

    N-Carbamoyl-l-amino-acid amidohydrolases (l-N-carbamoylases; EC 3.5.1.87) hydrolyze the carbon–nitrogen bond of the ureido group in N-carbamoyl-l-α-amino acids. These enzymes are commonly used in the production of optically pure natural and non-natural l-amino acids using the ‘hydantoinase process’. Recombinant l-N-carbamoylase from Geobacillus stearothermophilus CECT43 has been expressed, purified and crystallized by hanging-drop vapour diffusion. X-­ray data were collected to a resolution of 2.75 Å. The crystals belonged to space group P21212, with unit-cell parameters a = 103.2, b = 211.7, c = 43.1 Å and two subunits in the asymmetric unit. PMID:19052368

  1. 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

  2. Crystallization and preliminary crystallographic analysis of GanB, a GH42 intracellular β-galactosidase from Geobacillus stearothermophilus.

    PubMed

    Solomon, Hodaya V; Tabachnikov, Orly; Feinberg, Hadar; Govada, Lata; Chayen, Naomi E; Shoham, Yuval; Shoham, Gil

    2013-10-01

    Geobacillus stearothermophilus T-6 is a Gram-positive thermophilic soil bacterium that contains a multi-enzyme system for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. The bacterium uses a number of endo-acting extracellular enzymes that break down the high-molecular-weight polysaccharides into decorated oligosaccharides. These oligosaccharides enter the cell and are further hydrolyzed into sugar monomers by a set of intracellular glycoside hydrolases. One of these intracellular degrading enzymes is GanB, a glycoside hydrolase family 42 β-galactosidase capable of hydrolyzing short β-1,4-galactosaccharides to galactose. GanB and related enzymes therefore play an important part in the hemicellulolytic utilization system of many microorganisms which use plant biomass for growth. The interest in the biochemical characterization and structural analysis of these enzymes stems from their potential biotechnological applications. GanB from G. stearothermophilus T-6 has recently been cloned, overexpressed, purified, biochemically characterized and crystallized in our laboratory as part of its complete structure-function study. The best crystals obtained for this enzyme belong to the primitive orthorhombic space group P2₁2₁2₁, with average crystallographic unit-cell parameters of a=71.84, b=181.35, c=196.57 Å. Full diffraction data sets to 2.45 and 2.50 Å resolution have been collected for both the wild-type enzyme and its E323A nucleophile catalytic mutant, respectively, as measured from flash-cooled crystals at 100 K using synchrotron radiation. These data are currently being used for the full three-dimensional crystal structure determination of GanB.

  3. Crystallization and preliminary crystallographic analysis of a family 43 β-d-xylosidase from Geobacillus stearothermophilus T-6

    SciTech Connect

    Brüx, Christian; Niefind, Karsten; Ben-David, Alon; Leon, Maya; Shoham, Gil; Shoham, Yuval; Schomburg, Dietmar

    2005-12-01

    The crystallization and preliminary X-ray analysis of a β-d-xylosidase from G. stearothermophilus T-6, a family 43 glycoside hydrolase, is described. Native and catalytic inactive mutants of the enzymes were crystallized in two different space groups, orthorhombic P2{sub 1}2{sub 1}2 and tetragonal P4{sub 1}2{sub 1}2 (or the enantiomorphic space group P4{sub 3}2{sub 1}2), using a sensitive cryoprotocol. The latter crystal form diffracted X-rays to a resolution of 2.2 Å. β-d-Xylosidases (EC 3.2.1.37) are hemicellulases that cleave single xylose units from the nonreducing end of xylooligomers. In this study, the crystallization and preliminary X-ray analysis of a β-d-xylosidase from Geobacillus stearothermophilus T-6 (XynB3), a family 43 glycoside hydrolase, is described. XynB3 is a 535-amino-acid protein with a calculated molecular weight of 61 891 Da. Purified recombinant native and catalytic inactive mutant proteins were crystallized and cocrystallized with xylobiose in two different space groups, P2{sub 1}2{sub 1}2 (unit-cell parameters a = 98.32, b = 99.36, c = 258.64 Å) and P4{sub 1}2{sub 1}2 (or the enantiomorphic space group P4{sub 3}2{sub 1}2; unit-cell parameters a = b = 140.15, c = 233.11 Å), depending on the detergent. Transferring crystals to cryoconditions required a very careful protocol. Orthorhombic crystals diffract to 2.5 Å and tetragonal crystals to 2.2 Å.

  4. Crystallization and preliminary crystallographic analysis of Abp, a GH27 β-L-arabinopyranosidase from Geobacillus stearothermophilus.

    PubMed

    Lansky, Shifra; Salama, Rachel; Solomon, Vered H; Belrhali, Hassan; Shoham, Yuval; Shoham, Gil

    2013-06-01

    Geobacillus stearothermophilus T-6 is a thermophilic soil bacterium that possesses an extensive system for the utilization of hemicellulose. The bacterium produces a small number of endo-acting extracellular enzymes that cleave high-molecular-weight hemicellulolytic polymers into short decorated oligosaccharides, which are further hydrolysed into the respective sugar monomers by a battery of intracellular glycoside hydrolases. One of these intracellular processing enzymes is β-L-arabinopyranosidase (Abp), which is capable of removing β-L-arabinopyranose residues from naturally occurring arabino-polysaccharides. As arabino-polymers constitute a significant part of the hemicellulolytic content of plant biomass, their efficient enzymatic degradation presents an important challenge for many potential biotechnological applications. This aspect has led to an increasing interest in the biochemical characterization and structural analysis of this and related hemicellulases. Abp from G. stearothermophilus T-6 has recently been cloned, overexpressed, purified, biochemically characterized and crystallized in our laboratory, as part of its complete structure-function study. The best crystals obtained for this enzyme belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with average unit-cell parameters a = 107.7, b = 202.2, c = 287.3 Å. Full diffraction data sets to 2.3 Å resolution have been collected for both the wild-type enzyme and its D197A catalytic mutant from flash-cooled crystals at 100 K, using synchrotron radiation. These data are currently being used for a high-resolution three-dimensional structure determination of Abp.

  5. Efficient synthesis and secretion of a thermophilic alpha-amylase by protein-producing Bacillus brevis 47 carrying the Bacillus stearothermophilus amylase gene.

    PubMed Central

    Tsukagoshi, N; Iritani, S; Sasaki, T; Takemura, T; Ihara, H; Idota, Y; Yamagata, H; Udaka, S

    1985-01-01

    Bacillus subtilis and Bacillus brevis 47-5, carrying the Bacillus stearothermophilus alpha-amylase gene on pUB110 (pBAM101), synthesized the same alpha-amylase as the donor strain as determined by the enzyme's thermal stability and NH2-terminal amino acid sequence. Regardless of the host, the 34-amino acid signal peptide of the enzyme was processed at exactly the same site between two alanine residues. B. brevis 47-5(pBAM101) secreted the enzyme most efficiently of the hosts examined, 100, 15, and 5 times more than B. stearothermophilus, Escherichia coli HB101(pH1301), and B. subtilis 1A289(pBAM101), respectively. The efficient secretion of the enzyme in B. brevis 47-5(pBAM101) was suggested to be due to the unique properties of the cell wall of this organism. Images PMID:2999073

  6. 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

  7. [Regulation of citrate synthese in bacteria: Comparison of the action of various effectors on the enzymes of Rhodospirillum rurbum and Bacillus stearothermophilus].

    PubMed

    Higa, A I; Massarini, E; Cazzulo, J J

    1976-01-01

    A comparative study of the citrate synthases purified from the facultatively photosynthetic bacterium Rhodospirillum rubrum (Gram negative) and the thermophile Bacillus stearothermophilus (Gram positive) was made. The citrate synthase from R. rubrum was activated by KCl (6-fold at 0.1 M KCl) and, less effectively, by NaCl and NH4Cl. Its molecular weight was about 300,000. The enzyme was strongly inhibited by NADH, and this inhibition was counteracted by AMP. The citrate synthase from B. stearothermophilus was little affected by KCl, NaCl and NH4Cl, all of which activated by about 25% at 0.1 M. Its molecular weight was ca 100,000. The enzyme was not affected by NADH or AMP. Both citrate synthases were insensitive to alpah-oxoglutarate concentrations up to 5 mM, and were inhibited by ATP; the B. stearothermophilus enzyme was more strongly inhibited than the R. rubrum enzyme. In both cases the ATP inhibition was strictly competitive towards acetyl-CoA and non-competitive towards oxaloacetate. Both enzymes, in spite of the peculiar physiological properties of their bacterial sources, followed the close correlation between the properties of the citrate synthase and the taxonomical position of the microorganism, proposed by Weitzman and his co-workers.

  8. The Geobacillus stearothermophilus V iscS Gene, Encoding Cysteine Desulfurase, Confers Resistance to Potassium Tellurite in Escherichia coli K-12

    PubMed Central

    Tantaleán, Juan C.; Araya, Manuel A.; Saavedra, Claudia P.; Fuentes, Derie E.; Pérez, José M.; Calderón, Iván L.; Youderian, Philip; Vásquez, Claudio C.

    2003-01-01

    Many eubacteria are resistant to the toxic oxidizing agent potassium tellurite, and tellurite resistance involves diverse biochemical mechanisms. Expression of the iscS gene from Geobacillus stearothermophilus V, which is naturally resistant to tellurite, confers tellurite resistance in Escherichia coli K-12, which is naturally sensitive to tellurite. The G. stearothermophilus iscS gene encodes a cysteine desulfurase. A site-directed mutation in iscS that prevents binding of its pyridoxal phosphate cofactor abolishes both enzyme activity and its ability to confer tellurite resistance in E. coli. Expression of the G. stearothermophilus iscS gene confers tellurite resistance in tellurite-hypersensitive E. coli iscS and sodA sodB mutants (deficient in superoxide dismutase) and complements the auxotrophic requirement of an E. coli iscS mutant for thiamine but not for nicotinic acid. These and other results support the hypothesis that the reduction of tellurite generates superoxide anions and that the primary targets of superoxide damage in E. coli are enzymes with iron-sulfur clusters. PMID:13129955

  9. 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 ...

  10. Structure-function relationships in Gan42B, an intracellular GH42 β-galactosidase from Geobacillus stearothermophilus.

    PubMed

    Solomon, Hodaya V; Tabachnikov, Orly; Lansky, Shifra; Salama, Rachel; Feinberg, Hadar; Shoham, Yuval; Shoham, Gil

    2015-12-01

    Geobacillus stearothermophilus T-6 is a Gram-positive thermophilic soil bacterium that contains a battery of degrading enzymes for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. A 9.4 kb gene cluster has recently been characterized in G. stearothermophilus that encodes a number of galactan-utilization elements. A key enzyme of this degradation system is Gan42B, an intracellular GH42 β-galactosidase capable of hydrolyzing short β-1,4-galactosaccharides into galactose units, making it of high potential for various biotechnological applications. The Gan42B monomer is made up of 686 amino acids, and based on sequence homology it was suggested that Glu323 is the catalytic nucleophile and Glu159 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Gan42B (at 2.45 Å resolution) and its catalytic mutant E323A (at 2.50 Å resolution), as determined by X-ray crystallography, are reported. These structures demonstrate that the three-dimensional structure of the Gan42B monomer generally correlates with the overall fold observed for GH42 proteins, consisting of three main domains: an N-terminal TIM-barrel domain, a smaller mixed α/β domain, and the smallest all-β domain at the C-terminus. The two catalytic residues are located in the TIM-barrel domain in a pocket-like active site such that their carboxylic functional groups are about 5.3 Å from each other, consistent with a retaining mechanism. The crystal structure demonstrates that Gan42B is a homotrimer, resembling a flowerpot in general shape, in which each monomer interacts with the other two to form a cone-shaped tunnel cavity in the centre. The cavity is ∼35 Å at the wide opening and ∼5 Å at the small opening and ∼40 Å in length. The active sites are situated at the interfaces between the monomers, so that every two neighbouring monomers participate in the formation of each of the three active

  11. Glucose-6-Phosphate Dehydrogenase Revisited

    PubMed Central

    O'Connell, Jerome T.; Henderson, Alfred R.

    1984-01-01

    Hemolytic diseases associated with drugs have been recognized since antiquity. Many of these anemias have been associated with oxidizing agents and deficiencies in the intraerythrocytic enzyme glucose-6-phosphate dehydrogenase. This paper outlines the discovery, prevalence, and variants of this enzyme. Methods of diagnosis of associated anemias are offered. PMID:6502728

  12. Crystallization and preliminary crystallographic analysis of a family 43 β-d-xylosidase from Geobacillus stearothermophilus T-6

    PubMed Central

    Brüx, Christian; Niefind, Karsten; Ben-David, Alon; Leon, Maya; Shoham, Gil; Shoham, Yuval; Schomburg, Dietmar

    2005-01-01

    β-d-Xylosidases (EC 3.2.1.37) are hemicellulases that cleave single xylose units from the nonreducing end of xylooligomers. In this study, the crystallization and preliminary X-ray analysis of a β-d-xylosidase from Geobacillus stearothermophilus T-6 (XynB3), a family 43 glycoside hydrolase, is described. XynB3 is a 535-amino-acid protein with a calculated molecular weight of 61 891 Da. Purified recombinant native and catalytic inactive mutant proteins were crystallized and cocrystallized with xylobiose in two different space groups, P21212 (unit-cell parameters a = 98.32, b = 99.36, c = 258.64 Å) and P41212 (or the enantiomorphic space group P43212; unit-cell parameters a = b = 140.15, c = 233.11 Å), depending on the detergent. Transferring crystals to cryoconditions required a very careful protocol. Orthorhombic crystals diffract to 2.5 Å and tetragonal crystals to 2.2 Å. PMID:16511233

  13. How to Switch Off a Histidine Kinase: Crystal Structure of Geobacillus Stearothermophilus KinB with the Inhibitor Sda

    SciTech Connect

    Bick, M.; Lamour, V; Rajashankar, K; Gordiyenko, Y; Robinson, C; Darst, S

    2009-01-01

    Entry to sporulation in bacilli is governed by a histidine kinase phosphorelay, a variation of the predominant signal transduction mechanism in prokaryotes. Sda directly inhibits sporulation histidine kinases in response to DNA damage and replication defects. We determined a 2.0-Angstroms-resolution X-ray crystal structure of the intact cytoplasmic catalytic core [comprising the dimerization and histidine phosphotransfer domain (DHp domain), connected to the ATP binding catalytic domain] of the Geobacillus stearothermophilus sporulation kinase KinB complexed with Sda. Structural and biochemical analyses reveal that Sda binds to the base of the DHp domain and prevents molecular transactions with the DHp domain to which it is bound by acting as a simple molecular barricade. Sda acts to sterically block communication between the catalytic domain and the DHp domain, which is required for autophosphorylation, as well as to sterically block communication between the response regulator Spo0F and the DHp domain, which is required for phosphotransfer and phosphatase activities.

  14. Thermostability enhancement and change in starch hydrolysis profile of the maltohexaose-forming amylase of Bacillus stearothermophilus US100 strain

    PubMed Central

    Ben Ali, Mamdouh; Khemakhem, Bassem; Robert, Xavier; Haser, Richard; Bejar, Samir

    2005-01-01

    The implications of Asn315 and Val450 in the atypical starch hydrolysis profile of Bacillus stearothermophilus Amy (α-amylase) US100 have been suggested previously [Ben Ali, Mhiri, Mezghani and Bejar (2001) Enzyme Microb. Tech. 28, 537–542]. In order to confirm this hypothesis, three mutants were generated. Of these two have a single mutation, N315D or V450G, whereas the third contains both mutations. Analysis of the starch breakdown-profile of these three mutants, as well as of the wild-type, allowed us to conclude that each single mutation induces a small variation in the hydrolysis product. However, the major end product produced by the double mutant shifts from maltopentaose/maltohexaose to maltose/maltotriose, confirming the involvement of these two residues in starch hydrolysis. The superimposition of AmyUS100 model with that of Bacillus licheniformis shows in AmyUS100 an additional loop containing residues Ile214 and Gly215. Remarkably, the deletion of these two residues increases the half-life at 100 °C from 15 min to approx. 70 min. Moreover, this engineered amylase requires less calcium, 25 p.p.m. instead of 100 p.p.m., to reach maximal thermostability. PMID:16197365

  15. Modification of ascorbic acid using transglycosylation activity of Bacillus stearothermophilus maltogenic amylase to enhance its oxidative stability.

    PubMed

    Bae, Hee-Kyung; Lee, Soo-Bok; Park, Cheon-Seok; Shim, Jae-Hoon; Lee, Hye-Young; Kim, Myo-Jeong; Baek, Jin-Sook; Roh, Hoe-Jin; Choi, Jin-Hwan; Choe, Eun-Ok; Ahn, Dong-Uk; Park, Kwan-Hwa

    2002-05-22

    Ascorbic acid (1), a natural antioxidant, was modified by employing transglycosylation activity of Bacillus stearothermophilus maltogenic amylase with maltotriose and acarbose as donor molecules to enhance its oxidative stability. The transglycosylation reaction with maltotriose as donor created mono- and di-glycosyl transfer products with an alpha-(1,6)-glycosidic linkage. In addition, two acarviosine-glucosyl transfer products were generated when transglycosylation was performed with acarbose as a donor. All transfer products were observed by TLC and HPLC, and purified by Q-sepharose anion exchange and Biogel P-2 gel permeation chromatographies. LC/MS and (13)C NMR analyses revealed that the structures of the transfer products were 6-O-alpha-D-glucosyl- (2) and 6-O-alpha-D-maltosyl-ascorbic acids (3) in the reaction of maltotriose, and 6-O-alpha-acarviosine-D-glucosyl- (4) and 2-O-alpha-acarviosine-D-glucosyl ascorbic acids (5) in the reaction of acarbose. The stability of the transglycosylated ascorbic acid derivatives was greatly enhanced against oxidation by Cu(2+) ion and ascorbate oxidase. Among them, compound 3 proved to be the most stable against in vitro oxidation. The antioxidant effects of glycosyl-derivatives of ascorbic acid on the lipid oxidation in cooked chicken breast meat patties indicated that they had antioxidant activities similar to that of ascorbic acid. It is suggested that the transglycosylated ascorbic acids can possibly be applied as effective antioxidants with improved stability in food, cosmetic, and other applications.

  16. Improving the thermostability and enhancing the Ca(2+) binding of the maltohexaose-forming α-amylase from Bacillus stearothermophilus.

    PubMed

    Li, Zhu; Duan, Xuguo; Wu, Jing

    2016-03-20

    The thermostability of the maltohexaose-forming α-amylase from Bacillus stearothermophilus (AmyMH) without added Ca(2+) was improved through structure-based rational design in this study. Through comparison of a homologous model structure of AmyMH with the crystal structure of the thermostable α-amylase from Bacillus licheniformis, Ser242, which located at the beginning of fourth α-helix of the central (β/α)8 barrel was selected for mutation to improve thermostability. In addition, an amide-containing side chain (Asn193) and a loop in domain B (ΔIG mutation), which have been proven to be important for thermostability in corresponding position of other α-amylases, were also investigated. Five mutants carrying the mutations ΔIG, N193F, S242A, ΔIG/N193F, and ΔIG/N193F/S242A were generated and their proteins characterized. The most thermostable mutant protein, ΔIG/N193F/S242A, exhibited a 26-fold improvement in half-life at 95°C compared to the wild-type enzyme without added Ca(2+). Mutant ΔIG/N193F/S242A also exhibited substantially better activity and stability in the presence of the chelator EDTA, demonstrating enhanced Ca(2+) binding. These results suggest that mutant ΔIG/N193F/S242A has potential for use in the industrial liquefaction of starch.

  17. Aptamer biosensor for sensitive detection of toxin A of Clostridium difficile using gold nanoparticles synthesized by Bacillus stearothermophilus.

    PubMed

    Luo, Peng; Liu, Yi; Xia, Yun; Xu, Huajian; Xie, Guoming

    2014-04-15

    A sensitive electrochemical biosensor was developed to detect toxin A (TOA) of Clostridium difficile based on an aptamer selected by the systematic evolution of ligands using exponential enrichment and gold nanoparticles (GNPS) synthesized by Bacillus stearothermophilus. The thiolated single-stranded DNA used as the capture probe (CP) was first self-assembled on a Nafion-thionine-GNPS-modified screen-printed electrode (SPE) through an Au-thiol interaction. The horseradish peroxidase (HRP)-labeled aptamer probe (AP) was then hybridized to the complementary oligonucleotide of CP to form an aptamer-DNA duplex. In the absence of TOA, the aptamer-DNA duplex modified the electrode surface with HRP, so that an amperometric response was induced based on the electrocatalytic properties of thionine. This was mediated by the electrons that were generated in the enzymatic reaction of hydrogen peroxide under HRP catalysis. After the specific recognition of TOA, an aptamer-TOA complex was produced rather than the aptamer-DNA duplex, forcing the HRP-labeled AP to dissociate from the electrode surface, which reduced the catalytic capacity of HRP and reduced the response current. The reduction in the response current correlated linearly with the concentration of TOA in the range of 0-200 ng/mL. The detection limit was shown to be 1 nM for TOA. This biosensor was applied to the analysis of TOA and showed good selectivity, reproducibility, stability, and accuracy.

  18. The Bacillus stearothermophilus NUB36 surA gene encodes a thermophilic sucrase related to Bacillus subtilis SacA.

    PubMed

    Li, Y; Ferenci, T

    1996-07-01

    The complete nucleotide sequence of the surA gene, encoding a sucrase from Bacillus stearothermophilus NUB36, was determined. surA was composed of 1338 bp and encoded 445 amino acid residues. The deduced polypeptide of M(r) 51519 showed strong sequence similarity to sucrose and sucrose phosphate hydrolases from Bacillus subtilis, Klebsiella pneumoniae and Vibrio alginolyticus, and contained the 'sucrose box' residues thought to be important for catalysis of the transfer of fructose from sucrose. The enzyme was partially purified using affinity chromotography from extracts of Escherichia coli containing the cloned surA. SurA displayed an optimum temperature for sucrose hydrolysis of 55 degrees C and high stability. The M(r) of SurA determined by gel filtration was 105,000, which suggested that the active form of the enzyme is a dimer. SurA exhibited an apparent Km of 40 mM for sucrose but, unlike the homologous B. subtilis enzyme, had no detectable sucrose phosphate hydrolase activity.

  19. Hazard Inherent in Microbial Tracers: Reduction of Risk by the Use of Bacillus stearothermophilus Spores in Aerobiology

    PubMed Central

    Sattar, Syed A.; Synek, E. J.; Westwood, J. C. N.; Neals, Pierrette

    1972-01-01

    The use of a „biological tracer” forms an essential part of many aerobiological experiments. Where release of such tracers is likely to result in deliberate or inadvertent human exposure, safety becomes a primary consideration in the selection of the tracer organism. Of the three most commonly used organisms, namely Bacillus subtilis, Escherichia coli, and Serratia marcescens, only the first comes near to satisfying the need for nonpathogenicity and even it has been incriminated as a cause of human infection, sometimes with a fatal outcome. The relevant characteristics of B. stearothermophilus were, therefore, investigated. Because it can grow only at elevated temperatures (minimum 41 C; optimum 56 C), it should not pose a threat to human health and this view is supported by experimental evidence to be presented. It is extremely easy to grow and maintain in the laboratory, and spore suspensions are easily prepared and stored. It withstands the stresses of aerosolization and sampling and its stability in the aerosol state compares favorably with that of B. subtilis var. niger. PMID:4557557

  20. The S-Layer Proteins of Two Bacillus stearothermophilus Wild-Type Strains Are Bound via Their N-Terminal Region to a Secondary Cell Wall Polymer of Identical Chemical Composition

    PubMed Central

    Egelseer, Eva Maria; Leitner, Karl; Jarosch, Marina; Hotzy, Christoph; Zayni, Sonja; Sleytr, Uwe B.; Sára, Margit

    1998-01-01

    Two Bacillus stearothermophilus wild-type strains were investigated regarding a common recognition and binding mechanism between the S-layer protein and the underlying cell envelope layer. The S-layer protein from B. stearothermophilus PV72/p6 has a molecular weight of 130,000 and assembles into a hexagonally ordered lattice. The S-layer from B. stearothermophilus ATCC 12980 shows oblique lattice symmetry and is composed of subunits with a molecular weight of 122,000. Immunoblotting, peptide mapping, N-terminal sequencing of the whole S-layer protein from B. stearothermophilus ATCC 12980 and of proteolytic cleavage fragments, and comparison with the S-layer protein from B. stearothermophilus PV72/p6 revealed that the two S-layer proteins have identical N-terminal regions but no other extended structurally homologous domains. In contrast to the heterogeneity observed for the S-layer proteins, the secondary cell wall polymer isolated from peptidoglycan-containing sacculi of the different strains showed identical chemical compositions and comparable molecular weights. The S-layer proteins could bind and recrystallize into the appropriate lattice type on native peptidoglycan-containing sacculi from both organisms but not on those extracted with hydrofluoric acid, leading to peptidoglycan of the A1γ chemotype. Affinity studies showed that only proteolytic cleavage fragments possessing the complete N terminus of the mature S-layer proteins recognized native peptidoglycan-containing sacculi as binding sites or could associate with the isolated secondary cell wall polymer, while proteolytic cleavage fragments missing the N-terminal region remained unbound. From the results obtained in this study, it can be concluded that S-layer proteins from B. stearothermophilus wild-type strains possess an identical N-terminal region which is responsible for anchoring the S-layer subunits to a secondary cell wall polymer of identical chemical composition. PMID:9515918

  1. The S-layer from Bacillus stearothermophilus DSM 2358 functions as an adhesion site for a high-molecular-weight amylase.

    PubMed Central

    Egelseer, E; Schocher, I; Sára, M; Sleytr, U B

    1995-01-01

    The S-layer lattice from Bacillus stearothermophilus DSM 2358 completely covers the cell surface and exhibits oblique symmetry. During growth of B. stearothermophilus DSM 2358 on starch medium, three amylases with molecular weights of 58,000, 98,000, and 184,000 were secreted into the culture fluid, but only the high-molecular-weight enzyme was found to be cell associated. Studies of interactions between cell wall components and amylases revealed no affinity of the high-molecular-weight amylase to isolated peptidoglycan. On the other hand, this enzyme was always found to be associated with S-layer self-assembly products or S-layer fragments released during preparation of spheroplasts by treatment of whole cells with lysozyme. The molar ratio of S-layer subunits to the bound amylase was approximately 8:1, which corresponded to one enzyme molecule per four morphological subunits. Immunoblotting experiments with polyclonal antisera against the high-molecular-weight amylase revealed a strong immunological signal in response to the enzyme but no cross-reaction with the S-layer protein or the smaller amylases. Immunogold labeling of whole cells with anti-amylase antiserum showed that the high-molecular-weight amylase is located on the outer face of the S-layer lattice. Because extraction of the amylase was possible without disintegration of the S-layer lattice into its constituent subunits, it can be excluded that the enzyme is incorporated into the crystal lattice and participates in the self-assembly process. Affinity experiments strongly suggest the presence of a specific recognition mechanism between the amylase molecules and S-layer protein domains either exposed on the outermost surface or inside the pores. In summary, results obtained in this study confirmed that the S-layer protein from B. stearothermophilus DSM 2358 functions as an adhesion site for a high-molecular-weight amylase. PMID:7533757

  2. The identification of the acid-base catalyst of alpha-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase.

    PubMed

    Shallom, Dalia; Belakhov, Valery; Solomon, Dmitry; Gilead-Gropper, Sara; Baasov, Timor; Shoham, Gil; Shoham, Yuval

    2002-03-13

    The alpha-L-arabinofuranosidase from Geobacillus stearothermophilus T-6 (AbfA T-6) belongs to the retaining family 51 glycoside hydrolases. The conserved Glu175 was proposed to be the acid-base catalytic residue. AbfA T-6 exhibits residual activity towards aryl beta-D-xylopyranosides. This phenomenon was used to examine the catalytic properties of the putative acid-base mutant E175A. Data from kinetic experiments, pH profiles, azide rescue, and the identification of the xylopyranosyl azide product provide firm support to the assignment of Glu175 as the acid-base catalyst of AbfA T-6.

  3. A New Family of Carbohydrate Esterases Is Represented by a GDSL Hydrolase/Acetylxylan Esterase from Geobacillus stearothermophilus*

    PubMed Central

    Alalouf, Onit; Balazs, Yael; Volkinshtein, Margarita; Grimpel, Yael; Shoham, Gil; Shoham, Yuval

    2011-01-01

    Acetylxylan esterases hydrolyze the ester linkages of acetyl groups at positions 2 and/or 3 of the xylose moieties in xylan and play an important role in enhancing the accessibility of xylanases to the xylan backbone. The hemicellulolytic system of the thermophilic bacterium Geobacillus stearothermophilus T-6 comprises a putative acetylxylan esterase gene, axe2. The gene product belongs to the GDSL hydrolase family and does not share sequence homology with any of the carbohydrate esterases in the CAZy Database. The axe2 gene is induced by xylose, and the purified gene product completely deacetylates xylobiose peracetate (fully acetylated) and hydrolyzes the synthetic substrates 2-naphthyl acetate, 4-nitrophenyl acetate, 4-methylumbelliferyl acetate, and phenyl acetate. The pH profiles for kcat and kcat/Km suggest the existence of two ionizable groups affecting the binding of the substrate to the enzyme. Using NMR spectroscopy, the regioselectivity of Axe2 was directly determined with the aid of one-dimensional selective total correlation spectroscopy. Methyl 2,3,4-tri-O-acetyl-β-d-xylopyranoside was rapidly deacetylated at position 2 or at positions 3 and 4 to give either diacetyl or monoacetyl intermediates, respectively; methyl 2,3,4,6-tetra-O-acetyl-β-d-glucopyranoside was initially deacetylated at position 6. In both cases, the complete hydrolysis of the intermediates occurred at a much slower rate, suggesting that the preferred substrate is the peracetate sugar form. Site-directed mutagenesis of Ser-15, His-194, and Asp-191 resulted in complete inactivation of the enzyme, consistent with their role as the catalytic triad. Overall, our results show that Axe2 is a serine acetylxylan esterase representing a new carbohydrate esterase family. PMID:21994937

  4. Characterization of quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus: enzymatic activity and active site structure.

    PubMed

    Terasaka, Erina; Okada, Norihiro; Sato, Nozomi; Sako, Yoshihiko; Shiro, Yoshitsugu; Tosha, Takehiko

    2014-07-01

    Nitric oxide reductase (NOR) catalyzes the reduction of nitric oxide to generate nitrous oxide. We recently reported on the crystal structure of a quinol-dependent NOR (qNOR) from Geobacillus stearothermophilus [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238-246], and suggested that a water channel from the cytoplasm, which is not observed in cytochrome c-dependent NOR (cNOR), functions as a pathway transferring catalytic protons. Here, we further investigated the functional and structural properties of qNOR, and compared the findings with those for cNOR. The pH optimum for the enzymatic reaction of qNOR was in the alkaline range, whereas Pseudomonas aeruginosa cNOR showed a higher activity at an acidic pH. The considerably slower reduction rate, and a correlation of the pH dependence for enzymatic activity and the reduction rate suggest that the reduction process is the rate-determining step for the NO reduction by qNOR, while the reduction rate for cNOR was very fast and therefore is unlikely to be the rate-determining step. A close examination of the heme/non-heme iron binuclear center by resonance Raman spectroscopy indicated that qNOR has a more polar environment at the binuclear center compared with cNOR. It is plausible that a water channel enhances the accessibility of the active site to solvent water, creating a more polar environment in qNOR. This structural feature could control certain properties of the active site, such as redox potential, which could explain the different catalytic properties of the two NORs. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

  5. Electron spin-lattice relaxation of the (4Fe-4S) ferredoxin from B. stearothermophilus. Comparison with other iron proteins

    NASA Astrophysics Data System (ADS)

    Bertrand, Patrick; Gayda, Jean-Pierre; Rao, K. Krishna

    1982-05-01

    The temperature dependence of the electron spin-lattice relaxation time T1 of the (4Fe-4S) ferredoxin from Bacillus stearothermophilus is studied in the range 1.2 to 40 K. This dependence is similar to that observed for the (2Fe-2S) ferredoxin from Spirulina maxima and can be interpreted with the same relaxation processes [J.P. Gayda, P. Bertrand, A. Deville, C. More, G. Roger, J.F. Gibson, and R. Cammack, Biochim. Biophys. Acta 581, 15 (1979)]. In particular, between 4 and 15 K, the data are well fitted by a second-order Raman process involving three-dimensional phonons, with a Debye temperature of about 60 K (45 cm-1). This would give an estimation of the highest frequency of the vibrations which can propagate through the three-dimensional proteinic medium. In the highest temperature range (T≳30 K) the results are interpreted with an Orbach process involving an excited level of energy 120 cm-1. This process could be induced by the localized vibrations of the active site. Finally, these results are compared to those recently reported for some hemoproteins [H.J. Stapleton, J.P. Allen, C.P. Flynn, D.G. Stinson, and S.R. Kurtz, Phys. Rev. Lett. 45, 1456 (1980)]. Below 15 K, the temperature dependence of T1 for these samples is similar to that observed for the iron-sulfur proteins and may be interpreted in the same way. Our interpretation is compared to the fractal model proposed by Stapleton et al.

  6. Characterization of retinaldehyde dehydrogenase 3

    PubMed Central

    Graham, Caroline E.; Brocklehurst, Keith; Pickersgill, Richard W.; Warren, Martin J.

    2005-01-01

    RALDH3 (retinal dehydrogenase 3) was characterized by kinetic and binding studies, protein engineering, homology modelling, ligand docking and electrostatic-potential calculations. The major recognition determinant of an RALDH3 substrate was shown to be an eight-carbon chain bonded to the aldehyde group whose kinetic influence (kcat/Km at pH 8.5) decreases when shortened or lengthened. Surprisingly, the β-ionone ring of all-trans-retinal is not a major recognition site. The dissociation constants (Kd) of the complexes of RALDH3 with octanal, NAD+ and NADH were determined by intrinsic tryptophan fluorescence. The similarity of the Kd values for the complexes with NAD+ and with octanal suggests a random kinetic mechanism for RALDH3, in contrast with the ordered sequential mechanism often associated with aldehyde dehydrogenase enzymes. Inhibition of RALDH3 by tri-iodothyronine binding in competition with NAD+, predicted by the modelling, was established kinetically and by immunoprecipitation. Mechanistic implications of the kinetically influential ionizations with macroscopic pKa values of 5.0 and 7.5 revealed by the pH-dependence of kcat are discussed. Analogies with data for non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans, together with the present modelled structure of the thioacyl RALDH3, suggest (a) that kcat characterizes deacylation of this intermediate for specific substrates and (b) the assignment of the pKa of the major ionization (approximating to 7.5) to the perturbed carboxy group of Glu280 whose conjugate base is envisaged as supplying general base catalysis to attack of a water molecule. The macroscopic pKa of the minor ionization (5.0) is considered to approximate to that of the carboxy group of Glu488. PMID:16241904

  7. Cellobiose dehydrogenase in cellulose degradation

    SciTech Connect

    Eriksson, L.; Igarashi, Kiyohiko; Samejima, Masahiro

    1996-10-01

    Cellobiose dehydrogenase is produced by a variety of fungi. Although it was already discovered during the 70`s, it`s role in cellulose and lignin degradation is yet ambiguous. The enzyme contains both heme and FAD as prosthetic groups, and seems to have a domain specifically designed to bind the enzyme to cellulose. It`s affinity to amorphous cellulose is higher than to crystalline cellulose. We will report on the binding behavior of the enzyme, its usefulness in elucidation of cellulose structures and also, possibilities for applications such as its use in measuring individual and synergistic mechanisms for cellulose degradation by endo- and exo-glucanases.

  8. Predictive model to describe the combined effect of pH and NaCl on apparent heat resistance of Bacillus stearothermophilus.

    PubMed

    Periago, P M; Fernández, P S; Salmerón, M C; Martínez, A

    1998-10-20

    The combined effect of pH and NaCl on the apparent thermal resistance of Bacillus stearothermophilus ATCC 12980 spores was studied. Spores were heated at different temperatures (115-125 degrees C) in mushroom substrate, acidified using glucono-delta-lactone to different pH levels (from 5.75 to 6.7), which contained concentrations of NaCl that ranged from 0.5 to 3% (w/v). The recovery medium was acidified to the same pH level and contained the same NaCl concentration as the heating menstruum. A factorial experimental design allowed a predictive model to be developed, which described the combined effect of heating temperature, pH and NaCl on the thermal resistance of B. stearothermophilus spores. Predictions from the model provided a valid description of the data used to generate the model, and agreed with observations from the literature and from an independent experiment performed using asparagus and bean substrates.

  9. Study of the influence of sporulation conditions on heat resistance of Geobacillus stearothermophilus used in the development of biological indicators for steam sterilization.

    PubMed

    Guizelini, Belquis P; Vandenberghe, Luciana P S; Sella, Sandra Regina B R; Soccol, Carlos Ricardo

    2012-12-01

    Biological indicators are important tools in infection control via sterilization process monitoring. The use of a standardized spore crop with a well-defined heat resistance will guarantee the quality of a biological indicator. Ambient factors during sporulation can affect spore characteristics and properties, including heat resistance. The aim of this study is to evaluate the main sporulation factors responsible for heat resistance in Geobacillus stearothermophilus, a useful biological indicator for steam sterilization. A sequence of a three-step optimization of variables (initial pH, nutrient concentration, tryptone, peptone, beef extract, yeast extract, manganese sulfate, magnesium sulfate, calcium chloride and potassium phosphate) was carried out to screen those that have a significant influence on heat resistance of produced spores. The variable exerting greatest influence on G. stearothermophilus heat resistance during sporulation was found to be the initial pH. Lower nutrient concentration and alkaline pH around 8.5 tended to enhance decimal reduction time at 121 °C (D(121°C)). A central composite design enabled a fourfold enhancement in heat resistance, and the model obtained accurately describes positive pH and negative manganese sulfate concentration influence on spore heat resistance.

  10. Characterization of the Bacillus stearothermophilus manganese superoxide dismutase gene and its ability to complement copper/zinc superoxide dismutase deficiency in Saccharomyces cerevisiae.

    PubMed Central

    Bowler, C; Van Kaer, L; Van Camp, W; Van Montagu, M; Inzé, D; Dhaese, P

    1990-01-01

    Recombinant clones containing the manganese superoxide dismutase (MnSOD) gene of Bacillus stearothermophilus were isolated with an oligonucleotide probe designed to match a part of the previously determined amino acid sequence. Complementation analyses, performed by introducing each plasmid into a superoxide dismutase-deficient mutant of Escherichia coli, allowed us to define the region of DNA which encodes the MnSOD structural gene and to identify a promoter region immediately upstream from the gene. These data were subsequently confirmed by DNA sequencing. Since MnSOD is normally restricted to the mitochondria in eucaryotes, we were interested (i) in determining whether B. stearothermophilus MnSOD could function in eucaryotic cytosol and (ii) in determining whether MnSOD could replace the structurally unrelated copper/zinc superoxide dismutase (Cu/ZnSOD) which is normally found there. To test this, the sequence encoding bacterial MnSOD was cloned into a yeast expression vector and subsequently introduced into a Cu/ZnSOD-deficient mutant of the yeast Saccharomyces cerevisiae. Functional expression of the protein was demonstrated, and complementation tests revealed that the protein was able to provide tolerance at wild-type levels to conditions which are normally restrictive for this mutant. Thus, in spite of the evolutionary unrelatedness of these two enzymes, Cu/ZnSOD can be functionally replaced by MnSOD in yeast cytosol. Images FIG. 2 FIG. 4 FIG. 5 PMID:2407726

  11. Three-dimensional structure of a variant `Termamyl-like' Geobacillus stearothermophilus α-amylase at 1.9 Å resolution.

    PubMed

    Offen, Wendy A; Viksoe-Nielsen, Anders; Borchert, Torben V; Wilson, Keith S; Davies, Gideon J

    2015-01-01

    The enzyme-catalysed degradation of starch is central to many industrial processes, including sugar manufacture and first-generation biofuels. Classical biotechnological platforms involve steam explosion of starch followed by the action of endo-acting glycoside hydrolases termed α-amylases and then exo-acting α-glucosidases (glucoamylases) to yield glucose, which is subsequently processed. A key enzymatic player in this pipeline is the `Termamyl' class of bacterial α-amylases and designed/evolved variants thereof. Here, the three-dimensional structure of one such Termamyl α-amylase variant based upon the parent Geobacillus stearothermophilus α-amylase is presented. The structure has been solved at 1.9 Å resolution, revealing the classical three-domain fold stabilized by Ca2+ and a Ca2+-Na+-Ca2+ triad. As expected, the structure is similar to the G. stearothermophilus α-amylase but with main-chain deviations of up to 3 Å in some regions, reflecting both the mutations and differing crystal-packing environments.

  12. Coproduction of thermostable amylase and beta-galactosidase enzymes by Geobacillus stearothermophilus SAB-40: aplication of Plackett-Burman design to evaluate culture requirements affecting enzyme production.

    PubMed

    Solimam, Nadia A

    2008-04-01

    A locally isolated thermophile, Geobacillus sp. SAB-40, producing thermostable extracellular amylase constitutively and an induced intracellular beta-galactosidase was characterized and identified based on 16S rRNA sequencing. A phylogenetic analysis then revealed its closeness to Geobacillus stearothermophilus. To evaluate the effect of the culture conditions on the coproduction of both enzymes by G. stearothermophilus SAB-40, a Plackett-Burman fractional factorial design was applied to determine the impact of twenty variables. Among the tested variables, CaCl2, the incubation time, MgSO4.7H2O, and tryptone were found to be the most significant for encouraging amylase production. Lactose was found to promote beta-galactosidase production, whereas starch had a significantly negative effect on lactase production. Based on a statistical analysis, a preoptimized medium attained the maximum production of amylase and beta-galactosidase at 23.29 U/ml/min and 12,958 U/mg biomass, respectively, which was 3- and 2-fold higher than the yield of amylase and lactase obtained with the basal medium, respectively.

  13. 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...

  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, 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...

  16. 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...

  17. Glucose-6-Phosphate Dehydrogenase Deficiency.

    PubMed

    Luzzatto, Lucio; Nannelli, Caterina; Notaro, Rosario

    2016-04-01

    G6PD is a housekeeping gene expressed in all cells. Glucose-6-phosphate dehydrogenase (G6PD) is part of the pentose phosphate pathway, and its main physiologic role is to provide NADPH. G6PD deficiency, one of the commonest inherited enzyme abnormalities in humans, arises through one of many possible mutations, most of which reduce the stability of the enzyme and its level as red cells age. G6PD-deficient persons are mostly asymptomatic, but they can develop severe jaundice during the neonatal period and acute hemolytic anemia when they ingest fava beans or when they are exposed to certain infections or drugs. G6PD deficiency is a global health issue.

  18. 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.

  19. 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

  20. Modeling the combined effect of pH and temperature on the heat resistance of Bacillus stearothermophilus spores heated in a multicomponent food extract.

    PubMed

    Tejedor, W; Rodrigo, M; Martínez, A

    2001-10-01

    The combined effect of pH and temperature on the heat resistance of Bacillus stearothermophilus spores heated in an extract of complex food was studied. The results showed that, in general, reducing the pH reduced the heat resistance of the spores. Similarly, the value for the D parameter in the nonacidified extract was between 30 and 70% lower than the one obtained with double-distilled water. This result once again shows the importance of the substrate in inactivation studies of microorganisms. The experimental data were used to carry out a comparison of two predictive mathematical models of inactivation, one based on a multiparametric regression obtained in this study and the other obtained from the bibliography and based on a linear-Bigelow equation. Both models predict reasonably well, although the multiparametric model presented a slightly better accuracy factor (1.11) than the one obtained with the linear-Bigelow equation (1.13).

  1. Structural basis for thermostability revealed through the identification and characterization of a highly thermostable phosphotriesterase-like lactonase from Geobacillus stearothermophilus

    SciTech Connect

    Hawwa, Renda; Aikens, John; Turner, Robert J.; Santarsiero, Bernard D.; Mescar, Andrew D.

    2009-08-31

    A new enzyme homologous to phosphotriesterase was identified from the bacterium Geobacillus stearothermophilus (GsP). This enzyme belongs to the amidohydrolase family and possesses the ability to hydrolyze both lactone and organophosphate (OP) compounds, making it a phosphotriesterase-like lactonase (PLL). GsP possesses higher OP-degrading activity than recently characterized PLLs, and it is extremely thermostable. GsP is active up to 100 C with an energy of activation of 8.0 kcal/mol towards ethyl paraoxon, and it can withstand an incubation temperature of 60 C for two days. In an attempt to understand the thermostability of PLLs, the X-ray structure of GsP was determined and compared to those of existing PLLs. Based upon a comparative analysis, a new thermal advantage score and plot was developed and reveals that a number of different factors contribute to the thermostability of PLLs.

  2. Purification, crystallization and preliminary crystallographic analysis of Gan1D, a GH1 6-phospho-β-galactosidase from Geobacillus stearothermophilus T1

    PubMed Central

    Lansky, Shifra; Zehavi, Arie; Dann, Roie; Dvir, Hay; Belrhali, Hassan; Shoham, Yuval; Shoham, Gil

    2014-01-01

    Geobacillus stearothermophilus T1 is a Gram-positive thermophilic soil bacterium that contains an extensive system for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. The bacterium uses a number of extracellular enzymes that break down the high-molecular-weight polysaccharides into short oligosaccharides, which enter the cell and are further hydrolyzed into sugar monomers by dedicated intracellular glycoside hydrolases. The interest in the biochemical characterization and structural analysis of these proteins originates mainly from the wide range of their potential biotechnological applications. Studying the different hemicellulolytic utilization systems in G. stearothermophilus T1, a new galactan-utilization gene cluster was recently identified, which encodes a number of proteins, one of which is a GH1 putative 6-phospho-β-galactosidase (Gan1D). Gan1D has recently been cloned, overexpressed, purified and crystallized as part of its comprehensive structure–function study. The best crystals obtained for this enzyme belonged to the triclinic space group P1, with average crystallographic unit-cell parameters of a = 67.0, b = 78.1, c = 92.1 Å, α = 102.4, β = 93.5, γ = 91.7°. A full diffraction data set to 1.33 Å resolution has been collected for the wild-type enzyme, as measured from flash-cooled crystals at 100 K, using synchrotron radiation. These data are currently being used for the detailed three-dimensional crystal structure analysis of Gan1D. PMID:24637762

  3. ISBst12, a novel type of insertion-sequence element causing loss of S-layer-gene expression in Bacillus stearothermophilus ATCC 12980.

    PubMed

    Egelseer, E M; Idris, R; Jarosch, M; Danhorn, T; Sleytr, U B; Sára, M

    2000-09-01

    The cell surface of the surface layer (S-layer)-carrying strain of Bacillus stearothermophilus ATCC 12980 is completely covered with an oblique lattice composed of the S-layer protein SbsC. In the S-layer-deficient strain, theS-layer gene sbsC was still present but was interrupted by a novel type of insertion sequence (IS) element designated ISBst12. The insertion site was found to be located within the coding region of the sbsC gene, 199 bp downstream from the translation start of SbsC. ISBst12 is 1612 bp long, bounded by 16 bp imperfect inverted repeats and flanked by a directly repeated 8 bp target sequence. ISBst12 contains an ORF of 1446 bp and is predicted to encode a putative transposase of 482 aa with a calculated theoretical molecular mass of 55562 Da and an isoelectric point of 9.13. The putative transposase does not exhibit a typical DDE motif but displays aHis-Arg-Tyr triad characteristic of the active site of integrases from the bacteriophage lambda Int family. Furthermore, two overlapping leucine-zipper motifs were identified at the N-terminal part of the putative transposase. As revealed by Southern blotting, ISBst12 was present in multiple copies in the S-layer-deficient strain as well as in the S-layer-carrying strain. Northern blotting indicated that S-layer gene expression is already inhibited at the transcriptional level, since no sbsC-specific transcript could be identified in the S-layer-deficient strain. By using PCR, ISBst12 was also detected in B. stearothermophilus PV72/p6, in its oxygen-induced strain variant PV72/p2 and in the S-layer-deficient strain PV72/T5.

  4. Genetics Home Reference: pyruvate dehydrogenase deficiency

    MedlinePlus

    ... conversion is essential to begin the series of chemical reactions that produce energy for cells. The pyruvate dehydrogenase ... E3, each of which performs part of the chemical reaction that converts pyruvate to acetyl-CoA. In addition, ...

  5. Genetics Home Reference: dihydropyrimidine dehydrogenase deficiency

    MedlinePlus

    ... of the skin on the palms and soles (hand-foot syndrome); shortness of breath; and hair loss may also ... dehydrogenase deficiency , with its early-onset neurological symptoms, is a rare disorder. Its prevalence is ...

  6. Isocitrate dehydrogenase mutations in gliomas

    PubMed Central

    Waitkus, Matthew S.; Diplas, Bill H.; Yan, Hai

    2016-01-01

    Over the last decade, extraordinary progress has been made in elucidating the underlying genetic causes of gliomas. In 2008, our understanding of glioma genetics was revolutionized when mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) were identified in the vast majority of progressive gliomas and secondary glioblastomas (GBMs). IDH enzymes normally catalyze the decarboxylation of isocitrate to generate α-ketoglutarate (αKG), but recurrent mutations at Arg132 of IDH1 and Arg172 of IDH2 confer a neomorphic enzyme activity that catalyzes reduction of αKG into the putative oncometabolite D-2-hydroxyglutate (D2HG). D2HG inhibits αKG-dependent dioxygenases and is thought to create a cellular state permissive to malignant transformation by altering cellular epigenetics and blocking normal differentiation processes. Herein, we discuss the relevant literature on mechanistic studies of IDH1/2 mutations in gliomas, and we review the potential impact of IDH1/2 mutations on molecular classification and glioma therapy. PMID:26188014

  7. Structure-specificity relationships in Abp, a GH27 β-L-arabinopyranosidase from Geobacillus stearothermophilus T6.

    PubMed

    Lansky, Shifra; Salama, Rachel; Solomon, Hodaya V; Feinberg, Hadar; Belrhali, Hassan; Shoham, Yuval; Shoham, Gil

    2014-11-01

    L-Arabinose sugar residues are relatively abundant in plants and are found mainly in arabinan polysaccharides and in other arabinose-containing polysaccharides such as arabinoxylans and pectic arabinogalactans. The majority of the arabinose units in plants are present in the furanose form and only a small fraction of them are present in the pyranose form. The L-arabinan-utilization system in Geobacillus stearothermophilus T6, a Gram-positive thermophilic soil bacterium, has recently been characterized, and one of the key enzymes was found to be an intracellular β-L-arabinopyranosidase (Abp). Abp, a GH27 enzyme, was shown to remove β-L-arabinopyranose residues from synthetic substrates and from the native substrates sugar beet arabinan and larch arabinogalactan. The Abp monomer is made up of 448 amino acids, and based on sequence homology it was suggested that Asp197 is the catalytic nucleophile and Asp255 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Abp (at 2.28 Å resolution) and its catalytic mutant Abp-D197A with (at 2.20 Å resolution) and without (at 2.30 Å resolution) a bound L-arabinose product are reported as determined by X-ray crystallography. These structures demonstrate that the three-dimensional structure of the Abp monomer correlates with the general fold observed for GH27 proteins, consisting of two main domains: an N-terminal TIM-barrel domain and a C-terminal all-β domain. The two catalytic residues are located in the TIM-barrel domain, such that their carboxylic functional groups are about 5.9 Å from each other, consistent with a retaining mechanism. An isoleucine residue (Ile67) located at a key position in the active site is shown to play a critical role in the substrate specificity of Abp, providing a structural basis for the high preference of the enzyme towards arabinopyranoside over galactopyranoside substrates. The crystal structure demonstrates that Abp is a tetramer

  8. Regulation of heart muscle pyruvate dehydrogenase kinase

    PubMed Central

    Cooper, Ronald H.; Randle, Philip J.; Denton, Richard M.

    1974-01-01

    1. The activity of pig heart pyruvate dehydrogenase kinase was assayed by the incorporation of [32P]phosphate from [γ-32P]ATP into the dehydrogenase complex. There was a very close correlation between this incorporation and the loss of pyruvate dehydrogenase activity with all preparations studied. 2. Nucleoside triphosphates other than ATP (at 100μm) and cyclic 3′:5′-nucleotides (at 10μm) had no significant effect on kinase activity. 3. The Km for thiamin pyrophosphate in the pyruvate dehydrogenase reaction was 0.76μm. Sodium pyrophosphate, adenylyl imidodiphosphate, ADP and GTP were competitive inhibitors against thiamin pyrophosphate in the dehydrogenase reaction. 4. The Km for ATP of the intrinsic kinase assayed in three preparations of pig heart pyruvate dehydrogenase was in the range 13.9–25.4μm. Inhibition by ADP and adenylyl imidodiphosphate was predominantly competitive, but there was nevertheless a definite non-competitive element. Thiamin pyrophosphate and sodium pyrophosphate were uncompetitive inhibitors against ATP. It is suggested that ADP and adenylyl imidodiphosphate inhibit the kinase mainly by binding to the ATP site and that the adenosine moiety may be involved in this binding. It is suggested that thiamin pyrophosphate, sodium pyrophosphate, adenylyl imidodiphosphate and ADP may inhibit the kinase by binding through pyrophosphate or imidodiphosphate moieties at some site other than the ATP site. It is not known whether this is the coenzyme-binding site in the pyruvate dehydrogenase reaction. 5. The Km for pyruvate in the pyruvate dehydrogenase reaction was 35.5μm. 2-Oxobutyrate and 3-hydroxypyruvate but not glyoxylate were also substrates; all three compounds inhibited pyruvate oxidation. 6. In preparations of pig heart pyruvate dehydrogenase free of thiamin pyrophosphate, pyruvate inhibited the kinase reaction at all concentrations in the range 25–500μm. The inhibition was uncompetitive. In the presence of thiamin pyrophosphate

  9. Characterization of succinate dehydrogenase and alpha-glycerophosphate dehydrogenase in pancreatic islets.

    PubMed

    Lenzen, S; Panten, U

    1983-12-01

    Succinate dehydrogenase activities in homogenates of rat and ob/ob mouse pancreatic islets were only 13% of the activities in homogenates of liver and were also several times lower than in homogenates of pancreatic acinar tissue. This indicates that the content of mitochondria in pancreatic islet cells is very low. The very low activity of succinate dehydrogenase is in agreement with the low mitochondrial volume in the cytoplasmic ground substance of pancreatic islet cells as observed in morphometric studies. This may represent the poor equipment of pancreatic islet cells with electron transport chains and thus provide a regulatory role for the generation of reducing equivalents and chemical energy for the regulation of insulin secretion. The activities of succinate dehydrogenase in tissue homogenates of pancreatic islets, pancreatic acinar tissue, and liver were significantly inhibited by malonate and diazoxide but not by glucose, mannoheptulose, streptozotocin, or verapamil. Tolbutamide inhibited only pancreatic islet succinate dehydrogenase significantly, providing evidence for a different behavior of pancreatic islet cell mitochondria. Therefore diazoxide and tolbutamide may affect pancreatic islet function through their effects on succinate dehydrogenase activity. The activities of alpha-glycerophosphate dehydrogenase in homogenates of pancreatic islets and liver from rats and ob/ob mice were in the same range, while activities in homogenates of pancreatic acinar tissue were lower. None of the test agents affected alpha-glycerophosphate dehydrogenase activity. Thus the results provide no support for the recent contention that alpha-glycerophosphate dehydrogenase activity may be critical for the regulation of insulin secretion.

  10. Modification of rock/fluid and fluid/fluid interfaces during MEOR processes, using two biosurfactant producing strains of Bacillus stearothermophilus SUCPM#14 and Enterobacter cloacae: a mechanistic study.

    PubMed

    Sarafzadeh, Pegah; Zeinolabedini Hezave, Ali; Mohammadi, Sahar; Niazi, Ali; Ayatollahi, Shahab

    2014-05-01

    During any microbial enhanced oil recovery process, both cells and the metabolic products of bacteria govern the tertiary oil recovery efficiency. However, very accurate examination is needed to find the functionality of these tiny creatures at different reservoir conditions. In this regard, the effect of cell structure on ultimate microbial recovery efficiency which is the most dominant mechanism based on the microorganism types (gram-negative or gram-positive) was systematically investigated. At the first stage, possible different active mechanisms using Bacillus stearothermophilus SUCPM#14 strain were tested using specially designed injection protocol, in situ and ex situ core flooding experiments, interfacial tension, viscosity, pH and Amott wettability index measurements. At the second stage, comparing functionality of B. stearothermophilus SUCPM#14 (a gram-positive type) with the previously examined strain namely Enterobacter cloacae as a gram-negative type, proposed this hypothesis that the cell structure significantly affects the interfacial behaviors. New designed protocols were utilized to check the individual effects of cells, bioproducts and interaction of these together on the oil/water and also fluids/rock interfaces. The final results showed that the cells of B. stearothermophilus SUCPM#14 adhere more into the oil/water interface compared to E. cloacae and change its rheological properties; e.g. its elastic properties which affect the ultimate microbial oil recovery efficiency. Eventually, contradicting results revealed that biosurfactant produced by E. cloacae was able to considerably reduce the interfacial tension and alter the wettability of the rock (to neutral conditions) while biosurfactant produced by B. stearothermophilus SUCPM#14 was not very effective.

  11. 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.

  12. Sorbitol dehydrogenase: structure, function and ligand design.

    PubMed

    El-Kabbani, O; Darmanin, C; Chung, R P-T

    2004-02-01

    Sorbitol dehydrogenase (SDH), a member of the medium-chain dehydrogenase/reductase protein family and the second enzyme of the polyol pathway of glucose metabolism, converts sorbitol to fructose strictly using NAD(+) as coenzyme. SDH is expressed almost ubiquitously in all mammalian tissues. The enzyme has attracted considerable interest due to its implication in the development of diabetic complications and thus its tertiary structure may facilitate the development of drugs for the treatment of diabetes sufferers. Modelling studies suggest that SDH is structurally homologous to mammalian alcohol dehydrogenase with respect to conserved zinc binding motif and a hydrophobic substrate-binding pocket. Recently, the three-dimensional (3-D) structure of a mammalian SDH was solved, and it was found that while the overall 3-D structures of SDH and alcohol dehydrogenase are similar, the zinc coordination in the active sites of the two enzymes is different. The available structural and biochemical information of SDH are currently being utilized in a structure-based approach to develop drugs for the treatment or prevention of the complications of diabetes. This review provides an overview of the recent advances in the structure, function and drug development fields of sorbitol dehydrogenase.

  13. Fundamental molecular differences between alcohol dehydrogenase classes.

    PubMed Central

    Danielsson, O; Atrian, S; Luque, T; Hjelmqvist, L; Gonzàlez-Duarte, R; Jörnvall, H

    1994-01-01

    Two types of alcohol dehydrogenase in separate protein families are the "medium-chain" zinc enzymes (including the classical liver and yeast forms) and the "short-chain" enzymes (including the insect form). Although the medium-chain family has been characterized in prokaryotes and many eukaryotes (fungi, plants, cephalopods, and vertebrates), insects have seemed to possess only the short-chain enzyme. We have now also characterized a medium-chain alcohol dehydrogenase in Drosophila. The enzyme is identical to insect octanol dehydrogenase. It is a typical class III alcohol dehydrogenase, similar to the corresponding human form (70% residue identity), with mostly the same residues involved in substrate and coenzyme interactions. Changes that do occur are conservative, but Phe-51 is of functional interest in relation to decreased coenzyme binding and increased overall activity. Extra residues versus the human enzyme near position 250 affect the coenzyme-binding domain. Enzymatic properties are similar--i.e., very low activity toward ethanol (Km beyond measurement) and high selectivity for formaldehyde/glutathione (S-hydroxymethylglutathione; kcat/Km = 160,000 min-1.mM-1). Between the present class III and the ethanol-active class I enzymes, however, patterns of variability differ greatly, highlighting fundamentally separate molecular properties of these two alcohol dehydrogenases, with class III resembling enzymes in general and class I showing high variation. The gene coding for the Drosophila class III enzyme produces an mRNA of about 1.36 kb that is present at all developmental stages of the fly, compatible with the constitutive nature of the vertebrate enzyme. Taken together, the results bridge a previously apparent gap in the distribution of medium-chain alcohol dehydrogenases and establish a strictly conserved class III enzyme, consistent with an important role for this enzyme in cellular metabolism. Images PMID:8197167

  14. Kinetic analysis about the effects of neutral salts on the thermal stability of yeast alcohol dehydrogenase.

    PubMed

    Ikegaya, Kazuo

    2005-03-01

    The effects of salts on the rate constants of inactivation by heat of yeast alcohol dehydrogenase (YADH) at 60.0 degrees C were measured. Different effects were observed at low and high salt concentrations. At high concentrations, some salts had stabilizing effects, while others were destabilizing. The effects of salts in the high concentration range examined can be described as follows: (decreased thermal stability) NaClO(4) < NaI = (C(2)H(5))(4)NBr < NH(4)Br < NaBr = KBr = CsBr = (no addition) < (CH(3))(4)NBr < KCl < KF < Na(2)SO(4) (increased thermal stability). The decreasing effect of NaClO(4) on YADH controlled the thermal stability of the enzyme absolutely and was not compensated by the addition of Na(2)SO(4), a salt which stabilized the enzyme. However, Na(2)SO(4) compensation did occur in response to the decrease in thermal stability caused by (C(2)H(5))(4)NBr. The rate constants of inactivation by heat (k (in)) of the enzyme were measured at various temperatures. Effective values of the thermodynamic activation parameters of thermal inactivation, activation of free energy (DeltaG (double dagger)), activation enthalpy (DeltaH (double dagger)), and activation entropy (DeltaS (double dagger)), were determined. The thermal stability of YADH in 0.8 M Na(2)SO(4) increased more than that of pyruvate kinase from Bacillus stearothermophilus, a moderate thermophile. The changes in the values of DeltaH (double dagger) and DeltaS (double dagger) were great and showed a general compensatory tendency, with the exception of in the case of NaClO(4). The temperature for the general compensation effect (T (c)) was approximately 123 degrees C. With Na(2)SO(4), the thermal stability of YADH at a temperature below T (c) was greater than that in the absence of salt due to the higher values of DeltaH (double dagger) and DeltaS (double dagger), respectively, and thus was an example of low-temperature enzymatic stabilization. With (C(2)H(5))(4)NBr, the thermal stability of YADH

  15. 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.

  16. [Interaction of succinate dehydrogenase and oxaloacetate].

    PubMed

    Kotliar, A B; Vinogradov, A D

    1984-04-01

    The equilibrium and rate constants for interaction of the reduced and oxidized membrane-bound succinate dehydrogenase (EC 1.3.99.1) with oxaloacetate were determined. The 10-fold decrease in the oxaloacetate affinity for the reduced enzyme was shown to be due to the 10-fold increase of the enzyme-inhibitor complex dissociation rate, which occurs upon its reduction. The rate of dissociation induced by succinate is 10 times higher than that induced by malonate in the submitochondrial particles, being equal in the soluble enzyme preparations. The rates of dissociation induced by malonate excess, or by the enzyme irreversibly utilizing oxaloacetate (transaminase in the presence of glutamate) are also equal. The data obtained suggest that succinate dehydrogenase interaction with succinate and oxaloacetate results from the competition for a single dicarboxylate-specific site. In submitochondrial particles all succinate dehydrogenase molecules are in redox equilibrium provided for by endogenous ubiquinone. No electronic equilibrium between the individual enzyme molecules exists, when succinate dehydrogenase is solubilized.

  17. Development of an amine dehydrogenase for synthesis of chiral amines.

    PubMed

    Abrahamson, Michael J; Vázquez-Figueroa, Eduardo; Woodall, Nicholas B; Moore, Jeffrey C; Bommarius, Andreas S

    2012-04-16

    A leucine dehydrogenase has been successfully altered through several rounds of protein engineering to an enantioselective amine dehydrogenase. Instead of the wild-type α-keto acid, the new amine dehydrogenase now accepts the analogous ketone, methyl isobutyl ketone (MIBK), which corresponds to exchange of the carboxy group by a methyl group to produce chiral (R)-1,3-dimethylbutylamine.

  18. [Thermal stability of lactate dehydrogenase and alcohol dehydrogenase incorporated into highly concentrated gels].

    PubMed

    Kulis, Iu Iu

    1979-03-01

    The rate constants for inactivation of lactate dehydrogenase and alcohol dehydrogenase in solution at 65 degrees C (pH 7,5) are 0,72 and 0,013 min-1, respectively. The enzyme incorporation into acrylamide gels results in immobilized enzymes, whose residual activity is 18--25% of the original one. In 6,7% gels the rate of thermal inactivation for lactate dehydrogenase is decreased nearly 10-fold, whereas the inactivation rate for alcohol dehydrogenase is increased 4,6-fold as compared to the soluble enzymes. In 14% and 40% gels the inactivation constants for lactate dehydrogenase are 6,3.10(-3) and 5,9.10(-4) min-1, respectively. In 60% gels the thermal inactivation of lactate dehydrogenase is decelerated 3600-fold as compared to the native enzyme. The enthalpy and enthropy for the inactivation of the native enzyme are equal to 62,8 kcal/mole and 116,9 cal/(mole.grad.) for the native enzyme and those of gel-incorporated (6,7%) enzyme -- 38,7 kcal/mole and 42 cal/(mole.grad.), respectively. The thermal stability of alcohol dehydrogenase in 60% gels is increased 12-fold. To prevent gel swelling, methacrylic acid and allylamine were added to the matrix, with subsequent treatment by dicyclohexylcarbodiimide. The enzyme activity of the modified gels is 2,7--3% of that for the 6,7% gels. The stability of lactate dehydrogenase in such gels is significantly increased. A mechanism of stabilization of the subunit enzymes in highly concentrated gels is discussed.

  19. Dehydrogenase and Oxoreductase Activities of Porcine Placental 11Beta-Hydroxysteroid Dehydrogenase

    DTIC Science & Technology

    2016-06-07

    dehydrogenase (IIB-HSD) were measured in tissue fragment cultures on day 75 of gestation. Dehydrogenase activity was over fivefold greater than oxoreductase...oxoreductase activities in porcine placentae under physiological conditions using placental explant culture and endogenous concentrations of coenzymes and...f!M range). In human placental tissue fragments at midterm and late pregnancy ( 12, 18) and in trophoblast cell cultures from term placentae ( 41

  20. 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.

  1. Properties of formate dehydrogenase in Methanobacterium formicicum

    SciTech Connect

    Schauer, N.L.; Ferry, J.G.

    1982-04-01

    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..mu..mol of methyl viologen per min per mg of protein and 8.2 ..mu..mol of coenzyme F/sub 420/ per min per mg of protein. The apparent K/sub m/ for 7,8-didemethyl-8-hydroxy-5-deazariboflavin, a hydrolytic derivative of coenzyme F/sub 420/, was 10-fold greater (63 ..mu..M) than for coenzyme F/sub 420/ (6 ..mu..M). The purified enzyme also reduced flavin mononucleotide (K/sub m/ = 13 ..mu..M) and flavin adenine dinucleotide (K/sub m/ = 25 ..mu..M) with formate, but did not reduce NAD/sup +/ or NADP/sup +/. The reduction of NADP/sup +/ with formate required formate dehydrogenase, coenzyme F/sub 420/, and coenzyme F/sub 420/:NADP/sup +/ oxidoreductase. The formate dehydrogenase had an optimal pH of 7.9 when assayed with the physiological electron acceptor coenzyme F/sub 420/. The optimal reaction rate occurred at 55/sup 0/C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (K/sub i/ = 6 ..mu..M), azide (K/sub i/ = 39 ..mu..M),..cap alpha..,..cap 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.

  2. Application of artificial neural networks to describe the combined effect of pH and NaCl on the heat resistance of Bacillus stearothermophilus.

    PubMed

    Esnoz, A; Periago, P M; Conesa, R; Palop, A

    2006-02-01

    A model for prediction of bacterial spore inactivation was developed. The influence of temperature, pH and NaCl on the heat resistance of Bacillus stearothermophilus spores was described using low-complexity, black box models based on artificial neural networks. Literature data were used to build and train the neural network, and new experimental data were used to evaluate it. The neural network models gave better predictions than the classical quadratic response surface model in all the experiments tried. When the neural networks were evaluated using new experimental data, also good predictions were obtained, providing fail-safe predictions of D values in all cases. The weights and biases values of neurons of the neural network that gave the best results are presented, so the reader can use the model for their own purposes. The use of this non-linear modelling technique makes it possible to describe more accurately interacting effects of environmental factors when compared with classical predictive microbial models.

  3. Comparative studies of S-layer proteins from Bacillus stearothermophilus strains expressed during growth in continuous culture under oxygen-limited and non-oxygen-limited conditions.

    PubMed Central

    Sára, M; Sleytr, U B

    1994-01-01

    The specific properties of S-layer proteins from three different Bacillus stearothermophilus strains revealing oblique, square, or hexagonal lattice symmetry were preserved during growth in continuous culture on complex medium only under oxygen-limited conditions in which glucose was used as the sole carbon source. When oxygen limitation was relieved, amino acids became metabolized, cell density increased, and different S-layer proteins from wild-type strains became rapidly replaced by a new common type of S-layer protein with an apparent subunit molecular weight of 97,000 which assembled into an identical oblique (p2) lattice type. During switching from wild-type strains to variants, patches of the S-layer lattices characteristics for wild-type strains, granular regions, and areas with oblique lattice symmetry could be observed on the surface of individual cells from all organisms. The granular regions apparently consisted of mixtures of the S-layer proteins from the wild-type strains and the newly synthesized p2 S-layer proteins from the variants. S-layer proteins from wild-type strains possessed identical N-terminal regions but led to quite different cleavage products upon peptide mapping, indicating that they are encoded by different genes. Chemical analysis including N-terminal sequencing and peptide mapping showed that the oblique S-layer lattices synthesized under increased oxygen supply were composed of identical protein species. Images PMID:7961489

  4. Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from Geobacillus stearothermophilus by Site-Directed Mutagenesis at the Active Site

    PubMed Central

    Ma, Yi; Zhang, Beilei; Wang, Meng; Ou, Yanghui

    2016-01-01

    The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1.543 (Bst DNA polymerase) with 5′-3′ DNA polymerase activity while in absence of 5′-3′ exonuclease activity possesses high thermal stability and polymerase activity. Bst DNA polymerase was employed in isothermal multiple self-matching initiated amplification (IMSA) which amplified the interest sequence with high selectivity and was widely applied in the rapid detection of human epidemic diseases. However, the detailed information of commercial Bst DNA polymerase is unpublished and well protected by patents, which makes the high price of commercial kits. In this study, wild-type Bst DNA polymerase (WT) and substitution mutations for improving the efficiency of DNA polymerization were expressed and purified in E. coli. Site-directed substitutions of four conserved residues (Gly310, Arg412, Lys416, and Asp540) in the activity site of Bst DNA polymerase influenced efficiency of polymerizing dNTPs. The substitution of residue Gly310 by alanine or leucine and residue Asp540 by glutamic acid increased the efficiency of polymerase activity. All mutants with higher polymerizing efficiency were employed to complete the rapid detection of EV71-associated hand, foot, and mouth disease (HFMD) by IMSA approach with relatively shorter period which is suitable for the primary diagnostics setting in rural and underdeveloped areas. PMID:27981047

  5. Secretory expression of thermostable alkaline protease from Bacillus stearothermophilus FI by using native signal peptide and α-factor secretion signal in Pichia pastoris.

    PubMed

    Latiffi, Amaliawati Ahmad; Salleh, Abu Bakar; Rahman, Raja Noor Zaliha Raja Abd; Oslan, Siti Nurbaya; Basri, Mahiran

    2013-01-01

    The thermostable alkaline protease from Bacillus stearothermophilus F1 has high potential for industrial applications, and attempt to produce the enzyme in yeast for higher yield was undertaken. Secretory expression of F1 protease through yeast system could improve enzyme's capability, thus simplifying the purification steps. Mature and full genes of F1 protease were cloned into Pichia pastoris expression vectors (pGAPZαB and pPICZαB) and transformed into P. pastoris strains (GS115 and SMD1168H) via electroporation method. Recombinant F1 protease under regulation constitutive GAP promoter revealed that the highest expression was achieved after 72 h cultivation. While inducible AOX promoter showed that 0.5% (v/v) methanol was the best to induce expression. It was proven that constitutive expression strategy was better than inducible system. The α-secretion signal from the plasmid demonstrated higher secretory expression level of F1 protease as compared to native Open Reading Frame (ORF) in GS115 strain (GE6GS). Production medium YPTD was found to be the best for F1 protease expression with the highest yield of 4.13 U/mL. The protein was expressed as His-tagged fusion protein with a size about 34 kDa.

  6. "Enzymogenesis": classical liver alcohol dehydrogenase origin from the glutathione-dependent formaldehyde dehydrogenase line.

    PubMed Central

    Danielsson, O; Jörnvall, H

    1992-01-01

    Analysis of the activity and structure of lower vertebrate alcohol dehydrogenases reveals that relationships between the classical liver and yeast enzymes need not be continuous. Both the ethanol activity of class I-type alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) and the glutathione-dependent formaldehyde activity of the class III-type enzyme [formaldehyde:NAD+ oxidoreductase (glutathione-formylating), EC 1.2.1.1] are present in liver down to at least the stage of bony fishes (cod liver: ethanol activity, 3.4 units/mg of protein in one enzyme; formaldehyde activity, 4.5 units/mg in the major form of another enzyme). Structural analysis of the latter protein reveals it to be a typical class III enzyme, with limited variation from the mammalian form and therefore with stable activity and structure throughout much of the vertebrate lineage. In contrast, the classical alcohol dehydrogenase (the class I enzyme) appears to be the emerging form, first in activity and later also in structure. The class I activity is present already in the piscine line, whereas the overall structural-type enzyme is not observed until amphibians and still more recent vertebrates. Consequently, the class I/III duplicatory origin appears to have arisen from a functional class III form, not a class I form. Therefore, ethanol dehydrogenases from organisms existing before this duplication have origins separate from those leading to the "classical" liver alcohol dehydrogenases. The latter now often occur in isozyme forms from further gene duplications and have a high rate of evolutionary change. The pattern is, however, not simple and we presently find in cod the first evidence for isozymes also within a class III alcohol dehydrogenase. Overall, the results indicate that both of these classes of vertebrate alcohol dehydrogenase are important and suggest a protective metabolic function for the whole enzyme system. Images PMID:1409630

  7. Purification of arogenate dehydrogenase from Phenylobacterium immobile.

    PubMed

    Mayer, E; Waldner-Sander, S; Keller, B; Keller, E; Lingens, F

    1985-01-07

    Phenylobacterium immobile, a bacterium which is able to degrade the herbicide chloridazon, utilizes for L-tyrosine synthesis arogenate as an obligatory intermediate which is converted in the final biosynthetic step by a dehydrogenase to tyrosine. This enzyme, the arogenate dehydrogenase, has been purified for the first time in a 5-step procedure to homogeneity as confirmed by electrophoresis. The Mr of the enzyme that consists of two identical subunits amounts to 69000 as established by gel electrophoresis after cross-linking the enzyme with dimethylsuberimidate. The Km values were 0.09 mM for arogenate and 0.02 mM for NAD+. The enzyme has a high specificity with respect to its substrate arogenate.

  8. 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.

  9. 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.

  10. Relationships within the aldehyde dehydrogenase extended family.

    PubMed Central

    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. PMID:10210192

  11. Identification of Two Binding Domains, One for Peptidoglycan and Another for a Secondary Cell Wall Polymer, on the N-Terminal Part of the S-Layer Protein SbsB from Bacillus stearothermophilus PV72/p2

    PubMed Central

    Sára, Margit; Egelseer, Eva M.; Dekitsch, Christine; Sleytr, Uwe B.

    1998-01-01

    First studies on the structure-function relationship of the S-layer protein from B. stearothermophilus PV72/p2 revealed the coexistence of two binding domains on its N-terminal part, one for peptidoglycan and another for a secondary cell wall polymer (SCWP). The peptidoglycan binding domain is located between amino acids 1 to 138 of the mature S-layer protein comprising a typical S-layer homologous domain. The SCWP binding domain lies between amino acids 240 to 331 and possesses a high serine plus glycine content. PMID:9852032

  12. 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

  13. Calcium alginate matrix increases the stability and recycling capability of immobilized endo-β-1,4-xylanase from Geobacillus stearothermophilus KIBGE-IB29.

    PubMed

    Bibi, Zainab; Qader, Shah Ali Ul; Aman, Afsheen

    2015-07-01

    Exploration of microbial pool from extremely diversified ecosystem is significantly important for various industrial applications. Bacterial communities from extreme habitats including volcanic vents, hot springs, and industrial sectors are eagerly explored for the isolation of thermophiles. Geobacillus stearothermophilus KIBGE-IB29, isolated from blast furnace site of a steel processing industry, is capable of producing thermostable endo-β-1,4-xylanase. In the current study, this enzyme was immobilized within calcium alginate beads using entrapment technique. Amalgamation of sodium alginate (40.0 gL(-1)) and calcium chloride (0.4 M) was used for the formation of immobilized beads. It was observed that temperature (50 °C) and pH (7.0) optima of immobilized enzyme remained same, but enzyme-substrate reaction time increased from 5.0 to 30.0 min as compared to free enzyme. Diffusion limit of high molecular weight xylan (corncob) caused a decline in V max of immobilized enzyme from 4773 to 203.7 U min(-1), whereas K m value increased from 0.5074 to 0.5722 mg ml(-1) with reference to free enzyme. Immobilized endo-β-1,4-xylanase showed its stability even at high temperatures as compared to free enzyme and retained 18 and 9 % residual activity at 70 and 80 °C, respectively. Immobilized enzyme also exhibited sufficient recycling efficiency up to five reaction cycles which indicated that this enzyme can be a plausible candidate in paper and pulp industry.

  14. Immobilization of BSA, enzymes and cells of Bacillus stearothermophilus onto cellulose polygalacturonic acid and starch based graft copolymers containing maleic arhydride

    SciTech Connect

    Beddows, C.G.; Gil, M.H.; Guthrie, J.T.

    1986-01-01

    Poly(maleic anhydride styrene) graft copolymers of cellulose, pectin polygalacturonic acid salt, calcium polygalacturonate, and starch were prepared and used to immobilize proteins. The cellulose grafts coupled quite appreciable quantities of acid phosphatase, glucose oxidase, and trypsin. However, the general retention of activity was somewhat disappointing. Further investigation with acid phosphatase showed that the amount of enzyme immobilized increased as the amount of anhydride in the graft copolymer increased but no such relationship existed for the enzymic activity. The cellulose graft copolymers were hydrolyzed and it appeared that the carboxyl group aided adsorption of the enzyme. Attempts to couple acid phosphatase using CMC through the free carboxyl groups, created by hydrolysis, gave only a small increase in the extent of protein coupling. However, the unhydrolyzed system gave a useful degree of immobilization of cells of Bacillus stearothermophilus, as did a poly(maleic anhydride/styrene)-cocellulose system. Attempts to improve the activity by using grafts based on other polysaccharide supports met with mixed success. Pectin products were soluble. Polygalacturonic acid products were partially soluble and extremely high levels of enzymic activity were obtained. This was probably due in part to the hydrophilic nature of the system, which also encouraged absorption of the enzyme. Attempts were made to reduce the solubility by using the calcium pectinate salt. Immobilization of acid phosphatase and trypsin resulted in increased protein coupling but relatively poor activities were attained. Calcium polygalacturonate was used to prepare an insoluble graft copolymeric system containing acrylonitrile-comaleic anhydride. The resulting gels gave excellent coupling with acid phosphatase which had a very good retention of activity.

  15. Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol

    PubMed Central

    Dror, Adi; Shemesh, Einav; Dayan, Natali

    2014-01-01

    The abilities of enzymes to catalyze reactions in nonnatural environments of organic solvents have opened new opportunities for enzyme-based industrial processes. However, the main drawback of such processes is that most enzymes have a limited stability in polar organic solvents. In this study, we employed protein engineering methods to generate a lipase for enhanced stability in methanol, which is important for biodiesel production. Two protein engineering approaches, random mutagenesis (error-prone PCR) and structure-guided consensus, were applied in parallel on an unexplored lipase gene from Geobacillus stearothermophilus T6. A high-throughput colorimetric screening assay was used to evaluate lipase activity after an incubation period in high methanol concentrations. Both protein engineering approaches were successful in producing variants with elevated half-life values in 70% methanol. The best variant of the random mutagenesis library, Q185L, exhibited 23-fold-improved stability, yet its methanolysis activity was decreased by one-half compared to the wild type. The best variant from the consensus library, H86Y/A269T, exhibited 66-fold-improved stability in methanol along with elevated thermostability (+4.3°C) and a 2-fold-higher fatty acid methyl ester yield from soybean oil. Based on in silico modeling, we suggest that the Q185L substitution facilitates a closed lid conformation that limits access for both the methanol and substrate excess into the active site. The enhanced stability of H86Y/A269T was a result of formation of new hydrogen bonds. These improved characteristics make this variant a potential biocatalyst for biodiesel production. PMID:24362426

  16. Isolation of two physiologically induced variant strains of Bacillus stearothermophilus NRS 2004/3a and characterization of their S-layer lattices.

    PubMed Central

    Sára, M; Pum, D; Küpcü, S; Messner, P; Sleytr, U B

    1994-01-01

    During growth of Bacillus stearothermophilus NRS 2004/3a in continuous culture on complex medium, the chemical properties of the S-layer glycoprotein and the characteristic oblique lattice were maintained only if glucose was used as the sole carbon source. With increased aeration, amino acids were also metabolized, accompanied by liberation of ammonium and by changes in the S-layer protein. Depending on the stage of fermentation at which oxygen limitation was relieved, two different variants, one with a more delicate oblique S-layer lattice (variant 3a/V1) and one with a square S-layer lattice (variant 3a/V2), were isolated. During the switch from the wild-type strain to a variant or from variant 3a/V2 to variant 3a/V1, monolayers of two types of S-layer lattices could be demonstrated on the surfaces of single cells. S-layer proteins from variants had different molecular sizes and a significantly lower carbohydrate content than S-layer proteins from the wild-type strain did. Although the S-layer lattices from the wild-type and variant strains showed quite different protein mass distributions in two- and three-dimensional reconstructions, neither the amino acid composition nor the pore size, as determined by permeability studies, was significantly changed. Peptide mapping and N-terminal sequencing results strongly indicated that the three S-layer proteins are encoded by different genes and are not derived from a universal precursor form. Images PMID:8300538

  17. Evidence that the N-terminal part of the S-layer protein from Bacillus stearothermophilus PV72/p2 recognizes a secondary cell wall polymer.

    PubMed Central

    Ries, W; Hotzy, C; Schocher, I; Sleytr, U B; Sára, M

    1997-01-01

    The S-layer of Bacillus stearothermophilus PV72/p2 shows oblique lattice symmetry and is composed of identical protein subunits with a molecular weight of 97,000. The isolated S-layer subunits could bind and recrystallize into the oblique lattice on native peptidoglycan-containing sacculi which consist of peptidoglycan of the A1gamma chemotype and a secondary cell wall polymer with an estimated molecular weight of 24,000. The secondary cell wall polymer could be completely extracted from peptidoglycan-containing sacculi with 48% HF, indicating the presence of phosphodiester linkages between the polymer chains and the peptidoglycan backbone. The cell wall polymer was composed mainly of GlcNAc and ManNAc in a molar ratio of 4:1, constituted about 20% of the peptidoglycan-containing sacculus dry weight, and was also detected in the fraction of the S-layer self-assembly products. Extraction experiments and recrystallization of the whole S-layer protein and proteolytic cleavage fragments confirmed that the secondary cell wall polymer is responsible for anchoring the S-layer subunits by the N-terminal part to the peptidoglycan-containing sacculi. In addition to this binding function, the cell wall polymer was found to influence the in vitro self-assembly of the guanidinium hydrochloride-extracted S-layer protein. Chemical modification studies further showed that the secondary cell wall polymer does not contribute significant free amino or carboxylate groups to the peptidoglycan-containing sacculi. PMID:9190804

  18. Inhibitory effect of disulfiram (Antabuse) on alcohol dehydrogenase activity.

    PubMed

    Carper, W R; Dorey, R C; Beber, J H

    1987-10-01

    We investigated the effect of disulfiram (Antabuse) on the activity of alcohol dehydrogenase (EC 1.1.1.1) in vitro. We observed a time-dependent inhibition of this dehydrogenase by disulfiram and diethyldithiocarbamate similar to that obtained for aldehyde dehydrogenase (EC 1.2.1.3). These results suggest a possible explanation for various side effects observed in the clinical use of Antabuse.

  19. A lipoamide dehydrogenase from Neisseria meningitidis has a lipoyl domain.

    PubMed

    Bringas, R; Fernandez, J

    1995-04-01

    A protein of molecular weight of 64 kDa (p64k) found in the outer membrane of Neisseria meningitidis shows a high degree of homology with both the lipoyl domain of the acetyltransferase and the entire sequence of the lipoamide dehydrogenase, the E2 and E3 components of the dehydrogenase multienzyme complexes, respectively. The alignment of the p64k with lipoyl domains and lipoamide dehydrogenases from different species is presented. The possible implications of this protein in binding protein-dependent transport are discussed. This is the first lipoamide dehydrogenase reported to have a lipoyl domain.

  20. Inhibition of membrane-bound succinate dehydrogenase by disulfiram.

    PubMed

    Jay, D

    1991-04-01

    The effect of disulfiram on succinate oxidase and succinate dehydrogenase activities of beef heart submitochondrial particles was studied. Results show that disulfiram inhibits both functions. Succinate and malonate suppress the inhibitory action of disulfiram when succinate dehydrogenase is stabilized in an active conformation. Disulfiram is not able to inhibit the enzyme when succinate dehydrogenase is inactivated by oxaloacetate. The inhibitory effect of disulfiram is reverted by the addition of dithiothreitol. From these results, it is proposed that disulfiram inhibits the utilization of succinate by a direct modification of an -SH group located in the catalytically active site of succinate dehydrogenase.

  1. 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...

  2. 21 CFR 862.1500 - Malic dehydrogenase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 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...

  3. 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...

  4. 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...

  5. Placental glucose dehydrogenase polymorphism in Koreans.

    PubMed

    Kim, Y J; Paik, S G; Park, H Y

    1994-12-01

    The genetic polymorphism of placental glucose dehydrogenase (GDH) was investigated in 300 Korean placentae using horizontal starch gel electrophoresis. The allele frequencies for GDH1, GDH2 and GDH3 were 0.537, 0.440 and 0.005, respectively, which were similar to those in Japanese. We also observed an anodal allele which was similar to the GDH4 originally reported in Chinese populations at a low frequency of 0.015. An additional new cathodal allele (named GDH6) was observed in the present study with a very low frequency of 0.003.

  6. Green Synthesis of D-1,2,4-Butanetriol from D-Glucose

    DTIC Science & Technology

    2009-01-01

    Figure 1. Microbial synthesis of D-xylonic acid from D-glucose. (a) carbohydrate phosphotransferase (ptsG, crr); (b) D-glucose 6-phosphate dehydrogenase...coli WY9/pWN7.126B. (a) carbohydrate phosphotransferase (ptsG, crr); (b) D-glucose 6-phosphate dehydrogenase (zwf); (c) 6- phosphogluconate...controlled conditions. Recently, applying reaction engineering to various fermentation parameters led to a substantial improvement in the product D

  7. Isoenzyme characterization of Leishmania isolated from human cases with localized cutaneous leishmaniasis from the State of Campeche, Yucatan Peninsula, Mexico.

    PubMed

    Canto-Lara, S B; Cardenas-Maruffo, M F; Vargas-Gonzalez, A; Andrade-Narvaez, F

    1998-04-01

    Seventy-five isolates from the State of Campeche, Mexico, an area endemic for localized cutaneous leishmaniasis (LCL), were characterized by isoenzyme markers (glucose phosphate isomerase, mannose phospate isomerase, nucleoside hydrolase, phosphoglucomutase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase). Seventy (93.3%) were identified as Leishmania (Leishmania) mexicana and 5 (6.7%) as L. (Viannia) braziliensis. This is the first report of authochthonus human LCL caused by L. (V.) braziliensis in the State of Campeche, Yucatan Peninsula, Mexico.

  8. Kinetic mechanism of chicken liver xanthine dehydrogenase.

    PubMed Central

    Bruguera, P; Lopez-Cabrera, A; Canela, E I

    1988-01-01

    The kinetic behaviour of chicken-liver xanthine dehydrogenase (xanthine/NAD+ oxidoreductase; EC 1.2.1.37) has been studied. Steady-state results, obtained from a wide range of concentrations of substrates and products, were fitted by rational functions of degree 1:1, 1:2, 2:2 and 3:3 with respect to substrates, and 0:1, 1:1, 0:2 and 1:2 with regard to products, using a non-linear regression program which guarantees the fit. The goodness of fit was improved using a computer program that combines model discrimination, parameter refinement and sequential experimental design. The AIC and F tests were also used for model discrimination. For comparative purposes, the xanthine/oxygen oxidoreductase reaction was also studied. From the functions which give the maximum improvement, the complete rate equation was deduced. The significance of the terms was stated by the above methods. It was concluded that xanthine dehydrogenase requires a minimum mechanism of degree 1:1 for xanthine, 2:2 for NAD+, 1:1 for uric acid and 1:2 for NADH in the xanthine/NAD+ oxidoreductase reaction. These are the minimum degrees required but a rate equation of higher degree is not excluded. PMID:3422556

  9. Catecholamine regulation of lactate dehydrogenase in rat brain cell culture

    SciTech Connect

    Kumar, S.; McGinnis, J.F.; de Vellis, J.

    1980-03-25

    The mechanism of catecholamine induction of the soluble cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27) was studied in the rat glial tumor cell line, C6. Lactate dehydrogenase was partially purified from extracts of (/sup 3/H)leucine-labeled cells by affinity gel chromatography and quantitatively immunoprecipitated with anti-lactate dehydrogenase-5 IgG and with antilactate dehydrogenase-1 IgG. The immunoprecipitates were dissociated and electrophoresed on sodium dodecyl sulfate polyacrylamide gels. Using this methodology, the increased enzyme activity of lactate dehydrogenase in norepinephrine-treated C6 cells was observed to be concomitant with the increased synthesis of enzyme molecules. Despite the continued presence of norepinephrine, the specific increase in the rate of synthesis of lactate dehydrogenase was transient. It was first detected at 4 h, was maximum at 9 h, and returned to basal levels by 24 h. The half-life of lactate dehydrogenase enzyme activity was 36 h during the induction and 40 h during deinduction. The half-life for decay of /sup 3/H-labeled lactate dehydrogenase was 41 h. These observations suggest that the increase in lactate dehydrogenase activity in norepinephrine-treated cells does not involve any change in the rate of degradation. Norepinephrine increased the specific rate of synthesis of both lactate dehydrogenase-5 (a tetramer of four M subunits) and lactate dehydrogenase-1 (a tetramer of four H subunits), although to different extents. Since these subunits are coded for by two separate genes on separate chromosomes, it suggests that the regulatory mechanism involves at least two separate sites of action.

  10. Effect of Dimer Dissociation on Activity and Thermostability of the α-Glucuronidase from Geobacillus stearothermophilus: Dissecting the Different Oligomeric Forms of Family 67 Glycoside Hydrolases

    PubMed Central

    Shallom, Dalia; Golan, Gali; Shoham, Gil; Shoham, Yuval

    2004-01-01

    The oligomeric organization of enzymes plays an important role in many biological processes, such as allosteric regulation, conformational stability and thermal stability. α-Glucuronidases are family 67 glycosidases that cleave the α-1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid and xylose units as part of an array of hemicellulose-hydrolyzing enzymes. Currently, two crystal structures of α-glucuronidases are available, those from Geobacillus stearothermophilus (AguA) and from Cellvibrio japonicus (GlcA67A). Both enzymes are homodimeric, but surprisingly their dimeric organization is different, raising questions regarding the significance of dimerization for the enzymes' activity and stability. Structural comparison of the two enzymes suggests several elements that are responsible for the different dimerization organization. Phylogenetic analysis shows that the α-glucuronidases AguA and GlcA67A can be classified into two distinct subfamilies of bacterial α-glucuronidases, where the dimer-forming residues of each enzyme are conserved only within its own subfamily. It seems that the different dimeric forms of AguA and GlcA67A represent the two alternative dimeric organizations of these subfamilies. To study the biological significance of the dimerization in α-glucuronidases, we have constructed a monomeric form of AguA by mutating three of its interface residues (W328E, R329T, and R665N). The activity of the monomer was significantly lower than the activity of the wild-type dimeric AguA, and the optimal temperature for activity of the monomer was around 35°C, compared to 65°C of the wild-type enzyme. Nevertheless, the melting temperature of the monomeric protein, 72.9°C, was almost identical to that of the wild-type, 73.4°C. It appears that the dimerization of AguA is essential for efficient catalysis and that the dissociation into monomers results in subtle conformational changes in the structure which indirectly influence the active site region

  11. Influence of the Secondary Cell Wall Polymer on the Reassembly, Recrystallization, and Stability Properties of the S-Layer Protein from Bacillus stearothermophilus PV72/p2

    PubMed Central

    Sára, Margit; Dekitsch, Christine; Mayer, Harald F.; Egelseer, Eva M.; Sleytr, Uwe B.

    1998-01-01

    The high-molecular-weight secondary cell wall polymer (SCWP) from Bacillus stearothermophilus PV72/p2 is mainly composed of N-acetylglucosamine (GlcNAc) and N-acetylmannosamine (ManNAc) and is involved in anchoring the S-layer protein via its N-terminal region to the rigid cell wall layer. In addition to this binding function, the SCWP was found to inhibit the formation of self-assembly products during dialysis of the guanidine hydrochloride (GHCl)-extracted S-layer protein. The degree of assembly (DA; percent assembled from total S-layer protein) that could be achieved strongly depended on the amount of SCWP added to the GHCl-extracted S-layer protein and decreased from 90 to 10% when the concentration of the SCWP was increased from 10 to 120 μg/mg of S-layer protein. The SCWP kept the S-layer protein in the water-soluble state and favored its recrystallization on solid supports such as poly-l-lysine-coated electron microscopy grids. Derived from the orientation of the base vectors of the oblique S-layer lattice, the subunits had bound with their charge-neutral outer face, leaving the N-terminal region with the polymer binding domain exposed to the ambient environment. From cell wall fragments about half of the S-layer protein could be extracted with 1 M GlcNAc, indicating that the linkage type between the S-layer protein and the SCWP could be related to that of the lectin-polysaccharide type. Interestingly, GlcNAc had an effect on the in vitro self-assembly and recrystallization properties of the S-layer protein that was similar to that of the isolated SCWP. The SCWP generally enhanced the stability of the S-layer protein against endoproteinase Glu-C attack and specifically protected a potential cleavage site in position 138 of the mature S-layer protein. PMID:9696762

  12. Effect of dimer dissociation on activity and thermostability of the alpha-glucuronidase from Geobacillus stearothermophilus: dissecting the different oligomeric forms of family 67 glycoside hydrolases.

    PubMed

    Shallom, Dalia; Golan, Gali; Shoham, Gil; Shoham, Yuval

    2004-10-01

    The oligomeric organization of enzymes plays an important role in many biological processes, such as allosteric regulation, conformational stability and thermal stability. alpha-Glucuronidases are family 67 glycosidases that cleave the alpha-1,2-glycosidic bond between 4-O-methyl-D-glucuronic acid and xylose units as part of an array of hemicellulose-hydrolyzing enzymes. Currently, two crystal structures of alpha-glucuronidases are available, those from Geobacillus stearothermophilus (AguA) and from Cellvibrio japonicus (GlcA67A). Both enzymes are homodimeric, but surprisingly their dimeric organization is different, raising questions regarding the significance of dimerization for the enzymes' activity and stability. Structural comparison of the two enzymes suggests several elements that are responsible for the different dimerization organization. Phylogenetic analysis shows that the alpha-glucuronidases AguA and GlcA67A can be classified into two distinct subfamilies of bacterial alpha-glucuronidases, where the dimer-forming residues of each enzyme are conserved only within its own subfamily. It seems that the different dimeric forms of AguA and GlcA67A represent the two alternative dimeric organizations of these subfamilies. To study the biological significance of the dimerization in alpha-glucuronidases, we have constructed a monomeric form of AguA by mutating three of its interface residues (W328E, R329T, and R665N). The activity of the monomer was significantly lower than the activity of the wild-type dimeric AguA, and the optimal temperature for activity of the monomer was around 35 degrees C, compared to 65 degrees C of the wild-type enzyme. Nevertheless, the melting temperature of the monomeric protein, 72.9 degrees C, was almost identical to that of the wild-type, 73.4 degrees C. It appears that the dimerization of AguA is essential for efficient catalysis and that the dissociation into monomers results in subtle conformational changes in the structure

  13. A chemical proteomic probe for detecting dehydrogenases: catechol rhodanine.

    PubMed

    Ge, Xia; Sem, Daniel S

    2012-01-01

    Inherent complexity of the proteome often demands that it be studied as manageable subsets, termed subproteomes. A subproteome can be defined in a number of ways, although a pragmatic approach is to define it based on common features in an active site that lead to binding of a common small molecule ligand (e.g., a cofactor or a cross-reactive drug lead). The subproteome, so defined, can be purified using that common ligand tethered to a resin, with affinity chromatography. Affinity purification of a subproteome is described in the next chapter. That subproteome can then be analyzed using a common ligand probe, such as a fluorescent common ligand that can be used to stain members of the subproteome in a native gel. Here, we describe such a fluorescent probe, based on a catechol rhodanine acetic acid (CRAA) ligand that binds to dehydrogenases. The CRAA ligand is fluorescent and binds to dehydrogenases at pH > 7, and hence can be used effectively to stain dehydrogenases in native gels to identify what subset of proteins in a mixture are dehydrogenases. Furthermore, if one is designing inhibitors to target one or more of these dehydrogenases, the CRAA staining can be performed in a competitive assay format, with or without inhibitor, to assess the selectivity of the inhibitor for the targeted dehydrogenase. Finally, the CRAA probe is a privileged scaffold for dehydrogenases, and hence can easily be modified to increase affinity for a given dehydrogenase.

  14. Toxic Neuronal Death by Glyeraldehyde-3-Phosphate Dehydrogenase and Mitochondria

    DTIC Science & Technology

    2003-08-01

    Neuroreport, 10(5), 1149-1153. Sioud, M., & Jespersen, L. (1996). Enhancement of hammerhead ribozyme catalysis by glyceraldehyde-3-phosphate dehydrogenase...1996) Enhancemen t of hammerhead r ibozyme cata lysis by glycera ldehyde-3- phospha te dehydrogenase. J Mol Biol 257:775–789. Sirover MA (1997) Role of

  15. 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... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5560 Lactic dehydrogenase immunological test system. (a) Identification. A lactic...

  16. 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... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5560 Lactic dehydrogenase immunological test system. (a) Identification. A lactic...

  17. 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

  18. Characterization of the developmentally regulated Bacillus subtilis glucose dehydrogenase gene.

    PubMed Central

    Lampel, K A; Uratani, B; Chaudhry, G R; Ramaley, R F; Rudikoff, S

    1986-01-01

    The DNA sequence of the structural gene for glucose dehydrogenase (EC 1.1.1.47) of Bacillus subtilis was determined and comprises 780 base pairs. The subunit molecular weight of glucose dehydrogenase as deduced from the nucleotide sequence is 28,196, which agrees well with the subunit molecular weight of 31,500 as determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the 49 amino acids at the NH2 terminus of glucose dehydrogenase purified from sporulating B. subtilis cells matched the amino acid sequence derived from the DNA sequence. Glucose dehydrogenase was purified from an Escherichia coli strain harboring pEF1, a plasmid that contains the B. subtilis gene encoding glucose dehydrogenase. This enzyme has the identical amino acid sequence at the NH2 terminus as the B. subtilis enzyme. A putative ribosome-binding site, 5'-AGGAGG-3', which is complementary to the 3' end of the 16S rRNA of B. subtilis, was found 6 base pairs preceding the translational start codon of the structural gene of glucose dehydrogenase. No known promoterlike DNA sequences that are recognized by B. subtilis RNA polymerases were present immediately preceding the translational start site of the glucose dehydrogenase structural gene. The glucose dehydrogenase gene was found to be under sporulation control at the trancriptional level. A transcript of 1.6 kilobases hybridized to a DNA fragment within the structural gene of glucose dehydrogenase. This transcript was synthesized 3 h after the cessation of vegetative growth concomitant to the appearance of glucose dehydrogenase. Images PMID:3082854

  19. 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...

  20. 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).

  1. 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.

  2. 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.

  3. [Glucose-6-phosphate dehydrogenase deficiency in Japan].

    PubMed

    Kanno, Hitoshi; Ogura, Hiromi

    2015-07-01

    In the past 10 years, we have diagnosed congenital hemolytic anemia in 294 patients, approximately 33% of whom were found to have glucose-6-phosphate dehydrogenase (G6PD) deficiency. It is becoming more common for Japanese to marry people of other ethnic origins, such that G6PD deficiency is becoming more prevalent in Japan. Japanese G6PD deficiency tends to be diagnosed in the neonatal period due to severe jaundice, while G6PD-deficient patients with foreign ancestors tend to be diagnosed at the onset of an acute hemolytic crisis before the age of six. It is difficult to predict the clinical course of each patient by G6PD activity, reduced glutathione content, or the presence/absence of severe neonatal jaundice. We propose that both neonatal G6PD screening and systematic analyses of G6PD gene mutations may be useful for personalized management of patients with G6PD-deficient hemolytic anemia.

  4. 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.

  5. 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.

  6. 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.

  7. Cloning of the maltose phosphorylase gene from Bacillus sp. strain RK-1 and efficient production of the cloned gene and the trehalose phosphorylase gene from Bacillus stearothermophilus SK-1 in Bacillus subtilis.

    PubMed

    Inoue, Yasushi; Yasutake, Nozomu; Oshima, Yoshie; Yamamoto, Yoshie; Tomita, Tetsuji; Miyoshi, Shinsuke; Yatake, Tsuneya

    2002-12-01

    The maltose phosphorylase (MPase) gene of Bacillus sp. strain RK-1 was cloned by PCR with oligonucleotide primers designed on the basis of a partial N-terminal amino acid sequence of the purified enzyme. The MPase gene consisted of 2,655 bp encoding a theoretical protein with a Mr of 88,460, and had no secretion signal sequence, although most of the MPase activity was detected in the culture supernatant of RK-1. This cloned MPase gene and the trehalose phosphorylase (TPase) gene from Bacillus stearothermophilus SK-1 were efficiently expressed intracellularly under the control of the Bacillus amyloliquefaciens alpha-amylase promoter in Bacillus subtilis. The production yields were estimated to be more than 2 g of enzyme per liter of medium, about 250 times the production of the original strains, in a simple shake flask. About 60% of maltose was converted into trehalose by the simultaneous action of both enzymes produced in B. subtilis.

  8. Priapism and glucose-6-phosphate dehydrogenase deficiency: An underestimated correlation?

    PubMed

    De Rose, Aldo Franco; Mantica, Guglielmo; Tosi, Mattia; Bovio, Giulio; Terrone, Carlo

    2016-10-05

    Priapism is a rare clinical condition characterized by a persistent erection unrelated to sexual excitement. Often the etiology is idiopathic. Three cases of priapism in glucose-6-phosphate dehydrogenase (G6PD) deficiency patients have been described in literature. We present the case of a 39-year-old man with glucose- 6-phosphate dehydrogenase deficiency, who reached out to our department for the arising of a non-ischemic priapism without arteriolacunar fistula. We suggest that the glucose-6-phosphate dehydrogenase deficiency could be an underestimated risk factor for priapism.

  9. Protein engineering reveals ancient adaptive replacements in isocitrate dehydrogenase

    PubMed Central

    Dean, Antony M.; Golding, G. Brian

    1997-01-01

    Evolutionary analysis indicates that eubacterial NADP-dependent isocitrate dehydrogenases (EC 1.1.1.42) first evolved from an NAD-dependent precursor about 3.5 billion years ago. Selection in favor of utilizing NADP was probably a result of niche expansion during growth on acetate, where isocitrate dehydrogenase provides 90% of the NADPH necessary for biosynthesis. Amino acids responsible for differing coenzyme specificities were identified from x-ray crystallographic structures of Escherichia coli isocitrate dehydrogenase and the distantly related Thermus thermophilus NAD-dependent isopropylmalate dehydrogenase. Site-directed mutagenesis at sites lining the coenzyme binding pockets has been used to invert the coenzyme specificities of both enzymes. Reconstructed ancestral sequences indicate that these replacements are ancestral. Hence the adaptive history of molecular evolution is amenable to experimental investigation. PMID:9096353

  10. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of lactate dehydrogenase isoenzymes are used in the diagnosis and treatment of liver diseases, such as viral hepatitis,...

  11. 21 CFR 862.1500 - Malic dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... is a device that is intended to measure the activity of the enzyme malic dehydrogenase in serum and... diseases, myocardial infarctions, cancer, and blood disorders such as myelogenous (produced in the...

  12. 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...

  13. 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...

  14. 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...

  15. 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...

  16. 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...

  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 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 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...

  19. 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...

  20. 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...

  1. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 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. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 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...

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

    PubMed

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

    2001-05-01

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

  5. Carbon Monoxide Dehydrogenase Activity in Bradyrhizobium japonicum

    PubMed Central

    Lorite, María J.; Tachil, Jörg; Sanjuán, Juán; Meyer, Ortwin; Bedmar, Eulogio J.

    2000-01-01

    Bradyrhizobium japonicum strain 110spc4 was capable of chemolithoautotrophic growth with carbon monoxide (CO) as a sole energy and carbon source under aerobic conditions. The enzyme carbon monoxide dehydrogenase (CODH; EC 1.2.99.2) has been purified 21-fold, with a yield of 16% and a specific activity of 58 nmol of CO oxidized/min/mg of protein, by a procedure that involved differential ultracentrifugation, anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The purified enzyme gave a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis and had a molecular mass of 230,000 Da. The 230-kDa enzyme was composed of large (L; 75-kDa), medium (M; 28.4-kDa), and small (S; 17.2-kDa) subunits occurring in heterohexameric (LMS)2 subunit composition. The 75-kDa polypeptide exhibited immunological cross-reactivity with the large subunit of the CODH of Oligotropha carboxidovorans. The B. japonicum enzyme contained, per mole, 2.29 atoms of Mo, 7.96 atoms of Fe, 7.60 atoms of labile S, and 1.99 mol of flavin. Treatment of the enzyme with iodoacetamide yielded di(carboxamidomethyl)molybdopterin cytosine dinucleotide, identifying molybdopterin cytosine dinucleotide as the organic portion of the B. japonicum CODH molybdenum cofactor. The absorption spectrum of the purified enzyme was characteristic of a molybdenum-containing iron-sulfur flavoprotein. PMID:10788353

  6. Carbon monoxide dehydrogenase activity in Bradyrhizobium japonicum.

    PubMed

    Lorite, M J; Tachil, J; Sanjuán, J; Meyer, O; Bedmar, E J

    2000-05-01

    Bradyrhizobium japonicum strain 110spc4 was capable of chemolithoautotrophic growth with carbon monoxide (CO) as a sole energy and carbon source under aerobic conditions. The enzyme carbon monoxide dehydrogenase (CODH; EC 1.2.99.2) has been purified 21-fold, with a yield of 16% and a specific activity of 58 nmol of CO oxidized/min/mg of protein, by a procedure that involved differential ultracentrifugation, anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The purified enzyme gave a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis and had a molecular mass of 230,000 Da. The 230-kDa enzyme was composed of large (L; 75-kDa), medium (M; 28.4-kDa), and small (S; 17.2-kDa) subunits occurring in heterohexameric (LMS)(2) subunit composition. The 75-kDa polypeptide exhibited immunological cross-reactivity with the large subunit of the CODH of Oligotropha carboxidovorans. The B. japonicum enzyme contained, per mole, 2.29 atoms of Mo, 7.96 atoms of Fe, 7.60 atoms of labile S, and 1.99 mol of flavin. Treatment of the enzyme with iodoacetamide yielded di(carboxamidomethyl)molybdopterin cytosine dinucleotide, identifying molybdopterin cytosine dinucleotide as the organic portion of the B. japonicum CODH molybdenum cofactor. The absorption spectrum of the purified enzyme was characteristic of a molybdenum-containing iron-sulfur flavoprotein.

  7. 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

  8. 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.

  9. Herbicidal Activity of an Isopropylmalate Dehydrogenase Inhibitor.

    PubMed Central

    Wittenbach, V. A.; Teaney, P. W.; Hanna, W. S.; Rayner, D. R.; Schloss, J. V.

    1994-01-01

    Isopropylmalate dehydrogenase (IPMDH) is the third enzyme specific to leucine biosynthesis. It catalyzes the oxidative decarboxylation of 3-isopropylmalate (3-IPM) to 2-ketoisocaproic acid. The partially purified enzyme from pea (Pisum sativum L.) shows a broad pH optimum of 7.8 to 9.1 and has Km values for 3-IPM and NAD of 18 and 40 [mu]M, respectively. O-Isobutenyl oxalylhydroxamate (O-IbOHA) has been discovered to be an excellent inhibitor of the pea IPMDH, with an apparent inhibitor constant of 5 nM. As an herbicide, O-IbOHA showed only moderate activity on a variety of broadleaf and grass species. We characterized the herbicidal activity of O-IbOHA on corn (Zea mays L.), a sensitive species; giant foxtail (Setaria faberi) and morning glory (Ipomoea purpurea [L.] Roth), moderately tolerant species; and soybean [Glycine max L. Merr.), a tolerant species. Differences in tolerance among the species were not due to differences in the sensitivity of IPMDH. Studies with [14C]O-IbOHA suggested that uptake and translocation were not major limitations for herbicidal activity, nor were they determinants of tolerance. Moreover, metabolism could not account for the difference in tolerance of corn, foxtail, and morning glory, although it might account for the tolerance of soybean. Herbicidal activity on all four species was correlated with the accumulation of 3-IPM in the plants. PMID:12232331

  10. Iodination of glyceraldehyde 3-phosphate dehydrogenase

    PubMed Central

    Thomas, Jean O.; Harris, J. Ieuan

    1970-01-01

    1. A high degree of homology in the positions of tyrosine residues in glyceraldehyde 3-phosphate dehydrogenase from lobster and pig muscle, and from yeast, prompted an examination of the reactivity of tyrosine residues in the enzyme. 2. Iodination of the enzyme from lobster muscle with low concentrations of potassium tri-[125I]-iodide led to the identification of tyrosine residues of differing reactivity. Tyrosine-46 appeared to be the most reactive in the native enzyme. 3. When the monocarboxymethylated enzyme was briefly treated with small amounts of iodine, iodination could be confined almost entirely to tyrosine-46 in the lobster enzyme; tyrosine-39 or tyrosine-42, or both, were also beginning to react. 4. These three tyrosine residues were also those that reacted most readily in the carboxymethylated pig and yeast enzymes. 5. The difficulties in attaining specific reaction of the native enzyme are considered. 6. The differences between our results and those of other workers are discussed. ImagesPLATE 1PLATE 2 PMID:5530750

  11. Human liver aldehyde dehydrogenase: coenzyme binding

    SciTech Connect

    Kosley, L.L.; Pietruszko, R.

    1987-05-01

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

  12. 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

  13. Kinetic and mechanistic studies of methylated liver alcohol dehydrogenase.

    PubMed Central

    Tsai, C S

    1978-01-01

    Reductive methylation of lysine residues activates liver alcohol dehydrogenase in the oxidation of primary alcohols, but decreases the activity of the enzyme towards secondary alcohols. The modification also desensitizes the dehydrogenase to substrate inhibition at high alcohol concentrations. Steady-state kinetic studies of methylated liver alcohol dehydrogenase over a wide range of alcohol concentrations suggest that alcohol oxidation proceeds via a random addition of coenzyme and substrate with a pathway for the formation of the productive enzyme-NADH-alcohol complex. To facilitate the analyses of the effects of methylation on liver alcohol dehydrogenase and factors affecting them, new operational kinetic parameters to describe the results at high substrate concentration were introduced. The changes in the dehydrogenase activity on alkylation were found to be associated with changes in the maximum velocities that are affected by the hydrophobicity of alkyl groups introduced at lysine residues. The desensitization of alkylated liver alcohol dehydrogenase to substrate inhibition is identified with a decrease in inhibitory Michaelis constants for alcohols and this is favoured by the steric effects of substituents at the lysine residues. PMID:697732

  14. Functional characterization of the phosphorylating D-glyceraldehyde 3-phosphate dehydrogenase from the archaeon Methanothermus fervidus by comparative molecular modelling and site-directed mutagenesis.

    PubMed

    Talfournier, F; Colloc'h, N; Mornon, J P; Branlant, G

    1999-10-01

    Phosphorylating archaeal D-glyceraldehyde 3-phosphate dehydrogenases (GraP-DHs) share only 15-20% identity with their glycolytic bacterial and eukaryotic counterparts. Unlike the latter which are NAD-specific, archaeal GraP-DHs exhibit a dual-cofactor specificity with a marked preference for NADP. In the present study, we have constructed a three-dimensional model of the Methanothermus fervidus GraP-DH based upon the X-ray structures of the Bacillus stearothermophilus and Escherichia coli GraP-DHs. The overall structure of the archaeal enzyme is globally similar to homology modelling-derived structures, in particular for the cofactor binding domain, which might adopt a classical Rossmann fold. M. fervidus GraP-DH can be considered as a dimer of dimers which exhibits negative and positive cooperativity in binding the coenzymes NAD and NADP, respectively. As expected, the differences between the model and the templates are located mainly within the loops. Based on the predictions derived from molecular modelling, site-directed mutagenesis was performed to characterize better the cofactor binding pocket and the catalytic domain. The Lys32Ala, Lys32Glu and Lys32Asp mutants led to a drastic increase in the Km value for NADP (i.e. 165-, 500- and 1000-fold, respectively), thus demonstrating that the invariant Lys32 residue is one of the most important determinants favouring the adenosine 2'-PO42- binding of NADP. The involvement of the side chain of Asn281, which was postulated to play a role equivalent to that of the Asn313 of bacterial and eukaryotic GraP-DHs in fixing the position of the nicotinamide ring in a syn orientation [Fabry, S. & Hensel, R. (1988) Gene 64, 189-197], was ruled out. Most of the amino acids involved in catalysis and in substrate recognition in bacterial and eukaryotic GraP-DHs are not conserved in the archaeal enzyme except for the essential Cys149. Inspection of our model suggests that side chains of invariant residues Asn150, Arg176, Arg177 and

  15. Effect of fermented sea tangle on the alcohol dehydrogenase and acetaldehyde dehydrogenase in Saccharomyces cerevisiae.

    PubMed

    Cha, Jae-Young; Jeong, Jae-Jun; Yang, Hyun-Ju; Lee, Bae-Jin; Cho, Young-Su

    2011-08-01

    Sea tangle, a kind of brown seaweed, was fermented with Lactobacillus brevis BJ-20. The gamma-aminobutyric acid (GABA) content in fermented sea tangle (FST) was 5.56% (w/w) and GABA in total free amino acid of FST was 49.5%. The effect of FST on the enzyme activities and mRNA protein expression of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) involved in alcohol metabolism in Saccharomyces cerevisiae was investigated. Yeast was cultured in YPD medium supplemented with different concentrations of FST powder [0, 0.4, 0.8, and 1.0% (w/v)] for 18 h. FST had no cytotoxic effect on the yeast growth. The highest activities and protein expressions of ADH and ALDH from the cell-free extracts of S. cerevisiae were evident with the 0.4% and 0.8% (w/v) FST-supplemented concentrations, respectively. The highest concentrations of GABA as well as minerals (Zn, Ca, and Mg) were found in the cell-free extracts of S. cerevisiae cultured in medium supplemented with 0.4% (w/v) FST. The levels of GABA, Zn, Ca, and Mg in S. cerevisiae were strongly correlated with the enzyme activities of ADH and ALDH in yeast. These results indicate that FST can enhance the enzyme activities and protein expression of ADH and ALDH in S. cerevisiae.

  16. The Carbon Monoxide Dehydrogenase from Desulfovibrio vulgaris.

    PubMed

    Hadj-Saïd, Jessica; Pandelia, Maria-Eirini; Léger, Christophe; Fourmond, Vincent; Dementin, Sébastien

    2015-12-01

    Ni-containing Carbon Monoxide Dehydrogenases (CODHs) catalyze the reversible conversion between CO and CO₂and are involved in energy conservation and carbon fixation. These homodimeric enzymes house two NiFeS active sites (C-clusters) and three accessory [4Fe-4S] clusters. The Desulfovibrio vulgaris (Dv) genome contains a two-gene CODH operon coding for a CODH (cooS) and a maturation protein (cooC) involved in nickel insertion in the active site. According to the literature, the question of the precise function of CooC as a chaperone folding the C-cluster in a form which accommodates free nickel or as a mere nickel donor is not resolved. Here, we report the biochemical and spectroscopic characterization of two recombinant forms of the CODH, produced in the absence and in the presence of CooC, designated CooS and CooS(C), respectively. CooS contains no nickel and cannot be activated, supporting the idea that the role of CooC is to fold the C-cluster so that it can bind nickel. As expected, CooS(C) is Ni-loaded, reversibly converts CO and CO₂, displays the typical Cred1 and Cred2 EPR signatures of the C-cluster and activates in the presence of methyl viologen and CO in an autocatalytic process. However, Ni-loaded CooS(C) reaches maximum activity only upon reductive treatment in the presence of exogenous nickel, a phenomenon that had not been observed before. Surprisingly, the enzyme displays the Cred1 and Cred2 signatures whether it has been activated or not, showing that this activation process of the Ni-loaded Dv CODH is not associated with structural changes at the active site.

  17. Isocitrate dehydrogenase mutations in myeloid malignancies

    PubMed Central

    Medeiros, B C; Fathi, A T; DiNardo, C D; Pollyea, D A; Chan, S M; Swords, R

    2017-01-01

    Alterations to genes involved in cellular metabolism and epigenetic regulation are implicated in the pathogenesis of myeloid malignancies. Recurring mutations in isocitrate dehydrogenase (IDH) genes are detected in approximately 20% of adult patients with acute myeloid leukemia (AML) and 5% of adults with myelodysplastic syndromes (MDS). IDH proteins are homodimeric enzymes involved in diverse cellular processes, including adaptation to hypoxia, histone demethylation and DNA modification. The IDH2 protein is localized in the mitochondria and is a critical component of the tricarboxylic acid (also called the ‘citric acid' or Krebs) cycle. Both IDH2 and IDH1 (localized in the cytoplasm) proteins catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). Mutant IDH enzymes have neomorphic activity and catalyze reduction of α-KG to the (R) enantiomer of 2-hydroxyglutarate, which is associated with DNA and histone hypermethylation, altered gene expression and blocked differentiation of hematopoietic progenitor cells. The prognostic significance of mutant IDH (mIDH) is controversial but appears to be influenced by co-mutational status and the specific location of the mutation (IDH1-R132, IDH2-R140, IDH2-R172). Treatments specifically or indirectly targeted to mIDH are currently under clinical investigation; these therapies have been generally well tolerated and, when used as single agents, have shown promise for inducing responses in some mIDH patients when used as first-line treatment or in relapsed or refractory AML or MDS. Use of mIDH inhibitors in combination with drugs with non-overlapping mechanisms of action is especially promising, as such regimens may address the clonal heterogeneity and the multifactorial pathogenic processes involved in mIDH myeloid malignancies. Advances in mutational analysis have made testing more rapid and convenient, and less expensive; such testing should become part of routine diagnostic workup and repeated at

  18. Succinate Dehydrogenase Gene Mutations in Cardiac Paragangliomas

    PubMed Central

    Martucci, Victoria L.; Emaminia, Abbas; del Rivero, Jaydira; Lechan, Ronald M.; Magoon, Bindiya T.; Galia, Analyza; Fojo, Tito; Leung, Steve; Lorusso, Roberto; Jimenez, Camilo; Shulkin, Barry L.; Audibert, Jennifer L.; Adams, Karen T.; Rosing, Douglas R.; Vaidya, Anand; Dluhy, Robert G.; Horvath, Keith A.; Pacak, Karel

    2015-01-01

    Pheochromocytomas and paragangliomas are chromaffin cell tumors arising from neuroendocrine cells. At least one third of paragangliomas are related to germline mutations in one of 17 genes. While these tumors can occur throughout the body, cardiac paragangliomas are very rare, accounting for less than 0.3% of mediastinal tumors. The purpose of this study was to determine the clinical characteristics of patients with cardiac paragangliomas, particularly focusing on their genetic backgrounds. A retrospective chart analysis of fifteen patients with cardiac paraganglioma was performed to determine clinical presentation, genetic background, diagnostic work-up, and outcomes. The average age at diagnosis was 41.9 years. Typical symptoms of paraganglioma (e.g., hypertension, sweating, palpitations, headache) were reported at initial presentation in 13 patients (86.7%); the remaining 2, as well as 4 symptomatic patients, initially presented with cardiac-specific symptoms (e.g., chest pain, dyspnea). Genetic testing was done in 13 cases (86.7%); 10 (76.9%) were positive for mutations in succinate dehydrogenase (SDHx) subunits B, C, or D. Thirteen cases (86.7%) underwent surgery to remove the paraganglioma with no intraoperative morbidity or mortality; one additional patient underwent surgical resection but experienced intraoperative complications after removal of the tumor due to comorbities and did not survive. SDHx mutations are known to be associated with mediastinal locations and malignant behavior of paragangliomas. In this report, we extend the locations of predominantly SDHx-related paragangliomas to cardiac tumors. In conclusion, cardiac paragangliomas are frequently associated with underlying SDHx germline mutations, suggesting a need for genetic testing of all patients with this rare tumor. PMID:25896150

  19. 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.

  20. 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

  1. Properties and subunit structure of pig heart pyruvate dehydrogenase.

    PubMed

    Hamada, M; Hiraoka, T; Koike, K; Ogasahara, K; Kanzaki, T

    1976-06-01

    Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by sedimentation equilibrium methods. The enzyme was dissociated into two subunits (alpha and beta), with estimated molecular weights of 41,000 (alpha) and 36,000 (beta), respectively, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The subunits were separated by phosphocellulose column chromatography and their chemical properties were examined. The subunit structure of the pyruvate dehydrogenase was assigned as alpha2beta2. The content of right-handed alpha-helix in the enzyme molecule was estimated to be about 29 and 28% by optical rotatory dispersion and by circular dichroism, respectively. The enzyme contained no thiamine-PP, and its dehydrogenase activity was completely dependent on added thiamine-PP and partially dependent on added Mg2+ and Ca2+. The Km value of pyruvate dehydrogenase for thiamine diphosphate was estimated to be 6.5 X 10(-5) M in the presence of Mg2+ or Ca2+. The enzyme showed highly specific activity for thiamine-PP dependent oxidation of both pyruvate and alpha-ketobutyrate, but it also showed some activity with alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketoisovalerate. The pyruvate dehydrogenase activity was strongly inhibited by bivalent heavy metal ions and by sulfhydryl inhibitors; and the enzyme molecule contained 27 moles of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive sulfhydryl groups and a total of 36 moles of sulfhydryl groups. The inhibitory effect of p-chloromercuribenzoate was prevented by preincubating the enzyme with thiamine-PP plus pyruvate. The structure of pyruvate dehydrogenase necessary for formation of the complex is also reported.

  2. Metabolism of Red Beet Slices I. Effects of Washing 1

    PubMed Central

    Reed, D. J.; Kolattukudy, P. E.

    1966-01-01

    The changes in relative participation of pathways of glucose catabolism in red beet slices during washing have been examined using specifically 14C labeled glucoses. Washing of these slices brings about an increase in participation of the pentose phosphate pathway. The composition of the washing medium influences slightly the extent of change in pathway participation. The activity level of certain enzymes participating in the initial stages of glucose catabolism has been measured in fresh and washed beet slices. Fresh slices which barely metabolized gluconate were found to have very little 6-phosphogluconate dehydrogenase activity. Washing brings about a dramatic increase in 6-phosphogluconate dehydrogenase activity and this increase was accompanied by a marked increase in the ability of the slices to metabolize gluconate. In red beet slices the TPNH generated via pentose phosphate pathway appears to be utilized for biosynthetic reductions rather than as respiratory substrate. PMID:16656302

  3. Direct transfer of NADH between alpha-glycerol phosphate dehydrogenase and lactate dehydrogenase: fact or misinterpretation?

    PubMed

    Srivastava, D K; Smolen, P; Betts, G F; Fukushima, T; Spivey, H O; Bernhard, S A

    1989-09-01

    Following the criticism by Chock and Gutfreund [Chock, P.B. & Gutfreund, H. (1988) Proc. Natl. Acad. Sci. USA 85, 8870-8874], that our proposal of direct transfer of NADH between glycerol-3-phosphate dehydrogenase (alpha-glycerol phosphate dehydrogenase, alpha-GDH; EC 1.1.1.8) and L-lactate dehydrogenase (LDH; EC 1.1.1.27) was based on a misinterpretation of the kinetic data, we have reinvestigated the transfer mechanism between this enzyme pair. By using the "enzyme buffering" steady-state kinetic technique [Srivastava, D.K. & Bernhard, S.A. (1984) Biochemistry 23, 4538-4545], we examined the mechanism (random diffusion vs. direct transfer) of transfer of NADH between rabbit muscle alpha-GDH and pig heart LDH. The steady-state data reveal that the LDH-NADH complex and the alpha-GDH-NADH complex can serve as substrate for the alpha-GDH-catalyzed reaction and the LDH-catalyzed reaction, respectively. This is consistent with the direct-transfer mechanism and inconsistent with a mechanism in which free NADH is the only competent substrate for either enzyme-catalyzed reaction. The discrepancy between this conclusion and that of Chock and Gutfreund comes from (i) their incorrect measurement of the Km for NADH in the alpha-GDH-catalyzed reaction, (ii) inadequate design and range of the steady-state kinetic experiments, and (iii) their qualitative assessment of the prediction of the direct-transfer mechanism. Our transient kinetic measurements for the transfer of NADH from alpha-GDH to LDH and from LDH to alpha-GDH show that both are slower than predicted on the basis of free equilibration of NADH through the aqueous environment. The decrease in the rate of equilibration of NADH between alpha-GDH and LDH provides no support for the random-diffusion mechanism; rather, it suggests a direct interaction between enzymes that modulates the transfer rate of NADH. Thus, contrary to Chock and Gutfreund's conclusion, all our experimental data compel us to propose, once again, that

  4. 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

  5. Purification and characterization of limonoate dehydrogenase from Rhodococcus fascians.

    PubMed

    Humanes, L; López-Ruiz, A; Merino, M T; Roldán, J M; Diez, J

    1997-09-01

    Limonoate dehydrogenase from Rhodococcus fascians has been purified to electrophoretic homogeneity by a procedure that consists of ion-exchange, hydrophobic, and affinity chromatography. The native enzyme has a molecular mass of around 128,000 Da and appears to be composed of four similar subunits (30,000 Da each). The isoelectric point is 4.9 as determined by isoelectric focusing. The homogeneous enzyme was used to determine the NH2-terminal amino acid sequence. The enzyme was purified from cells grown in either fructose or limonoate as a carbon source. Limonoate dehydrogenase activity was higher in limonoate-grown cultures. Additionally, the enzyme preparations differed in their affinity for limonoids but not for NAD+. In all cases limonoate dehydrogenase exhibited a higher catalytic rate and stronger affinity for limonoate A-ring lactone than for disodium limonoate, the limonoid traditionally used for in vitro activity assays. Our data confirm previous reports proposing that limonoate A-ring lactone is the physiological substrate for limonoate dehydrogenase. The increase in limonoate dehydrogenase activity observed in limonoate-grown cultures appears to be caused by a rise in protein levels, since chloramphenicol prevented such an effect.

  6. 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.

  7. Radial immunodiffusion and immunoelectrophoresis compared for identifying autoantibodies to lactate dehydrogenase in human serum.

    PubMed

    Harff, G A; Backer, E T

    1990-12-14

    Variant electrophoretic patterns of lactate dehydrogenase isoenzymes were studied. By radial immunodiffusion and immunoelectrophoresis, immunoglobulin and light chain class of autoantibodies to lactate dehydrogenase were identified in nine sera: seven of these sera demonstrated IgG (5 lambda, 2 kappa) autoantibodies to lactate dehydrogenase, the other two demonstrated IgA (both kappa) autoantibodies to lactate dehydrogenase, the other two demonstrated IgA (both kappa) autoantibodies to lactate dehydrogenase. We conclude that radial immunodiffusion and immunoelectrophoresis are equally effective for identifying auto-antibodies to lactate dehydrogenase in serum. Radial immunodiffusion, however, is easier to perform than immunoelectrophoresis.

  8. 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

  9. The Pyruvate Dehydrogenase Complexes: Structure-based Function and Regulation*

    PubMed Central

    Patel, Mulchand S.; Nemeria, Natalia S.; Furey, William; Jordan, Frank

    2014-01-01

    The pyruvate dehydrogenase complexes (PDCs) from all known living organisms comprise three principal catalytic components for their mission: E1 and E2 generate acetyl-coenzyme A, whereas the FAD/NAD+-dependent E3 performs redox recycling. Here we compare bacterial (Escherichia coli) and human PDCs, as they represent the two major classes of the superfamily of 2-oxo acid dehydrogenase complexes with different assembly of, and interactions among components. The human PDC is subject to inactivation at E1 by serine phosphorylation by four kinases, an inactivation reversed by the action of two phosphatases. Progress in our understanding of these complexes important in metabolism is reviewed. PMID:24798336

  10. Purification of xanthine dehydrogenase and sulfite oxidase from chicken liver.

    PubMed

    Ratnam, K; Brody, M S; Hille, R

    1996-05-01

    Xanthine dehydrogenase and sulfite oxidase from chicken liver are oxomolybdenum enzymes which catalyze the oxidation of xanthine to uric acid and sulfite to sulfate, respectively. Independent purification protocols have been previously described for both enzymes. Here we describe a procedure by which xanthine dehydrogenase and sulfite oxidase are purified simultaneously from the same batch of fresh chicken liver. Also, unlike the protocols described earlier, this procedure avoids the use of acetone extraction as well as a heat step, thus minimizing damage to the molybdenum centers of the enzymes.

  11. The α-ketoglutarate dehydrogenase complex in cancer metabolic plasticity.

    PubMed

    Vatrinet, Renaud; Leone, Giulia; De Luise, Monica; Girolimetti, Giulia; Vidone, Michele; Gasparre, Giuseppe; Porcelli, Anna Maria

    2017-01-01

    Deregulated metabolism is a well-established hallmark of cancer. At the hub of various metabolic pathways deeply integrated within mitochondrial functions, the α-ketoglutarate dehydrogenase complex represents a major modulator of electron transport chain activity and tricarboxylic acid cycle (TCA) flux, and is a pivotal enzyme in the metabolic reprogramming following a cancer cell's change in bioenergetic requirements. By contributing to the control of α-ketoglutarate levels, dynamics, and oxidation state, the α-ketoglutarate dehydrogenase is also essential in modulating the epigenetic landscape of cancer cells. In this review, we will discuss the manifold roles that this TCA enzyme and its substrate play in cancer.

  12. Differential energetic metabolism during Trypanosoma cruzi differentiation. I. Citrate synthase, NADP-isocitrate dehydrogenase, and succinate dehydrogenase.

    PubMed

    Adroher, F J; Osuna, A; Lupiañez, J A

    1988-11-15

    The activities of the mitochondrial enzymes citrate synthase (citrate oxaloacetatelyase, EC 4.1.3.7), NADP-linked isocitrate dehydrogenase (threo-Ds-isocitrate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42), and succinate dehydrogenase (succinate: FAD oxidoreductase, EC 1.3.99.1) as well as their kinetic behavior in the two developmental forms of Trypanosoma cruzi at insect vector stage, epimastigotes and infective metacyclic trypomastigotes, were studied. The results presented in this work clearly demonstrate a higher mitochondrial metabolism in the metacyclic forms as is shown by the extraordinary enhanced activities of metacyclic citrate synthase, isocitrate dehydrogenase, and succinate dehydrogenase. In epimastigotes, the specific activities of citrate synthase at variable concentrations of oxalacetate and acetyl-CoA were 24.6 and 26.6 mU/mg of protein, respectively, and the Michaelis constants were 7.88 and 6.84 microM for both substrates. The metacyclic enzyme exhibited the following kinetic parameters: a specific activity of 228.4 mU/mg and Km of 3.18 microM for oxalacetate and 248.5 mU/mg and 2.75 microM, respectively, for acetyl-CoA. NADP-linked isocitrate dehydrogenase specific activities for epimastigotes and metacyclics were 110.2 and 210.3 mU/mg, whereas the apparent Km's were 47.9 and 12.5 microM, respectively. No activity for the NAD-dependent isozyme was found in any form of T. cruzi differentiation. The particulated succinate dehydrogenase showed specific activities of 8.2 and 39.1 mU/mg for epimastigotes and metacyclic trypomastigotes, respectively, although no significant changes in the Km (0.46 and 0.48 mM) were found. The cellular role and the molecular mechanism that probably take place during this significant shift in the mitochondrial metabolism during the T. cruzi differentiation have been discussed.

  13. Phanerochaete chrysosporium Cellobiohydrolase and Cellobiose Dehydrogenase Transcripts in Wood

    PubMed Central

    Vallim, Marcelo A.; Janse, Bernard J. H.; Gaskell, Jill; Pizzirani-Kleiner, Aline A.; Cullen, Daniel

    1998-01-01

    The transcripts of structurally related cellobiohydrolase genes in Phanerochaete chrysosporium-colonized wood chips were quantified. The transcript patterns obtained were dramatically different from the transcript patterns obtained previously in defined media. Cellobiose dehydrogenase transcripts were also detected, which is consistent with the hypothesis that such transcripts play an important role in cellulose degradation. PMID:9572973

  14. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 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...

  15. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 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...

  16. 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 ...

  17. Genetics Home Reference: 2-methylbutyryl-CoA dehydrogenase deficiency

    MedlinePlus

    ... down proteins from food into smaller parts called amino acids. Amino acids can be further processed to provide energy for ... methylbutyryl-CoA dehydrogenase deficiency cannot process a particular amino acid called isoleucine. Most cases of 2-methylbutyryl-CoA ...

  18. Distribution of the Pyruvate Dehydrogenase Complex in Developing Soybean Cotyledons

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The somewhat surprising report that storage proteins and oil are non-uniformly distributed in the cotyledons of developing soybeans prompted us to determine the spatial distribution of the mitochondrial and plastidial forms of the pyruvate dehydrogenase complex (PDC). It has been proposed that pla...

  19. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... test system is a device intended to measure the activity of the enzyme isocitric dehydrogenase in serum... disease such as viral hepatitis, cirrhosis, or acute inflammation of the biliary tract; pulmonary disease...), and diseases associated with pregnancy. (b) Classification. Class I (general controls). The device...

  20. Efficiency of superoxide anions in the inactivation of selected dehydrogenases

    NASA Astrophysics Data System (ADS)

    Rodacka, Aleksandra; Serafin, Eligiusz; Puchala, Mieczyslaw

    2010-09-01

    The most ubiquitous of the primary reactive oxygen species, formed in all aerobes, is the superoxide free radical. It is believed that the superoxide anion radical shows low reactivity and in oxidative stress it is regarded mainly as an initiator of more reactive species such as rad OH and ONOO -. In this paper, the effectiveness of inactivation of selected enzymes by radiation-generated superoxide radicals in comparison with the effectiveness of the other products of water radiolysis is examined. We investigate three enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH). We show that the direct contribution of the superoxide anion radical to GAPDH and ADH inactivation is significant. The effectiveness of the superoxide anion in the inactivation of GAPDH and ADG was only 2.4 and 2.8 times smaller, respectively, in comparison with hydroxyl radical. LDH was practically not inactivated by the superoxide anion. Despite the fact that the studied dehydrogenases belong to the same class of enzymes (oxidoreductases), all have a similar molecular weight and are tetramers, their susceptibility to free-radical damage varies. The differences in the radiosensitivity of the enzymes are not determined by the basic structural parameters analyzed. A significant role in inactivation susceptibility is played by the type of amino acid residues and their localization within enzyme molecules.

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

    PubMed

    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.

  2. NADP+-Preferring d-Lactate Dehydrogenase from Sporolactobacillus inulinus

    PubMed Central

    Zhu, Lingfeng; Xu, Xiaoling; Wang, Limin; Ma, Yanhe

    2015-01-01

    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 Asn174 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. PMID:26150461

  3. 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...

  4. 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...

  5. 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...

  6. 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...

  7. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... hemolytic anemia associated with a glucose-6-phosphate dehydrogenase deficiency. This generic device... 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...

  8. 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.

  9. 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.

  10. 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

  11. Inducible UDP-glucose dehydrogenase from French bean (Phaseolus vulgaris L.) locates to vascular tissue and has alcohol dehydrogenase activity.

    PubMed

    Robertson, D; Smith, C; Bolwell, G P

    1996-01-01

    UDP-glucose dehydrogenase is responsible for channelling UDP-glucose into the pool of UDP-sugars utilized in the synthesis of wall matrix polysaccharides and glycoproteins. It has been purified to homogeneity from suspension-cultured cells of French bean by a combination of hydrophobic-interaction chromatography, gel filtration and dye-ligand chromatography. The enzyme had a subunit of Mr 40,000. Km values were measured for UDP-glucose as 5.5 +/- 1.4 mM and for NAD+ as 20 +/- 3 microM. It was subject to inhibition by UDP-xylose. UDP-glucose dehydrogenase activity co-purified with alcohol dehydrogenase activity from suspension-cultured cells, elicitor-treated cells and elongating hypocotyls, even when many additional chromatographic steps were employed subsequently. The protein from each source was resolved into virtually identical patterns of isoforms on two-dimensional isoelectric focusing/PAGE. However, a combination of peptide mapping and sequence analysis, gel analysis using activity staining and kinetic analysis suggests that both activities are a function of the same protein. An antibody was raised and used to immunolocalize UDP-glucose dehydrogenase to developing xylem and phloem of French bean hypocotyl. Together with data published previously, these results are consistent with an important role in the regulation of carbon flux into wall matrix polysaccharides.

  12. 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.

  13. Molecular determinants of the cofactor specificity of ribitol dehydrogenase, a short-chain dehydrogenase/reductase.

    PubMed

    Moon, Hee-Jung; Tiwari, Manish Kumar; Singh, Ranjitha; Kang, Yun Chan; Lee, Jung-Kul

    2012-05-01

    Ribitol dehydrogenase from Zymomonas mobilis (ZmRDH) catalyzes the conversion of ribitol to d-ribulose and concomitantly reduces NAD(P)(+) to NAD(P)H. A systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-directed mutagenesis of the screened residues, was used to study the molecular determinants of the cofactor specificity of ZmRDH. A homologous conserved amino acid, Ser156, in the substrate-binding pocket of the wild-type ZmRDH was identified as an important residue affecting the cofactor specificity of ZmRDH. Further insights into the function of the Ser156 residue were obtained by substituting it with other hydrophobic nonpolar or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered the cofactor specificity of ZmRDH toward NAD(+) (S156D, [k(cat)/K(m)(,NAD)]/[k(cat)/K(m)(,NADP)] = 10.9, where K(m)(,NAD) is the K(m) for NAD(+) and K(m)(,NADP) is the K(m) for NADP(+)). In contrast, the mutants containing positively charged amino acids (His, Lys, or Arg) at position 156 showed a higher efficiency with NADP(+) as the cofactor (S156H, [k(cat)/K(m)(,NAD)]/[k(cat)/K(m)(,NADP)] = 0.11). These data, in addition to those of molecular dynamics and isothermal titration calorimetry studies, suggest that the cofactor specificity of ZmRDH can be modulated by manipulating the amino acid residue at position 156.

  14. Expression of lactate dehydrogenase C correlates with poor prognosis in renal cell carcinoma.

    PubMed

    Hua, Yibo; Liang, Chao; Zhu, Jundong; Miao, Chenkui; Yu, Yajie; Xu, Aimin; Zhang, Jianzhong; Li, Pu; Li, Shuang; Bao, Meiling; Yang, Jie; Qin, Chao; Wang, Zengjun

    2017-03-01

    Lactate dehydrogenase C is an isoenzyme of lactate dehydrogenase and a member of the cancer-testis antigens family. In this study, we aimed to investigate the expression and functional role of lactate dehydrogenase C and its basic mechanisms in renal cell carcinoma. First, a total of 133 cases of renal cell carcinoma samples were analysed in a tissue microarray, and Kaplan-Meier survival curve analyses were performed to investigate the correlation between lactate dehydrogenase C expression and renal cell carcinoma progression. Lactate dehydrogenase C protein levels and messenger RNA levels were significantly upregulated in renal cell carcinoma tissues, and the patients with positive lactate dehydrogenase C expression had a shorter progression-free survival, indicating the oncogenic role of lactate dehydrogenase C in renal cell carcinoma. In addition, further cytological experiments demonstrated that lactate dehydrogenase C could prompt renal cell carcinoma cells to produce lactate, and increase metastatic and invasive potential of renal cell carcinoma cells. Furthermore, lactate dehydrogenase C could induce the epithelial-mesenchymal transition process and matrix metalloproteinase-9 expression. In summary, these findings showed lactate dehydrogenase C was associated with poor prognosis in renal cell carcinoma and played a pivotal role in the migration and invasion of renal cell carcinoma cells. Lactate dehydrogenase C may act as a novel biomarker for renal cell carcinoma progression and a potential therapeutic target for the treatment of renal cell carcinoma.

  15. A Set of Activators and Repressors Control Peripheral Glucose Pathways in Pseudomonas putida To Yield a Common Central Intermediate▿

    PubMed Central

    del Castillo, Teresa; Duque, Estrella; Ramos, Juan L.

    2008-01-01

    Pseudomonas putida KT2440 channels glucose to the central Entner-Doudoroff intermediate 6-phosphogluconate through three convergent pathways. The genes for these convergent pathways are clustered in three independent regions on the host chromosome. A number of monocistronic units and operons coexist within each of these clusters, favoring coexpression of catabolic enzymes and transport systems. Expression of the three pathways is mediated by three transcriptional repressors, HexR, GnuR, and PtxS, and by a positive transcriptional regulator, GltR-2. In this study, we generated mutants in each of the regulators and carried out transcriptional assays using microarrays and transcriptional fusions. These studies revealed that HexR controls the genes that encode glucokinase/glucose 6-phosphate dehydrogenase that yield 6-phosphogluconate; the genes for the Entner-Doudoroff enzymes that yield glyceraldehyde-3-phosphate and pyruvate; and gap-1, which encodes glyceraldehyde-3-phosphate dehydrogenase. GltR-2 is the transcriptional regulator that controls specific porins for the entry of glucose into the periplasmic space, as well as the gtsABCD operon for glucose transport through the inner membrane. GnuR is the repressor of gluconate transport and gluconokinase responsible for the conversion of gluconate into 6-phosphogluconate. PtxS, however, controls the enzymes for oxidation of gluconate to 2-ketogluconate, its transport and metabolism, and a set of genes unrelated to glucose metabolism. PMID:18245293

  16. A specific radiochemical assay for pyrroline-5-carboxylate dehydrogenase.

    PubMed

    Small, C; Jones, M E

    1987-03-01

    Previous studies of pyrroline-5-carboxylate dehydrogenase have been conducted using a spectrophotometric method to monitor substrate-dependent NAD(P)H production. For the assay of the mammalian enzyme, the spectrophotometric assay was found to be unacceptable for kinetic studies as the production of NAD(P)H was nonlinear with time and protein concentration. An assay which measures radiolabeled glutamate production by this enzyme in the presence of NAD+ from radiolabeled pyrroline-5-carboxylate has been developed. Separation of substrate from product is achieved by column chromatography using Dowex 50 cation-exchange resin. The product isolated by this procedure was identified as glutamate. This new assay is linear with time and protein concentration and gives reproducible results. The assay is not influenced by competing enzyme activities, such as glutamate dehydrogenase, in a liver homogenate so that quantitative conversion of pyrroline-5-carboxylate to glutamate is observed.

  17. Regional development of glutamate dehydrogenase in the rat brain.

    PubMed

    Leong, S F; Clark, J B

    1984-07-01

    The development of glutamate dehydrogenase enzyme activity in rat brain regions has been followed from the late foetal stage to the adult and through to the aged (greater than 2 years) adult. In the adult brain the enzyme activity was greatest in the medulla oblongata and pons greater than midbrain = hypothalamus greater than cerebellum = striatum = cortex. In the aged adult brain, glutamate dehydrogenase activity was significantly lower in the medulla oblongata and pons when compared to the 90-day-old adult value, but not in other regions. The enzyme-specific activity of nonsynaptic (free) mitochondria purified from the medulla oblongata and pons of 90-day-old animals was about twice that of mitochondria purified from the striatum and the cortex. The specific activity of the enzyme in synaptic mitochondria purified from the above three brain regions, however, remained almost constant.

  18. Parasite Lactate Dehydrogenase for Diagnosis of Plasmodium Falciparum. Phase II.

    DTIC Science & Technology

    1997-04-01

    Diagnosis of Plasmodium Falciparum PRINCIPAL INVESTIGATOR: Robert C. Piper, Ph.D. CONTRACTING ORGANIZATION: Flow, Incorporated Portland, Oregon 97201...Phase 11 (24 Mar 95 - 23 Mar 97) 4. TITLE AND SUBTITLE 5. FUNDING NUMBERS Parasite Lactate Dehydrogenase for Diagnosis of Plasmodium Falciparum DAMD...that infected patients become ill. Four species of Plasmodium infect humans. P. falciparum accounts for -85 % of the world’s malaria. P. falciparum is

  19. Reappraisal of the Regulation of Lactococcal l-Lactate Dehydrogenase

    PubMed Central

    van Niel, Ed W. J.; Palmfeldt, Johan; Martin, Rani; Paese, Marco; Hahn-Hägerdal, Bärbel

    2004-01-01

    Lactococcal lactate dehydrogenases (LDHs) are coregulated at the substrate level by at least two mechanisms: the fructose-1,6-biphosphate/phosphate ratio and the NADH/NAD ratio. Among the Lactococcus lactis species, there are strains that are predominantly regulated by the first mechanism (e.g., strain 65.1) or by the second mechanism (e.g., strain NCDO 2118). A more complete model of the kinetics of the regulation of lactococcal LDH is discussed. PMID:15006814

  20. Inhibition of membrane-bound succinate dehydrogenase by fluorescamine.

    PubMed

    Jay, D; Jay, E G; Garcia, C

    1993-12-01

    Fluorescamine rapidly inactivated membrane-bound succinate dehydrogenase. The inhibition of the enzyme by this reagent was prevented by succinate and malonate, suggesting that the group modified by fluorescamine was located at the active site. The modification of the active site sulfhydryl group by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) did not alter the inhibitory action of fluorescamine. However, the protective effect of malonate against fluorescamine inhibition was abolished in the enzyme modified at the thiol.

  1. 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

  2. Recent advances in biotechnological applications of alcohol dehydrogenases.

    PubMed

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

    2017-02-01

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

  3. Purification and characterization of dimeric dihydrodiol dehydrogenase from dog liver.

    PubMed

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

    1994-09-01

    High NADP(+)-linked dihydrodiol dehydrogenase activity was detected in dog liver cytosol, from which a dimeric enzyme composed of M(r) 39,000 subunits was purified to homogeneity. The enzyme oxidized trans-cyclohexanediol, and trans-dihydrodiols of benzene and naphthalene, the [1R,2R]-isomers of which were selectively oxidized. In the reverse reaction in the presence of NADPH as a coenzyme, the enzyme reduced alpha-dicarbonyl compounds, such as methylglyoxal, 3-deoxyglucosone, and diacetyl, and some compounds with a carbonyl group, such as glyceraldehyde, lactaldehyde, and acetoin. 4-Hydroxyphenylketones and ascorbates inhibited the enzyme. The results of steady-state kinetic analyses indicated that the reaction proceeds through an ordered bi bi mechanism with the coenzyme binding to the free enzyme, and suggested that the inhibitors bind to the enzyme-NADP+ binary complex. The dimeric enzyme was detected in liver and kidney of dog, and was immunochemically similar to the dimeric enzymes from monkey kidney, rabbit lens, and pig liver. The sequences (total 127 amino acid residues) of eight peptides derived on enzymatic digestion of the dog liver enzyme did not show significant similarity with the primary structures of members of the aldo-keto reductase and short chain dehydrogenase superfamilies, which include monomeric dihydrodiol dehydrogenases and carbonyl reductase, respectively.

  4. Daidzin: a potent, selective inhibitor of human mitochondrial aldehyde dehydrogenase.

    PubMed

    Keung, W M; Vallee, B L

    1993-02-15

    Human mitochondrial aldehyde dehydrogenase (ALDH-I) is potently, reversibly, and selectively inhibited by an isoflavone isolated from Radix puerariae and identified as daidzin, the 7-glucoside of 4',7-dihydroxyisoflavone. Kinetic analysis with formaldehyde as substrate reveals that daidzin inhibits ALDH-I competitively with respect to formaldehyde with a Ki of 40 nM, and uncompetitively with respect to the coenzyme NAD+. The human cytosolic aldehyde dehydrogenase isozyme (ALDH-II) is nearly 3 orders of magnitude less sensitive to daidzin inhibition. Daidzin does not inhibit human class I, II, or III alcohol dehydrogenases, nor does it have any significant effect on biological systems that are known to be affected by other isoflavones. Among more than 40 structurally related compounds surveyed, 12 inhibit ALDH-I, but only prunetin and 5-hydroxydaidzin (genistin) combine high selectivity and potency, although they are 7- to 15-fold less potent than daidzin. Structure-function relationships have established a basis for the design and synthesis of additional ALDH inhibitors that could both be yet more potent and specific.

  5. 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

  6. Mitochondrial alpha-ketoglutarate dehydrogenase complex generates reactive oxygen species.

    PubMed

    Starkov, Anatoly A; Fiskum, Gary; Chinopoulos, Christos; Lorenzo, Beverly J; Browne, Susan E; Patel, Mulchand S; Beal, M Flint

    2004-09-08

    Mitochondria-produced reactive oxygen species (ROS) are thought to contribute to cell death caused by a multitude of pathological conditions. The molecular sites of mitochondrial ROS production are not well established but are generally thought to be located in complex I and complex III of the electron transport chain. We measured H(2)O(2) production, respiration, and NADPH reduction level in rat brain mitochondria oxidizing a variety of respiratory substrates. Under conditions of maximum respiration induced with either ADP or carbonyl cyanide p-trifluoromethoxyphenylhydrazone,alpha-ketoglutarate supported the highest rate of H(2)O(2) production. In the absence of ADP or in the presence of rotenone, H(2)O(2) production rates correlated with the reduction level of mitochondrial NADPH with various substrates, with the exception of alpha-ketoglutarate. Isolated mitochondrial alpha-ketoglutarate dehydrogenase (KGDHC) and pyruvate dehydrogenase (PDHC) complexes produced superoxide and H(2)O(2). NAD(+) inhibited ROS production by the isolated enzymes and by permeabilized mitochondria. We also measured H(2)O(2) production by brain mitochondria isolated from heterozygous knock-out mice deficient in dihydrolipoyl dehydrogenase (Dld). Although this enzyme is a part of both KGDHC and PDHC, there was greater impairment of KGDHC activity in Dld-deficient mitochondria. These mitochondria also produced significantly less H(2)O(2) than mitochondria isolated from their littermate wild-type mice. The data strongly indicate that KGDHC is a primary site of ROS production in normally functioning mitochondria.

  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. Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase.

    PubMed

    Madiraju, Anila K; Erion, Derek M; Rahimi, Yasmeen; Zhang, Xian-Man; Braddock, Demetrios T; Albright, Ronald A; Prigaro, Brett J; Wood, John L; Bhanot, Sanjay; MacDonald, Michael J; Jurczak, Michael J; Camporez, Joao-Paulo; Lee, Hui-Young; Cline, Gary W; Samuel, Varman T; Kibbey, Richard G; Shulman, Gerald I

    2014-06-26

    Metformin is considered to be one of the most effective therapeutics for treating type 2 diabetes because it specifically reduces hepatic gluconeogenesis without increasing insulin secretion, inducing weight gain or posing a risk of hypoglycaemia. For over half a century, this agent has been prescribed to patients with type 2 diabetes worldwide, yet the underlying mechanism by which metformin inhibits hepatic gluconeogenesis remains unknown. Here we show that metformin non-competitively inhibits the redox shuttle enzyme mitochondrial glycerophosphate dehydrogenase, resulting in an altered hepatocellular redox state, reduced conversion of lactate and glycerol to glucose, and decreased hepatic gluconeogenesis. Acute and chronic low-dose metformin treatment effectively reduced endogenous glucose production, while increasing cytosolic redox and decreasing mitochondrial redox states. Antisense oligonucleotide knockdown of hepatic mitochondrial glycerophosphate dehydrogenase in rats resulted in a phenotype akin to chronic metformin treatment, and abrogated metformin-mediated increases in cytosolic redox state, decreases in plasma glucose concentrations, and inhibition of endogenous glucose production. These findings were replicated in whole-body mitochondrial glycerophosphate dehydrogenase knockout mice. These results have significant implications for understanding the mechanism of metformin's blood glucose lowering effects and provide a new therapeutic target for type 2 diabetes.

  9. Characterization of two β-decarboxylating dehydrogenases from Sulfolobus acidocaldarius.

    PubMed

    Takahashi, Kento; Nakanishi, Fumika; Tomita, Takeo; Akiyama, Nagisa; Lassak, Kerstin; Albers, Sonja-Verena; Kuzuyama, Tomohisa; Nishiyama, Makoto

    2016-11-01

    Sulfolobus acidocaldarius, a hyperthermoacidophilic archaeon, possesses two β-decarboxylating dehydrogenase genes, saci_0600 and saci_2375, in its genome, which suggests that it uses these enzymes for three similar reactions in lysine biosynthesis through 2-aminoadipate, leucine biosynthesis, and the tricarboxylic acid cycle. To elucidate their roles, these two genes were expressed in Escherichia coli in the present study and their gene products were characterized. Saci_0600 recognized 3-isopropylmalate as a substrate, but exhibited slight and no activity for homoisocitrate and isocitrate, respectively. Saci_2375 exhibited distinct and similar activities for isocitrate and homoisocitrate, but no detectable activity for 3-isopropylmalate. These results suggest that Saci_0600 is a 3-isopropylmalate dehydrogenase for leucine biosynthesis and Saci_2375 is a dual function enzyme serving as isocitrate-homoisocitrate dehydrogenase. The crystal structure of Saci_0600 was determined as a closed-form complex that binds 3-isopropylmalate and Mg(2+), thereby revealing the structural basis for the extreme thermostability and novel-type recognition of the 3-isopropyl moiety of the substrate.

  10. 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.

  11. Asp295 stabilizes the active-site loop structure of pyruvate dehydrogenase, facilitating phosphorylation of Ser292 by pyruvate dehydrogenase-kinase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have developed an invitro system for detailed analysis of reversible phosphorylation of the plant mitochondrial pyruvate dehydrogenase complex, comprising recombinant Arabidopsis thaliana a2b2-hetero tetrameric pyruvate dehydrogenase (E1) plus A.thaliana E1-kinase (AtPDK). Upon addition of MgATP...

  12. Levels of Alpha-Glycerophosphate Dehydrogenase, Triosephosphate Isomerase and Lactic Acid Dehydrogenase in Muscles of the Cockroach, ’Periplaneta americana’ L.,

    DTIC Science & Technology

    The level of alpha-glycerophosphate dehydrogenase is slightly higher in leg muscle than in thoracic muscle of the American cockroach, Periplaneta ... americana . Triosephosphate isomerase in leg muscle is about twice that of thoracic muscle. There is little lactic acid dehydrogenase in both muscles. (Author)

  13. Cloning, sequencing and mutagenesis of the genes for aromatic amine dehydrogenase from Alcaligenes faecalis and evolution of amine dehydrogenases.

    PubMed

    Chistoserdov, A Y

    2001-08-01

    The nucleotide sequence of the aromatic amine utilization (aau) gene region from Alcaligenes faecalis contained nine genes (orf-1, aauBEDA, orf-2, orf-3, orf-4 and hemE) transcribed in the same direction. The aauB and aauA genes encode the periplasmic aromatic amine dehydrogenase (AADH) large and small subunit polypeptides, respectively, and were homologous to mauB and mauA, the genes for the large and small subunits of methylamine dehydrogenase (MADH). aauE and aauD are homologous to mauE and mauD and apparently carry out the same function of transport and folding of the small subunit polypeptide in the periplasm. No analogues of the mauF, mauG, mauL, mauM and mauN genes responsible for biosynthesis of tryptophan tryptophylquinone (the prosthetic group of amine dehydrogenases) were found in the aau cluster. orf-2 was predicted to encode a small periplasmic monohaem c-type cytochrome. No biological function can be assigned to polypeptides encoded by orf-1, orf-3 and orf-4 and mutations in these genes appeared to be lethal. Mutants generated by insertions into mauD were not able to use phenylethylamine, tyramine and tryptamine as a source of carbon and phenylethylamine, 3'-hydroxytyramine (dopamine) and tyramine as a source of nitrogen, indicating that AADH is the only enzyme involved in utilization of primary amines in A. faecalis. AADH genes are present in Alcaligenes xylosoxydans subsp. xylosoxydans, but not in other beta- and gamma-proteobacteria. Phylogenetic analysis of amine dehydrogenases (MADH and AADH) indicated that AADH and MADH evolutionarily diverged before separation of proteobacteria into existing subclasses.

  14. Methodological problems in the histochemical demonstration of succinate semialdehyde dehydrogenase activity.

    PubMed

    Bernocchi, G; Barni, S

    1983-12-01

    Methodological aspects of the histochemical technique for the demonstration of succinate semialdehyde dehydrogenase activity (EC 1.2.1.24) (indicative of the degradative step of gamma-aminobutyric acid catabolism) have been analysed in rat Purkinje neurons, where gamma-aminobutyric acid has been shown to be a neurotransmitter, and in hepatocytes, where it is metabolized. During a histochemical incubation for the enzyme, artefacts of succinate dehydrogenase activity and the 'nothing dehydrogenase' reaction are produced. Inhibition of these artefacts by the addition of two inhibitors, malonate and p-hydroxybenzaldehyde, revealed specific reaction products. Formazan granules, which can be ascribed only to specific succinate semialdehyde dehydrogenase activity, are obtained by adding malonate to the incubation medium in order to inhibit both succinate dehydrogenase activity and nothing dehydrogenase. The formation of these granules is completely inhibited by p-hydroxybenzaldehyde, an inhibitor of succinate semialdehyde dehydrogenase activity. Different levels of succinate semialdehyde dehydrogenase activity were noted in Purkinje neurons. This activity was also found in hepatocytes, mostly in the portal area, but with a lesser degree of intensity and specificity. Indeed, non-specific formazan granules were still produced, because of the 'nothing dehydrogenase' reaction, even in the presence of malonate. Thus, a malonate-insensitive 'nothing dehydrogenase' reaction seems to be present in neural and hepatic tissues.

  15. Human dehydrogenase/reductase (SDR family) member 11 is a novel type of 17β-hydroxysteroid dehydrogenase.

    PubMed

    Endo, Satoshi; Miyagi, Namiki; Matsunaga, Toshiyuki; Hara, Akira; Ikari, Akira

    2016-03-25

    We report characterization of a member of the short-chain dehydrogenase/reductase superfamily encoded in a human gene, DHRS11. The recombinant protein (DHRS11) efficiently catalyzed the conversion of the 17-keto group of estrone, 4- and 5-androstenes and 5α-androstanes into their 17β-hydroxyl metabolites with NADPH as a coenzyme. In contrast, it exhibited reductive 3β-hydroxysteroid dehydrogenase activity toward 5β-androstanes, 5β-pregnanes, 4-pregnenes and bile acids. Additionally, DHRS11 reduced α-dicarbonyls (such as diacetyl and methylglyoxal) and alicyclic ketones (such as 1-indanone and loxoprofen). The enzyme activity was inhibited in a mixed-type manner by flavonoids, and competitively by carbenoxolone, glycyrrhetinic acid, zearalenone, curcumin and flufenamic acid. The expression of DHRS11 mRNA was observed widely in human tissues, most abundantly in testis, small intestine, colon, kidney and cancer cell lines. Thus, DHRS11 represents a novel type of 17β-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution.

  16. Structural Insights into the Drosophila melanogaster Retinol Dehydrogenase, a Member of the Short-Chain Dehydrogenase/Reductase Family

    PubMed Central

    Hofmann, Lukas; Tsybovsky, Yaroslav; Alexander, Nathan S.; Babino, Darwin; Leung, Nicole Y.; Montell, Craig; Banerjee, Surajit; von Lintig, Johannes; Palczewski, Krzysztof

    2016-01-01

    The 11-cis-retinylidene chromophore of visual pigments isomerizes upon interaction with a photon, initiating a downstream cascade of signaling events that ultimately lead to visual perception. 11-cis-Retinylidene is regenerated through enzymatic transformations collectively called the visual cycle. The first and rate-limiting enzymatic reaction within this cycle, i.e., the reduction of all-trans-retinal to all-trans-retinol, is catalyzed by retinol dehydrogenases. Here, we determined the structure of Drosophila melanogaster photoreceptor retinol dehydrogenase (PDH) isoform C that belongs to the short-chain dehydrogenase/reductase (SDR) family. This is the first reported structure of a SDR that possesses this biologically important activity. Two crystal structures of the same enzyme grown under different conditions revealed a novel conformational change of the NAD+ cofactor, likely representing a change during catalysis. Amide hydrogen–deuterium exchange of PDH demonstrated changes in the structure of the enzyme upon dinucleotide binding. In D. melanogaster, loss of PDH activity leads to photoreceptor degeneration that can be partially rescued by transgenic expression of human RDH12. Based on the structure of PDH, we analyzed mutations causing Leber congenital amaurosis 13 in a homology model of human RDH12 to obtain insights into the molecular basis of RDH12 disease-causing mutations. PMID:27809489

  17. High substrate specificity of ipsdienol dehydrogenase (IDOLDH), a short-chain dehydrogenase from Ips pini bark beetles.

    PubMed

    Figueroa-Teran, Rubi; Pak, Heidi; Blomquist, Gary J; Tittiger, Claus

    2016-09-01

    Ips spp. bark beetles use ipsdienol, ipsenol, ipsdienone and ipsenone as aggregation pheromone components and pheromone precursors. For Ips pini, the short-chain oxidoreductase ipsdienol dehydrogenase (IDOLDH) converts (-)-ipsdienol to ipsdienone, and thus likely plays a role in determining pheromone composition. In order to further understand the role of IDOLDH in pheromone biosynthesis, we compared IDOLDH to its nearest functionally characterized ortholog with a solved structure: human L-3-hydroxyacyl-CoA dehydrogenase type II/ amyloid-β binding alcohol dehydrogenase (hHADH II/ABAD), and conducted functional assays of recombinant IDOLDH to determine substrate and product ranges and structural characteristics. Although IDOLDH and hHADH II/ABAD had only 35% sequence identity, their predicted tertiary structures had high identity. We found IDOLDH is a functional homo-tetramer. In addition to oxidizing (-)-ipsdienol, IDOLDH readily converted racemic ipsenol to ipsenone, and stereo-specifically reduced both ketones to their corresponding (-)-alcohols. The (+)-enantiomers were never observed as products. Assays with various substrate analogs showed IDOLDH had high substrate specificity for (-)-ipsdienol, ipsenol, ipsenone and ipsdienone, supporting that IDOLDH functions as a pheromone-biosynthetic enzyme. These results suggest that different IDOLDH orthologs and or activity levels contribute to differences in Ips spp. pheromone composition.

  18. 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.

  19. Chirality of the hydrogen transfer to the coenzyme catalyzed by ribitol dehydrogenase from Klebsiella pneumoniae and D-mannitol 1-phosphate dehydrogenase from Escherichia coli.

    PubMed

    Alizade, M A; Gaede, K; Brendel, K

    1976-08-01

    The stereochemistry of the hydrogen transfer to NAD catalyzed by ribitol dehydrogenase (ribitol:NAD 2-oxidoreductase, EC 1.1.1.56) from Klebsiella pneumoniae and D-mannitol-1-phosphate dehydrogenase (D-mannitol-1-phosphate:NAD 2-oxidoreductase, EC 1.1.1.17) from Escherichia coli was investigated. [4-3H]NAD was enzymatically reduced with nonlabelled ribitol in the presence of ribitol dehydrogenase and with nonlabelled D-mannitol 1-phosphate and D-mannitol 1-phosphate dehydrogenase, respectively. In both cases the [4-3H]-NADH produced was isolated and the chirality at the C-4 position determined. It was found that after the transfer of hydride, the label was in both reactions exclusively confined to the (4R) position of the newly formed [4-3H]NADH. In order to explain these results, the hydrogen transferred from the nonlabelled substrates to [4-3H]NAD must have entered the (4S) position of the nicotinamide ring. These data indicate for both investigated inducible dehydrogenases a classification as B or (S) type enzymes. Ribitol also can be dehydrogenated by the constitutive A-type L-iditol dehydrogenase (L-iditol:NAD 5-oxidoreductase, EC 1.1.1.14) from sheep liver. When L-iditol dehydrogenase utilizes ribitol as hydrogen donor, the same A-type classification for this oxidoreductase, as expected, holds true. For the first time, opposite chirality of hydrogen transfer to NAD in one organic reaction--ribitol + NAD = D-ribu + NADH + H--is observed when two different dehydrogenases, the inducible ribitol dehydrogenase from K. pneumoniae and the constitutive L-iditol dehydrogenase from sheep liver, are used as enzymes. This result contradicts the previous generalization that the chirality of hydrogen transfer to the coenzyme for the same reaction is independent of the source of the catalyzing enzyme.

  20. [Class III alcohol dehydrogenase and its role in the human body].

    PubMed

    Jelski, Wojciech; Sani, Tufik Alizade; Szmitkowski, Maciej

    2006-01-01

    Class III alcohol dehydrogenase is composed of two chi subunits, encoded by the ADH5 gene and existing in all tissues examined. It possesses a great ability to metabolize long-chain alcohols, while its capacity to oxidize ethanol is very limited. The amino-acid sequence homology and identical structural and kinetic properties indicate that class III alcohol dehydrogenase and formaldehyde dehydrogenase are identical enzymes. ADH III plays a significant role in the metabolism of formaldehyde in the human body.

  1. [Dihydropirymidine dehydrogenase (DPD)--a toxicity marker for 5-fluorouracil?].

    PubMed

    Jedrzychowska, Adriana; Dołegowska, Barbara

    2013-01-01

    In proceedings relating to patients suffering from cancer, an important step is predicting response and toxicity to treatment. Depending on the type of cancer, physicians use the generally accepted schema of treatment, for example pharmacotherapy. 5-fluorouracil (5-FU) is the most widely used anticancer drug in chemotherapy for colon, breast, and head and neck cancer. Patients with dihydropyrimidine dehydrogenase (DPD) deficiency, which is responsible for the metabolism of 5-FU, may experience severe side effects during treatment, and even death. In many publications the need for determining the activity of DPD is discussed, which would protect the patient from the numerous side effects of treatment. However, in practice these assays are not done routinely, despite the high demand. In most cases, a genetic test is used to detect changes in the gene encoding DPD (such as in the USA), but because of the large number of mutations the genetic test cannot be used as a screening test. Dihydropyrimidine dehydrogenase activity has been shown to have high variability among the general population, with an estimated proportion of at least 3-5% of individuals showing low or deficient DPD activity. In this publication we presents data about average dihydropirymidine dehydrogenase activity in various populations of the world (e.g. Japan, Ghana, Great Britain) including gender differences and collected information about the possibility of determination of DPD activity in different countries. Detection of reduced DPD activity in patients with planned chemotherapy will allow a lower dosage of 5-FU or alternative treatment without exposing them to adverse reactions.

  2. Cancer-associated isocitrate dehydrogenase mutations induce mitochondrial DNA instability.

    PubMed

    Kingsbury, Joanne M; Shamaprasad, Nachiketha; Billmyre, R Blake; Heitman, Joseph; Cardenas, Maria E

    2016-08-15

    A major advance in understanding the progression and prognostic outcome of certain cancers, such as low-grade gliomas, acute myeloid leukaemia, and chondrosarcomas, has been the identification of early-occurring mutations in the NADP(+)-dependent isocitrate dehydrogenase genes IDH1 and IDH2 These mutations result in the production of the onco-metabolite D-2-hydroxyglutarate (2HG), thought to contribute to disease progression. To better understand the mechanisms of 2HG pathophysiology, we introduced the analogous glioma-associated mutations into the NADP(+ )isocitrate dehydrogenase genes (IDP1, IDP2, IDP3) in Saccharomyces cerevisiae Intriguingly, expression of the mitochondrial IDP1(R148H) mutant allele results in high levels of 2HG production as well as extensive mtDNA loss and respiration defects. We find no evidence for a reactive oxygen-mediated mechanism mediating this mtDNA loss. Instead, we show that 2HG production perturbs the iron sensing mechanisms as indicated by upregulation of the Aft1-controlled iron regulon and a concomitant increase in iron levels. Accordingly, iron chelation, or overexpression of a truncated AFT1 allele that dampens transcription of the iron regulon, suppresses the loss of respirative capacity. Additional suppressing factors include overexpression of the mitochondrial aldehyde dehydrogenase gene ALD5 or disruption of the retrograde response transcription factor RTG1 Furthermore, elevated α-ketoglutarate levels also suppress 2HG-mediated respiration loss; consistent with a mechanism by which 2HG contributes to mtDNA loss by acting as a toxic α-ketoglutarate analog. Our findings provide insight into the mechanisms that may contribute to 2HG oncogenicity in glioma and acute myeloid leukaemia progression, with the promise for innovative diagnostic and prognostic strategies and novel therapeutic modalities.

  3. 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

    Burdette, D; Zeikus, J G

    1994-08-15

    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.

  4. 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

  5. Buformin suppresses the expression of glyceraldehyde 3-phosphate dehydrogenase.

    PubMed

    Yano, Akiko; Kubota, Masafumi; Iguchi, Kazuhiro; Usui, Shigeyuki; Hirano, Kazuyuki

    2006-05-01

    The biguanides metformin and buformin, which are clinically used for diabetes mellitus, are known to improve resistance to insulin in patients. Biguanides were reported to cause lactic acidosis as a side effect. Since the mechanism of the side effect still remains obscure, we have examined genes whose expression changes by treating HepG2 cells with buformin in order to elucidate the mechanisms of the side effect. A subtraction cDNA library was constructed by the method of suppressive subtractive hybridization and the screening of the library was performed with cDNA probes prepared from HepG2 cells treated with or without buformin for 12 h. The expression of the gene and the protein obtained by the screening was monitored by real-time RT-PCR with specific primers and Western blotting with specific antibody. The amounts of ATP and NAD+ were determined with luciferase and alcohol dehydrogenase, respectively. We found that expression of the glyceraldehyde 3-phosphate dehydrogenase (GAPD) gene was suppressed by treating HepG2 cells with 0.25 mM buformin for 12 h as a result of the library screening. The decrease in the expression depended on the treatment period. The amount of GAPD protein also decreased simultaneously with the suppression of the gene expression by the treatment with buformin. The amount of ATP and NAD+ in the HepG2 cells treated with buformin decreased to 10 and 20% of the control, respectively. These observations imply that the biguanide causes deactivation of the glycolytic pathway and subsequently the accumulation of pyruvate and NADH and a decrease in NAD+. Therefore, the reaction equilibrium catalyzed by lactate dehydrogenase leans towards lactate production and this may result in lactic acidosis.

  6. Identification, Cloning, and Characterization of l-Phenylserine Dehydrogenase from Pseudomonas syringae NK-15

    PubMed Central

    Ueshima, Sakuko; Muramatsu, Hisashi; Nakajima, Takanori; Yamamoto, Hiroaki; Kato, Shin-ichiro; Misono, Haruo; Nagata, Shinji

    2010-01-01

    The gene encoding d-phenylserine dehydrogenase from Pseudomonas syringae NK-15 was identified, and a 9,246-bp nucleotide sequence containing the gene was sequenced. Six ORFs were confirmed in the sequenced region, four of which were predicted to form an operon. A homology search of each ORF predicted that orf3 encoded l-phenylserine dehydrogenase. Hence, orf3 was cloned and overexpressed in Escherichia coli cells and recombinant ORF3 was purified to homogeneity and characterized. The purified ORF3 enzyme showed l-phenylserine dehydrogenase activity. The enzymological properties and primary structure of l-phenylserine dehydrogenase (ORF3) were quite different from those of d-phenylserine dehydrogenase previously reported. l-Phenylserine dehydrogenase catalyzed the NAD+-dependent oxidation of the β-hydroxyl group of l-β-phenylserine. l-Phenylserine and l-threo-(2-thienyl)serine were good substrates for l-phenylserine dehydrogenase. The genes encoding l-phenylserine dehydrogenase and d-phenylserine dehydrogenase, which is induced by phenylserine, are located in a single operon. The reaction products of both enzymatic reactions were 2-aminoacetophenone and CO2. PMID:21048868

  7. [The role of hepatic and erythrocyte aldehyde dehydrogenase in the development of burn toxemia in rats].

    PubMed

    Solov'eva, A G

    2009-01-01

    The study was designed to examine catalytic properties of non-specific aldehyde dehydrogenase from rat liver and erythrocyte as the main markers of endogenous intoxication after burn. Enzymatic activity was assayed from changes in the rate of NADH synthesis during acetaldehyde oxidation. Burn was shown to decrease it both in the liver and in erythrocytes which resulted in the accumulation of toxic aldehydes and the development of intoxication. Simultaneous fall in alcohol dehydrogenase and lactate dehydrogenase activities is supposed to contribute to the decrease of aldehyde dehydrogenase activity as a result of thermal injury.

  8. Ribitol dehydrogenase of Klebsiella aerogenes. Sequence and properties of wild-type and mutant strains.

    PubMed Central

    Dothie, J M; Giglio, J R; Moore, C B; Taylor, S S; Hartley, B S

    1985-01-01

    Evidence is presented for the sequence of 249 amino acids in ribitol dehydrogenase-A from Klebsiella aerogenes. Continuous culture on xylitol yields strains that superproduce 'wild-type' enzyme but mutations appear to have arisen in this process. Other strains selected by such continuous culture produce enzymes with increased specific activity for xylitol but without loss of ribitol activity. One such enzyme, ribitol dehydrogenase-D, has Pro-196 for Gly-196. Another, ribitol dehydrogenase-B, has a different mutation. PMID:3904726

  9. 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.

  10. [Effects of H2-blockers on alcohol dehydrogenase (ADH) activity].

    PubMed

    Jelski, Wojciech; Orywal, Karolina; Szmitkowski, Maciej

    2008-12-01

    First-pass metabolism (FPM) of alcohol is demonstrated by lower blood alcohol concentrations after oral than intravenous administration of the same dose. FPM occurs predominantly in the stomach and has been attributed to class IV of alcohol dehydrogenase (ADH) isoenzyme localizated in the gastric mucosa. A number of factors that influence on gastric ADH activity and thereby modulate FPM have been identified. These include age, sex, ethnicity, concentrations and amounts of alcohol consumed and drugs. Several H2-receptor antagonists, including cimetidine and ranitidine, inhibit gastric ADH activity and reduce FPM, resulting in higher blood alcohol concentrations after H2-blockers administration.

  11. Drug-induced haemolysis in glucose-6-phosphate dehydrogenase deficiency.

    PubMed Central

    Chan, T K; Todd, D; Tso, S C

    1976-01-01

    People with the variants of glucose-6-phosphate dehydrogenase (GPD) deficiency common in the southern Chinese (Canton, B(-)Chinese, and Hong Kong-Pokfulam) have a moderate shortening of red-cell survival but no anaemia when they are in the steady state. With a cross-transfusion technique, primaquine, nitrofurantoin, and large doses of aspirin were found to aggravate the haemolysis while sulphamethoxazole did so only in some people. Individual differences in drug metabolism may be the reason for this. Many commonly used drugs reported to accentuate haemolysis in GPD deficiency did not shorten red-cell survival. PMID:990860

  12. Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose

    PubMed Central

    Wang, Qingzhao; Ingram, Lonnie O.; Shanmugam, K. T.

    2011-01-01

    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

  13. 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.

  14. Amino acid substitutions at glutamate-354 in dihydrolipoamide dehydrogenase of Escherichia coli lower the sensitivity of pyruvate dehydrogenase to NADH.

    PubMed

    Sun, Zhentao; Do, Phi Minh; Rhee, Mun Su; Govindasamy, Lakshmanan; Wang, Qingzhao; Ingram, Lonnie O; Shanmugam, K T

    2012-05-01

    Pyruvate dehydrogenase (PDH) of Escherichia coli is inhibited by NADH. This inhibition is partially reversed by mutational alteration of the dihydrolipoamide dehydrogenase (LPD) component of the PDH complex (E354K or H322Y). Such a mutation in lpd led to a PDH complex that was functional in an anaerobic culture as seen by restoration of anaerobic growth of a pflB, ldhA double mutant of E. coli utilizing a PDH- and alcohol dehydrogenase-dependent homoethanol fermentation pathway. The glutamate at position 354 in LPD was systematically changed to all of the other natural amino acids to evaluate the physiological consequences. These amino acid replacements did not affect the PDH-dependent aerobic growth. With the exception of E354M, all changes also restored PDH-dependent anaerobic growth of and fermentation by an ldhA, pflB double mutant. The PDH complex with an LPD alteration E354G, E354P or E354W had an approximately 20-fold increase in the apparent K(i) for NADH compared with the native complex. The apparent K(m) for pyruvate or NAD(+) for the mutated forms of PDH was not significantly different from that of the native enzyme. A structural model of LPD suggests that the amino acid at position 354 could influence movement of NADH from its binding site to the surface. These results indicate that glutamate at position 354 plays a structural role in establishing the NADH sensitivity of LPD and the PDH complex by restricting movement of the product/substrate NADH, although this amino acid is not directly associated with NAD(H) binding.

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

    PubMed Central

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

    2015-01-01

    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 kcat/Km 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 Kd value of 125 μM for Hyp, a value ~1600-fold lower than the Km 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

  16. Intracellular coagulation inhibits the extraction of proteins from Prochloron

    NASA Technical Reports Server (NTRS)

    Fall, R.; Lewin, R. A.; Fall, L. R.

    1983-01-01

    Protein extraction from the prokaryotic alga Prochloron LP (isolated from the ascidian host Lissoclinum patella) was complicated by an irreversible loss of cell fragility in the isolated algae. Accompanying this phenomenon, which is termed intracellular coagulation, was a redistribution of thylakoids around the cell periphery, a loss of photosynthetic O2 production, and a drastic decrease in the extractability of cell proteins. Procedures are described for the successful preparation and transport of cell extracts yielding the enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase as well as other soluble proteins.

  17. Dynamics in oxygen-induced changes in S-layer protein synthesis from Bacillus stearothermophilus PV72 and the S-layer-deficient variant T5 in continuous culture and studies of the cell wall composition.

    PubMed Central

    Sára, M; Kuen, B; Mayer, H F; Mandl, F; Schuster, K C; Sleytr, U B

    1996-01-01

    Stable synthesis of the hexagonally ordered (p6) S-layer protein from the wild-type strain of Bacillus stearothermophilus PV72 could be achieved in continuous culture on complex medium only under oxygen-limited conditions when glucose was used as the sole carbon source. Depending on the adaptation of the wild-type strain to low oxygen supply, the dynamics in oxygen-induced changes in S-layer protein synthesis was different when the rate of aeration was increased to a level that allowed dissimilation of amino acids. If oxygen supply was increased at the beginning of continuous culture, synthesis of the p6 S-layer protein from the wild-type strain (encoded by the sbsA gene) was immediately stopped and replaced by that of a new type of S-layer protein (encoded by the sbsB gene) which assembled into an oblique (p2) lattice. In cells adapted to a prolonged low oxygen supply, first, low-level p2 S-layer protein synthesis and second, synchronous synthesis of comparable amounts of both types of S-layer proteins could be induced by stepwise increasing the rate of aeration. The time course of changes in S-layer protein synthesis was followed up by immunogold labelling of whole cells. Synthesis of the p2 S-layer protein could also be induced in the p6-deficient variant T5. Hybridization data obtained by applying the radiolabelled N-terminal and C-terminal sbsA fragments and the N-terminal sbsB fragment to the genomic DNA of all the three organisms indicated that changes in S-layer protein synthesis were accompanied by chromosomal rearrangement. Chemical analysis of peptidoglycan-containing sacculi and extraction and recrystallization experiments revealed that at least for the wild-type strain, a cell wall polymer consisting of N-acetylglucosamine and glucose is responsible for binding of the p6 S-layer protein to the rigid cell wall layer. PMID:8606191

  18. Evidence that an N-terminal S-layer protein fragment triggers the release of a cell-associated high-molecular-weight amylase in Bacillus stearothermophilus ATCC 12980.

    PubMed Central

    Egelseer, E M; Schocher, I; Sleytr, U B; Sára, M

    1996-01-01

    During growth on starch medium, the S-layer-carrying Bacillus stearothermophilus ATCC 12980 and an S-layer-deficient variant each secreted three amylases, with identical molecular weights of 58,000, 122,000, and 184,000, into the culture fluid. Only the high-molecular-weight amylase (hmwA) was also identified as cell associated. Extraction and reassociation experiments showed that the hmwA had a high-level affinity to the peptidoglycan-containing layer and to the S-layer surface, but the interactions with the peptidoglycan-containing layer were stronger than those with the S-layer surface. For the S-layer-deficient variant, no changes in the amount of cell-associated and free hmwA could be observed during growth on starch medium, while for the S-layer-carrying strain, cell association of the hmwA strongly depended on the growth phase of the cells. The maximum amount of cell-associated hmwA was observed 3 h after inoculation, which corresponded to early exponential growth. The steady decrease in cell-associated hmwA during continued growth correlated with the appearance and the increasing intensity of a protein with an apparent molecular weight of 60,000 on sodium dodecyl sulfate gels. This protein had a high-level affinity to the peptidoglycan-containing layer and was identified as an N-terminal S-layer protein fragment which did not result from proteolytic cleavage of the whole S-layer protein but seems to be a truncated copy of the S-layer protein which is coexpressed with the hmwA under certain culture conditions. During growth on starch medium, the N-terminal S-layer protein fragment was integrated into the S-layer lattice, which led to the loss of its regular structure over a wide range and to the loss of amylase binding sites. Results obtained in the present study provide evidence that the N-terminal part of the S-layer protein is responsible for the anchoring of the subunits to the peptidoglycan-containing layer, while the surface-located C-terminal half

  19. Interaction of the crystalline bacterial cell surface layer protein SbsB and the secondary cell wall polymer of Geobacillus stearothermophilus PV72 assessed by real-time surface plasmon resonance biosensor technology.

    PubMed

    Mader, Christoph; Huber, Carina; Moll, Dieter; Sleytr, Uwe B; Sára, Margit

    2004-03-01

    The interaction between S-layer protein SbsB and the secondary cell wall polymer (SCWP) of Geobacillus stearothermophilus PV72/p2 was investigated by real-time surface plasmon resonance biosensor technology. The SCWP is an acidic polysaccharide that contains N-acetylglucosamine, N-acetylmannosamine, and pyruvic acid. For interaction studies, recombinant SbsB (rSbsB) and two truncated forms consisting of either the S-layer-like homology (SLH) domain (3SLH) or the residual part of SbsB were used. Independent of the setup, the data showed that the SLH domain was exclusively responsible for SCWP binding. The interaction was found to be highly specific, since neither the peptidoglycan nor SCWPs from other organisms nor other polysaccharides were recognized. Data analysis from that setup in which 3SLH was immobilized on a sensor chip and SCWP represented the soluble analyte was done in accordance with a model that describes binding of a bivalent analyte to a fixed ligand in terms of an overall affinity for all binding sites. The measured data revealed the presence of at least two binding sites on a single SCWP molecule with a distance of about 14 nm and an overall Kd of 7.7 x 10(-7) M. Analysis of data from the inverted setup in which the SCWP was immobilized on a sensor chip was done in accordance with an extension of the heterogeneous-ligand model, which indicated the existence of three binding sites with low (Kd = 2.6 x 10(-5) M), medium (Kd = 6.1 x 10(-8) M), and high (Kd = 6.7 x 10(-11) M) affinities. Since in this setup 3SLH was the soluble analyte and the presence of small amounts of oligomers in even monomeric protein solutions cannot be excluded, the high-affinity binding site may result from avidity effects caused by binding of at least dimeric 3SLH. Solution competition assays performed with both setups confirmed the specificity of the protein-carbohydrate interaction investigated.

  20. Structural organization of the human sorbitol dehydrogenase gene (SORD)

    SciTech Connect

    Iwata, T.; Carper, D.; Popescu, N.C.

    1995-03-01

    The primary structure of human sorbitol dehydrogenase (SORD) was determined by cDNA and genomic cloning. The nucleotide sequence of the mRNA covers 2471 bp including an open reading frame that yields a protein of 356 amino acid residues. The gene structure of SORD spans approximatley 30 kb divided into 9 exons and 8 introns. The gene was localized to chromosome 15q21.1 by in situ hybridization. Two transcription initiation sites were detected. Three Sp1 sites and a repetitive sequence (CAAA){sub 5} were observed in the 5{prime} noncoding region; no classical TATAA or CCAAT elements were found. The related alcohol dehydrogenases and {zeta}-crystallin have the same gene organization split by 8 introns, but no splice points coincide between SORD and these gene types. The deduced amino acid sequence of the SORD structure differs at a few positions from the directly determined protein sequence, suggesting allelic forms of the enzyme. High levels of SORD transcripts were observed in lens and kidney, as judged from Northern blot analysis. 42 refs., 7 figs., 1 tab.

  1. Interaction of mitochondrial malate dehydrogenase monomer with phospholipid vesicles.

    PubMed Central

    Webster, K A; Patel, H V; Freeman, K B; Papahadjopoulos, D

    1979-01-01

    The association between bovine and porcine mitochondrial malate dehydrogenase (EC 1.1.1.37) and phospholipid vesicles was investigated. At concentrations at which malate dehydrogenase exists as a dimer, entrapment within the aqueous compartment but not binding of the 14C-labelled enzyme was observed. The dissociated enzyme was labile to moderate heat and to p-chloromercuribenzoate, but in both cases inactivation was decreased by incubation with suspensions of charged phospholipid vesicles. This suggested an interaction between enzyme subunits and phospholipid, and this was confirmed by direct binding measurements and by studies that followed changes in the fluorescein-labelled enzyme. The circular-dichroism spectra of the enzyme indicated a high alpha-helix content, and suggested that a small conformational change occurred when the enzyme dissociated. Fluorescence data also suggested less-rigid molecules after dissociation. A possible mechanism, based on the flexibility of enzyme monomer and its interaction with phospholipids, by which mitochondrial matrix enzymes are specifically localized in cells, is discussed. PMID:435273

  2. 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

  3. Phytoestrogens as inhibitors of fungal 17beta-hydroxysteroid dehydrogenase.

    PubMed

    Kristan, Katja; Krajnc, Katja; Konc, Janez; Gobec, Stanislav; Stojan, Jure; Rizner, Tea Lanisnik

    2005-09-01

    Different phytoestrogens were tested as inhibitors of 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl), a member of the short-chain dehydrogenase/reductase superfamily. Phytoestrogens inhibited the oxidation of 100 microM 17beta-hydroxyestra-4-en-3-one and the reduction of 100 microM estra-4-en-3,17-dione, the best substrate pair known. The best inhibitors of oxidation, with IC(50) below 1 microM, were flavones hydroxylated at positions 3, 5 and 7: 3-hydroxyflavone, 3,7-dihydroxyflavone, 5,7-dihydroxyflavone (chrysin) and 5-hydroxyflavone, together with 5-methoxyflavone. The best inhibitors of reduction were less potent; 3-hydroxyflavone, 5-methoxyflavone, coumestrol, 3,5,7,4'-tetrahydroxyflavone (kaempferol) and 5-hydroxyflavone all had IC(50) values between 1 and 5 microM. Docking the representative inhibitors chrysin and kaempferol into the active site of 17beta-HSDcl revealed the possible binding mode, in which they are sandwiched between the nicotinamide moiety and Tyr212. The structural features of phytoestrogens, inhibitors of both oxidation and reduction catalyzed by the fungal 17beta-HSD, are similar to the reported structural features of phytoestrogen inhibitors of human 17beta-HSD types 1 and 2.

  4. Phytoestrogens as inhibitors of fungal 17beta-hydroxysteroid dehydrogenase.

    PubMed

    Kristan, Katja; Krajnc, Katja; Konc, Janez; Gobec, Stanislav; Stojan, Jure; Lanisnik Rizner, Tea

    2005-08-01

    Different phytoestrogens were tested as inhibitors of 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl), a member of the short-chain dehydrogenase/reductase superfamily. Phytoestrogens inhibited the oxidation of 100microM 17beta-hydroxyestra-4-en-3-one and the reduction of 100microM estra-4-en-3,17-dione, the best substrate pair known. The best inhibitors of oxidation, with IC(50) below 1microM, were flavones hydroxylated at positions 3, 5 and 7: 3-hydroxyflavone, 3,7-dihydroxyflavone, 5,7-dihydroxyflavone (chrysin) and 5-hydroxyflavone, together with 5-methoxyflavone. The best inhibitors of reduction were less potent; 3-hydroxyflavone, 5-methoxyflavone, coumestrol, 3,5,7,4'-tetrahydroxyflavone (kaempferol) and 5-hydroxyflavone, all had IC(50) values between 1 and 5microM. Docking the representative inhibitors chrysin and kaempferol into the active site of 17beta-HSDcl revealed the possible binding mode, in which they are sandwiched between the nicotinamide moiety and Tyr212. The structural features of phytoestrogens, inhibitors of both oxidation and reduction catalyzed by the fungal 17beta-HSD, are similar to the reported structural features of phytoestrogen inhibitors of human 17beta-HSD types 1 and 2.

  5. Phenylbutyrate Therapy for Pyruvate Dehydrogenase Complex Deficiency and Lactic Acidosis

    PubMed Central

    Ferriero, Rosa; Manco, Giuseppe; Lamantea, Eleonora; Nusco, Edoardo; Ferrante, Mariella I.; Sordino, Paolo; Stacpoole, Peter W.; Lee, Brendan; Zeviani, Massimo; Brunetti-Pierri, Nicola

    2014-01-01

    Lactic acidosis is a build-up of lactic acid in the blood and tissues, which can be due to several inborn errors of metabolism as well as nongenetic conditions. Deficiency of pyruvate dehydrogenase complex (PDHC) is the most common genetic disorder leading to lactic acidosis. Phosphorylation of specific serine residues of the E1α subunit of PDHC by pyruvate dehydrogenase kinase (PDK) inactivates the enzyme, whereas dephosphorylation restores PDHC activity. We found that phenylbutyrate enhances PDHC enzymatic activity in vitro and in vivo by increasing the proportion of unphosphorylated enzyme through inhibition of PDK. Phenylbutyrate given to C57B6/L wild-type mice results in a significant increase in PDHC enzyme activity and a reduction of phosphorylated E1α in brain, muscle, and liver compared to saline-treated mice. By means of recombinant enzymes, we showed that phenylbutyrate prevents phosphorylation of E1α through binding and inhibition of PDK, providing a molecular explanation for the effect of phenylbutyrate on PDHC activity. Phenylbutyrate increases PDHC activity in fibroblasts from PDHC-deficient patients harboring various molecular defects and corrects the morphological, locomotor, and biochemical abnormalities in the noam631 zebrafish model of PDHC deficiency. In mice, phenylbutyrate prevents systemic lactic acidosis induced by partial hepatectomy. Because phenylbutyrate is already approved for human use in other diseases, the findings of this study have the potential to be rapidly translated for treatment of patients with PDHC deficiency and other forms of primary and secondary lactic acidosis. PMID:23467562

  6. CYTOCHEMICAL LOCALIZATION OF TWO GLYCOLYTIC DEHYDROGENASES IN WHITE SKELETAL MUSCLE

    PubMed Central

    Fahimi, H. Dariush; Karnovsky, Morris J.

    1966-01-01

    The cytochemical localization, by conventional methods, of lactate and glyceraldehyde-3-phosphate dehydrogenases is limited, firstly, by the solubility of these enzymes in aqueous media and, secondly, by the dependence of the final electron flow from reduced nicotinamide-adenine dinucleotide (NADH) to the tetrazolium on tissue diaphorase activity: localization is therefore that of the diaphorase, which in rabbit adductor magnus is mitochondrial. NADH has been found to have great affinity to bind in the sarcoplasmic reticulum, and, therefore, if it is generated freely in the incubation media containing 2,2',5,5'-tetra-p-nitrophenyl-3,3'-(3,3'-dimethoxy-4,4'-phenylene)-ditetrazolium chloride (TNBT) and N-methyl phenazonium methyl sulfate (PMS), it can bind there and cause a false staining. Since such a production of NADH can readily occur in the incubation media for glycolytic dehydrogenases due to diffusion of these soluble enzymes from tissue sections, the prevention of enzyme solubilization is extremely important. Fixation in formaldehyde prevented such enzyme diffusion, while at the same time sufficient activity persisted to allow for adequate staining. The incubation media contained PMS, so that the staining system was largely independent of tissue diaphorase activity. Application of these methods to adductor magnus of rabbit revealed by light microscopy, for both enzymes, a fine network which was shown by electron microscopy to represent staining of the sarcoplasmic reticulum. Mitochondria also reacted. These findings add further support for the notion that the sarcoplasmic reticulum is probably involved in glycolytic activity. PMID:4288329

  7. Lactate Dehydrogenase Catalysis: Roles of Keto, Hydrated, and Enol Pyruvate

    NASA Astrophysics Data System (ADS)

    Meany, J. E.

    2007-09-01

    Many carbonyl substrates of oxidoreductase enzymes undergo hydration and enolization so that these substrate systems are partitioned between keto, hydrated (gem-diol), and enol forms in aqueous solution. Some oxidoreductase enzymes are subject to inhibition by high concentrations of substrate. For such enzymes, two questions arise pertaining to enzyme "substrate" interactions: (i) which form of the substrate system serves as the preferential substrate and (ii) which form acts to inhibit the enzyme? Thus the relative concentrations of the forms of these substrate systems (keto, hydrated, enol) may provide a form of metabolic control. In this light, the present article considers the reduction of pyruvate by lactate dehydrogenase in the presence of NADH. This reaction is inhibited by relatively high concentrations of pyruvate and the physiological significance of this inhibition has been a subject of controversy for many years. Summarized in this article are data from the literature pertaining to the interactions of keto, hydrated, and enol pyruvate with lactate dehydrogenase. Biochemistry instructors and their students are invited to review such pertinent articles so that they also may evaluate the possibility that the "substrate" inhibition of the isoenzymes in the heart muscle may be, under certain conditions, relevant as a form of metabolic control.

  8. 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

  9. 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.

  10. Crystal structure of a chimaeric bacterial glutamate dehydrogenase

    SciTech Connect

    Oliveira, Tânia; Sharkey, Michael A.; Engel, Paul C.; Khan, Amir R.

    2016-05-23

    Glutamate dehydrogenases (EC 1.4.1.2–4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate using NAD(P)+as a cofactor. The bacterial enzymes are hexameric, arranged with 32 symmetry, and each polypeptide consists of an N-terminal substrate-binding segment (domain I) followed by a C-terminal cofactor-binding segment (domain II). The catalytic reaction takes place in the cleft formed at the junction of the two domains. Distinct signature sequences in the nucleotide-binding domain have been linked to the binding of NAD+versusNADP+, but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies, as shown by the apo structure of glutamate dehydrogenase fromClostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia colienzyme has been determined in the apo state. The enzyme is fully functional and reveals possible determinants of interdomain flexibility at a hinge region following the pivot helix. The enzyme retains the preference for NADP+cofactor from the parentE. colidomain II, although there are subtle differences in catalytic activity.

  11. alpha-Ketoglutarate dehydrogenase mutant of Rhizobium meliloti.

    PubMed Central

    Duncan, M J; Fraenkel, D G

    1979-01-01

    A mutant of Rhizobium meliloti selected as unable to grow on L-arabinose also failed to grow on acetate or pyruvate. It grew, but slower than the parental strain, on many other carbon sources. Assay showed it to lack alpha-ketoglutarate dehydrogenase (kgd) activity, and revertants of normal growth phenotype contained the activity again. Other enzymes of the tricarboxylic acid cycle and of the glyoxylate cycle were present in both mutant and parent strains. Enzymes of pyruvate metabolism were also assayed. L-Arabinose degradation in R. meliloti was found to differ from the known pathway in R. japonicum, since the former strain lacked 2-keto-o-deoxy-L-arabonate aldolase but contained alpha-ketoglutarate semialdehyde dehydrogenase; thus, it is likely that R. meliloti has the L-arabinose pathway leading to alpha-ketoglutarate rather than the one to glycolaldehyde and pyruvate. This finding accounts for the L-arabinose negativity of the mutant. Resting cells of the mutant were able to metabolize the three substrates which did not allow growth. PMID:762018

  12. 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

  13. Proteomic and biochemical basis for enhanced growth yield of Enterobacter sp. LCR1 on insoluble phosphate medium.

    PubMed

    Kumar, Arvind; Rai, Lal Chand

    2015-01-01

    Proteomics and biochemical analyses were used to unravel the basis for higher growth yield of Enterobacter sp. LCR1 on insoluble phosphate medium compared to soluble. Proteomic analysis using 2-DE, MALDI-TOF/MS and LC-MS revealed the involvement of nine proteins. Down-regulation of fructose bisphosphate aldolase with decreased concentrations of glucose-6-phosphate and fructose-6-phosphate indicated diminished glycolysis. However, up-regulation of phosphoglycerate mutase, increase in the activities of 6-phosphogluconate dehydratase, 2-keto-3-deoxy-6-phosphogluconate aldolase and 6-phosphogluconate dehydrogenase suggested induction of Entner-Doudoroff and pentose phosphate pathways. These pathways generate sufficient energy from gluconic acid, which is also used for biosynthesis as indicated by up-regulation of elongation factor Tu, elongation factor G and protein disulfide isomerase. Increased reactive oxygen species (ROS) formation resulting from organic acid oxidation leads to overexpressed manganese superoxide dismutase and increased activities of catalase and ascorbate peroxidase. Thus the organism uses gluconate instead of glucose for energy, while alleviating extra ROS formation by oxidative defense enzymes.

  14. The Development of Leucine Dehydrogenase and Formate Dehydrogenase Bifunctional Enzyme Cascade Improves the Biosynthsis of L-tert-Leucine.

    PubMed

    Lu, Jixue; Zhang, Yonghui; Sun, Dongfang; Jiang, Wei; Wang, Shizhen; Fang, Baishan

    2016-11-01

    Leucine dehydrogenase (LDH) and formate dehydrogenase (FDH) were assembled together based on a high-affinity interaction between two different cohesins in a miniscaffoldin and corresponding dockerins in LDH and FDH. The miniscaffoldin with two enzymes was further absorbed by regenerated amorphous cellulose (RAC) to form a bifunctional enzyme complex (miniscaffoldin with LDH and FDH adsorbed by RAC, RSLF) in vitro. The enzymatic characteristics of the bifunctional enzyme complex and free enzymes mixture were systematically compared. The synthesis of L-tert-leucine by the RSLF and free enzyme mixture were compared under different concentrations of enzymes, coenzyme, and substrates. The initial L-tert-leucine production rate by RSLF was enhanced by 2-fold compared with that of the free enzyme mixture. Ninety-one grams per liter of L-tert-leucine with an enantiomeric purity of 99 % e.e. was obtained by RSLF multienzyme catalysis. The results indicated that the bifuntional enzyme complex based on cohesin-dockerin interaction has great potential in the synthesis of L-tert-leucine.

  15. 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.

  16. A 'random steady-state' model for the pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase enzyme complexes

    NASA Astrophysics Data System (ADS)

    Najdi, T. S.; Hatfield, G. W.; Mjolsness, E. D.

    2010-03-01

    The multienzyme complexes, pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, involved in the central metabolism of Escherichia coli consist of multiple copies of three different enzymes, E1, E2 and E3, that cooperate to channel substrate intermediates between their active sites. The E2 components form the core of the complex, while a mixture of E1 and E3 components binds to the core. We present a random steady-state model to describe catalysis by such multienzyme complexes. At a fast time scale, the model describes the enzyme catalytic mechanisms of substrate channeling at a steady state, by polynomially approximating the analytic solution of a biochemical master equation. At a slower time scale, the structural organization of the different enzymes in the complex and their random binding/unbinding to the core is modeled using methods from equilibrium statistical mechanics. Biologically, the model describes the optimization of catalytic activity by substrate sharing over the entire enzyme complex. The resulting enzymatic models illustrate the random steady state (RSS) for modeling multienzyme complexes in metabolic pathways.

  17. 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.

  18. 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.

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

    PubMed

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

    2015-01-02

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

  20. The influence of oxygen on radiation-induced structural and functional changes in glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase

    NASA Astrophysics Data System (ADS)

    Rodacka, Aleksandra; Serafin, Eligiusz; Bubinski, Michal; Krokosz, Anita; Puchala, Mieczyslaw

    2012-07-01

    Proteins are major targets for oxidative damage due to their abundance in cells and high reactivity with free radicals. In the present study we examined the influence of oxygen on radiation-induced inactivation and structural changes of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH). We chose these two enzymes because they occur at high concentrations and participate in the most important processes in organisms; furthermore, they show considerable similarity in their structure. Protein solutions were irradiated with X-rays in doses ranging from 0.1 to 0.7 kGy, in air and N2O. The much higher radiation inactivation of GAPDH as compared to LDH is correlated with substantially greater structural changes in this protein, mainly involving the loss of free thiol groups (-SH). Of lesser importance in the differentiation of the radiosensitivity of the studied enzymes are tryptophan residues. Molecular oxygen, present during irradiation, increased to a significantly greater extent the inactivation and structural changes of GAPDH than that of LDH. The results suggest that the greater effect of oxygen on GAPDH is due to the higher efficiency of the superoxide radical, the higher amount of hydroperoxides generated, and the higher degree of unfolding of this protein.

  1. Molecular characterization of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824.

    PubMed Central

    Nair, R V; Bennett, G N; Papoutsakis, E T

    1994-01-01

    A gene (aad) coding for an aldehyde/alcohol dehydrogenase (AAD) was identified immediately upstream of the previously cloned ctfA (J. W. Cary, D. J. Petersen, E. T. Papoutsakis, and G. N. Bennett, Appl. Environ. Microbiol. 56:1576-1583, 1990) of Clostridium acetobutylicum ATCC 824 and sequenced. The 2,619-bp aad codes for a 96,517-Da protein. Primer extension analysis identified two transcriptional start sites 83 and 243 bp upstream of the aad start codon. The N-terminal section of AAD shows homology to aldehyde dehydrogenases of bacterial, fungal, mammalian, and plant origin, while the C-terminal section shows homology to alcohol dehydrogenases of bacterial (which includes three clostridial alcohol dehydrogenases) and yeast origin. AAD exhibits considerable amino acid homology (56% identity) over its entire sequence to the trifunctional protein encoded by adhE from Escherichia coli. Expression of aad from a plasmid in C. acetobutylicum showed that AAD, which appears as a approximately 96-kDa band in denaturing protein gels, provides elevated activities of NADH-dependent butanol dehydrogenase, NAD-dependent acetaldehyde dehydrogenase and butyraldehyde dehydrogenase, and a small increase in NADH-dependent ethanol dehydrogenase. A 957-bp open reading frame that could potentially encode a 36,704-Da protein was identified upstream of aad. Images PMID:8300540

  2. The crystal structure of SDR-type pyridoxal 4-dehydrogenase of Mesorhizobium loti.

    PubMed

    Chu, Huy Nhat; Kobayashi, Jun; Mikami, Bunzo; Yagi, Toshiharu

    2011-01-01

    Pyridoxal 4-dehydrogenase catalyzes the irreversible oxidation of pyridoxal to 4-pyridoxolactone and is involved in degradation pathway I of pyridoxine, a vitamin B(6) compound. Its crystal structure was elucidated for the first time. Molecular replacement with (S)-1-phenylthanol dehydrogenase (PDB code 2EW8) was adopted to determine the tertiary structure of the NAD(+)-bound enzyme.

  3. Cloning and mRNA Expression of NADH Dehydrogenase during Ochlerotatus taeniorhynchus Development and Pesticide Response

    Technology Transfer Automated Retrieval System (TEKTRAN)

    NADH dehydrogenase, the largest of the respiratory complexes, is the first enzyme of the mitochondrial electron transport chain. We have cloned and sequenced cDNA of NADH dehydrogenase gene from Ochlerotatus (Ochlerotatus) taeniorhynchus (Wiedemann) adult (GeneBank Accession number: FJ458415). The ...

  4. AROMATIC METABOLISM IN PLANTS. I. A STUDY OF THE PREPHENATE DEHYDROGENASE FROM BEAN PLANTS,

    DTIC Science & Technology

    Prephenate dehydrogenase (prephenate: NADP(+) oxidoreductase (decarboxylating)) was isolated from cotyledons of wax bean ( Phaseolus vulgaris L. var...mung bean ( Phaseolus aureus Roxb.). A study was made of the variation in the amount of prephenate dehydrogenase and aromatic amino acid transaminase in

  5. 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

  6. Heterogeneous expression of protein and mRNA in pyruvate dehydrogenase deficiency.

    PubMed Central

    Wexler, I D; Kerr, D S; Ho, L; Lusk, M M; Pepin, R A; Javed, A A; Mole, J E; Jesse, B W; Thekkumkara, T J; Pons, G

    1988-01-01

    Deficiency of pyruvate dehydrogenase [pyruvate:lipoamide 2-oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1], the first component of the pyruvate dehydrogenase complex, is associated with lactic acidosis and central nervous system dysfunction. Using both specific antibodies to pyruvate dehydrogenase and cDNAs coding for its two alpha and beta subunits, we characterized pyruvate dehydrogenase deficiency in 11 patients. Three different patterns were found on immunologic and RNA blot analyses. (i) Seven patients had immunologically detectable crossreactive material for the alpha and beta proteins of pyruvate dehydrogenase. (ii) Two patients had no detectable crossreactive protein for either the alpha or beta subunit but had normal amounts of mRNA for both alpha and beta subunits. (iii) The remaining two patients also had no detectable crossreactive protein but had diminished amounts of mRNA for the alpha subunit of pyruvate dehydrogenase only. These results indicate that loss of pyruvate dehydrogenase activity may be associated with either absent or catalytically inactive proteins, and in those cases in which this enzyme is absent, mRNA for one of the subunits may also be missing. When mRNA for one of the subunits is lacking, both protein subunits are absent, suggesting that a mutation affecting the expression of one of the subunit proteins causes the remaining uncomplexed subunit to be unstable. The results show that several different mutations account for the molecular heterogeneity of pyruvate dehydrogenase deficiency. Images PMID:3140238

  7. 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.

  8. Structural and functional properties of a yeast xylitol dehydrogenase, a Zn2+-containing metalloenzyme similar to medium-chain sorbitol dehydrogenases.

    PubMed Central

    Lunzer, R; Mamnun, Y; Haltrich, D; Kulbe, K D; Nidetzky, B

    1998-01-01

    The NAD+-dependent xylitol dehydrogenase from the xylose-assimilating yeast Galactocandida mastotermitis has been purified in high yield (80%) and characterized. Xylitol dehydrogenase is a heteronuclear multimetal protein that forms homotetramers and contains 1 mol of Zn2+ ions and 6 mol of Mg2+ ions per mol of 37.4 kDa protomer. Treatment with chelating agents such as EDTA results in the removal of the Zn2+ ions with a concomitant loss of enzyme activity. The Mg2+ ions are not essential for activity and are removed by chelation or extensive dialysis without affecting the stability of the enzyme. Results of initial velocity studies at steady state for d-sorbitol oxidation and d-fructose reduction together with the characteristic patterns of product inhibition point to a compulsorily ordered Theorell-Chance mechanism of xylitol dehydrogenase in which coenzyme binds first and leaves last. At pH 7.5, the binding of NADH (Ki approximately 10 microM) is approx. 80-fold tighter than that of NAD+. Polyhydroxyalcohols require at least five carbon atoms to be good substrates of xylitol dehydrogenase, and the C-2 (S), C-3 (R) and C-4 (R) configuration is preferred. Therefore xylitol dehydrogenase shares structural and functional properties with medium-chain sorbitol dehydrogenases. PMID:9806889

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

    SciTech Connect

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

    1991-03-19

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

  10. Mutation of Arg-115 of human class III alcohol dehydrogenase: a binding site required for formaldehyde dehydrogenase activity and fatty acid activation.

    PubMed Central

    Engeland, K; Höög, J O; Holmquist, B; Estonius, M; Jörnvall, H; Vallee, B L

    1993-01-01

    The origin of the fatty acid activation and formaldehyde dehydrogenase activity that distinguishes human class III alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) from all other alcohol dehydrogenases has been examined by site-directed mutagenesis of its Arg-115 residue. The Ala- and Asp-115 mutant proteins were expressed in Escherichia coli and purified by affinity chromatography and ion-exchange HPLC. The activities of the recombinant native and mutant enzymes toward ethanol are essentially identical, but mutagenesis greatly decreases the kcat/Km values for glutathione-dependent formaldehyde oxidation. The catalytic efficiency for the Asp variant is < 0.1% that of the unmutated enzyme, due to both a higher Km and a lower kcat value. As with the native enzyme, neither mutant can oxidize methanol, be saturated by ethanol, or be inhibited by 4-methylpyrazole; i.e., they retain these class III characteristics. In contrast, however, their activation by fatty acids, another characteristic unique to class III alcohol dehydrogenase, is markedly attenuated. The Ala mutant is activated only slightly, but the Asp mutant is not activated at all. The results strongly indicate that Arg-115 in class III alcohol dehydrogenase is a component of the binding site for activating fatty acids and is critical for the binding of S-hydroxymethylglutathione in glutathione-dependent formaldehyde dehydrogenase activity. PMID:8460164

  11. 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.

  12. Pyruvate Dehydrogenase Complex Activity in Normal and Deficient Fibroblasts

    PubMed Central

    Sheu, Kwan-Fu Rex; Hu, Chii-Whei C.; Utter, Merton F.

    1981-01-01

    Pyruvate dehydrogenase complex (PDC) activity in human skin fibroblasts appears to be regulated by a phosphorylation-dephosphorylation mechanism, as is the case with other animal cells. The enzyme can be activated by pretreating the cells with dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, before they are disrupted for measurement of PDC activity. With such treatment, the activity reaches 5-6 nmol/min per mg of protein at 37°C with fibroblasts from infants. Such values represent an activation of about 5-20-fold over those observed with untreated cells. That this assay, based on [1-14C]pyruvate decarboxylation, represents a valid measurement of the overall PDC reaction is shown by the dependence of 14CO2 production on the presence of thiamin-PP, coenzyme A (CoA), Mg++, and NAD+. Also, it has been shown that acetyl-CoA and 14CO2 are formed in a 1:1 ratio. A similar degree of activation of PDC can also be achieved by adding purified pyruvate dehydrogenase phosphatase and high concentrations of Mg++ and Ca++, or in some cases by adding the metal ions alone to the cell homogenate after disruption. These results strongly suggest that activation is due to dephosphorylation. Addition of NaF, which inhibits dephosphorylation, leads to almost complete loss of PDC activity. Assays of completely activated PDC were performed on two cell lines originating from patients reported to be deficient in this enzyme (Blass, J. P., J. Avigan, and B. W. Ublendorf. 1970. J. Clin. Invest. 49: 423-432; Blass, J. P., J. D. Schuman, D. S. Young, and E. Ham. 1972. J. Clin. Invest. 51: 1545-1551). Even after activation with DCA, fibroblasts from the patients showed values of only 0.1 and 0.3 nmol/min per mg of protein. A familial study of one of these patients showed that both parents exhibited activity in fully activated cells about half that of normal values, whereas cells from a sibling appeared normal. These results demonstrate the inheritance nature of PDC deficiency

  13. 17 beta-hydroxysteroid dehydrogenase activity in canine pancreas

    SciTech Connect

    Mendoza-Hernandez, G.; Lopez-Solache, I.; Rendon, J.L.; Diaz-Sanchez, V.; Diaz-Zagoya, J.C.

    1988-04-15

    The mitochondrial fraction of the dog pancreas showed NAD(H)-dependent enzyme activity of 17 beta-hydroxysteroid dehydrogenase. The enzyme catalyzes oxidoreduction between androstenedione and testosterone. The apparent Km value of the enzyme for androstenedione was 9.5 +/- 0.9 microM, the apparent Vmax was determined as 0.4 nmol mg-1 min-1, and the optimal pH was 6.5. In phosphate buffer, pH 7.0, maximal rate of androstenedione reduction was observed at 37 degrees C. The oxidation of testosterone by the enzyme proceeded at the same rate as the reduction of the androstenedione at a pH of 6.8-7.0. The apparent Km value and the optimal pH of the enzyme for testosterone were 3.5 +/- 0.5 microM and 7.5, respectively.

  14. Conjugated bilirubin in neonates with glucose-6-phosphate dehydrogenase deficiency.

    PubMed

    Kaplan, M; Rubaltelli, F F; Hammerman, C; Vilei, M T; Leiter, C; Abramov, A; Muraca, M

    1996-05-01

    We used a system capable of measuring conjugated bilirubin and its monoconjugated and diconjugated fractions in serum to assess bilirubin conjugation in 29 glucose-6-phosphate dehydrogenase (G6PD)-deficient, term, male newborn infants and 35 control subjects; all had serum bilirubin levels > or = 256 mumol/L (15 mg/dI). The median value for diconjugated bilirubin was lower in the G6PD-deficient neonates than in control subjects (0.06 (range 0.00 to 1.84) vs 0.21 (range 0.00 to 1.02) mumol/L, p = 0.006). Diglucuronide was undetectable in 11 (38.9%) of the G6PD-deficient infants versus 3 (8.6%) of the control subjects (p = 0.015). These findings imply a partial defect of bilirubin conjugation not previously demonstrated in G6PD-deficient newborn infants.

  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. Separation of turkey lactate dehydrogenase isoenzymes using isoelectric focusing technique.

    PubMed

    Heinová, Dagmar; Kostecká, Zuzana; Csank, Tomáš

    2016-01-01

    Native polyacrylamide gel electrophoresis at pH 8.8 did not allow to separate lactate dehydrogenase (LDH) isoenzymes of turkey origin. Five electrophoretically distinguishable forms of the enzyme were detected in serum and tissues of turkey using IEF technique in a pH range of 3-9. Generally, three different groups were seen: (i) those having an anodic domination (heart, kidney, pancreas, and erythrocytes) with mainly LDH-1 fraction, (ii) those having a cathodic domination (breast muscle and serum) with prevalence of LDH-5, and (iii) those with a more uniform distribution (liver, spleen, lung, and brain). The specific enzyme activity was the highest in the breast muscle, followed by heart muscle, and brain. Low activities were detected in serum, kidney, and liver.

  17. 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.

  18. [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.

  19. Aldehyde dehydrogenase is used by cancer cells for energy metabolism

    PubMed Central

    Kang, Joon Hee; Lee, Seon-Hyeong; Hong, Dongwan; Lee, Jae-Seon; Ahn, Hee-Sung; Ahn, Ju-Hyun; Seong, Tae Wha; Lee, Chang-Hun; Jang, Hyonchol; Hong, Kyeong Man; Lee, Cheolju; Lee, Jae-Ho; Kim, Soo-Youl

    2016-01-01

    We found that non-small-cell lung cancer (NSCLC) cells express high levels of multiple aldehyde dehydrogenase (ALDH) isoforms via an informatics analysis of metabolic enzymes in NSCLC and immunohistochemical staining of NSCLC clinical tumor samples. Using a multiple reaction-monitoring mass spectrometry analysis, we found that multiple ALDH isozymes were generally abundant in NSCLC cells compared with their levels in normal IMR-90 human lung cells. As a result of the catalytic reaction mediated by ALDH, NADH is produced as a by-product from the conversion of aldehyde to carboxylic acid. We hypothesized that the NADH produced by ALDH may be a reliable energy source for ATP production in NSCLC. This study revealed that NADH production by ALDH contributes significantly to ATP production in NSCLC. Furthermore, gossypol, a pan-ALDH inhibitor, markedly reduced the level of ATP. Gossypol combined with phenformin synergistically reduced the ATP levels, which efficiently induced cell death following cell cycle arrest. PMID:27885254

  20. 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

  1. Environmental inhibition of 11beta-hydroxysteroid dehydrogenase.

    PubMed

    Reidenberg, M M

    2000-04-03

    Gossypol, a polyphenolic compound from cotton seed, caused hypokalemia in some men receiving it in a trial of its contraceptive activity. Searching for the mechanism for its hypokalemic action led to the observation that it inhibited 11beta-hydroxysteroid dehydrogenase. This would enhance mineralocorticoid effect in the kidney. Many other polyphenols also inhibit this enzyme including those in grapefruit juice. Ingesting 1-2 l of grapefruit juice inhibited this enzyme in two men in a clinical experiment. Tea polyphenols inhibit this enzyme and add to the inhibition caused by gossypol. Men in China have lower serum potassium values than men elsewhere and this is due to the environment, presumably the diet, in China. The importance of dietary and other exogenous inhibitors of this enzyme in electrolyte metabolism remains to be determined.

  2. 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

  3. 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.

  4. Suicidal dephosphorylation of thiamine pyrophosphate coupled with pyruvate dehydrogenase complex.

    PubMed

    Strumilo, Slawomir; Dobrzyn, Pawel; Czerniecki, Jan; Tylicki, Adam

    2004-12-01

    Earlier it was noted that purified pyruvate dehydrogenase complex (PDC) produced by "Sigma" usually contains almost saturating amounts of thiamine pyrophosphate (ThPP). In this communication we present the observation that the endogenous ThPP coupled to PDC is dephosphorylated while staying at -10 degrees C, because in the enzyme preparation thiamine monophosphate and un-phosphorylated thiamine appear (HPLC determination). Under the same conditions exogenous ThPP is not dephosphorylated despite contact with the PDC preparation. This may suggest that interactions of some active groups of the enzyme with molecules of endogenous ThPP leads to break-up of the phosphoesters bonds, and destruction of the coenzyme. Decrease of PDC activity during storage is not in proportion with the degree of ThPP dephosphorylation. However the observed instability of PDC activity may be a consequence of the spontaneous process of its coenzyme autodestruction.

  5. 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.

  6. Encapsulation of alcohol dehydrogenase in mannitol by spray drying.

    PubMed

    Shiga, Hirokazu; Joreau, Hiromi; Neoh, Tze Loon; Furuta, Takeshi; Yoshii, Hidefumi

    2014-03-24

    The retention of the enzyme activity of alcohol dehydrogenase (ADH) has been studied in various drying processes such as spray drying. The aim of this study is to encapsulate ADH in mannitol, either with or without additive in order to limit the thermal denaturation of the enzyme during the drying process. The retention of ADH activity was investigated at different drying temperatures. When mannitol was used, the encapsulated ADH was found inactive in all the dried powders. This is presumably due to the quick crystallization of mannitol during spray drying that resulted in the impairment of enzyme protection ability in comparison to its amorphous form. Maltodextin (dextrose equivalent = 11) was used to reduce the crystallization of mannitol. The addition of maltodextrin increased ADH activity and drastically changed the powder X-ray diffractogram of the spray-dried powders.

  7. Hemolytic anemia caused by glucose-6-phosphate dehydrogenase deficiency.

    PubMed

    Olivares, N; Medina, C; Sánchez-Corona, J; Rivas, F; Rivera, H; Hernández, A; Delgado, J L; Ibarra, B; Cantú, J M; Vaca, G; Martínez, C

    1979-01-01

    Results are reported concerning quantitation of glucose -6- phosphate dehydrogenase (G6PD) enzyme activity where in one of the members of a family a clinical diagnosis of acute hemolytic anemia due to G6PD deficiency had been established. In the propositus, G6PD levels were found to be less than 10 per cent thus confirming diagnosis; the same enzymatic deficiency was identified in one of the siblings without a history of hematologic pathology and in a maternal cousin with a history of neonatal jaundice as well as two obliged carriers. Electrophoretical enzyme phenotype was similar to A variant in three affected males. Advantages of prevention and medical care possible with early diagnosis of G6PD deficiency are discussed.

  8. 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.

  9. Sequential injection analysis of ethanol using immobilized alcohol dehydrogenase

    SciTech Connect

    Hedenfalk, M.; Mattiasson, B.

    1996-05-01

    A Sequential Injection (SI) system was used to analyze the ethanol concentration in fermentation broth. The method is based on the use of immobilized NAD{sup +} dependent alcohol dehydrogenase. A non-linear standard curve for ethanol (range 0.25-100 mM) was used to determine the concentration in fermentation broth and the results correlated well with HPLC measurements. The assay time was 140 s, 0.5 {mu}mol of cofactor was used for each determination, and the relative standard deviation was less than 6% when analyzing fermentation samples. The assay system is very stable and makes it possible to reduce the cofactor consumption while keeping the system set up simple.

  10. Alcohol dehydrogenase polymorphism in barrel cactus populations of Drosophila mojavensis.

    PubMed

    Cleland, S; Hocutt, G D; Breitmeyer, C M; Markow, T A; Pfeiler, E

    1996-07-01

    Starch gel electrophoresis revealed that the alcohol dehydrogenase (ADH-2) locus was polymorphic in two populations (from Agua Caliente, California and the Grand Canyon, Arizona) of cactophilic Drosophila mojavensis that utilize barrel cactus (Ferocactus acanthodes) as a host plant. Electromorphs representing products of a slow (S) and a fast (F) allele were found in adult flies. The frequency of the slow allele was 0.448 in flies from Agua Caliente and 0.659 in flies from the Grand Canyon. These frequencies were intermediate to those of the low (Baja California peninsula, Mexico) and high (Sonora, Mexico and southern Arizona) frequency Adh-2S populations of D. mojavensis that utilize different species of host cacti.

  11. 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.

  12. 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

  13. Expanding the clinical spectrum of 3-phosphoglycerate dehydrogenase deficiency

    PubMed Central

    Tabatabaie, L.; Klomp, L. W. J.; Rubio-Gozalbo, M. E.; Spaapen, L. J. M.; Haagen, A. A. M.; Dorland, L.

    2010-01-01

    3-Phosphoglycerate dehydrogenase (3-PGDH) deficiency is considered to be a rare cause of congenital microcephaly, infantile onset of intractable seizures and severe psychomotor retardation. Here, we report for the first time a very mild form of genetically confirmed 3-PGDH deficiency in two siblings with juvenile onset of absence seizures and mild developmental delay. Amino acid analysis showed serine values in CSF and plasma identical to what is observed in the severe infantile form. Both patients responded favourably to relatively low dosages of serine supplementation with cessation of seizures, normalisation of their EEG abnormalities and improvement of well-being and behaviour. These cases illustrate that 3-PGDH deficiency can present with mild symptoms and should be considered as a treatable disorder in the differential diagnosis of mild developmental delay and seizures. Synopsis: we present a novel mild phenotype in patients with 3-PGDH deficiency. PMID:21113737

  14. [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%.

  15. Biofuel cell anode: NAD +/glucose dehydrogenase-coimmobilized ketjenblack electrode

    NASA Astrophysics Data System (ADS)

    Miyake, T.; Oike, M.; Yoshino, S.; Yatagawa, Y.; Haneda, K.; Kaji, H.; Nishizawa, M.

    2009-09-01

    We have studied the coimmobilization of glucose dehydrogenase (GDH) and its cofactor, oxidized nicotinamide adenine dinucleotide (NAD +), on a ketjenblack (KB) electrode as a step toward a biofuel cell anode that works without mediators. A KB electrode was first treated with a sulfuric acid/nitric acid/water mixture to lower the overvoltage for NADH oxidation, and was next chemically modified with NAD + and GDH. The improved GDH/NAD +/KB electrode is found to oxidize glucose around 0 V vs. Ag/AgCl. A biofuel cell constructed with a bilirubin oxidase-immobilized KB cathode showed a maximum power density of 52 μW/cm 2 at 0.3 V.

  16. Ketonic diet in the management of pyruvate dehydrogenase deficiency.

    PubMed

    Falk, R E; Cederbaum, S D; Blass, J P; Gibson, G E; Kark, R A; Carrel, R E

    1976-11-01

    Two brothers, aged 11 years 6 months and 2 years 3 months, with psychomotor and growth retardation, episodes of weakness, ataxia, ophthalmoplegia, and elevated levels of blood pyruvate were shown to have a deficiency in the pyruvate dehydrogenase complex (PDH). When they ate a diet high enough in fats to cause ketonemia but not acidosis, there was a fall in blood pyruvate levels, a decrease in the frequency and severity of the episodes of neurological deterioration, an increased rate of growth and development in the younger brother, and increased strength and endurance in the older one. The possibility of dietary treatment makes the early diagnosis of PDH deficiency more important. Determination of blood pyruvate and lactate levels following a standard glucose meal (glucose-pyruvate test) appears to be the most reliable screening test for this condition.

  17. Lactate dehydrogenase A silencing in IDH mutant gliomas

    PubMed Central

    Chesnelong, Charles; Chaumeil, Myriam M.; Blough, Michael D.; Al-Najjar, Mohammad; Stechishin, Owen D.; Chan, Jennifer A.; Pieper, Russell O.; Ronen, Sabrina M.; Weiss, Samuel; Luchman, H. Artee; Cairncross, J. Gregory

    2014-01-01

    Background Mutations of the isocitrate dehydrogenase 1 and 2 gene (IDH1/2) were initially thought to enhance cancer cell survival and proliferation by promoting the Warburg effect. However, recent experimental data have shown that production of 2-hydroxyglutarate by IDH mutant cells promotes hypoxia-inducible factor (HIF)1α degradation and, by doing so, may have unexpected metabolic effects. Methods We used human glioma tissues and derived brain tumor stem cells (BTSCs) to study the expression of HIF1α target genes in IDH mutant (mt) and IDH wild-type (wt) tumors. Focusing thereafter on the major glycolytic enzyme, lactate dehydrogenase A (LDHA), we used standard molecular methods and pyrosequencing-based DNA methylation analysis to identify mechanisms by which LDHA expression was regulated in human gliomas. Results We found that HIF1α-responsive genes, including many essential for glycolysis (SLC2A1, PDK1, LDHA, SLC16A3), were underexpressed in IDHmt gliomas and/or derived BTSCs. We then demonstrated that LDHA was silenced in IDHmt derived BTSCs, including those that did not retain the mutant IDH1 allele (mIDHwt), matched BTSC xenografts, and parental glioma tissues. Silencing of LDHA was associated with increased methylation of the LDHA promoter, as was ectopic expression of mutant IDH1 in immortalized human astrocytes. Furthermore, in a search of The Cancer Genome Atlas, we found low expression and high methylation of LDHA in IDHmt glioblastomas. Conclusion To our knowledge, this is the first demonstration of downregulation of LDHA in cancer. Although unexpected findings, silencing of LDHA and downregulation of several other glycolysis essential genes raise the intriguing possibility that IDHmt gliomas have limited glycolytic capacity, which may contribute to their slow growth and better prognosis. PMID:24366912

  18. Biochemical and structural characterization of Plasmodium falciparum glutamate dehydrogenase 2.

    PubMed

    Zocher, Kathleen; Fritz-Wolf, Karin; Kehr, Sebastian; Fischer, Marina; Rahlfs, Stefan; Becker, Katja

    2012-05-01

    Glutamate dehydrogenases (GDHs) play key roles in cellular redox, amino acid, and energy metabolism, thus representing potential targets for pharmacological interventions. Here we studied the functional network provided by the three known glutamate dehydrogenases of the malaria parasite Plasmodium falciparum. The recombinant production of the previously described PfGDH1 as hexahistidyl-tagged proteins was optimized. Additionally, PfGDH2 was cloned, recombinantly produced, and characterized. Like PfGDH1, PfGDH2 is an NADP(H)-dependent enzyme with a specific activity comparable to PfGDH1 but with slightly higher K(m) values for its substrates. The three-dimensional structure of hexameric PfGDH2 was solved to 3.1 Å resolution. The overall structure shows high similarity with PfGDH1 but with significant differences occurring at the subunit interface. As in mammalian GDH1, in PfGDH2 the subunit-subunit interactions are mainly assisted by hydrogen bonds and hydrophobic interactions, whereas in PfGDH1 these contacts are mediated by networks of salt bridges and hydrogen bonds. In accordance with this, the known bovine GDH inhibitors hexachlorophene, GW5074, and bithionol were more effective on PfGDH2 than on PfGDH1. Subcellular localization was determined for all three plasmodial GDHs by fusion with the green fluorescent protein. Based on our data, PfGDH1 and PfGDH3 are cytosolic proteins whereas PfGDH2 clearly localizes to the apicoplast, a plastid-like organelle specific for apicomplexan parasites. This study provides new insights into the structure and function of GDH isoenzymes of P. falciparum, which represent potential targets for the development of novel antimalarial drugs.

  19. Human liver class I alcohol dehydrogenase gammagamma isozyme: the sole cytosolic 3beta-hydroxysteroid dehydrogenase of iso bile acids.

    PubMed

    Marschall, H U; Oppermann, U C; Svensson, S; Nordling, E; Persson, B; Höög, J O; Jörnvall, H

    2000-04-01

    3beta-Hydroxy (iso) bile acids are formed during enterohepatic circulation from 3alpha-hydroxy bile acids and constitute normal compounds in plasma but are virtually absent in bile. Isoursodeoxycholic acid (isoUDCA) is a major metabolite of UDCA. In a recent study it was found that after administration of isoUDCA, UDCA became the major acid in bile. Thus, epimerization of the 3beta-hydroxy to a 3alpha-hydroxy group, catalyzed by 3beta-hydroxysteroid dehydrogenases (HSD) and 3-oxo-reductases must occur. The present study aims to characterize the human liver bile acid 3beta-HSD. Human liver cytosol and recombinant alcohol dehydrogenase (ADH) betabeta and gammagamma isozymes were subjected to native polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing. Activity staining with oxidized nicotinamide adenine dinucleotide (NAD(+)) or oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)) as cofactors and various iso bile acids as substrates was used to screen for 3beta-HSD activity. Reaction products were identified and quantified by gas chromotography/mass spectrometry (GC/MS). Computer-assisted substrate docking of isoUDCA to the active site of a 3-dimensional model of human class I gammagamma ADH was performed. ADH gammagamma isozyme was identified as the iso bile acid 3beta-HSD present in human liver cytosol, with NAD(+) as a cofactor. Values for k(cat)/K(m) were in the rank order isodeoxycholic acid (isoDCA), isochenodeoxycholic acid (isoCDCA), isoUDCA, and isolithocholic acid (isoLCA) (0.10, 0.09, 0.08, and 0. 05 min(-1) x micromol/L(-1), respectively). IsoUDCA fits as substrate to the 3-dimensional model of the active-site of ADH gammagamma. ADH gammagamma isozyme was defined as the only bile acid 3beta-HSD in human liver cytosol. Hydroxysteroid dehydrogenases are candidates for the binding and transport of 3alpha-hydroxy bile acids. We assume that ADH gammagamma isozyme is involved in cytosolic bile acid binding and transport processes as well.

  20. Microbial Oxidation of Methane and Methanol: Crystallization of Methanol Dehydrogenase and Properties of Holo- and Apo-Methanol Dehydrogenase from Methylomonas methanica

    PubMed Central

    Patel, Ramesh N.; Hou, Ching T.; Felix, Andre

    1978-01-01

    Procedures are described for the purification and crystallization of methanol dehydrogenase from the soluble fraction of the type I obligate methylotroph Methylomonas methanica strain S1. The crystallized enzyme is homogeneous as judged by acrylamide gel electrophoresis and ultracentrifugation. The enzyme had a high pH optimum (9.5) and required ammonium salt as an activator. In the presence of phenazine methosulfate as an electron acceptor, the enzyme catalyzed the oxidation of primary alcohols and formaldehyde. Secondary, tertiary, and aromatic alcohols were not oxidized. The molecular weight as well as subunit size of methanol dehydrogenase was 60,000, indicating that it is monomeric. The sedimentation constant (s20,w) was 3.1S. The amino acid composition of the crystallized enzyme is also presented. Antisera prepared against the crystalline enzyme were nonspecific; they cross-reacted with and inhibited the isofunctional enzyme from other obligate methylotrophic bacteria. The crystalline methanol dehydrogenase had an absorption peak at 350 nm in the visible region and weak fluorescence peaks at 440 and 470 nm due to the presence of a pteridine derivative as the prosthetic group. A procedure was developed for the preparation of apo-methanol dehydrogenase. The molecular weights, sedimentation constants, electrophoretic mobilities, and immunological properties of apo- and holo-methanol dehydrogenases are identical. Apo-methanol dehydrogenase lacked the absorption peak at 350 nm and the fluorescence peaks at 440 and 470 nm and was catalytically inactive. All attempts to reconstitute an active enzyme from apo-methanol dehydrogenase, using various pteridine derivatives, were unsuccessful. Images PMID:415046

  1. The molybdenum formylmethanofuran dehydrogenase operon and the tungsten formylmethanofuran dehydrogenase operon from Methanobacterium thermoautotrophicum. Structures and transcriptional regulation.

    PubMed

    Hochheimer, A; Linder, D; Thauer, R K; Hedderich, R

    1996-11-15

    Methanobacterium thermoautotrophicum contains a tungsten formylmethanofuran dehydrogenase (FwdABCD) and a molybdenum formylmethanofuran dehydrogenase (FmdABC). The fwdHFGDACB operon encoding the tungsten enzyme has recently been characterized. We report here on the structure and expression of the gene cluster encoding the molybdenum enzyme. This gene cluster is composed of three open reading frames (fmdECB). The fmdB gene was found to encode the molybdopterin-dinucleotide-binding subunit harboring the enzyme's active site; FmdB is thus functionally equivalent to FwdB. fmdC encodes a protein with sequence similarity to FwdC in its N-terminal part and with sequence similarity to FwdD in its C-terminal part; FmdC is thus functionally equivalent to FwdC and FwdD. Interestingly, the fmd operon lacks a gene fmdA encoding the subunit FmdA of the molybdenum enzyme. FmdA has the same apparent molecular mass and the same N-terminal amino acid sequence as FwdA and only one DNA sequence encoding for this N-terminal amino acid sequence was found in the M. thermoautotrophicum genome. It is therefore proposed that FmdA and FwdA are encoded by the same gene namely fwdA in the fwd operon. In agreement with this proposal is the finding that fwdA is expressed constitutively: northern-blot analysis of RNA from tungstate- and molybdate-grown cells of M. thermo-autotrophicum revealed that the fwdHFGDACB gene cluster is transcribed in the presence of either molybdate or tungstate in the growth medium whereas the fmdECB gene cluster was only transcribed when molybdate was present.

  2. Dimerization and enzymatic activity of fungal 17β-hydroxysteroid dehydrogenase from the short-chain dehydrogenase/reductase superfamily

    PubMed Central

    Kristan, Katja; Deluca, Dominga; Adamski, Jerzy; Stojan, Jure; Rižner, Tea Lanišnik

    2005-01-01

    Background 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17β-HSDcl) is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. SDR proteins usually function as dimers or tetramers and 17β-HSDcl is also a homodimer under native conditions. Results We have investigated here which secondary structure elements are involved in the dimerization of 17β-HSDcl and examined the importance of dimerization for the enzyme activity. Sequence similarity with trihydroxynaphthalene reductase from Magnaporthe grisea indicated that Arg129 and His111 from the αE-helices interact with the Asp121, Glu117 and Asp187 residues from the αE and αF-helices of the neighbouring subunit. The Arg129Asp and His111Leu mutations both rendered 17β-HSDcl monomeric, while the mutant 17β-HSDcl-His111Ala was dimeric. Circular dichroism spectroscopy analysis confirmed the conservation of the secondary structure in both monomers. The three mutant proteins all bound coenzyme, as shown by fluorescence quenching in the presence of NADP+, but both monomers showed no enzymatic activity. Conclusion We have shown by site-directed mutagenesis and structure/function analysis that 17β-HSDcl dimerization involves the αE and αF helices of both subunits. Neighbouring subunits are connected through hydrophobic interactions, H-bonds and salt bridges involving amino acid residues His111 and Arg129. Since the substitutions of these two amino acid residues lead to inactive monomers with conserved secondary structure, we suggest dimerization is a prerequisite for catalysis. A detailed understanding of this dimerization could lead to the development of compounds that will specifically prevent dimerization, thereby serving as a new type of inhibitor. PMID:16359545

  3. 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

  4. Dietary methimazole-induced hypothyroidism reduces hepatic lipid deposition by down-regulating lipogenesis and up-regulating lipolysis in Pelteobagrus fulvidraco.

    PubMed

    Chen, Qi-Liang; Luo, Zhi; Shi, Xi; Wu, Kun; Zhuo, Mei-Qin; Song, Yu-Feng; Hu, Wei

    2015-01-01

    The present study was conducted to investigate the effects and mechanisms of hypothyroidism, induced by administration of 0.2% methimazole through the food, on lipid metabolism in the liver of juvenile yellow catfish Pelteobagrus fulvidraco. To this end, yellow catfish were fed diets containing either 0 or 2g methimazole per kg of diet for 8weeks, respectively. The results showed that fish fed diet containing methimazole had a significant reduction in growth performance, plasma THs levels and hepatic lipid content. Meanwhile, methimazole treatment inhibited the activities of lipogenic enzymes (6-phosphogluconate dehydrogenase, glucose 6-phosphate dehydrogenase, malic enzyme, isocitrate dehydrogenase and fatty acid synthase) and the mRNA levels of genes involved in lipogenesis (6-phosphogluconate dehydrogenase, glucose 6-phosphate dehydrogenase, fatty acid synthase, acetyl-CoA carboxylase α, sterol-regulator element-binding protein-1 and liver X receptor), but increased lipolytic enzyme (carnitine palmitoyltransferase 1) activity and the expression of genes involved in lipolysis (carnitine palmitoyltransferase 1a, hormone-sensitive lipase and peroxisome proliferators-activated receptor α). Thus, our study indicated that dietary methimazole-induced hypothyroidism could disturb the normal processes of lipid metabolism at the enzymatic and molecular levels in yellow catfish, and the reduced hepatic lipid content by hypothyroidism was attributable to the down-regulation of lipogenesis and up-regulation of lipolysis.

  5. Biorhythms of activities of liver and blood dehydrogenases and changes in body weight of the rats feeding normal diet or excess of sugar substitutes.

    PubMed

    Petrovich, Yu A; Volozhin, A I; Zubtsov, V A; Kichenko, S M

    2007-12-01

    Biorhythms with higher levels of activity of sorbitol dehydrogenase and lactate dehydrogenase in blood plasma, specific activity of sorbitol dehydrogenase, lactate dehydrogenase, and malate dehydrogenase in the liver, and body weight of rats were more pronounced in the spring-summer period than in the autumn-winter period. These specific features were revealed in animals feeding a normal diet or food with 54 and 27% sugar substitute sorbitol. However, specific activity of glucose-6-phosphate dehydrogenase in the liver was higher in the autumn-winter period. Activity of sorbitol dehydrogenase in blood plasma increased by tens of times due to induction of sorbitol synthesis (substrate) in the liver. Sugar substitute xylitol is structurally similar to sorbitol, but is not the substrate for sorbitol dehydrogenase. However, the effect of xylitol on activities of lactate dehydrogenase, malate dehydrogenase, and glucose-6-phosphate dehydrogenase in the spring-summer period was similar to that of sorbitol.

  6. Peripartal changes in serum alkaline phosphatase activity and lactate dehydrogenase activity in dairy cows.

    PubMed Central

    Peter, A T; Bosu, W T; MacWilliams, P; Gallagher, S

    1987-01-01

    Peripartal serum alkaline phosphatase activity and lactate dehydrogenase activity were measured in 30 dairy cows in order to examine the association between retained fetal membranes and enzyme activity. Daily blood samples were obtained from pregnant cows, starting 15 days before the expected day of calving until eight days after parturition. Sera from 15 cows which retained fetal membranes longer than 24 hours and 15 cows which shed fetal membranes within six hours after parturition were analyzed for alkaline phosphatase and lactate dehydrogenase enzyme activities. Mean alkaline phosphatase enzyme activities ranged from 15.93 to 32.6 U/L in retained and nonretained placenta cows. There was a trend towards higher serum alkaline phosphatase activities in retained placenta cows but the differences were not significant among the groups (P greater than 0.05). Mean lactate dehydrogenase activities ranged from 307.2 to 438.86 U/L in nonretained and retained placenta cows. Lactate dehydrogenase enzyme activities in nonretained and retained placenta cows were similar (P greater than 0.05). The alkaline phosphatase and lactate dehydrogenase enzyme activities peaked at the time of parturition in both groups. However, the differences in alkaline phosphatase and lactate dehydrogenase activities on different days within non-retained and retained placenta cows were significant (P less than 0.05). Results indicate that prepartal changes in alkaline phosphatase and lactate dehydrogenase enzyme activities are not predictive of placental retention postpartum. PMID:3453274

  7. On the role of microsomal aldehyde dehydrogenase in metabolism of aldehydic products of lipid peroxidation.

    PubMed

    Antonenkov, V D; Pirozhkov, S V; Panchenko, L F

    1987-11-30

    To elucidate a possible role of membrane-bound aldehyde dehydrogenase in the detoxication of aldehydic products of lipid peroxidation, the substrate specificity of the highly purified microsomal enzyme was investigated. The aldehyde dehydrogenase was active with different aliphatic aldehydes including 4-hydroxyalkenals, but did not react with malonic dialdehyde. When Fe/ADP-ascorbate-induced lipid peroxidation of arachidonic acid was carried out in an in vitro system, the formation of products which react with microsomal aldehyde dehydrogenase was observed parallel with malonic dialdehyde accumulation.

  8. Binding region of alanopine dehydrogenase predicted by unbiased molecular dynamics simulations of ligand diffusion.

    PubMed

    Gohlke, Holger; Hergert, Ulrike; Meyer, Tatu; Mulnaes, Daniel; Grieshaber, Manfred K; Smits, Sander H J; Schmitt, Lutz

    2013-10-28

    Opine dehydrogenases catalyze the reductive condensation of pyruvate with L-amino acids. Biochemical characterization of alanopine dehydrogenase from Arenicola marina revealed that this enzyme is highly specific for L-alanine. Unbiased molecular dynamics simulations with a homology model of alanopine dehydrogenase captured the binding of L-alanine diffusing from solvent to a putative binding region near a distinct helix-kink-helix motif. These results and sequence comparisons reveal how mutations and insertions within this motif dictate the L-amino acid specificity.

  9. Protein-Associated Lipid of Bacillus stearothermophilus

    PubMed Central

    Card, George L.; Szuba, Joan C.; Shimizu, Marilyn

    1979-01-01

    The composition and patterns of metabolism of phospholipids isolated as part of a lipid-depleted membrane fragment (LDM fragment) and associated with the membrane adenosine triphosphatase complex have been compared with those of the bulk membrane phospholipid. The bulk lipid was extracted from washed membranes with sodium cholate. The LDM fragments, which contained a portion of the electron transport system and the membrane adenosine triphosphatase complex, were purified by chromatography with Sepharose 6B. The LDM fragment preparations contained 0.10 ± 0.02 μmol of lipid phosphorus per mg of protein, compared with 0.54 ± 0.05 μmol of lipid phosphorus per mg of protein for washed membranes. The phospholipid associated with the LDM fragments consisted of 78 ± 4% cardiolipin, 7 ± 1% phosphatidylglycerol, and 15 ± 3% phosphatidylethanolamine. Changes in the total membrane lipid composition (produced by culture conditions) did not alter the phospholipid composition of the LDM fragments. The adenosine triphosphate complex was separated from the other components of the LDM fragments by suspension of the fragments in 1% Triton X-100 and precipitation with antibody specific for the F1 component of the adenosine triphosphatase complex. The phospholipid isolated with the adenosine triphosphatase complex consisted of 86% cardiolipin, 8% phosphatidylglycerol, and 6% phosphatidylethanolamine. In pulse-chase experiments with 32P and [2-3H]glycerol, the labeling patterns of the phosphatididylglycerol and phosphatidylethanolamine associated with the LDM fragments were different from those of the bulk membrane phosphatidylglycerol and phosphatidylethanolamine. It was concluded that at least a portion of the phospholipid isolated with the LDM fragments was part of a native lipid-protein complex. PMID:159285

  10. AB104. Glucose-6 phospate dehydrogenase deficiency among mongolian neonates

    PubMed Central

    Batjargal, Khishigjargal; Nansal, Gerelmaa; Zagd, Gerelmaa; Ganbaatar, Erdenetuya

    2015-01-01

    Background and objective Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency in humans, affecting 400 million people worldwide and a high prevalence in persons of African, Middle Asian countries. The most common clinical manifestations are neonatal jaundice and acute hemolytic anemia, which is caused by the impairment of erythrocyte’s ability to remove harmful oxidative stress triggered by exogenous agents such as drugs, infection, or fava bean ingestion. Neonatal hyperbilirubinemia caused by G6PD is strongly associated with mortality and long-term neurodevelopmental impairment. The study aims to determine a level of G6PD in healthy neonates. Methods We obtained blood spot samples from 268 infants around 24-72 hours in their age who has unsuspected intranatal and neonatal disorders. Glucose 6 phosphate dehydrogenase “Perkin Elmer, Finland” level is determined by Victor 2D Fluorometer assay, developing of neonatal jaundice is examined by recall. Results The76.5% of all participants (n=205) was assessed 4.36±1.15 Ug/Hb in normal reference range of G6PD, other 23.5% (n=63) was 0.96±0.51 Ug/Hb with G6PD deficiency. In the both sex, 51.5% of male 0.88±0.46 Ug/Hb (n=33) and 47.6% of female (n=30) 0.97±0.55 Ug/Hb was assessed with G6PD deficiency. Developing Jaundice period in number of 63 neonates with G6PD deficiency, 86% of neonates (n=54) was in 1-4 days, 4% of neonates (n=3) was in 5-7 days and there is no sign of jaundice in 9% (n=6). Therefore neonates with G6PD deficiency, 53.9% (n=34) continued jaundice more than two weeks. Conclusions G6PD deficiency was determined in male neonates (51.5%) more than female (47.6%). The 76.5% of all participants (n=205) was assessed 4.36±1.15 Ug/Hb in normal reference range of G6PDH other 23.5% (n=63) of all participants was 0.96±0.51 Ug/Hb with G6PD deficiency. It shows that G6PD might be one potential risk of neonatal jaundice and hyperbilirubinemia in neonates in Mongolia.

  11. Heterozygosity of the sheep: Polymorphism of 'malic enzyme', isocitrate dehydrogenase (NADP+), catalase and esterase.

    PubMed

    Baker, C M; Manwell, C

    1977-04-01

    In contrast to other reports, it is found that the sheep has approximately as much enzyme variation as man. Most of the genetically interpretable enzyme variation in heart, liver, kidney and muscle from 52 sheep (Merinos or Merino crosses) is in the NADP-dependent dehydrogenases [two 'malic enzymes' and the supernatant isocitrate dehydrogenase (NADP+)] and in the esterases. Ten different loci for NAD-dependent dehydrogenases are electrophoretically monomorphic, as are five different NADH diaphorases from heart muscle and 15 different major proteins from skeletal muscle. It is highly statistically significant that NADP-dependent dehydrogenases and esterases are polymorphic but representatives of several other major classes of enzymes are not. The physiological significance of this polymorphism may be related to the role of these enzymes in growth and detoxication, sheep having been selected by man for faster growth, of wool or of carcass, and for grazing a wide variety of plants.

  12. Mitochondrial type II NAD(P)H dehydrogenases in fungal cell death

    PubMed Central

    Gonçalves, A. Pedro; Videira, Arnaldo

    2015-01-01

    During aerobic respiration, cells produce energy through oxidative phosphorylation, which includes a specialized group of multi-subunit complexes in the inner mitochondrial membrane known as the electron transport chain. However, this canonical pathway is branched into single polypeptide alternative routes in some fungi, plants, protists and bacteria. They confer metabolic plasticity, allowing cells to adapt to different environmental conditions and stresses. Type II NAD(P)H dehydrogenases (also called alternative NAD(P)H dehydrogenases) are non-proton pumping enzymes that bypass complex I. Recent evidence points to the involvement of fungal alternative NAD(P)H dehydrogenases in the process of programmed cell death, in addition to their action as overflow systems upon oxidative stress. Consistent with this, alternative NAD(P)H dehydrogenases are phylogenetically related to cell death - promoting proteins of the apoptosis-inducing factor (AIF)-family. PMID:28357279

  13. Identification and Characterization of an Inducible NAD(P)H Dehydrogenase from Red Beetroot Mitochondria.

    PubMed Central

    Menz, R. I.; Day, D. A.

    1996-01-01

    Exogenous NADH oxidation of mitochondria isolated from red beetroots (Beta vulgaris L.) increased dramatically upon slicing and aging the tissue. Anion-exchange chromatography of soluble fractions derived by sonication from fresh and aged beetroot mitochondria yielded three NADH dehydrogenase activity peaks. The third peak from aged beetroot mitochondria was separated into two activities by blue-affinity chromatography. One of these (the unbound peak) readily oxidized dihydrolipoamide, whereas the other (the bound peak) did not. The latter was an NAD(P)H dehydrogenase with high quinone and ferricyanide reductase activity and was absent from fresh beet mitochondria. Further affinity chromatography of the NAD(P)H dehydrogenase indicated enrichment of a 58-kD polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We propose that this 58-kD protein is the inducible, external NADH dehydrogenase. PMID:12226415

  14. Identification and Characterization of an Inducible NAD(P)H Dehydrogenase from Red Beetroot Mitochondria.

    PubMed

    Menz, R. I.; Day, D. A.

    1996-10-01

    Exogenous NADH oxidation of mitochondria isolated from red beetroots (Beta vulgaris L.) increased dramatically upon slicing and aging the tissue. Anion-exchange chromatography of soluble fractions derived by sonication from fresh and aged beetroot mitochondria yielded three NADH dehydrogenase activity peaks. The third peak from aged beetroot mitochondria was separated into two activities by blue-affinity chromatography. One of these (the unbound peak) readily oxidized dihydrolipoamide, whereas the other (the bound peak) did not. The latter was an NAD(P)H dehydrogenase with high quinone and ferricyanide reductase activity and was absent from fresh beet mitochondria. Further affinity chromatography of the NAD(P)H dehydrogenase indicated enrichment of a 58-kD polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We propose that this 58-kD protein is the inducible, external NADH dehydrogenase.

  15. Structural Biology of Proteins of the Multi-enzyme Assembly Human Pyruvate Dehydrogenase Complex

    NASA Technical Reports Server (NTRS)

    2003-01-01

    Objectives and research challenges of this effort include: 1. Need to establish Human Pyruvate Dehydrogenase Complex protein crystals; 2. Need to test value of microgravity for improving crystal quality of Human Pyruvate Dehydrogenase Complex protein crystals; 3. Need to improve flight hardware in order to control and understand the effects of microgravity on crystallization of Human Pyruvate Dehydrogenase Complex proteins; 4. Need to integrate sets of national collaborations with the restricted and specific requirements of flight experiments; 5. Need to establish a highly controlled experiment in microgravity with a rigor not yet obtained; 6. Need to communicate both the rigor of microgravity experiments and the scientific value of results obtained from microgravity experiments to the national community; and 7. Need to advance the understanding of Human Pyruvate Dehydrogenase Complex structures so that scientific and commercial advance is identified for these proteins.

  16. Glucose-6-phosphate dehydrogenase deficiency presented with convulsion: a rare case.

    PubMed

    Merdin, Alparslan; Avci, Fatma; Guzelay, Nihal

    2014-01-29

    Red blood cells carry oxygen in the body and Glucose-6-Phosphate Dehydrogenase protects these cells from oxidative chemicals. If there is a lack of Glucose-6-Phosphate Dehydrogenase, red blood cells can go acute hemolysis. Convulsion is a rare presentation for acute hemolysis due to Glucose-6-Phosphate Dehydrogenase deficiency. Herein, we report a case report of a Glucose-6-Phosphate Dehydrogenase deficiency diagnosed patient after presentation with convulsion. A 70 year-old woman patient had been hospitalized because of convulsion and fatigue. She has not had similar symptoms before. She had ingested fava beans in the last two days. Her hypophyseal and brain magnetic resonance imaging were normal. Blood transfusion was performed and the patient recovered.

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

    EPA Science Inventory

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

  18. Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis

    DOEpatents

    Ingram, Lonnie O.; Conway, Tyrrell

    1992-01-01

    The alcohol dehydrogenase II gene from Zymomonas mobilis has been cloned and sequenced. This gene can be expressed at high levels in other organisms to produce acetaldehyde or to convert acetaldehyde to ethanol.

  19. The Effect of Salinity on the Malic Dehydrogenase of Pea Roots 1

    PubMed Central

    Hason-Porath, Edna; Poljakoff-Mayber, Alexandra

    1969-01-01

    Effect of salinity on malate dehydrogenase activity was studied. Pea root tips contain 2 different malate dehydrogenases. One is located in the particulate, mitochondrial fraction, the other in the soluble, cytoplasmic fraction. Both can act when coupled with either NAD or NADP. Growing plants in Na2SO4 salinated medium did not affect the pattern of the malate dehydrogenases in the root tips. Growing plants in NaCl salinated media resulted in the appearance of a new, third isoenzyme. This new isoenzyme was located in the cytoplasmic fraction. Salinity of both types, when present in growth medium, induced increases in the NADP coupled activity of the mitochondrial malate dehydrogenase. The NAD coupled activity, however, was depressed except in the cytoplasmic fraction of plants grown in media salinated with NaCl to 1 atmosphere. Addition of either of the salts to assay media of enzymes, isolated from plants grown in non salinated substrate, did not have any significant effect. PMID:16657152

  20. Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action

    DOE PAGES

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

    2015-01-07

    Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacylmore » intermediate and an NAD+-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp3-to-sp2 transition during enzyme-mediated oxidation.« less

  1. Crystal structure of dihydroorotate dehydrogenase from Leishmania major.

    PubMed

    Cordeiro, Artur T; Feliciano, Patricia R; Pinheiro, Matheus P; Nonato, M Cristina

    2012-08-01

    Dihydroorotate dehydrogenase (DHODH) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and has been exploited as the target for therapy against proliferative and parasitic diseases. In this study, we report the crystal structures of DHODH from Leishmania major, the species of Leishmania associated with zoonotic cutaneous leishmaniasis, in its apo form and in complex with orotate and fumarate molecules. Both orotate and fumarate were found to bind to the same active site and exploit similar interactions, consistent with a ping-pong mechanism described for class 1A DHODHs. Analysis of LmDHODH structures reveals that rearrangements in the conformation of the catalytic loop have direct influence on the dimeric interface. This is the first structural evidence of a relationship between the dimeric form and the catalytic mechanism. According to our analysis, the high sequence and structural similarity observed among trypanosomatid DHODH suggest that a single strategy of structure-based inhibitor design can be used to validate DHODH as a druggable target against multiple neglected tropical diseases such as Leishmaniasis, Sleeping sickness and Chagas' diseases.

  2. Leucaena sp. recombinant cinnamyl alcohol dehydrogenase: purification and physicochemical characterization.

    PubMed

    Patel, Parth; Gupta, Neha; Gaikwad, Sushama; Agrawal, Dinesh C; Khan, Bashir M

    2014-02-01

    Cinnamyl alcohol dehydrogenase is a broad substrate specificity enzyme catalyzing the final step in monolignol biosynthesis, leading to lignin formation in plants. Here, we report characterization of a recombinant CAD homologue (LlCAD2) isolated from Leucaena leucocephala. LlCAD2 is 80 kDa homo-dimer associated with non-covalent interactions, having substrate preference toward sinapaldehyde with Kcat/Km of 11.6×10(6) (M(-1) s(-1)), and a possible involvement of histidine at the active site. The enzyme remains stable up to 40 °C, with the deactivation rate constant (Kd(*)) and half-life (t1/2) of 0.002 and 5h, respectively. LlCAD2 showed optimal activity at pH 6.5 and 9 for reduction and oxidation reactions, respectively, and was stable between pH 7 and 9, with the deactivation rate constant (Kd(*)) and half-life (t1/2) of 7.5×10(-4) and 15 h, respectively. It is a Zn-metalloenzyme with 4 Zn(2+) per dimer, however, was inhibited in presence of externally supplemented Zn(2+) ions. The enzyme was resistant to osmolytes, reducing agents and non-ionic detergents.

  3. 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

  4. Pyruvate dehydrogenase kinase regulates hepatitis C virus replication

    PubMed Central

    Jung, Gwon-Soo; Jeon, Jae-Han; Choi, Yeon-Kyung; Jang, Se Young; Park, Soo Young; Kim, Sung-Woo; Byun, Jun-Kyu; Kim, Mi-Kyung; Lee, Sungwoo; Shin, Eui-Cheol; Lee, In-Kyu; Kang, Yu Na; Park, Keun-Gyu

    2016-01-01

    During replication, hepatitis C virus (HCV) utilizes macromolecules produced by its host cell. This process requires host cellular metabolic reprogramming to favor elevated levels of aerobic glycolysis. Therefore, we evaluated whether pyruvate dehydrogenase kinase (PDK), a mitochondrial enzyme that promotes aerobic glycolysis, can regulate HCV replication. Levels of c-Myc, hypoxia-inducible factor-1α (HIF-1α), PDK1, PDK3, glucokinase, and serine biosynthetic enzymes were compared between HCV-infected and uninfected human liver and Huh-7.5 cells infected with or without HCV. Protein and mRNA expression of c-Myc, HIF-1α, and glycolytic enzymes were significantly higher in HCV-infected human liver and hepatocytes than in uninfected controls. This increase was accompanied by upregulation of serine biosynthetic enzymes, suggesting cellular metabolism was altered toward facilitated nucleotide synthesis essential for HCV replication. JQ1, a c-Myc inhibitor, and dichloroacetate (DCA), a PDK inhibitor, decreased the expression of glycolytic and serine synthetic enzymes in HCV-infected hepatocytes, resulting in suppressed viral replication. Furthermore, when co-administered with IFN-α or ribavirin, DCA further inhibited viral replication. In summary, HCV reprograms host cell metabolism to favor glycolysis and serine biosynthesis; this is mediated, at least in part, by increased PDK activity, which provides a surplus of nucleotide precursors. Therefore, blocking PDK activity might have therapeutic benefits against HCV replication. PMID:27471054

  5. Metabolism of the novel IMP dehydrogenase inhibitor benzamide riboside.

    PubMed

    Jäger, Walter; Salamon, Alexandra; Szekeres, Thomas

    2002-04-01

    Benzamide riboside (BR) is a novel anticancer agent exhibiting pronounced activity against several human tumor cell lines via the inhibition of inosine 5'-monophosphate dehydrogenase (IMPDH) that catalyzes the formation of xanthine 5'-monophosphate from inosine 5'-monophosphate and nicotinamide adenine dinucleotide, thereby restricting the biosynthesis of guanylates. Phosphorylation of BR to its 5'-monophosphate derivative appears to be ubiquitous in most cells catalyzed by the enzymes, adenosine kinase, nicotinamide nucleoside kinase and 5' nucleotidase. BR 5'-monophosphate is then converted to the active metabolite benzamide adenine dinucleotide (BAD) by NMN adenylyltransferase, the rate-limiting enzyme in the biosynthesis of NAD. As BAD is more potent in the inhibition of IMPDH than BR and BR 5'-monophosphate, cytotoxicity of BR is closely connected with intercellular metabolism to BAD. However, intracellular BAD level is also affected by BADase activity, a phosphodiesterase which hydrolyzes BAD to BR-5'-monophosphate and AMP. A recent study demonstrates enzymatic deamination of BR to non-cytotoxic benzene carboxylic acid (BR-COOH) as the main hepatic BR biotransformation product in rat liver. As the IMPDH inhibitors tiazofurin and ribavirin exhibit predominant accumulation and biotransformation in liver, hepatic metabolism may be an important factor also for BR activation and inactivation and should be considered in human liver during cancer therapy when BR is used as a single drug or in combination with other anticancer agents.

  6. Function, kinetic properties, crystallization, and regulation of microbial malate dehydrogenase*

    PubMed Central

    Takahashi-Íñiguez, Tóshiko; Aburto-Rodríguez, Nelly; Vilchis-González, Ana Laura; Flores, María Elena

    2016-01-01

    Malate dehydrogenase (MDH) is an enzyme widely distributed among living organisms and is a key protein in the central oxidative pathway. It catalyzes the interconversion between malate and oxaloacetate using NAD+ or NADP+ as a cofactor. Surprisingly, this enzyme has been extensively studied in eukaryotes but there are few reports about this enzyme in prokaryotes. It is necessary to review the relevant information to gain a better understanding of the function of this enzyme. Our review of the data generated from studies in bacteria shows much diversity in their molecular properties, including weight, oligomeric states, cofactor and substrate binding affinities, as well as differences in the direction of the enzymatic reaction. Furthermore, due to the importance of its function, the transcription and activity of this enzyme are rigorously regulated. Crystal structures of MDH from different bacterial sources led to the identification of the regions involved in substrate and cofactor binding and the residues important for the dimer-dimer interface. This structural information allows one to make direct modifications to improve the enzyme catalysis by increasing its activity, cofactor binding capacity, substrate specificity, and thermostability. A comparative analysis of the phylogenetic reconstruction of MDH reveals interesting facts about its evolutionary history, dividing this superfamily of proteins into two principle clades and establishing relationships between MDHs from different cellular compartments from archaea, bacteria, and eukaryotes.

  7. Retinol Dehydrogenases Regulate Vitamin A Metabolism for Visual Function

    PubMed Central

    Sahu, Bhubanananda; Maeda, Akiko

    2016-01-01

    The visual system produces visual chromophore, 11-cis-retinal from dietary vitamin A, all-trans-retinol making this vitamin essential for retinal health and function. These metabolic events are mediated by a sequential biochemical process called the visual cycle. Retinol dehydrogenases (RDHs) are responsible for two reactions in the visual cycle performed in retinal pigmented epithelial (RPE) cells, photoreceptor cells and Müller cells in the retina. RDHs in the RPE function as 11-cis-RDHs, which oxidize 11-cis-retinol to 11-cis-retinal in vivo. RDHs in rod photoreceptor cells in the retina work as all-trans-RDHs, which reduce all-trans-retinal to all-trans-retinol. Dysfunction of RDHs can cause inherited retinal diseases in humans. To facilitate further understanding of human diseases, mouse models of RDHs-related diseases have been carefully examined and have revealed the physiological contribution of specific RDHs to visual cycle function and overall retinal health. Herein we describe the function of RDHs in the RPE and the retina, particularly in rod photoreceptor cells, their regulatory properties for retinoid homeostasis and future therapeutic strategy for treatment of retinal diseases. PMID:27879662

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

    PubMed

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

    2013-01-08

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

  9. Computational design of glutamate dehydrogenase in Bacillus subtilis natto.

    PubMed

    Chen, Li-Li; Wang, Jia-Le; Hu, Yu; Qian, Bing-Jun; Yao, Xiao-Min; Wang, Jing-Fang; Zhang, Jian-Hua

    2013-04-01

    Bacillus subtilis natto is widely used in industry to produce natto, a traditional and popular Japanese soybean food. However, during its secondary fermentation, high amounts of ammonia are released to give a negative influence on the flavor of natto. Glutamate dehydrogenase (GDH) is a key enzyme for the ammonia produced and released, because it catalyzes the oxidative deamination of glutamate to alpha-ketoglutarate using NAD(+) or NADP(+) as co-factor during carbon and nitrogen metabolism processes. To solve this problem, we employed multiple computational methods model and re-design GDH from Bacillus subtilis natto. Firstly, a structure model of GDH with cofactor NADP(+) was constructed by threading and ab initio modeling. Then the substrate glutamate were flexibly docked into the structure model to form the substrate-binding mode. According to the structural analysis of the substrate-binding mode, Lys80, Lys116, Arg196, Thr200, and Ser351 in the active site were found could form a significant hydrogen bonding network with the substrate, which was thought to play a crucial role in the substrate recognition and position. Thus, these residues were then mutated into other amino acids, and the substrate binding affinities for each mutant were calculated. Finally, three single mutants (K80A, K116Q, and S351A) were found to have significant decrease in the substrate binding affinities, which was further supported by our biochemical experiments.

  10. Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action

    SciTech Connect

    Huo, Lu; Davis, Ian; Liu, Fange; Andi, Babak; Esaki, Shingo; Iwaki, Hiroaki; Hasegawa, Yoshie; Orville, Allen M.; Liu, Aimin

    2015-01-07

    Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NAD+-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp3-to-sp2 transition during enzyme-mediated oxidation.

  11. Immobilization and enzymatic properties of Bacillus megaterium glucose dehydrogenase

    SciTech Connect

    Baron, M.; Fontana, J.D.; Guimaraes, M.F.; Woodward, J.

    1996-12-31

    The enzymatic production of hydrogen gas from renewable sources of energy; e.g., cellulose, starch, lactose, can be obtained by coupling the reactions catalyzed by glucose dehydrogenase (GDH) and hydrogenase. In order to enhance the thermostability of GDH from Bacillus megaterium, the enzyme was immobilized by ionic adsorption using the polycationic polymer DEAE-(dextran)Sephadex. The effect of enzyme concentration on immobilization showed a tendency to increase the activity of the immobilized enzyme with the increase of the amount of added GDH. When the enzyme: support ratio was 15.97 U: 100 mg, the immobilization yield was 84.76%. The enzymatic profiles for the immobilized GDH were a little different when compared to those for free enzyme with respect to the effects of pH and temperature. Concerning the effect of incubation time carried at pH 7.5 and at 40{degrees}C, the maximum production of reduced coenzyme by the immobilized enzyme was reached within 4 h and it was maintained up to 16 h without loss of enzymatic activity. The coupling of the immobilized GDH activity with that for free alkaline cellulose (Novozym. 342) demonstrated the possibility for obtaining reduced coenzyme from the cellulose hydrolysis and the immobilized GDH could be reassayed 10 times maintaining its enzyme activity.

  12. Stabilization and reutilization of Bacillus megaterium glucose dehydrogenase by immobilization

    SciTech Connect

    Baron, M. |; Fontana, J.D.; Guimaraes, M.F.; Woodward, J.

    1997-12-31

    Glucose dehydrogenase (GDH) from Bacillus megaterium was immobilized using aminopropyl controlled-pore silica (CPS, average pore sizes of 170 and 500 {angstrom}) as a support and glutaraldehyde as a bifunctional crosslinking agent. The CPS-immobilized enzyme could be reused 12 times and the best results were obtained using aminopropyl CPS-500 and bovine serum albumin as a feeder for stabilizing the protein layer on the support. DEAE-Sephadex (A-25 and A-50) was also used as a support for immobilizing GDH, with yields of around 42% for A-25 and 25-30% for A-50. The effect of pH on the immobilization procedure showed pH 6.5 to be better than pH 7.5 with respect to the recovery of enzyme activity. Both preparations of DEAE-Sephadex immobilized GDH could be reused several times and were thermostable at 400{degrees}C for 7 h. The kinetic parameters as Michaelis constant and maximum rate were determined for the immobilized enzyme and compared with those for the freeform. 9 refs., 6 figs., 3 tabs.

  13. Xanthine dehydrogenase to xanthine oxidase conversion in ischemic rat intestine

    SciTech Connect

    McKelvey, T.G.; Engerson, T.D.; Elmore, C.R.; Jones, H.P. )

    1990-02-26

    The ischemic conversion of the NADH-producing xanthine dehydrogenase (XDH) to an oxidase form, that produces both superoxide radical and hydrogen peroxide, has been proposed as an important step in initiating oxygen radical-mediated ischemia-reperfusion injury. It has also been reported that two forms of converted oxidase are produced in ischemic rat liver; a reversible xanthine oxidase produced through sulfhydryl oxidation, that can be reconverted to XDH by incubation with 10mM dithiothreitol (Dtt) at 37{degrees}C, and a Dtt-irreversible oxidase produced via proteolysis. The authors report that increased oxidase in the ischemic rat intestine results from significant increases in both the Dtt-reversible and Dtt-irreversible forms of xanthine oxidase. Total oxidase activity (Irreversible + Dtt-reversible) was 19% of the total enzyme activity (XDH + XO) in control ileum and distal jejunum, increased to 26% after 1 hour of ischemia at 37{degrees}C, and significantly to 36% after 1.5 hours. After 3 hours 73% of the activity was in the oxidase form. Irreversible oxidase comprised 15% of the total activity in control intestine, significantly increased to 25% after 2 hours, and further to 42% after 3 hours. Dtt-reversible oxidase was 3% of the total activity in controls, increased to 13% after 1.5 hours, and significantly to 29% after 2 hours.

  14. The gene encoding proline dehydrogenase modulates sensorimotor gating in mice.

    PubMed

    Gogos, J A; Santha, M; Takacs, Z; Beck, K D; Luine, V; Lucas, L R; Nadler, J V; Karayiorgou, M

    1999-04-01

    Hemizygous cryptic deletions of the q11 band of human chromosome 22 have been associated with a number of psychiatric and behavioural phenotypes, including schizophrenia. Here we report the isolation and characterization of PRODH, a human homologue of Drosophila melanogaster sluggish-A (slgA), which encodes proline dehydrogenase responsible for the behavioural phenotype of the slgA mutant. PRODH is localized at chromosome 22q11 in a region deleted in some psychiatric patients. We also isolated the mouse homologue of slgA (Prodh), identified a mutation in this gene in the Pro/Re hyperprolinaemic mouse strain and found that these mice have a deficit in sensorimotor gating accompanied by regional neurochemical alterations in the brain. Sensorimotor gating is a neural filtering process that allows attention to be focused on a given stimulus, and is affected in patients with neuropsychiatric disorders. Furthermore, several lines of evidence suggest that proline may serve as a modulator of synaptic transmission in the mammalian brain. Our observations, in conjunction with the chromosomal location of PRODH, suggest a potential involvement of this gene in the 22q11-associated psychiatric and behavioural phenotypes.

  15. Inactivation of Bakers' yeast glucose-6-phosphate dehydrogenase by aluminum

    SciTech Connect

    Cho, Sungwoo; Joshi, J.G. )

    1989-04-18

    Preincubation of yeast glucose-6-phosphate dehydrogenase (G6PD) with Al(III) produced an inactive enzyme containing 1 mol of Al(III)/mol of enzyme subunit. None of the enzyme-bound Al(III) was dissociated by dialysis against 10 mM Tris-HCl, pH 7.0, containing 0.2 mM EDTA at 4{degree}C for 24 h. Citrate, NADP{sup +}, EDTA, or NaF protected the enzyme against the Al(III) inactivation. The Al(III)-inactivated enzyme, however, was completely reactivated only by citrate and NaF. The dissociation constant for the enzyme-aluminum complex was calculated to be 4 {times} 10{sup {minus}6} M with NaF, a known reversible chelator for aluminum. Modification of histidine and lysine residues of the enzyme with diethyl pyrocarbonate and acetylsalicylic acid, respectively, inactivated the enzyme. However, the modified enzyme still bound 1 mol of Al(III)/mol of enzyme subunit. Circular dichroism studies showed that the binding of Al(III) to the enzyme induced a decrease in {alpha}-helix and {beta}-sheet and an increase in random coil. Therefore, it is suggested that inactivation of G6PD by Al(III) is due to the conformational change induced by Al(III) binding.

  16. Regulation of L-threonine dehydrogenase in somatic cell reprogramming.

    PubMed

    Han, Chuanchun; Gu, Hao; Wang, Jiaxu; Lu, Weiguang; Mei, Yide; Wu, Mian

    2013-05-01

    Increasing evidence suggests that metabolic remodeling plays an important role in the regulation of somatic cell reprogramming. Threonine catabolism mediated by L-threonine dehydrogenase (TDH) has been recognized as a specific metabolic trait of mouse embryonic stem cells. However, it remains unknown whether TDH-mediated threonine catabolism could regulate reprogramming. Here, we report TDH as a novel regulator of somatic cell reprogramming. Knockdown of TDH inhibits, whereas induction of TDH enhances reprogramming efficiency. Moreover, microRNA-9 post-transcriptionally regulates the expression of TDH and thereby inhibits reprogramming efficiency. Furthermore, protein arginine methyltransferase (PRMT5) interacts with TDH and mediates its post-translational arginine methylation. PRMT5 appears to regulate TDH enzyme activity through both methyltransferase-dependent and -independent mechanisms. Functionally, TDH-facilitated reprogramming efficiency is further enhanced by PRMT5. These results suggest that TDH-mediated threonine catabolism controls somatic cell reprogramming and indicate the importance of post-transcriptional and post-translational regulation of TDH.

  17. Incidence and Geographic Distribution of Succinic Semialdehyde Dehydrogenase (SSADH) Deficiency.

    PubMed

    Attri, Savita Verma; Singhi, Pratibha; Wiwattanadittakul, Natrujee; Goswami, Jyotindra N; Sankhyan, Naveen; Salomons, Gajja S; Roullett, Jean-Baptiste; Hodgeman, Ryan; Parviz, Mahsa; Gibson, K Michael; Pearl, Phillip L

    2016-11-05

    The incidence of succinic semialdehyde dehydrogenase (SSADH) deficiency, an autosomal recessive inherited disorder of GABA degradation, is unknown. Upon a recent diagnosis of a new family of affected fraternal twins from the Punjabi ethnic group of India, case ascertainment from the literature and our database was done to determine the number of confirmed cases along with their geographic distribution. The probands presented with global developmental delay, infantile onset epilepsy, and a persistent neurodevelopmental disorder upon diagnosis at 10 years of age with intellectual disability, expressive aphasia, and behavioral problems most prominent for hyperactivity. Gamma-hydroxybutyric aciduria and homozygous ALDH5A1 c.608C>T; p.Pro203Leu mutations were confirmed. Identification of all available individual cases with clinical details available including geographic or ethnic origin revealed 182 patients from 40 countries, with the largest number of patients reported from the USA (24%), Turkey (10%), China (7%), Saudi Arabia (6%), and Germany (5%). This study provides an accounting of all published cases of confirmed SSADH deficiency and provides data useful in planning further studies of this rare inborn error of metabolism.

  18. Natural history of succinic semialdehyde dehydrogenase deficiency through adulthood

    PubMed Central

    Lewis, Evan Cole; De Meulemeester, Christine; Chakraborty, Pranesh; Gibson, K. Michael; Torres, Carlos; Guberman, Alan; Salomons, Gajja S.; Jakobs, Cornelis; Ali-Ridha, Andre; Parviz, Mahsa; Pearl, Phillip L.

    2015-01-01

    Objective: The natural history of succinic semialdehyde dehydrogenase (SSADH) deficiency in adulthood is unknown; we elucidate the clinical manifestations of the disease later in life. Methods: A 63-year-old man with long-standing intellectual disability was diagnosed with SSADH deficiency following hospitalization for progressive decline, escalating seizures, and prolonged periods of altered consciousness. We present a detailed review of his clinical course and reviewed our SSADH deficiency database adult cohort to derive natural history information. Results: Of 95 patients in the database for whom age at diagnosis is recorded, there are 40 individuals currently aged 18 years or older. Only 3 patients were diagnosed after age 18 years. Of 25 adults for whom data are available after age 18, 60% have a history of epilepsy. Predominant seizure types are generalized tonic-clonic, absence, and myoclonic. EEGs showed background slowing or generalized epileptiform discharges in two-thirds of adults for whom EEG data were collected. History of psychiatric symptoms was prominent, with frequent anxiety, sleep disturbances, and obsessive-compulsive disorder. Conclusions: We identified patients older than 18 years with SSADH deficiency in our database following identification and review of a patient diagnosed in the seventh decade of life. The illness had a progressive course with escalating seizures in the index case, with fatality at age 63. Diagnosis in adulthood is rare. Epilepsy is more common in the adult than the pediatric SSADH deficiency cohort; neuropsychiatric morbidity remains prominent. PMID:26268900

  19. Aldehyde dehydrogenase 2 inhibits inflammatory response and regulates atherosclerotic plaque

    PubMed Central

    Wei, Shu-jian; Zhang, Ming-xiang; Wang, Xu-ping; Yuan, Qiu-huan; Xue, Li; Wang, Jia-li; Cui, Zhao-qiang; Zhang, Yun; Xu, Feng; Chen, Yu-guo

    2016-01-01

    Previous studies demonstrated that aldehyde dehydrogenase 2 (ALDH2) rs671 polymorphism, which eliminates ALDH2 activity down to 1%-6%, is a susceptibility gene for coronary disease. Here we investigated the underlying mechanisms based on our prior clinical and experimental studies. Male apoE−/− mice were transfected with GFP, ALDH2-overexpression and ALDH2-RNAi lentivirus respectively (n=20 each) after constrictive collars were placed around the right common carotid arteries. Consequently, ALDH2 gene silencing led to an increased en face plaque area, more unstable plaque with heavier accumulation of lipids, more macrophages, less smooth muscle cells and collagen, which were associated with aggravated inflammation. However, ALDH2 overexpression displayed opposing effects. We also found that ALDH2 activity decreased in atherosclerotic plaques of human and aged apoE−/− mice. Moreover, in vitro experiments with human umbilical vein endothelial cells further illustrated that, inhibition of ALDH2 activity resulted in elevating inflammatory molecules, an increase of nuclear translocation of NF-κB, and enhanced phosphorylation of NF-κB p65, AP-1 c-Jun, Jun-N terminal kinase and p38 MAPK, while ALDH2 activation could trigger contrary effects. These findings suggested that ALDH2 can influence plaque development and vulnerability, and inflammation via MAPK, NF-κB and AP-1 signaling pathways. PMID:27191745

  20. Short-chain dehydrogenases/reductases (SDR): the 2002 update.

    PubMed

    Oppermann, Udo; Filling, Charlotta; Hult, Malin; Shafqat, Naeem; Wu, Xiaoqiu; Lindh, Monica; Shafqat, Jawed; Nordling, Erik; Kallberg, Yvonne; Persson, Bengt; Jörnvall, Hans

    2003-02-01

    Short-chain dehydrogenases/reductases (SDR) form a large, functionally heterogeneous protein family presently with about 3000 primary and about 30 3D structures deposited in databases. Despite low sequence identities between different forms (about 15-30%), the 3D structures display highly similar alpha/beta folding patterns with a central beta-sheet, typical of the Rossmann-fold. Based on distinct sequence motifs functional assignments and classifications are possible, making it possible to build a general nomenclature system. Recent mutagenetic and structural studies considerably extend the knowledge on the general reaction mechanism, thereby establishing a catalytic tetrad of Asn-Ser-Tyr-Lys residues, which presumably form the framework for a proton relay system including the 2'-OH of the nicotinamide ribose, similar to the mechanism found in horse liver ADH. Based on their cellular functions, several SDR enzymes appear as possible and promising pharmacological targets with application areas spanning hormone-dependent cancer forms or metabolic diseases such as obesity and diabetes, and infectious diseases.

  1. Succinic semialdehyde dehydrogenase deficiency: lessons from mice and men.

    PubMed

    Pearl, P L; Gibson, K M; Cortez, M A; Wu, Y; Carter Snead, O; Knerr, I; Forester, K; Pettiford, J M; Jakobs, C; Theodore, W H

    2009-06-01

    Succinic semialdehyde dehydrogenase (SSADH) deficiency, a disorder of GABA degradation with subsequent elevations in brain GABA and GHB, is a neurometabolic disorder with intellectual disability, epilepsy, hypotonia, ataxia, sleep disorders, and psychiatric disturbances. Neuroimaging reveals increased T2-weighted MRI signal usually affecting the globus pallidus, cerebellar dentate nucleus, and subthalamic nucleus, and often cerebral and cerebellar atrophy. EEG abnormalities are usually generalized spike-wave, consistent with a predilection for generalized epilepsy. The murine phenotype is characterized by failure-to-thrive, progressive ataxia, and a transition from generalized absence to tonic-clonic to ultimately fatal convulsive status epilepticus. Binding and electrophysiological studies demonstrate use-dependent downregulation of GABA(A) and (B) receptors in the mutant mouse. Translational human studies similarly reveal downregulation of GABAergic activity in patients, utilizing flumazenil-PET and transcranial magnetic stimulation for GABA(A) and (B) activity, respectively. Sleep studies reveal decreased stage REM with prolonged REM latencies and diminished percentage of stage REM. An ad libitum ketogenic diet was reported as effective in the mouse model, with unclear applicability to the human condition. Acute application of SGS-742, a GABA(B) antagonist, leads to improvement in epileptiform activity on electrocorticography. Promising mouse data using compounds available for clinical use, including taurine and SGS-742, form the framework for human trials.

  2. Aldehyde dehydrogenases in cancer stem cells: potential as therapeutic targets

    PubMed Central

    Clark, David W.

    2016-01-01

    Resistance to current chemotherapeutic or radiation-based cancer treatment strategies is a serious concern. Cancer stem cells (CSCs) are typically able to evade treatment and establish a recurrent tumor or metastasis, and it is these that lead to the majority of cancer deaths. Therefore, a major current goal is to develop treatment strategies that eliminate the resistant CSCs as well as the bulk tumor cells in order to achieve complete disease clearance. Aldehyde dehydrogenases (ALDHs) are important for maintenance and differentiation of stem cells as well as normal development. There is expanding evidence that ALDH expression increases in response to therapy and promotes chemoresistance and survival mechanisms in CSCs. This perspective will discuss a paper by Cojoc and colleagues recently published in Cancer Research, that indicates ALDHs play a key role in resistance to radiation therapy and tumor recurrence in prostate cancer. The authors suggest that ALDHs are a potential therapeutic target for treatment prostate cancer patients to limit radiation resistance and disease recurrence. The findings are consistent with work from other cancers showing ALDHs are major contributors of CSC signaling and resistance to anti-cancer treatments. This perspective will address representative work concerning the validity of ALDH and the associated retinoic acid signaling pathway as chemotherapeutic targets for prostate as well as other cancers. PMID:28149880

  3. Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action

    PubMed Central

    Huo, Lu; Davis, Ian; Liu, Fange; Andi, Babak; Esaki, Shingo; Iwaki, Hiroaki; Hasegawa, Yoshie; Orville, Allen M.; Liu, Aimin

    2015-01-01

    Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NAD+-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp3-to-sp2 transition during enzyme-mediated oxidation. PMID:25565451

  4. Functional characterization of a vanillin dehydrogenase in Corynebacterium glutamicum.

    PubMed

    Ding, Wei; Si, Meiru; Zhang, Weipeng; Zhang, Yaoling; Chen, Can; Zhang, Lei; Lu, Zhiqiang; Chen, Shaolin; Shen, Xihui

    2015-01-27

    Vanillin dehydrogenase (VDH) is a crucial enzyme involved in the degradation of lignin-derived aromatic compounds. Herein, the VDH from Corynebacterium glutamicum was characterized. The relative molecular mass (Mr) determined by SDS-PAGE was ~51 kDa, whereas the apparent native Mr values revealed by gel filtration chromatography were 49.5, 92.3, 159.0 and 199.2 kDa, indicating the presence of dimeric, trimeric and tetrameric forms. Moreover, the enzyme showed its highest level of activity toward vanillin at pH 7.0 and 30°C, and interestingly, it could utilize NAD(+) and NADP(+) as coenzymes with similar efficiency and showed no obvious difference toward NAD(+) and NADP(+). In addition to vanillin, this enzyme exhibited catalytic activity toward a broad range of substrates, including p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, o-phthaldialdehyde, cinnamaldehyde, syringaldehyde and benzaldehyde. Conserved catalytic residues or putative cofactor interactive sites were identified based on sequence alignment and comparison with previous studies, and the function of selected residues were verified by site-directed mutagenesis analysis. Finally, the vdh deletion mutant partially lost its ability to grow on vanillin, indicating the presence of alternative VDH(s) in Corynebacterium glutamicum. Taken together, this study contributes to understanding the VDH diversity from bacteria and the aromatic metabolism pathways in C. glutamicum.

  5. Human choline dehydrogenase: medical promises and biochemical challenges.

    PubMed

    Salvi, Francesca; Gadda, Giovanni

    2013-09-15

    Human choline dehydrogenase (CHD) is located in the inner membrane of mitochondria primarily in liver and kidney and catalyzes the oxidation of choline to glycine betaine. Its physiological role is to regulate the concentrations of choline and glycine betaine in the blood and cells. Choline is important for regulation of gene expression, the biosynthesis of lipoproteins and membrane phospholipids and for the biosynthesis of the neurotransmitter acetylcholine; glycine betaine plays important roles as a primary intracellular osmoprotectant and as methyl donor for the biosynthesis of methionine from homocysteine, a required step for the synthesis of the ubiquitous methyl donor S-adenosyl methionine. Recently, CHD has generated considerable medical attention due to its association with various human pathologies, including male infertility, homocysteinuria, breast cancer and metabolic syndrome. Despite the renewed interest, the biochemical characterization of the enzyme has lagged behind due to difficulties in the obtainment of purified, active and stable enzyme. This review article summarizes the medical relevance and the physiological roles of human CHD, highlights the biochemical knowledge on the enzyme, and provides an analysis based on the comparison of the protein sequence with that of bacterial choline oxidase, for which structural and biochemical information is available.

  6. Not only students can express alcohol dehydrogenase: goldfish can too!

    PubMed

    Chamberland, Valérie; Rioux, Pierre

    2010-12-01

    This article describes a novel approach to study the metabolic regulation of the respiratory system in vertebrates that suits physiology lessons for undergraduate students. It consists of an experimental demonstration of the goldfish's (Carassius auratus) adaptations to anoxia. The goldfish is one of the few vertebrates showing strong enzymatic plasticity for the expression of alcohol dehydrogenase (ADH), which allows it to survive long periods of severe anoxia. Therefore, we propose two simple laboratory exercises in which students are first asked to characterize the distribution of ADH isozymes in the goldfish by performing cellulose acetate electrophoresis. The second part of this laboratory lesson is the determination of liver glycogen. To further student comprehension, an interspecies comparative component is integrated, in which the same subjects are studied in an anoxia-sensitive species, the brook charr (Salvelinus fontinalis). ADH in goldfish is restricted to skeletal muscles, where it catalyzes alcoholic fermentation, permitting ethanol excretion through the gills and therefore preventing lactate acidosis caused by sustained glycolysis during anoxia. Electrophoresis also reveals the occurrence of a liver isozyme in the brook charr, which ADH catalyzes in the opposite pathway, allowing the usual ethanol degradation. As for the liver glycogen assay, it shows largely superior content in the goldfish liver compared with the brook charr, providing goldfish with a sustained energy supply during anoxia. The results of this laboratory exercise clearly demonstrate several physiological strategies developed by goldfish to cope with such a crucial environmental challenge as oxygen depletion.

  7. Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene.

    PubMed Central

    Goldberg, D A

    1980-01-01

    The alcohol dehydrogenase (ADH; alcohol: NAD+ oxidoreductase, EC 1.1.1.1) gene (Adh) of Drosophila melanogaster was isolated by utilizing a mutant strain in which the Adh locus is deleted. Adult RNA from wild-type flies was enriched in ADH sequences by gel electrophoresis and then used to prepare labeled cDNA for screening a bacteriophage lambda library of genomic Drosophila DNA. Of the clones that hybridized in the initial screen, one clone was identified that hybridized with labeled cDNA prepared from a wild-type Drosophila strain but did not hybridize with cDNA prepared from an Adh deletion strain. This clone was shown to contain ADH structural gene sequences by three criteria: in situ hybridization, in vitro translation of mRNA selected by hybridization to the cloned DNA, and comparison of the ADH protein sequence with a nucleotide sequence derived from the cloned DNA. Comparison of the restriction site maps from clones of three different wild-type Drosophila strains revealed the presence of a 200-nucleotide sequence in one strain that was absent from the other two strains. The ADH mRNA sequences were located within the cloned DNA by hybridization mapping experiments. Two intervening sequences were identified within Adh by S1 nuclease mapping experiments. Images PMID:6777776

  8. Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity

    PubMed Central

    Mathias, Rommel A.; Greco, Todd M.; Oberstein, Adam; Budayeva, Hanna G.; Chakrabarti, Rumela; Rowland, Elizabeth A.; Kang, Yibin; Shenk, Thomas; Cristea, Ileana M.

    2014-01-01

    Summary Sirtuins (SIRTs) are critical enzymes that govern genome regulation, metabolism, and aging. Despite conserved deacetylase domains, mitochondrial SIRT4 and SIRT5 have little to no deacetylase activity, and a robust catalytic activity for SIRT4 has been elusive. Here, we establish SIRT4 as a cellular lipoamidase that regulates the pyruvate dehydrogenase complex (PDH). Importantly, SIRT4 catalytic efficiency for lipoyl- and biotinyl-lysine modifications is superior to its deacetylation activity. PDH, which converts pyruvate to acetyl-CoA, has been known to be primarily regulated by phosphorylation of its E1 component. We determine that SIRT4 enzymatically hydrolyzes the lipoamide cofactors from the E2 component dihydrolipoyllysine acetyltransferase (DLAT), diminishing PDH activity. We demonstrate SIRT4-mediated regulation of DLAT lipoyl levels and PDH activity in cells and in vivo, in mouse liver. Furthermore, metabolic flux switching via glutamine stimulation induces SIRT4 lipoamidase activity to inhibit PDH, highlighting SIRT4 as a guardian of cellular metabolism. PMID:25525879

  9. Aldehyde dehydrogenase 2 in cardiac protection: a new therapeutic target?

    PubMed Central

    Budas, Grant R; Disatnik, Marie- Hélène; Mochly-Rosen, Daria

    2010-01-01

    Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is emerging as a key enzyme involved in cytoprotection in the heart. ALDH2 mediates both the detoxification of reactive aldehydes such as acetaldehyde and 4-hydroxy-2-nonenal (4-HNE) and the bioactivation of nitroglycerin (GTN) to nitric oxide (NO). In addition, chronic nitrate treatment results in ALDH2 inhibition and contributes to nitrate tolerance. Our lab recently identified ALDH2 to be a key mediator of endogenous cytoprotection. We reported that ALDH2 is phosphorylated and activated by the survival kinase protein kinase C epsilon (PKCε) and found a strong inverse correlation between ALDH2 activity and infarct size. We also identified a small molecule ALDH2 activator (Alda-1) which reduces myocardial infarct size induced by ischemia/reperfusion in vivo. In this review, we discuss evidence that ALDH2 is a key mediator of endogenous survival signaling in the heart, suggest possible cardioprotective mechanisms mediated by ALDH2, and discuss potential clinical implications of these findings. PMID:20005475

  10. Accelerated Lactate Dehydrogenase Activity Potentiates Osteoclastogenesis via NFATc1 Signaling

    PubMed Central

    Kim, Jin Man; Kwon, So Hyun; Lee, Seoung Hoon; Lee, Soo Young; Jeong, Daewon

    2016-01-01

    Osteoclasts seem to be metabolic active during their differentiation and bone-resorptive activation. However, the functional role of lactate dehydrogenase (LDH), a tetrameric enzyme consisting of an A and/or B subunit that catalyzes interconversion of pyruvate to lactate, in RANKL-induced osteoclast differentiation is not known. In this study, RANKL treatment induced gradual gene expression and activation of the LDH A2B2 isotype during osteoclast differentiation as well as the LDH A1B3 and B4 isotypes during osteoclast maturation after pre-osteoclast formation. Glucose consumption and lactate production in growth media were accelerated during osteoclast differentiation, together with enhanced expression of H+-lactate co-transporter and increased extracellular acidification, demonstrating that glycolytic metabolism was stimulated during differentiation. Further, oxygen consumption via mitochondria was stimulated during osteoclast differentiation. On the contrary, depletion of LDH-A or LDH-B subunit suppressed both glycolytic and mitochondrial metabolism, resulting in reduced mature osteoclast formation via decreased osteoclast precursor fusion and down-regulation of the osteoclastogenic critical transcription factor NFATc1 and its target genes. Collectively, our findings suggest that RANKL-induced LDH activation stimulates glycolytic and mitochondrial respiratory metabolism, facilitating mature osteoclast formation via osteoclast precursor fusion and NFATc1 signaling. PMID:27077737

  11. Green tea polyphenols modulate insulin secretion by inhibiting glutamate dehydrogenase.

    PubMed

    Li, Changhong; Allen, Aron; Kwagh, Jae; Doliba, Nicolai M; Qin, Wei; Najafi, Habiba; Collins, Heather W; Matschinsky, Franz M; Stanley, Charles A; Smith, Thomas J

    2006-04-14

    Insulin secretion by pancreatic beta-cells is stimulated by glucose, amino acids, and other metabolic fuels. Glutamate dehydrogenase (GDH) has been shown to play a regulatory role in this process. The importance of GDH was underscored by features of hyperinsulinemia/hyperammonemia syndrome, where a dominant mutation causes the loss of inhibition by GTP and ATP. Here we report the effects of green tea polyphenols on GDH and insulin secretion. Of the four compounds tested, epigallocatechin gallate (EGCG) and epicatechin gallate were found to inhibit GDH with nanomolar ED(50) values and were therefore found to be as potent as the physiologically important inhibitor GTP. Furthermore, we have demonstrated that EGCG inhibits BCH-stimulated insulin secretion, a process that is mediated by GDH, under conditions where GDH is no longer inhibited by high energy metabolites. EGCG does not affect glucose-stimulated insulin secretion under high energy conditions where GDH is probably fully inhibited. We have further shown that these compounds act in an allosteric manner independent of their antioxidant activity and that the beta-cell stimulatory effects are directly correlated with glutamine oxidation. These results demonstrate that EGCG, much like the activator of GDH (BCH), can facilitate dissecting the complex regulation of insulin secretion by pharmacologically modulating the effects of GDH.

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

    PubMed Central

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

    2013-01-01

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

  13. Modulation of lactate dehydrogenase isozymes by modified base queuine.

    PubMed

    Pathak, C; Vinayak, Manjula

    2005-09-01

    The modified base queuine is a nutrient factor for lower and higher eukaryotes except yeast. It is synthesized in eubacteria and inserted into the wobble position of specific tRNAs (tRNA(GUN)) in exchange of guanine at position 34. The tRNAs of Q family are completely modified in terminally differentiated somatic cells. However, mainly free queuine is present in embryonic and fast proliferating cells, tRNA remains Q deficient. Lactate dehydrogenase (LDH) A mRNA and LDH A protein is known to increase when cells are grown in hypoxic conditions. In the present study, the level of LDH isozymes is analyzed in different tissues of normal and cancerous (DLA) mice and the effect of queuine treatment on LDH isozyme is observed. LDH A isozyme is shown to increase in serum and liver of DLA mice. The level and activity of LDH A decreases on queuine treatment. In skeletal muscle and heart, LDH A isozyme decreases while LDH B increases in DLA mice. Queuine administration leads to change back towards normal. In case of brain, LDH A increases but LDH B decreases in DLA mice. Queuine treatment leads to decrease in A4 anaerobic isozymes of LDH. The results suggest that queuine suppresses anaerobic glycolytic pathway, which leads to tumor suppression of DLA mice.

  14. Glucose-6-phosphate dehydrogenase deficiency: the added value of cytology.

    PubMed

    Roelens, Marie; Dossier, Claire; Fenneteau, Odile; Couque, Nathalie; Da Costa, Lydie

    2016-06-01

    We report the case of a 2 year-old boy hospitalized into the emergency room for influenza pneumonia infection. The evolution was marked by a respiratory distress syndrome, a severe hemolytic anemia, associated with thrombocytopenia and kidney failure. First, a diagnosis of hemolytic uremic syndrome (HUS) has been judiciously suggested due to the classical triad: kidney failure, hemolytic anemia and thrombocytopenia. But, strikingly, blood smears do not exhibit schizocytes, but instead ghosts and hemighosts, some characteristic features of a glucose-6-phosphate dehydrogenase deficiency. Our hypothesis has been confirmed by enzymatic dosage and molecular biology. The unusual initial aplastic feature of this anemia could be the result of a transient erythroblastopenia due to the viral agent, at the origin of the G6PD crisis on a background of a major erythrocyte anti-oxydant enzyme defect. This case of G6PD defect points out the continuously importance of the cytology, which was able to redirect the diagnosis by the hemighost and ghost detection.

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

    PubMed

    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.

  16. Phosphorylation of the pyruvate dehydrogenase complex isolated from Ascaris suum

    SciTech Connect

    Thissen, J.; Komuniecki, R.

    1987-05-01

    The pyruvate dehydrogenase complex (PDC) from body wall muscle of the porcine nematode, Ascaris suum, plays a pivotal role in anaerobic mitochondrial metabolism. As in mammalian mitochondria, PDC activity is inhibited by the phosphorylation of the ..cap alpha..PDH subunit, catalyzed by an associated PDH/sub a/ kinase. However, in contrast to PDC's isolated from all other eukaryotic sources, phosphorylation decreases the mobility of the ..cap alpha..PDH subunit on SDS-PAGE and permits the separation of the phosphorylated and nonphosphorylated ..cap alpha..PDH's. Phosphorylation and the inactivation of the Ascaris PDC correspond directly, and the additional phosphorylation that occurs after complete inactivation in mammalian PDC's is not observed. The purified ascarid PDC incorporates 10 nmoles /sup 32/P/mg P. Autoradiography of the radiolabeled PDC separated by SDS-PAGE yields a band which corresponds to the phosphorylated ..cap alpha..PDH and a second, faint band which is present only during the first three minutes of PDC inactivation, intermediate between the phosphorylated and nonphosphorylated ..cap alpha..PDH subunit. Tryptic digests of the /sup 32/P-PDC yields one major phosphopeptide, when separated by HPLC, and its amino acid sequence currently is being determined.

  17. RECIPIENT PRETRANSPLANT INOSINE MONOPHOSPHATE DEHYDROGENASE ACTIVITY IN NONMYELOABLATIVE HCT

    PubMed Central

    Bemer, Meagan J.; Risler, Linda J.; Phillips, Brian R.; Wang, Joanne; Storer, Barry E.; Sandmaier, Brenda M.; Duan, Haichuan; Raccor, Brianne S.; Boeckh, Michael J.; McCune, Jeannine S.

    2014-01-01

    Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5’- monophosphate (IMP) to xanthosine 5’-monophosphate (XMP). We developed a highly sensitive liquid chromatography–mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNC) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T-cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation, but not with chronic GVHD, relapse, non-relapse mortality, or overall mortality. We conclude that quantitation of the recipient’s pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient’s sensitivity to MMF, but confirmatory studies are needed. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients. PMID:24923537

  18. An animal model of human aldehyde dehydrogenase deficiency

    SciTech Connect

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

    1994-09-01

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

  19. Determinants of performance in the isocitrate dehydrogenase of Escherichia coli.

    PubMed Central

    Dean, A. M.; Shiau, A. K.; Koshland, D. E.

    1996-01-01

    The substrate specificity of the NADP-dependent isocitrate dehydrogenase of Escherichia coli was investigated by combining site-directed mutagenesis and utilization of alternative substrates. A comparison of the kinetics of the wild-type enzyme with 2R-malate reveals that the gamma-carboxylate of 2R,3S-isocitrate contributes a factor of 12,000,000 to enzyme performance. Analysis of kinetic data compiled for 10 enzymes and nine different substrates reveals that a factor of 1,650 can be ascribed to the hydrogen bond formed between S113 and the gamma-carboxylate of bound isocitrate, a factor of 150 to the negative charge of the gamma-carboxylate, and a factor of 50 for the gamma-methyl. These results are entirely consistent with X-ray structures of Michaelis complexes that show a hydrogen bond positions the gamma-carboxylate of isocitrate so that a salt bridge can form to the nicotinamide ring of NADP. PMID:8745412

  20. Reactivity of the sulfhydryl groups of soluble succinate dehydrogenase.

    PubMed

    Vinogradov, A D; Gavrikova, E V; Zuevsky, V V

    1976-04-01

    Soluble succinate dehydrogenase prepared by butanol extraction reacts with N-ethylmaleimide according to first-order kinetics with respect to both remaining active enzyme and the inhibitor concentration. Binding of the sulfhydryl groups of the enzyme prevents its alkylation by N-ethylmaleimide and inhibition by oxaloacetate. A kinetic analysis of the inactivation of alkylating reagent in the presence of succinate or malonate suggests that N-ethylmaleimide acts as a site-directed inhibitor. The apparent first-order rate constant of alkylation increases between pH 5.8 and 7.8 indicating a pKa value for the enzyme sulfhydryl group equal to 7.0 at 22 degrees C in 50 mM Tris-sufate buffer. Certain anions (phosphate, citrate, maleate and acetate) decrease the reactivity of the enzyme towards the alkylating reagent. Succinate/phenazine methosulfate reductase activity measured in the presence of a saturating concentration of succinate shows the same pH-dependence as the alkylation rate by N-ethylmaleimide. The mechanism of the first step of succinate oxidation, including a nucleophilic attack of substrate by the active-site sulfhydryl group, is discussed.

  1. Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes

    SciTech Connect

    Castonguay, Zachary; Auger, Christopher; Thomas, Sean C.; Chahma, M’hamed; Appanna, Vasu D.

    2014-11-07

    Highlights: • Nuclear LDH is up-regulated under oxidative stress. • SIRT1 is co-immunoprecipitated bound to nuclear LDH. • Nuclear LDH is involved in histone deacetylation and epigenetics. - Abstract: It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD{sup +}), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H{sub 2}O{sub 2}) in the culture medium. Under oxidative stress, the NAD{sup +} generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD{sup +} reveals an intricate link between metabolism and the processing of genetic information.

  2. Sequence variation of alcohol dehydrogenase (Adh) paralogs in cactophilic Drosophila.

    PubMed Central

    Matzkin, Luciano M; Eanes, Walter F

    2003-01-01

    This study focuses on the population genetics of alcohol dehydrogenase (Adh) in cactophilic Drosophila. Drosophila mojavensis and D. arizonae utilize cactus hosts, and each host contains a characteristic mixture of alcohol compounds. In these Drosophila species there are two functional Adh loci, an adult form (Adh-2) and a larval and ovarian form (Adh-1). Overall, the greater level of variation segregating in D. arizonae than in D. mojavensis suggests a larger population size for D. arizonae. There are markedly different patterns of variation between the paralogs across both species. A 16-bp intron haplotype segregates in both species at Adh-2, apparently the product of an ancient gene conversion event between the paralogs, which suggests that there is selection for the maintenance of the intron structure possibly for the maintenance of pre-mRNA structure. We observe a pattern of variation consistent with adaptive protein evolution in the D. mojavensis lineage at Adh-1, suggesting that the cactus host shift that occurred in the divergence of D. mojavensis from D. arizonae had an effect on the evolution of the larval expressed paralog. Contrary to previous work we estimate a recent time for both the divergence of D. mojavensis and D. arizonae (2.4 +/- 0.7 MY) and the age of the gene duplication (3.95 +/- 0.45 MY). PMID:12586706

  3. Yeast cell-based analysis of human lactate dehydrogenase isoforms.

    PubMed

    Mohamed, Lulu Ahmed; Tachikawa, Hiroyuki; Gao, Xiao-Dong; Nakanishi, Hideki

    2015-12-01

    Human lactate dehydrogenase (LDH) has attracted attention as a potential target for cancer therapy and contraception. In this study, we reconstituted human lactic acid fermentation in Saccharomyces cerevisiae, with the goal of constructing a yeast cell-based LDH assay system. pdc null mutant yeast (mutated in the endogenous pyruvate decarboxylase genes) are unable to perform alcoholic fermentation; when grown in the presence of an electron transport chain inhibitor, pdc null strains exhibit a growth defect. We found that introduction of the human gene encoding LDHA complemented the pdc growth defect; this complementation depended on LDHA catalytic activity. Similarly, introduction of the human LDHC complemented the pdc growth defect, even though LDHC did not generate lactate at the levels seen with LDHA. In contrast, the human LDHB did not complement the yeast pdc null mutant, although LDHB did generate lactate in yeast cells. Expression of LDHB as a red fluorescent protein (RFP) fusion yielded blebs in yeast, whereas LDHA-RFP and LDHC-RFP fusion proteins exhibited cytosolic distribution. Thus, LDHB exhibits several unique features when expressed in yeast cells. Because yeast cells are amenable to genetic analysis and cell-based high-throughput screening, our pdc/LDH strains are expected to be of use for versatile analyses of human LDH.

  4. Single amino acid polymorphism in aldehyde dehydrogenase gene superfamily.

    PubMed

    Priyadharshini Christy, J; George Priya Doss, C

    2015-01-01

    The aldehyde dehydrogenase gene superfamily comprises of 19 genes and 3 pseudogenes. These superfamily genes play a vital role in the formation of molecules that are involved in life processes, and detoxification of endogenous and exogenous aldehydes. ALDH superfamily genes associated mutations are implicated in various diseases, such as pyridoxine-dependent seizures, gamma-hydroxybutyric aciduria, type II Hyperprolinemia, Sjogren-Larsson syndrome including cancer and Alzheimer's disease. Accumulation of large DNA variations data especially Single Amino acid Polymorphisms (SAPs) in public databases related to ALDH superfamily genes insisted us to conduct a survey on the disease associated mutations and predict their function impact on protein structure and function. Overall this study provides an update and highlights the importance of pathogenic mutations in associated diseases. Using KD4v and Project HOPE a computational based platform, we summarized all the deleterious properties of SAPs in ALDH superfamily genes by the providing valuable insight into structural alteration rendered due to mutation. We hope this review might provide a way to define the deleteriousness of a SAP and helps to understand the molecular basis of the associated disease and also permits precise diagnosis and treatment in the near future.

  5. Triazaspirodimethoxybenzoyls as Selective Inhibitors of Mycobacterial Lipoamide Dehydrogenase

    SciTech Connect

    Bryk, Ruslana; Arango, Nancy; Venugopal, Aditya; Warren, J. David; Park, Yun-Hee; Patel, Mulchand S.; Lima, Christopher D.; Nathan, Carl

    2010-06-25

    Mycobacterium tuberculosis (Mtb) remains the leading single cause of death from bacterial infection. Here we explored the possibility of species-selective inhibition of lipoamide dehydrogenase (Lpd), an enzyme central to Mtb's intermediary metabolism and antioxidant defense. High-throughput screening of combinatorial chemical libraries identified triazaspirodimethoxybenzoyls as high-nanomolar inhibitors of Mtb's Lpd that were noncompetitive versus NADH, NAD{sup +}, and lipoamide and >100-fold selective compared to human Lpd. Efficacy required the dimethoxy and dichlorophenyl groups. The structure of an Lpd-inhibitor complex was resolved to 2.42 {angstrom} by X-ray crystallography, revealing that the inhibitor occupied a pocket adjacent to the Lpd NADH/NAD{sup +} binding site. The inhibitor did not overlap with the adenosine moiety of NADH/NAD{sup +} but did overlap with positions predicted to bind the nicotinamide rings in NADH and NAD{sup +} complexes. The dimethoxy ring occupied a deep pocket adjacent to the FAD flavin ring where it would block coordination of the NADH nicotinamide ring, while the dichlorophenyl group occupied a more exposed pocket predicted to coordinate the NAD{sup +} nicotinamide. Several residues that are not conserved between the bacterial enzyme and its human homologue were predicted to contribute both to inhibitor binding and to species selectivity, as confirmed for three residues by analysis of the corresponding mutant Mtb Lpd proteins. Thus, nonconservation of residues lining the electron-transfer tunnel in Mtb Lpd can be exploited for development of species-selective Lpd inhibitors.

  6. Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii.

    PubMed

    Guo, Qi; Gakhar, Lokesh; Wickersham, Kyle; Francis, Kevin; Vardi-Kilshtain, Alexandra; Major, Dan T; Cheatum, Christopher M; Kohen, Amnon

    2016-05-17

    The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system for studies on the role of protein dynamics in catalysis, but so far these studies have been limited by the availability of structural information. Second, CbFDH and its mutants can be used in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in commercial development. Here, we report the crystallization and structural determination of both holo- and apo-CbFDH. The free-energy barrier for the catalyzed reaction was computed and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: the apoenzyme crystal was obtained under cocrystallization conditions with a saturating concentration of both the cofactor (NAD(+)) and inhibitor (azide), which has a nanomolar dissociation constant. It was found that the fraction of the apoenzyme present in the solution is less than 1.7 × 10(-7) (i.e., the solution is 99.9999% holoenzyme). This is an extreme case where the crystal structure represents an insignificant fraction of the enzyme in solution, and a mechanism rationalizing this phenomenon is presented.

  7. Nitrated carbon nanoblisters for high-performance glucose dehydrogenase bioanodes.

    PubMed

    de Souza, João C P; Iost, Rodrigo M; Crespilho, Frank N

    2016-03-15

    Recently, many strategies are being explored for efficiently wiring glucose dehydrogenase (GDh) enzymes capable of glucose (fuel) oxidation. For instance, the use of GDh NAD(+)-dependent for glucose oxidation is of great interest in biofuel cell technology because the enzyme are unaffected by the presence of molecular oxygen commonly present in electrolyte. Here we present the fabrication of flexible carbon fibers modified with nitrated carbon nanoblisters and their application as high-performance GDh bioanodes. These bioelectrodes could electro-oxidize glucose at -360 mV (vs. Ag/AgClsat) in the presence of a molecular oxygen saturated electrolyte with current densities higher than 1.0 mAcm(-2) at 0.0 V. It is corroborated by open circuit potential, where a potential stabilization occurs at -150 mV in a long term stability current-transient experiment. This value is in agreement with the quasi-steady current obtained at very low scan rate (0.1 mVs(-1)), where the onset potential for glucose oxidation is -180 mV. X-ray photoelectron spectroscopy and scanning electron microscopy revealed that the nitrated blisters and edge-like carbon structures, enabling highly efficient enzyme immobilization and low overpotential for electron transfer, allowing for glucose oxidation with potential values close to the thermodynamic cofactor.

  8. Glutamate dehydrogenase 1 and SIRT4 regulate glial development.

    PubMed

    Komlos, Daniel; Mann, Kara D; Zhuo, Yue; Ricupero, Christopher L; Hart, Ronald P; Liu, Alice Y-C; Firestein, Bonnie L

    2013-03-01

    Congenital hyperinsulinism/hyperammonemia (HI/HA) syndrome is caused by an activation mutation of glutamate dehydrogenase 1 (GDH1), a mitochondrial enzyme responsible for the reversible interconversion between glutamate and α-ketoglutarate. The syndrome presents clinically with hyperammonemia, significant episodic hypoglycemia, seizures, and frequent incidences of developmental and learning defects. Clinical research has implicated that although some of the developmental and neurological defects may be attributed to hypoglycemia, some characteristics cannot be ascribed to low glucose and as hyperammonemia is generally mild and asymptomatic, there exists the possibility that altered GDH1 activity within the brain leads to some clinical changes. GDH1 is allosterically regulated by many factors, and has been shown to be inhibited by the ADP-ribosyltransferase sirtuin 4 (SIRT4), a mitochondrially localized sirtuin. Here we show that SIRT4 is localized to mitochondria within the brain. SIRT4 is highly expressed in glial cells, specifically astrocytes, in the postnatal brain and in radial glia during embryogenesis. Furthermore, SIRT4 protein decreases in expression during development. We show that factors known to allosterically regulate GDH1 alter gliogenesis in CTX8 cells, a novel radial glial cell line. We find that SIRT4 and GDH1 overexpression play antagonistic roles in regulating gliogenesis and that a mutant variant of GDH1 found in HI/HA patients accelerates the development of glia from cultured radial glia cells.

  9. Asparaginyl deamidation in two glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae.

    PubMed

    DeLuna, Alexander; Quezada, Héctor; Gómez-Puyou, Armando; González, Alicia

    2005-03-25

    The non-enzymatic deamidation of asparaginyl residues is a major source of spontaneous damage of several proteins under physiological conditions. In many cases, deamidation and isoaspartyl formation alters the biological activity or stability of the native polypeptide. Rates of deamidation of particular residues depend on many factors including protein structure and solvent exposure. Here, we investigated the spontaneous deamidation of the two NADP-glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae, which have different kinetic properties and are differentially expressed in this yeast. Our results show that Asn54, present in Gdh3p but missing in the GDH1-encoded homologue, is readily deamidated in vitro under alkaline conditions. Relative to the native enzyme, deamidated Gdh3p shows reduced protein stability. The different deamidation rates of the two isoenzymes could explain to some extent, the relative in vivo instability of the allosteric Gdh3p enzyme, compared to that of Gdh1p. It is thus possible that spontaneous asparaginyl modification could play a role in the metabolic regulation of ammonium assimilation and glutamate biosynthesis.

  10. Regulation by ammonium of glutamate dehydrogenase (NADP+) from Saccharomyces cerevisiae.

    PubMed

    Bogonez, E; Satrústegui, J; Machado, A

    1985-06-01

    The activity of glutamate dehydrogenase (NADP+) (EC 1.4.1.4; NADP-GDH) of Saccharomyces cerevisiae is decreased under conditions in which intracellular ammonia concentrations increases. A high internal ammonia concentration can be obtained (a) by increasing the ammonium sulphate concentration in the culture medium, and (b) by growing the yeast either in acetate + ammonia media, where the pH of the medium rises during growth, or in heavily buffered glucose + ammonia media at pH 7.5. Under these conditions cellular oxoglutarate concentrations do not vary and changes in NADP-GDH activity appear to provide a constant rate of oxoglutarate utilization. The following results suggest that the decrease in NADP-GDH activity in ammonia-accumulating yeast cells is brought about by repression of synthesis: (i) after a shift to high ammonium sulphate concentrations, the number of units of activity per cell decreased as the inverse of cell doubling; and (ii) the rate of degradation of labelled NADP-GDH was essentially the same in ammonia-accumulating yeast cells and in controls, whereas the synthesis constant was much lower in the ammonia-accumulating cells than in the controls.

  11. The role of glutamate dehydrogenase in mammalian ammonia metabolism.

    PubMed

    Spanaki, Cleanthe; Plaitakis, Andreas

    2012-01-01

    Glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and ammonia. High levels of GDH activity is found in mammalian liver, kidney, brain, and pancreas. In the liver, GDH reaction appears to be close-to-equilibrium, providing the appropriate ratio of ammonia and amino acids for urea synthesis in periportal hepatocytes. In addition, GDH produces glutamate for glutamine synthesis in a small rim of pericentral hepatocytes. Hence, hepatic GDH can be either a source for ammonia or an ammonia scavenger. In the kidney, GDH function produces ammonia from glutamate to control acidosis. In the human, the presence of two differentially regulated isoforms (hGDH1 and hGDH2) suggests a complex role for GDH in ammonia homeostasis. Whereas hGDH1 is sensitive to GTP inhibition, hGDH2 has dissociated its function from GTP control. Furthermore, hGDH2 shows a lower optimal pH than hGDH1. The hGDH2 enzyme is selectively expressed in human astrocytes and Sertoli cells, probably facilitating metabolic recycling processes essential for their supportive role. Here, we report that hGDH2 is also expressed in the epithelial cells lining the convoluted tubules of the renal cortex. As hGDH2 functions more efficiently under acidotic conditions without the operation of the GTP energy switch, its presence in the kidney may increase the efficacy of the organ to maintain acid base equilibrium.

  12. Alcohol dehydrogenase and an inactivator from rice seedlings

    SciTech Connect

    Shimomura, S.; Beevers, H.

    1983-01-01

    Alcohol dehydrogenase (ADH) was measured in the various organs of rice seedlings (Oryza sativa) growing in air. In extracts from ungerminated seeds, the ADH is stable, but in extracts from seedlings more than 2 days old the enzyme initially present loses activity in a time- and temperature-dependent fashion, due to the presence of an inactivating component which increases with age in roots and shoots. The inactivation can be prevented completely by dithiothreitol, and when this is included in the extraction medium the apparent loss of total ADH in roots and shoots with age is not observed. In seedlings grown in N/sub 2/, ADA levels in coleoptile extracts are higher than those in air, the enzyme is stable, and no inactivator can be detected. When seedlings grown for 5 days in air were transferred to N/sub 2/ for 3 days, ADA levels increased and there was a decline in inactivator activity. Transfer back to air after 1 day in N/sub 2/ led to loss of the accumulated ADH and increase in inactivator. These reciprocal changes and the fact that the inactivator is absent from coleoptiles of seedlings grown in N/sub 2/ appear to suggest a regulator role for the inactivator in vivo. However, it is clear that high levels of inactivator and ADH can exist in cells of seedlings grown in air for long periods without loss of enzyme activity, and it is argued that they must normally be separately compartmented.

  13. Psychotic mania in glucose-6-phosphate-dehydrogenase-deficient subjects

    PubMed Central

    Bocchetta, Alberto

    2003-01-01

    Background Glucose-6-phosphate dehydrogenase (G6PD) deficiency has been associated with acute psychosis, catatonic schizophrenia, and bipolar disorders by previous inconclusive reports. A particularly disproportionate rate of enzyme deficiency was found in manic schizoaffective patients from 662 lithium patients surveyed in Sardinia. The purpose of this study was to describe clinical characteristics which may be potentially associated with G6PD deficiency. Methods Characteristics of episodes, course of illness, family pattern of illness, laboratory tests, and treatment response of 29 G6PD-deficient subjects with a Research Diagnostic Criteria diagnosis of manic schizoaffective disorder were abstracted from available records. Results The most peculiar pattern was that of acute recurrent psychotic manic episodes, mostly characterized by loosening of associations, agitation, catatonic symptoms, and/or transient confusion, concurrent hyperbilirubinemia, positive psychiatric family history, and partial response to long-term lithium treatment. Conclusions A relationship between psychiatric disorder and G6PD deficiency is to be searched in the bipolar spectrum, particularly among patients with a history of acute episodes with psychotic and/or catatonic symptoms or with transient confusion. PMID:12844366

  14. SERUM VALUES OF ALKALINE PHOSPHATASE AND LACTATE DEHYDROGENASE IN OSTEOSARCOMA

    PubMed Central

    ZUMÁRRAGA, JUAN PABLO; BAPTISTA, ANDRÉ MATHIAS; ROSA, LUIS PABLO DE LA; CAIERO, MARCELO TADEU; CAMARGO, OLAVO PIRES DE

    2016-01-01

    ABSTRACT Objective: To study the relationship between the pre and post chemotherapy (CT) serum levels of alkaline phosphatase (AP) and lactate dehydrogenase (LDH), and the percentage of tumor necrosis (TN) found in specimens after the pre surgical CT in patients with osteosarcoma. Methods: Series of cases with retrospective evaluation of patients diagnosed with osteosarcoma. Participants were divided into two groups according to serum values of both enzymes. The values of AP and LDH were obtained before and after preoperative CT. The percentage of tumor necrosis (TN) of surgical specimens of each patient was also included. Results: One hundred and thirty seven medical records were included from 1990 to 2013. Both the AP as LDH decreased in the patients studied, being the higher in pre CT than post CT. The average LHD decrease was 795.12U/L and AP decrease was 437.40 U/L. The average TN was 34.10 %. There was no statistically significant correlation between the serums values and the percentage of tumoral necrosis. Conclusion: The serum levels values of AP and LDH are not good predictors for the chemotherapy-induced necrosis in patients with osteosarcoma. Level of Evidence IV, Case Series. PMID:27217815

  15. Triazaspirodimethoxybenzoyls as selective inhibitors of mycobacterial lipoamide dehydrogenase .

    PubMed

    Bryk, Ruslana; Arango, Nancy; Venugopal, Aditya; Warren, J David; Park, Yun-Hee; Patel, Mulchand S; Lima, Christopher D; Nathan, Carl

    2010-03-02

    Mycobacterium tuberculosis (Mtb) remains the leading single cause of death from bacterial infection. Here we explored the possibility of species-selective inhibition of lipoamide dehydrogenase (Lpd), an enzyme central to Mtb's intermediary metabolism and antioxidant defense. High-throughput screening of combinatorial chemical libraries identified triazaspirodimethoxybenzoyls as high-nanomolar inhibitors of Mtb's Lpd that were noncompetitive versus NADH, NAD(+), and lipoamide and >100-fold selective compared to human Lpd. Efficacy required the dimethoxy and dichlorophenyl groups. The structure of an Lpd-inhibitor complex was resolved to 2.42 A by X-ray crystallography, revealing that the inhibitor occupied a pocket adjacent to the Lpd NADH/NAD(+) binding site. The inhibitor did not overlap with the adenosine moiety of NADH/NAD(+) but did overlap with positions predicted to bind the nicotinamide rings in NADH and NAD(+) complexes. The dimethoxy ring occupied a deep pocket adjacent to the FAD flavin ring where it would block coordination of the NADH nicotinamide ring, while the dichlorophenyl group occupied a more exposed pocket predicted to coordinate the NAD(+) nicotinamide. Several residues that are not conserved between the bacterial enzyme and its human homologue were predicted to contribute both to inhibitor binding and to species selectivity, as confirmed for three residues by analysis of the corresponding mutant Mtb Lpd proteins. Thus, nonconservation of residues lining the electron-transfer tunnel in Mtb Lpd can be exploited for development of species-selective Lpd inhibitors.

  16. Site Saturation Mutagenesis Applications on Candida methylica Formate Dehydrogenase

    PubMed Central

    Özgün, Gülşah P.; Ordu, Emel B.; Tütüncü, H. Esra; Yelboğa, Emrah; Sessions, Richard B.

    2016-01-01

    In NADH regeneration, Candida methylica formate dehydrogenase (cmFDH) is a highly significant enzyme in pharmaceutical industry. In this work, site saturation mutagenesis (SSM) which is a combination of both rational design and directed evolution approaches is applied to alter the coenzyme specificity of NAD+-dependent cmFDH from NAD+ to NADP+ and increase its thermostability. For this aim, two separate libraries are constructed for screening a change in coenzyme specificity and an increase in thermostability. To alter the coenzyme specificity, in the coenzyme binding domain, positions at 195, 196, and 197 are subjected to two rounds of SSM and screening which enabled the identification of two double mutants D195S/Q197T and D195S/Y196L. These mutants increase the overall catalytic efficiency of NAD+ to 5.6 × 104-fold and 5 × 104-fold value, respectively. To increase the thermostability of cmFDH, the conserved residue at position 1 in the catalytic domain of cmFDH is subjected to SSM. The thermodynamic and kinetic results suggest that 8 mutations on the first residue can be tolerated. Among all mutants, M1L has the best residual activity after incubation at 60°C with 17%. These studies emphasize that SSM is an efficient method for creating “smarter libraries” for improving the properties of cmFDH. PMID:27847673

  17. Genetic control of alcohol dehydrogenase levels in Drosophila.

    PubMed

    Maroni, G

    1978-06-01

    Among the progeny of Drosophila flies heterozygous for two noncomplementing Adh-negative alleles, two individuals were found that had recovered appreciable alcohol dehydrogenase activity, thereby surviving the ethanol medium used as a screen. The most likely explanation is that these Adh-positive flies are the product of intracistronic recombination within the Adh locus. Judging by the distribution of outside markers, one of the crossovers would have been a conventional reciprocal exchange while the other appears to have been an instance of nonreciprocal recombination. The enzymes produced in strains derived from the original survivors can be easily distinguished from wild-type enzymes ADH-S and ADH-F on the basis of their sensitivity to denaturing agents. None of various physical and catalytic properties tested revealed differences between the enzymes of the survivor strains except that in one of them the level of activity is 55--65% of the other. Quantitative immunological determinations of ADH gave estimates of enzyme protein which are proportional to the measured activity levels. These results are interpreted to indicate that different amounts of ADH protein are being accumulated in the two strains.

  18. Structure of Plasmodium falciparum dihydroorotate dehydrogenase with a bound inhibitor.

    PubMed

    Hurt, Darrell E; Widom, Joanne; Clardy, Jon

    2006-03-01

    Membrane-associated dihydroorotate dehydrogenase (DHODH) is an antimalarial therapeutic target without an effective inhibitor. Studies on human DHODH (HsDHODH) led to a structural mechanistic model in which respiratory quinones bind in a tunnel formed by the highly variable N-terminus that leads to the flavin mononucleotide-binding site. The therapeutic agents leflunomide (Arava) and brequinar sodium inhibit HsDHODH by binding in this tunnel. Plasmodium falciparum DHODH (PfDHODH) and HsDHODH have markedly different sensitivities to the two drugs. To understand the structural basis of this differential sensitivity and begin a structure-based drug-design cycle for PfDHODH inhibitors, the three-dimensional structure (2.4 Angstroms, R = 20.1%) of PfDHODH bound to the active metabolite of leflunomide was determined by X-ray crystallography. Comparison of the structures of HsDHODH and PfDHODH reveals a completely different binding mode for the same inhibitor in these two catalytically identical enzymes and explains the previously observed species-specific preferential binding. Because no effective inhibitors have been described for PfDHODH, this structure provides critical insight for the design of potential antimalarials.

  19. New isozyme systems for maize (Zea mays L.): aconitate hydratase, adenylate kinase, NADH dehydrogenase, and shikimate dehydrogenase.

    PubMed

    Wendel, J F; Goodman, M M; Stuber, C W; Beckett, J B

    1988-06-01

    Electrophoretic variation and inheritance of four novel enzyme systems were studied in maize (Zea mays L.). A minimum of 10 genetic loci collectively encodes isozymes of aconitate hydratase (ACO; EC 4.2.1.3.), adenylate kinase (ADK; EC 2.7.4.3), NADH dehydrogenase (DIA; EC 1.6.99.-), and shikimate dehydrogenase (SAD; EC 1.1.1.25). At least four loci are responsible for the genetic control of ACO. Genetic data for two of the encoding loci, Aco1 and Aco4, demonstrated that at least two maize ACOs are active as monomers. Analysis of organellar preparations suggests that ACO1 and ACO4 are localized in the cytosolic and mitochondrial subcellular fractions, respectively. Maize ADK is encoded by a single nuclear locus, Adk1, governing monomeric enzymes that are located in the chloroplasts. Two cytosolic and two mitochondrial forms of DIA were electrophoretically resolved. Segregation analyses demonstrated that the two cytosolic isozymes are controlled by separate loci, Dia1 and Dia2, coding for products that are functional as monomers (DIA1) and dimers (DIA2). The major isozyme of SAD is apparently cytosolic, although an additional faintly staining plastid form may be present. Alleles at Sad1 are each associated with two bands that cosegregate in controlled crosses. Linkage analyses and crosses with B-A translocation stocks were effective in determining the map locations of six loci, including the previously described but unmapped locus Acp4. Several of these loci were localized to sparsely mapped regions of the genome. Dia2 and Acp4 were placed on the distal portion of the long arm of chromosome 1, 12.6 map units apart. Dia1 was localized to chromosome 2, 22.2 centimorgans (cM) from B1. Aco1 was mapped to chromosome 4, 6.2 cM from su1. Adk1 was placed on the poorly marked short arm of chromosome 6, 8.1 map units from rgd1. Less than 1% recombination was observed between Glu1 (on chromosome 10) and Sad1. In contrast to many other maize isozyme systems, there was little

  20. [Glutamate dehydrogenase activity of Bradyrhizobium japonicum in the presence of phytoregulators].

    PubMed

    Leonova, N O; Tytova, L V; Tantsiurenko, O V; Antypchuk, A F

    2006-01-01

    Influence of plant growth regulators ivin and emistim C, and flavonoids daidzein and quercetin on the glutamate dehydrogenase activity of soybean nodule bacteria, with contrasting symbiotic properties, were studied. It was shown that all used phytoregulators stimulated glutamate dehydrogenase activity of Bradyrhizobium japonicum 71t (the strain with highly efficient symbiotic properties) 1.2-4.9 times. Bradyrhizobium japonicum 21110 (the strain with inefficient symbiotic properties) diminished the enzyme activity in the presence of all phythoregulators except for ivin.

  1. Gene silencing in phlebotomine sand flies: Xanthine dehydrogenase knock down by dsRNA microinjections.

    PubMed

    Sant'Anna, Mauricio R; Alexander, Bruce; Bates, Paul A; Dillon, Rod J

    2008-06-01

    Lutzomyia longipalpis are vectors of medically important visceral leishmaniasis in South America. Blood-fed adult females digest large amounts of protein, and xanthine dehydrogenase is thought to be a key enzyme involved in protein catabolism through the production of urate. Large amounts of heme are also released during digestion with potentially damaging consequences, as heme can generate oxygen radicals that damage lipids, proteins and nucleic acids. However, urate is an antioxidant that may prevent such oxidative damage produced by heme. We investigated xanthine dehydrogenase by developing the RNAi technique for sand flies and used this technique to knock down the Lu. longipalpis xanthine dehydrogenase gene to evaluate its role in survival of adult females after blood feeding. The gene sequence of Lu. longipalpis xanthine dehydrogenase is described together with expression in different life cycle stages and RNAi knock down. Semi-quantitative RT-PCR of xanthine dehydrogenase expression showed a significant increase in expression after bloodmeal ingestion. Microinjection of dsRNA via the thorax of 1-day-old adult female sand flies resulted in approximately 40% reduction of xanthine dehydrogenase gene expression in comparison to flies injected with a control dsRNA. A significant reduction of urate in the whole body and excretions of Lu. longipalpis was observed after dsRNA xanthine dehydrogenase microinjection and feeding 96h later on rabbit blood. Sand flies injected with XDH dsRNA also exhibit significantly reduced life span in comparison with the mock-injected group when fed on sucrose or when rabbit blood fed, showing that urate could be indeed an important free radical scavenger in Lu. longipalpis. The demonstration of xanthine dehydrogenase knock down by dsRNA microinjection, low mortality of microinjected insects and the successful bloodfeeding of injected insects demonstrated the utility of RNAi as a tool for functional analysis of genes in phlebotomine

  2. Structural Basis for "Flip-Flop" Action of Human Pyruvate Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Ciszak, Ewa; Korotchkina, Lioubov; Dominiak, Paulina; Sidhu, Sukhdeep; Patel, Mulchand

    2003-01-01

    The derivative of vitamin B1, thiamin pyrophosphate is a cofactor of pyruvate dehydrogenase, a component enzyme of the mitochondrial pyruvate dehydrogenase multienzyme complex that plays a major role in directing energy metabolism in the cell. This cofactor is used to cleave the C(sup alpha)-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. In alpha(sub 2)beta(sub 2)-tetrameric human pyruvate dehydrogenase, there are two cofactor binding sites, each of them being a center of independently conducted, although highly coordinated enzymatic reactions. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites can now be understood based on the recently determined crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95A resolution. The structure of pyruvate dehydrogenase was determined using a combination of MAD phasing and molecular replacement followed by rounds of torsion-angles molecular-dynamics simulated-annealing refinement. The final pyruvate dehydrogenase structure included coordinates for all protein amino acids two cofactor molecules, two magnesium and two potassium ions, and 742 water molecules. The structure was refined to R = 0.202 and R(sub free) = 0.244. Our structural analysis of the enzyme folding and domain assembly identified a simple mechanism of this protein motion required for the conduct of catalytic action.

  3. Characteristics of external and internal NAD(P)H dehydrogenases in Hoya carnosa mitochondria.

    PubMed

    Hong, Hoang Thi Kim; Nose, Akihiro

    2012-12-01

    This study aims at characterizing NAD(P)H dehydrogenases on the inside and outside of the inner membrane of mitochondria of one phosphoenolpyruvate carboxykinase-crassulacean acid metabolism plant, Hoya carnosa. In crassulacean acid metabolism plants, NADH is produced by malate decarboxylation inside and outside mitochondria. The relative importance of mitochondrial alternative NADH dehydrogenases and their association was determined in intact-and alamethicin-permeabilized mitochondria of H. carnosa to discriminate between internal and external activities. The major findings in H. carnosa mitochondria are: (i) external NADPH oxidation is totally inhibited by DPI and totally dependent on Ca(2+), (ii) external NADH oxidation is partially inhibited by DPI and mainly dependent on Ca(2+), (iii) total NADH oxidation measured in permeabilized mitochondria is partially inhibited by rotenone and also by DPI, (iv) total NADPH oxidation measured in permeabilized mitochondria is partially dependent on Ca(2+) and totally inhibited by DPI. The results suggest that complex I, external NAD(P)H dehydrogenases, and internal NAD(P)H dehydrogenases are all linked to the electron transport chain. Also, the total measurable NAD(P)H dehydrogenases activity was less than the total measurable complex I activity, and both of these enzymes could donate their electrons not only to the cytochrome pathway but also to the alternative pathway. The finding indicated that the H. carnosa mitochondrial electron transport chain is operating in a classical way, partitioning to both Complex I and alternative Alt. NAD(P)H dehydrogenases.

  4. Decrease in nicotinamide adenine dinucleotide dehydrogenase is related to skin pigmentation.

    PubMed

    Nakama, Mitsuo; Murakami, Yuhko; Tanaka, Hiroshi; Nakata, Satoru

    2012-03-01

    Skin pigmentation is caused by various physical and chemical factors. It might also be influenced by changes in the physiological function of skin with aging. Nicotinamide adenine dinucleotide (NADH) dehydrogenase is an enzyme related to the mitochondrial electron transport system and plays a key role in cellular energy production. It has been reported that the functional decrease in this system causes Parkinson's disease. Another study reports that the amount of NADH dehydrogenase in heart and skeletal muscle decreases with aging. A similar decrease in the skin would probably affect its physiological function. However, no reports have examined the age-related change in levels of NADH dehydrogenase in human skin. In this study, we investigated this change and its effect on skin pigmentation using cultured human epidermal keratinocytes. The mRNA expression of NDUFA1, NDUFB7, and NDUFS2, subunits of NADH dehydrogenase, and its activity were significantly decreased in late passage keratinocytes compared to early passage cells. Conversely, the mRNA expression of melanocyte-stimulating cytokines, interleukin-1 alpha and endothelin 1, was increased in late passage cells. On the other hand, the inhibition of NADH dehydrogenase upregulated the mRNA expression of melanocyte-stimulating cytokines. Moreover, the level of NDUFB7 mRNA was lower in pigmented than in nonpigmented regions of skin in vivo. These results suggest the decrease in NADH dehydrogenase with aging to be involved in skin pigmentation.

  5. Hepatic alcohol dehydrogenase activity in alcoholic subjects with and without liver disease.

    PubMed Central

    Vidal, F; Perez, J; Morancho, J; Pinto, B; Richart, C

    1990-01-01

    Alcohol dehydrogenase activity was measured in samples of liver tissue from a group of alcoholic and non-alcoholic subjects to determine whether decreased liver alcohol dehydrogenase activity is a consequence of ethanol consumption or liver damage. The alcoholic patients were classified further into the following groups: control subjects with no liver disease (group 1), subjects with non-cirrhotic liver disease (group 2), and subjects with cirrhotic liver disease (group 3). The non-alcoholic subjects were also divided, using the same criteria, into groups 4, 5, and 6, respectively. The analysis of the results showed no significant differences when mean alcohol dehydrogenase activities of alcoholic and non-alcoholic patients with similar degrees of liver pathology were compared (groups 1 v 4, 2 v 5, and 3 v 6. Alcohol dehydrogenase activity was, however, severely reduced in patients with liver disease compared with control subjects. Our findings suggest that alcohol consumption does not modify hepatic alcohol dehydrogenase activity. The reduction in specific alcohol dehydrogenase activity in alcoholic liver disease is a consequence of liver damage. PMID:2379876

  6. The Crystal Structure of Aquifex aeolicus Prephenate Dehydrogenase Reveals the Mode of Tyrosine Inhibition

    SciTech Connect

    Sun, Warren; Shahinas, Dea; Bonvin, Julie; Hou, Wenjuan; Kimber, Matthew S.; Turnbull, Joanne; Christendat, Dinesh

    2009-08-14

    TyrA proteins belong to a family of dehydrogenases that are dedicated to l-tyrosine biosynthesis. The three TyrA subclasses are distinguished by their substrate specificities, namely the prephenate dehydrogenases, the arogenate dehydrogenases, and the cyclohexadienyl dehydrogenases, which utilize prephenate, l-arogenate, or both substrates, respectively. The molecular mechanism responsible for TyrA substrate selectivity and regulation is unknown. To further our understanding of TyrA-catalyzed reactions, we have determined the crystal structures of Aquifex aeolicus prephenate dehydrogenase bound with NAD(+) plus either 4-hydroxyphenylpyuvate, 4-hydroxyphenylpropionate, or l-tyrosine and have used these structures as guides to target active site residues for site-directed mutagenesis. From a combination of mutational and structural analyses, we have demonstrated that His-147 and Arg-250 are key catalytic and binding groups, respectively, and Ser-126 participates in both catalysis and substrate binding through the ligand 4-hydroxyl group. The crystal structure revealed that tyrosine, a known inhibitor, binds directly to the active site of the enzyme and not to an allosteric site. The most interesting finding though, is that mutating His-217 relieved the inhibitory effect of tyrosine on A. aeolicus prephenate dehydrogenase. The identification of a tyrosine-insensitive mutant provides a novel avenue for designing an unregulated enzyme for application in metabolic engineering.

  7. Reduced aldehyde dehydrogenase expression in preeclamptic decidual mesenchymal stem/stromal cells is restored by aldehyde dehydrogenase agonists

    PubMed Central

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

    2017-01-01

    High resistance to oxidative stress is a common feature of mesenchymal stem/stromal cells (MSC) and is associated with higher cell survival and ability to respond to oxidative damage. Aldehyde dehydrogenase (ALDH) activity is a candidate “universal” marker for stem cells. ALDH expression was significantly lower in decidual MSC (DMSC) isolated from preeclamptic (PE) patients. ALDH gene knockdown by siRNA transfection was performed to create a cell culture model of the reduced ALDH expression detected in PE-DMSC. We showed that ALDH activity in DMSC is associated with resistance to hydrogen peroxide (H2O2)-induced toxicity. Our data provide evidence that ALDH expression in DMSC is required for cellular resistance to oxidative stress. Furthermore, candidate ALDH activators were screened and two of the compounds were effective in upregulating ALDH expression. This study provides a proof-of-principle that the restoration of ALDH activity in diseased MSC is a rational basis for a therapeutic strategy to improve MSC resistance to cytotoxic damage. PMID:28205523

  8. Mechanism of activation of pyruvate dehydrogenase by dichloroacetate and other halogenated carboxylic acids

    PubMed Central

    Whitehouse, Sue; Cooper, Ronald H.; Randle, Philip J.

    1974-01-01

    1. Monochloroacetate, dichloroacetate, trichloroacetate, difluoroacetate, 2-chloropropionate, 2,2′-dichloropropionate and 3-chloropropionate were inhibitors of pig heart pyruvate dehydrogenase kinase. Dichloroacetate was also shown to inhibit rat heart pyruvate dehydrogenase kinase. The inhibition was mainly non-competitive with respect to ATP. The concentration required for 50% inhibition was approx. 100μm for the three chloroacetates, difluoroacetate and 2-chloropropionate and 2,2′-dichloropropionate. Dichloroacetamide was not inhibitory. 2. Dichloroacetate had no significant effect on the activity of pyruvate dehydrogenase phosphate phosphatase when this was maximally activated by Ca2+ and Mg2+. 3. Dichloroacetate did not increase the catalytic activity of purified pig heart pyruvate dehydrogenase. 4. Dichloroacetate, difluoroacetate, 2-chloropropionate and 2,2′-dichloropropionate increased the proportion of the active (dephosphorylated) form of pyruvate dehydrogenase in rat heart mitochondria with 2-oxoglutarate and malate as respiratory substrates. Similar effects of dichloroacetate were shown with kidney and fat-cell mitochondria. Glyoxylate, monochloroacetate and dichloroacetamide were inactive. 5. Dichloroacetate increased the proportion of active pyruvate dehydrogenase in the perfused rat heart, isolated rat diaphragm and rat epididymal fat-pads. Difluoroacetate and dichloroacetamide were also active in the perfused heart, but glyoxylate, monochloroacetate and trichloroacetate were inactive. 6. Injection of dichloroacetate into rats starved overnight led within 60 min to activation of pyruvate dehydrogenase in extracts from heart, psoas muscle, adipose tissue, kidney and liver. The blood concentration of lactate fell within 15 min to reach a minimum after 60 min. The blood concentration of glucose fell after 90 min and reached a minimum after 120 min. There was no significant change in plasma glycerol concentration. 7. In epididymal fatpads

  9. 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

  10. 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

  11. Function of formate dehydrogenases in Desulfovibrio vulgaris Hildenborough energy metabolism.

    PubMed

    da Silva, Sofia M; Voordouw, Johanna; Leitão, Cristina; Martins, Mónica; Voordouw, Gerrit; Pereira, Inês A C

    2013-08-01

    The genome of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough encodes three formate dehydrogenases (FDHs), two of which are soluble periplasmic enzymes (FdhAB and FdhABC3) and one that is periplasmic but membrane-associated (FdhM). FdhAB and FdhABC3 were recently shown to be the main enzymes present during growth with lactate, formate or hydrogen. To address the role of these two enzymes, ΔfdhAB and ΔfdhABC3, mutants were generated and studied. Different phenotypes were observed in the presence of either molybdenum or tungsten, since both enzymes were important for growth on formate in the presence of Mo, whereas in the presence of W only FdhAB played a role. Both ΔfdhAB and ΔfdhABC3 mutants displayed defects in growth with lactate and sulfate providing the first direct evidence for the involvement of formate cycling under these conditions. In support of this mechanism, incubation of concentrated cell suspensions of the mutant strains with lactate and limiting sulfate also gave elevated formate concentrations, as compared to the wild-type strain. In contrast, both mutants grew similarly to the wild-type with H2 and sulfate. In the absence of sulfate, the wild-type D. vulgaris cells produced formate when supplied with H2 and CO2, which resulted from CO2 reduction by the periplasmic FDHs. The conversion of H2 and CO2 to formate allows the reversible storage of reducing power in a much more soluble molecule. Furthermore, we propose this may be an expression of the ability of some sulfate-reducing bacteria to grow by hydrogen oxidation, in syntrophy with organisms that consume formate, but are less efficient in H2 utilization.

  12. Residues that influence coenzyme preference in the aldehyde dehydrogenases.

    PubMed

    González-Segura, Lilian; Riveros-Rosas, Héctor; Julián-Sánchez, Adriana; Muñoz-Clares, Rosario A

    2015-06-05

    To find out the residues that influence the coenzyme preference of aldehyde dehydrogenases (ALDHs), we reviewed, analyzed and correlated data from their known crystal structures and amino-acid sequences with their published kinetic parameters for NAD(P)(+). We found that the conformation of the Rossmann-fold loops participating in binding the adenosine ribose is very conserved among ALDHs, so that coenzyme specificity is mainly determined by the nature of the residue at position 195 (human ALDH2 numbering). Enzymes with glutamate or proline at 195 prefer NAD(+) because the side-chains of these residues electrostatically and/or sterically repel the 2'-phosphate group of NADP(+). But contrary to the conformational rigidity of proline, the conformational flexibility of glutamate may allow NADP(+)-binding in some enzymes by moving the carboxyl group away from the 2'-phosphate group, which is possible if a small neutral residue is located at position 224, and favored if the residue at position 53 interacts with Glu195 in a NADP(+)-compatible conformation. Of the residues found at position 195, only glutamate interacts with the NAD(+)-adenosine ribose; glutamine and histidine cannot since their side-chain points are opposite to the ribose, probably because the absence of the electrostatic attraction by the conserved nearby Lys192, or its electrostatic repulsion, respectively. The shorter side-chains of other residues-aspartate, serine, threonine, alanine, valine, leucine, or isoleucine-are distant from the ribose but leave room for binding the 2'-phosphate group. Generally, enzymes having a residue different from Glu bind NAD(+) with less affinity, but they can also bind NADP(+) even sometimes with higher affinity than NAD(+), as do enzymes containing Thr/Ser/Gln195. Coenzyme preference is a variable feature within many ALDH families, consistent with being mainly dependent on a single residue that apparently has no other structural or functional roles, and therefore can

  13. Prognostic value of preoperative serum lactate dehydrogenase in thymic carcinoma

    PubMed Central

    Yuan, Zu-Yang; Gao, Shu-Geng; Mu, Ju-Wei; Xue, Qi; Mao, You-Sheng; Wang, Da-Li; Zhao, Jun; Gao, Yu-Shun; Huang, Jin-Feng

    2016-01-01

    Background The prognostic value of serum lactate dehydrogenase (LDH) has been demonstrated in various solid tumors. We attempted to determine whether serum LDH was predictive of survival in thymic carcinoma after surgical resection. Methods Ninety-five patients with thymic carcinoma treated in our hospital between January 2005 and December 2015 were retrospectively enrolled. Serum LDH was measured before surgery and categorized as low or high relative to the upper limit of normal (ULN) (225 U/L). The relationships of serum LDH level and other clinical variables with survival were estimated by Cox regression and Kaplan-Meier survival analysis. Results Serum LDH levels were found to be significantly associated with overall survival (OS) and progression-free survival (PFS) of these patients. The 1-, 3-, and 5-year PFS were 76%, 51%, and 38%, and the 1-, 3- and 5-year OS were 97%, 75%, and 46%, respectively. Univariate analysis found that high serum LDH (>225 U/L) was associated with both lower OS [hazard ratio (HR) =2.710; 95% confidence interval (CI): 1.363–1.5.391; P=0.004] and PFS (HR =3.365; 95% CI: 1.776–6.374; P<0.001). Multivariate analysis found that high serum LDH was associated with lower PFS (HR =2.122; 95% CI: 1.056–4.267; P=0.035). Moreover, high LDH was significantly associated with advanced Masaoka stage (P=0.001). Conclusions High serum LDH (>225 U/L) was an independent predictor of decreased PFS in thymic carcinoma patients. It was also significantly associated with reduced OS, but was not an independent predictor of death in those patients. PMID:27746998

  14. Salivary lactate dehydrogenase and aminotransferases in diabetic patients

    PubMed Central

    Malicka, Barbara; Skoskiewicz-Malinowska, Katarzyna; Kaczmarek, Urszula

    2016-01-01

    Abstract Diabetes mellitus (DM) is a group of metabolic diseases resulting from impaired insulin secretion and/or action. DM is characterized by hyperglycemia that can lead to the dysfunction or damage of organs, including the salivary glands. The aim of this study was to compare the levels of salivary lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in diabetic patients. The study was approved by the Bioethics Committee of Wroclaw Medical University (Poland). The study comprised 90 adults of both sexes, aged 21 to 57 years. The patients were divided into 3 groups: type 1 diabetics (D1), type 2 diabetics (D2), and a healthy control group (C). Each group consisted of 30 age- and sex-matched subjects. Total protein (P, by Lowry method), LDH, AST, ALT (with Alpha Diagnostics kits), and salivary flow rate were measured in unstimulated mixed saliva. The level of glycosylated hemoglobin (HbA1c) was measured with DCA 2000 Reagent Kit. The obtained data were analyzed using the Mann–Whitney U test and the Spearman rank at a significance level of P < 0.05 with the use of STATISTICA 9.0 software. In comparison with C, D1 presented a significantly higher activity of LDH (P < 0.001), AST (P < 0.001), and ALT (P < 0.01), whereas D2 indicated higher levels of LDH (P < 0.001) and ALT (P < 0.05) compared with C. Comparing D1 to D2, approximately 3-fold higher activity of AST (P < 0.01) and approximately 4.5-fold higher activity of ALT (P < 0.01) was observed. Higher levels of salivary LDH, AST, and ALT in D1 compared with D2 and C confirm that salivary glands of D1 might be attributed to autoimmunological damage associated with the pathomechanism of DM. PMID:27893660

  15. The expression of succinate dehydrogenase in breast phyllodes tumor.

    PubMed

    Choi, Junjeong; Kim, Do Hee; Jung, WooHee; Koo, Ja Seung

    2014-10-01

    The purpose of this study is to investigate the expression of succinate dehydrogenase (SDH)A, SDHB, and HIF-1α in phyllodes tumors and the association with clinic-pathologic factors. Using tissue microarray (TMA) for 206 phyllodes tumor cases, we performed immunohistochemical stains for SDHA, SDHB, and HIF-1α and analyzed their expression in regard to clinicopathologic parameters of each case. The cases were comprised of 156 benign, 34 borderline, and 16 malignant phyllodes tumors. The expression of stromal SDHA and epithelial- and stromal- SDHB increased as the tumor progressed from benign to malignant (P⟨0.001). There were five stromal SDHA-negative cases and 31 stromal SDHB-negative cases. SDHB negativity was associated with a lower histologic grade (P=0.054) and lower stromal atypia (P=0.048). Univariate analysis revealed that a shorter disease free survival (DFS) was associated with stromal SDHB high-positivity (P=0.013) and a shorter overall survival (OS) was associated with high-positivity of stromal SDHA and SDHB (P⟨0.001 and P⟨0.001, respectively). The multivariate Cox analysis with the variables stromal cellularity, stromal atypia, stromal mitosis, stromal overgrowth, tumor margin, stromal SDHA expression, and stromal SDHB expression revealed that stromal overgrowth was associated with a shorter DFS (hazard ratio: 24.78, 95% CI: 3.126-196.5, P=0.002) and a shorter OS (hazard ratio: 176.7, 95% CI: 8.466-3691, P=0.001). In conclusion, Tumor grade is positively correlated with SDHA and SDHB expression in the tumor stroma in phyllodes tumors of the breast. This result may be attributed to the increased metabolic demand in high grade tumors.

  16. Short-chain dehydrogenases/reductases in cyanobacteria.

    PubMed

    Kramm, Anneke; Kisiela, Michael; Schulz, Rüdiger; Maser, Edmund

    2012-03-01

    The short-chain dehydrogenases/reductases (SDRs) represent a large superfamily of enzymes, most of which are NAD(H)-dependent or NADP(H)-dependent oxidoreductases. They display a wide substrate spectrum, including steroids, alcohols, sugars, aromatic compounds, and xenobiotics. On the basis of characteristic sequence motifs, the SDRs are subdivided into two main (classical and extended) and three smaller (divergent, intermediate, and complex) families. Despite low residue identities in pairwise comparisons, the three-dimensional structure among the SDRs is conserved and shows a typical Rossmann fold. Here, we used a bioinformatics approach to determine whether and which SDRs are present in cyanobacteria, microorganisms that played an important role in our ecosystem as the first oxygen producers. Cyanobacterial SDRs could indeed be identified, and were clustered according to the SDR classification system. Furthermore, because of the early availability of its genome sequence and the easy application of transformation methods, Synechocystis sp. PCC 6803, one of the most important cyanobacterial strains, was chosen as the model organism for this phylum. Synechocystis sp. SDRs were further analysed with bioinformatics tools, such as hidden Markov models (HMMs). It became evident that several cyanobacterial SDRs show remarkable sequence identities with SDRs in other organisms. These so-called 'homologous' proteins exist in plants, model organisms such as Drosophila melanogaster and Caenorhabditis  elegans, and even in humans. As sequence identities of up to 60% were found between Synechocystis and humans, it was concluded that SDRs seemed to have been well conserved during evolution, even after dramatic terrestrial changes such as the conversion of the early reducing atmosphere to an oxidizing one by cyanobacteria.

  17. Dysfunctional TCA-Cycle Metabolism in Glutamate Dehydrogenase Deficient Astrocytes.

    PubMed

    Nissen, Jakob D; Pajęcka, Kamilla; Stridh, Malin H; Skytt, Dorte M; Waagepetersen, Helle S

    2015-12-01

    Astrocytes take up glutamate in the synaptic area subsequent to glutamatergic transmission by the aid of high affinity glutamate transporters. Glutamate is converted to glutamine or metabolized to support intermediary metabolism and energy production. Glutamate dehydrogenase (GDH) and aspartate aminotransferase (AAT) catalyze the reversible reaction between glutamate and α-ketoglutarate, which is the initial step for glutamate to enter TCA cycle metabolism. In contrast to GDH, AAT requires a concomitant interconversion of oxaloacetate and aspartate. We have investigated the role of GDH in astrocyte glutamate and glucose metabolism employing siRNA mediated knock down (KD) of GDH in cultured astrocytes using stable and radioactive isotopes for metabolic mapping. An increased level of aspartate was observed upon exposure to [U-(13) C]glutamate in astrocytes exhibiting reduced GDH activity. (13) C Labeling of aspartate and TCA cycle intermediates confirmed that the increased amount of aspartate is associated with elevated TCA cycle flux from α-ketoglutarate to oxaloacetate, i.e. truncated TCA cycle. (13) C Glucose metabolism was elevated in GDH deficient astrocytes as observed by increased de novo synthesis of aspartate via pyruvate carboxylation. In the absence of glucose, lactate production from glutamate via malic enzyme was lower in GDH deficient astrocytes. In conclusions, our studies reveal that metabolism via GDH serves an important anaplerotic role by adding net carbon to the TCA cycle. A reduction in GDH activity seems to cause the astrocytes to up-regulate activity in pathways involved in maintaining the amount of TCA cycle intermediates such as pyruvate carboxylation as well as utilization of alternate substrates such as branched chain amino acids.

  18. Metabolic control analysis of eucaryotic pyruvate dehydrogenase multienzyme complex.

    PubMed

    Modak, Jayant; Deckwer, Wolf-Dieter; Zeng, An-Ping

    2002-01-01

    Metabolic control analysis (MCA) of pyruvate dehydrogenase multienzyme (PDH) complex of eucaryotic cells has been carried out using both in vitro and in vivo mechanistic models. Flux control coefficients (FCC) for the sensitivity of pyruvate decarboxylation rate to activities of various PDH complex reactions are determined. FCCs are shown to be strong functions of both pyruvate levels and various components of PDH complex. With the in vitro model, FCCs are shown to be sensitive to only the E1 component of the PDH complex at low pyruvate concentrations. At high pyruvate concentrations, the control is shared by all of the components, with E1 having a negative influence while the other three components, E2, X, and K, exert a positive control over the pyruvate decarboxylation rate. An unusual behavior of deactivation of the E1 component leading to higher net PDH activity is shown to be linked to the combined effect of protein X acylation and E1 deactivation. The steady-state analysis of the in vivo model reveals multiple steady state behavior of pyruvate metabolism with two stable and one unstable steady-states branches. FCCs also display multiplicity, showing completely different control distribution exerted by pyruvate and PDH components on three branches. At low pyruvate concentrations, pyruvate supply dominates the decarboxylation rate and PDH components do not exert any significant control. Reverse control distribution is observed at high pyruvate concentration. The effect of dilution due to cell growth on pyruvate metabolism is investigated in detail. While pyruvate dilution effects are shown to be negligible under all conditions, significant PDH complex dilution effects are observed under certain conditions. Comparison of in vitro and in vivo models shows that PDH components exert different degrees of control outside and inside the cells. At high pyruvate levels, PDH components are shown to exert a higher degree of control when reactions are taking place inside

  19. Characteristics and crystal structure of bacterial inosine-5'-monophosphate dehydrogenase.

    SciTech Connect

    Zhang, R.; Evans, G.; Rotella, F. J.; Westbrook, E. M.; Beno, D.; Huberman, E.; Joachimiak, A.; Collart, F. R.

    1999-01-01

    IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the first step unique to GTP synthesis. To provide a basis for the evaluation of IMPDH inhibitors as antimicrobial agents, we have expressed and characterized IMPDH from the pathogenic bacterium Streptococcus pyogenes. Our results show that the biochemical and kinetic characteristics of S. pyogenes IMPDH are similar to other bacterial IMPDH enzymes. However, the lack of sensitivity to mycophenolic acid and the K{sub m} for NAD (1180 {mu}M) exemplify some of the differences between the bacterial and mammalian IMPDH enzymes, making it an attractive target for antimicrobial agents. To evaluate the basis for these differences, we determined the crystal structure of the bacterial enzyme at 1.9 {angstrom} with substrate bound in the catalytic site. The structure was determined using selenomethionine-substituted protein and multiwavelength anomalous (MAD) analysis of data obtained with synchrotron radiation from the undulator beamline (19ID) of the Structural Biology Center at Argonne's Advanced Photon Source. S. pyogenes IMPDH is a tetramer with its four subunits related by a crystallographic 4-fold axis. The protein is composed of two domains: a TIM barrel domain that embodies the catalytic framework and a cystathione {beta}-synthase (CBS) dimer domain of so far unknown function. Using information provided by sequence alignments and the crystal structure, we prepared several site-specific mutants to examine the role of various active site regions in catalysis. These variants implicate the active site flap as an essential catalytic element and indicate there are significant differences in the catalytic environment of bacterial and mammalian IMPDH enzymes. Comparison of the structure of bacterial IMPDH with the known partial structures from eukaryotic organisms will provide an explanation of their distinct properties and contribute to the design of specific bacterial IMPDH inhibitors.

  20. Sequences and expression of pyruvate dehydrogenase genes from Pseudomonas aeruginosa.

    PubMed Central

    Rae, J L; Cutfield, J F; Lamont, I L

    1997-01-01

    A mutant of Pseudomonas aeruginosa, OT2100, which appeared to be defective in the production of the fluorescent yellow-green siderophore pyoverdine had been isolated previously following transposon mutagenesis (T. R. Merriman and I. L. Lamont, Gene 126:17-23, 1993). DNA from either side of the transposon insertion site was cloned, and the sequence was determined. The mutated gene had strong identity with the dihydrolipoamide acetyltransferase (E2) components of pyruvate dehydrogenase (PDH) from other bacterial species. Enzyme assays revealed that the mutant was defective in the E2 subunit of PDH, preventing assembly of a functional complex. PDH activity in OT2100 cell extracts was restored when extract from an E1 mutant was added. On the basis of this evidence, OT2100 was identified as an aceB or E2 mutant. A second gene, aceA, which is likely to encode the E1 component of PDH, was identified upstream from aceB. Transcriptional analysis revealed that aceA and aceB are expressed as a 5-kb polycistronic transcript from a promoter upstream of aceA. An intergenic region of 146 bp was located between aceA and aceB, and a 2-kb aceB transcript that originated from a promoter in the intergenic region was identified. DNA fragments upstream of aceA and aceB were shown to have promoter activities in P. aeruginosa, although only the aceA promoter was active in Escherichia coli. It is likely that the apparent pyoverdine-deficient phenotype of mutant OT2100 is a consequence of acidification of the growth medium due to accumulation of pyruvic acid in the absence of functional PDH. PMID:9171401

  1. 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

  2. IMP dehydrogenase from Pneumocystis carinii as a potential drug target.

    PubMed Central

    O'Gara, M J; Lee, C H; Weinberg, G A; Nott, J M; Queener, S F

    1997-01-01

    Mycophenolic acid, a specific inhibitor of IMP dehydrogenase (IMPDH; EC 1.1.1.205), is a potent inhibitor of Pneumocystis carinii growth in culture, suggesting that IMPDH may be a sensitive target for chemotherapy in this organism. The IMPDH gene was cloned as a first step to characterizing the enzyme and developing selective inhibitors. A 1.3-kb fragment containing a portion of the P. carinii IMPDH gene was amplified by PCR with two degenerate oligonucleotides based on conserved sequences in IMPDH from humans and four different microorganisms. Northern hybridization analysis showed the P. carinii IMPDH mRNA to be approximately 1.6 kb. The entire cDNA encoding P. carinii IMPDH was isolated and cloned. The deduced amino acid sequence of P. carinii IMPDH shared homology with bacterial (31 to 38%), protozoal (48 to 59%), mammalian (60 to 62%), and fungal (62%) IMPDH enzymes. The IMPDH cDNA was expressed by using a T7 expression system in an IMPDH-deficient strain of Escherichia coli (strain S phi 1101). E. coli S phi 1101 cells containing the P. carinii IMPDH gene were able to grow on medium lacking guanine, implying that the protein expressed in vivo was functional. Extracts of these E. coli cells contained IMPDH activity that had an apparent Km for IMP of 21.7 +/- 0.3 microM and an apparent Km for NAD of 314 +/- 84 microM (mean +/- standard error of the mean; n = 3), and the activity was inhibited by mycophenolic acid (50% inhibitory concentration, 24 microM; n = 2). PMID:8980752

  3. Purification and characterization of Plasmodium falciparum succinate dehydrogenase.

    PubMed

    Suraveratum, N; Krungkrai, S R; Leangaramgul, P; Prapunwattana, P; Krungkrai, J

    2000-02-05

    Succinate dehydrogenase (SDH), a Krebs cycle enzyme and complex II of the mitochondrial electron transport system was purified to near homogeneity from the human malarial parasite Plasmodium falciparum cultivated in vitro by FPLC on Mono Q, Mono S and Superose 6 gel filtration columns. The malarial SDH activity was found to be extremely labile. Based on Superose 6 FPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nondenaturing-PAGE analyses, it was demonstrated that the malarial enzyme had an apparent native molecular mass of 90 +/- 8 kDa and contained two major subunits with molecular masses of 55 +/- 6 and 35 +/- 4 kDa (n = 8). The enzymatic reaction required both succinate and coenzyme Q (CoQ) for its maximal catalysis with Km values of 3 and 0.2 microM, and k(cat) values of 0.11 and 0.06 min(-1), respectively. Catalytic efficiency of the malarial SDH for both substrates were found to be relatively low (approximately 600-5000 M(-1) s(-1)). Fumarate, malonate and oxaloacetate were found to inhibit the malarial enzyme with Ki values of 81, 13 and 12 microM, respectively. The malarial enzyme activity was also inhibited by substrate analog of CoQ, 5-hydroxy-2-methyl-1,4-naphthoquinone, with a 50% inhibitory concentration of 5 microM. The quinone had antimalarial activity against the in vitro growth of P. falciparum with a 50% inhibitory concentration of 0.27 microM and was found to completely inhibit oxygen uptake of the parasite at a concentration of 0.88 microM. A known inhibitor of mammalian mitochondrial SDH, 2-thenoyltrifluoroacetone. had no inhibitory effect on both the malarial SDH activity and the oxygen uptake of the parasite at a concentration of 50 microM. Many properties observed in the malarial SDH were found to be different from the host mammalian enzyme.

  4. Lactate dehydrogenase activity is inhibited by methylmalonate in vitro.

    PubMed

    Saad, Laura O; Mirandola, Sandra R; Maciel, Evelise N; Castilho, Roger F

    2006-04-01

    Methylmalonic acidemia (MMAemia) is an inherited metabolic disorder of branched amino acid and odd-chain fatty acid metabolism, involving a defect in the conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A. Systemic and neurological manifestations in this disease are thought to be associated with the accumulation of methylmalonate (MMA) in tissues and biological fluids with consequent impairment of energy metabolism and oxidative stress. In the present work we studied the effect of MMA and two other inhibitors of mitochondrial respiratory chain complex II (malonate and 3-nitropropionate) on the activity of lactate dehydrogenase (LDH) in tissue homogenates from adult rats. MMA potently inhibited LDH-catalyzed conversion of lactate to pyruvate in liver and brain homogenates as well as in a purified bovine heart LDH preparation. LDH was about one order of magnitude less sensitive to inhibition by MMA when catalyzing the conversion of pyruvate to lactate. Kinetic studies on the inhibition of brain LDH indicated that MMA inhibits this enzyme competitively with lactate as a substrate (K (i)=3.02+/-0.59 mM). Malonate and 3-nitropropionate also strongly inhibited LDH-catalyzed conversion of lactate to pyruvate in brain homogenates, while no inhibition was observed by succinate or propionate, when present in concentrations of up to 25 mM. We propose that inhibition of the lactate/pyruvate conversion by MMA contributes to lactate accumulation in blood, metabolic acidemia and inhibition of gluconeogenesis observed in patients with MMAemia. Moreover, the inhibition of LDH in the central nervous system may also impair the lactate shuttle between astrocytes and neurons, compromising neuronal energy metabolism.

  5. Mechanism of Thermal Adaptation in the Lactate Dehydrogenases.

    PubMed

    Peng, Huo-Lei; Egawa, Tsuyoshi; Chang, Eric; Deng, Hua; Callender, Robert

    2015-12-10

    The mechanism of thermal adaptation of enzyme function at the molecular level is poorly understood but is thought to lie within the structure of the protein or its dynamics. Our previous work on pig heart lactate dehydrogenase (phLDH) has determined very high resolution structures of the active site, via isotope edited IR studies, and has characterized its dynamical nature, via laser-induced temperature jump (T-jump) relaxation spectroscopy on the Michaelis complex. These particular probes are quite powerful at getting at the interplay between structure and dynamics in adaptation. Hence, we extend these studies to the psychrophilic protein cgLDH (Champsocephalus gunnari; 0 °C) and the extreme thermophile tmLDH (Thermotoga maritima LDH; 80 °C) for comparison to the mesophile phLDH (38-39 °C). Instead of the native substrate pyruvate, we utilize oxamate as a nonreactive substrate mimic for experimental reasons. Using isotope edited IR spectroscopy, we find small differences in the substate composition that arise from the detailed bonding patterns of oxamate within the active site of the three proteins; however, we find these differences insufficient to explain the mechanism of thermal adaptation. On the other hand, T-jump studies of reduced β-nicotinamide adenine dinucleotide (NADH) emission reveal that the most important parameter affecting thermal adaptation appears to be enzyme control of the specific kinetics and dynamics of protein motions that lie along the catalytic pathway. The relaxation rate of the motions scale as cgLDH > phLDH > tmLDH in a way that faithfully matches kcat of the three isozymes.

  6. Energy substrate metabolism in pyruvate dehydrogenase complex deficiency.

    PubMed

    Stenlid, Maria Halldin; Ahlsson, Fredrik; Forslund, Anders; von Döbeln, Ulrika; Gustafsson, Jan

    2014-11-01

    Pyruvate dehydrogenase (PDH) deficiency is an inherited disorder of carbohydrate metabolism, resulting in lactic acidosis and neurological dysfunction. In order to provide energy for the brain, a ketogenic diet has been tried. Both the disorder and the ketogenic therapy may influence energy production. The aim of the study was to assess hepatic glucose production, lipolysis and resting energy expenditure (REE) in an infant, given a ketogenic diet due to neonatal onset of the disease. Lipolysis and glucose production were determined for two consecutive time periods by constant-rate infusions of [1,1,2,3,3-²H₅]-glycerol and [6,6-²H²]-glucose. The boy had been fasting for 2.5 h at the start of the sampling periods. REE was estimated by indirect calorimetry. Rates of glucose production and lipolysis were increased compared with those of term neonates. REE corresponded to 60% of normal values. Respiratory quotient (RQ) was increased, indicating a predominance of glucose oxidation. Blood lactate was within the normal range. Several mechanisms may underlie the increased rates of glucose production and lipolysis. A ketogenic diet will result in a low insulin secretion and reduced peripheral and hepatic insulin sensitivity, leading to increased production of glucose and decreased peripheral glucose uptake. Surprisingly, RQ was high, indicating active glucose oxidation, which may reflect a residual enzyme activity, sufficient during rest. Considering this, a strict ketogenic diet might not be the optimal choice for patients with PDH deficiency. We propose an individualised diet for this group of patients aiming at the highest glucose intake that each patient will tolerate without elevated lactate levels.

  7. A ketogenic diet increases succinic dehydrogenase activity in aging cardiomyocytes.

    PubMed

    Balietti, Marta; Fattoretti, Patrizia; Giorgetti, Belinda; Casoli, Tiziana; Di Stefano, Giuseppina; Solazzi, Moreno; Platano, Daniela; Aicardi, Giorgio; Bertoni-Freddari, Carlo

    2009-08-01

    Impairment of energy metabolism and an increase of reactive oxygen species (ROS) production seem to play a major role in age-related apoptotic loss of cardiomyocytes. Succinic dehydrogenase (SDH) is an important marker of the mitochondrial capability to provide an adequate amount of ATP. Moreover, because of its unique redox properties, SDH activity contributes to maintain the reduced state of the ubiquinone pool. Recent reports have shown that ketone body intake improves cardiac metabolic efficiency and exerts a cardioprotective antioxidant action, we therefore performed a cytochemical investigation of SDH activity in cardiomyocytes of late-adult (19-month-old) rats fed for 8 weeks with a medium-chain triglycerides ketogenic diet (MCT-KD). Young, age-matched and old animals fed with a standard chow were used as controls. The overall area of the precipitates (PA) from SDH activity and the area of the SDH-positive mitochondria (MA) were measured. The percent ratios PA/MA and MA/total myocardial tissue area (MA/TA) were the parameters taken into account. We found that PA/MA was significantly higher in young control rats and in MCT-KD-fed rats versus late-adult and old control rats and in young control versus MCT-KD-fed rats. MA/TA of MCT-KD-fed rats was significantly higher versus age-matched and old control rats and tended to be higher versus young control rats; this parameter was significantly higher in young versus old control rats. Thus, MCT-KD intake partially recovers age-related decrease of SDH activity and increases the myocardial area occupied by metabolically active mitochondria. These effects might counteract metabolic alterations leading to apoptosis-induced myocardial atrophy and failure during aging.

  8. Targeting Aldehyde Dehydrogenase Cancer Stem Cells in Ovarian Cancer

    PubMed Central

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

    2010-01-01

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

  9. Biochemical analysis of the modular enzyme inosine 5'-monophosphate dehydrogenase.

    PubMed

    Nimmesgern, E; Black, J; Futer, O; Fulghum, J R; Chambers, S P; Brummel, C L; Raybuck, S A; Sintchak, M D

    1999-11-01

    Two prominent domains have been identified in the X-ray crystal structure of inosine-5'-monophosphate dehydrogenase (IMPDH), a core domain consisting of an alpha/beta barrel which contains the active site and an inserted subdomain whose structure is less well defined. The core domain encompassing amino acids 1-108 and 244-514 of wild-type human IMPDH (II) connected by the tetrapeptide linker Ile-Arg-Thr-Gly was expressed. The subdomain including amino acids 99-244 of human wild-type IMPDH (II) was expressed as a His-tagged fusion protein, where the His-tag was removable by enterokinase cleavage. These two proteins as well as wild-type human IMPDH (II), all proteins expressed in Escherichia coli, have been purified to apparent homogeneity. Both the wild-type and core domain proteins are tetrameric and have very similar enzymatic activities. In contrast, the subdomain migrates as a monomer or dimer on a gel filtration column and lacks enzymatic activity. Circular dichroism spectropolarimetry indicates that the core domain retains secondary structure very similar to full-length IMPDH, with 30% alpha-helix and 30% beta-sheet vs 33% alpha-helix and 29% beta-sheet for wild-type protein. Again, the subdomain protein is distinguished from both wild-type and core domain proteins by its content of secondary structure, with only 15% each of alpha-helix and beta-sheet. These studies demonstrate that the core domain of IMPDH expressed separately is both structurally intact and enzymatically active. The availability of the modules of IMPDH will aid in dissecting the architecture of this enzyme of the de novo purine nucleotide biosynthetic pathway, which is an important target for immunosuppressive and antiviral drugs.

  10. Phylogenetic analysis of vertebrate lactate dehydrogenase (LDH) multigene families.

    PubMed

    Li, Yi-Ju; Tsoi, Stephen C-M; Mannen, Hideyuka; Shoei-lung Li, Steven

    2002-05-01

    In this paper we analyzed 49 lactate dehydrogenase (LDH) sequences, mostly from vertebrates. The amino acid sequence differences were found to be larger for a human-killifish pair than a human-lamprey pair. This indicates that some protein sequence convergence may occur and reduce the sequence differences in distantly related species. We also examined transitions and transversions separately for several species pairs and found that the transitions tend to be saturated in the distantly related species pair, while transversions are increasing. We conclude that transversions maintain a conservative rate through the evolutionary time. Kimura's two-parameter model for multiple-hit correction on transversions only was used to derive a distance measure and then construct a neighbor-joining (NJ) tree. Three findings were revealed from the NJ tree: (i) the branching order of the tree is consistent with the common branch pattern of major vertebrates; (ii) Ldh-A and Ldh-B genes were duplicated near the origin of vertebrates; and (iii) Ldh-C and Ldh-A in mammals were produced by an independent gene duplication in early mammalian history. Furthermore, a relative rate test showed that mammalian Ldh-C evolved more rapidly than mammalian Ldh-A. Under a two-rate model, this duplication event was calibrated to be approximately 247 million years ago (mya), dating back to the Triassic period. Other gene duplication events were also discovered in Xenopus, the first duplication occurring approximately 60-70 mya in both Ldh-A and Ldh-B, followed by another recent gene duplication event, approximately 20 mya, in Ldh-B.

  11. Cloning, characterization, and engineering of fungal L-arabinitol dehydrogenases.

    PubMed

    Kim, Byoungjin; Sullivan, Ryan P; Zhao, Huimin

    2010-07-01

    L-Arabinitol 4-dehydrogenase (LAD) catalyzes the conversion of L-arabinitol to L-xylulose with concomitant NAD(+) reduction in fungal L-arabinose catabolism. It is an important enzyme in the development of recombinant organisms that convert L: -arabinose to fuels and chemicals. Here, we report the cloning, characterization, and engineering of four fungal LADs from Penicillium chrysogenum, Pichia guilliermondii, Aspergillus niger, and Trichoderma longibrachiatum, respectively. The LAD from P. guilliermondii was inactive, while the other three LADs were NAD(+)-dependent and showed high catalytic activities, with P. chrysogenum LAD being the most active. T. longibrachiatum LAD was the most thermally stable and showed the maximum activity in the temperature range of 55-65 degrees C with the other LADs showed the maximum activity in the temperature range of 40-50 degrees C. These LADs were active from pH 7 to 11 with an optimal pH of 9.4. Site-directed mutagenesis was used to alter the cofactor specificity of these LADs. In a T. longibrachiatum LAD mutant, the cofactor preference toward NADP(+) was increased by 2.5 x 10(4)-fold, whereas the cofactor preference toward NADP(+) of the P. chrysogenum and A. niger LAD mutants was also drastically improved, albeit at the expense of significantly reduced catalytic efficiencies. The wild-type LADs and their mutants with altered cofactor specificity could be used to investigate the functionality of the fungal L-arabinose pathways in the development of recombinant organisms for efficient microbial L-arabinose utilization.

  12. THE HEME BINDING PROPERTIES OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

    PubMed Central

    Hannibal, Luciana; Collins, Daniel; Brassard, Julie; Chakravarti, Ritu; Vempati, Rajesh; Dorlet, Pierre; Santolini, Jérôme; Dawson, John H.; Stuehr, Dennis J.

    2012-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that also functions in transcriptional regulation, oxidative stress, vesicular trafficking, and apoptosis. Because GAPDH is required for cellular heme insertion into inducible nitric oxide synthase (Chakravarti et al, PNAS 2010, 107(42):18004-9), we extensively characterized the heme binding properties of GAPDH. Substoichiometric amounts of ferric heme bound to GAPDH (1 heme per GAPDH tetramer) to form a low-spin complex with UV-visible maxima at 362, 418 and 537 nm, and when reduced to ferrous gave maxima at 424, 527 and 559 nm. Ferric heme association and dissociation rate constants at 10 °C were kon =17,800 M−1s−1 and koff1 = 7.0 × 10−3 s−1; koff2 = 3.3 × 10−4 s−1 respectively, giving approximate affinities of 19–390 nM. Ferrous heme bound more poorly to GAPDH and dissociated with a koff = 4.2 × 10−3 s−1. Magnetic circular dichroism (MCD), resonance Raman (rR) and EPR spectroscopic data on the ferric, ferrous, and ferrous-CO complexes of GAPDH showed that the heme is bis-ligated with His as the proximal ligand. The distal ligand in ferric complex was not displaced by CN− or N3− but in ferrous complex was displaceable by CO at a rate of 1.75 s−1 (for [CO]>0.2 mM). Studies with heme analogs revealed selectivity toward the coordinating metal and porphyrin ring structure. GAPDH-heme was isolated from bacteria induced to express rabbit GAPDH in the presence of δ-amino levulinic acid. Our finding of heme binding to GAPDH expands the protein’s potential roles. The strength, selectivity, reversibility, and redox sensitivity of heme binding to GAPDH is consistent with it performing heme sensing or heme chaperone-like functions in cells. PMID:22957700

  13. Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD(+).

    PubMed

    Malver, Olaf; Sebastian, Mina J; Oppenheimer, Norman J

    2014-11-01

    A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD(+) with a redox potential of -324mV and a 341nm λmax for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD(+) by primary alcohols. With HLADH 1-butanol has the highest Vmax at 49% that of β-NAD(+). The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon-hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD(+). In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD(+) by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD(+) has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases.

  14. The conserved Lysine69 residue plays a catalytic role in Mycobacterium tuberculosis shikimate dehydrogenase

    PubMed Central

    2009-01-01

    Background The shikimate pathway is an attractive target for the development of antitubercular agents because it is essential in Mycobacterium tuberculosis, the causative agent of tuberculosis, but absent in humans. M. tuberculosis aroE-encoded shikimate dehydrogenase catalyzes the forth reaction in the shikimate pathway. Structural and functional studies indicate that Lysine69 may be involved in catalysis and/or substrate binding in M. tuberculosis shikimate dehydrogenase. Investigation of the kinetic properties of mutant enzymes can bring important insights about the role of amino acid residues for M. tuberculosis shikimate dehydrogenase. Findings We have performed site-directed mutagenesis, steady-state kinetics, equilibrium binding measurements and molecular modeling for both the wild-type M. tuberculosis shikimate dehydrogenase and the K69A mutant enzymes. The apparent steady-state kinetic parameters for the M. tuberculosis shikimate dehydrogenase were determined; the catalytic constant value for the wild-type enzyme (50 s-1) is 68-fold larger than that for the mutant K69A (0.73 s-1). There was a modest increase in the Michaelis-Menten constant for DHS (K69A = 76 μM; wild-type = 29 μM) and NADPH (K69A = 30 μM; wild-type = 11 μM). The equilibrium dissociation constants for wild-type and K69A mutant enzymes are 32 (± 4) μM and 134 (± 21), respectively. Conclusion Our results show that the residue Lysine69 plays a catalytic role and is not involved in substrate binding for the M. tuberculosis shikimate dehydrogenase. These efforts on M. tuberculosis shikimate dehydrogenase catalytic mechanism determination should help the rational design of specific inhibitors, aiming at the development of antitubercular drugs. PMID:19917104

  15. Orchestration of enzymatic processing by thiazole/oxazole-modified microcin dehydrogenases.

    PubMed

    Melby, Joel O; Li, Xiangpo; Mitchell, Douglas A

    2014-01-21

    Thiazole/oxazole-modified microcins (TOMMs) comprise a structurally diverse family of natural products with varied bioactivities linked by the presence of posttranslationally installed thiazol(in)e and oxazol(in)e heterocycles. The detailed investigation of the TOMM biosynthetic enzymes from Bacillus sp. Al Hakam (Balh) has provided significant insight into heterocycle biosynthesis. Thiazoles and oxazoles are installed by the successive action of an ATP-dependent cyclodehydratase (C- and D-protein) and a FMN-dependent dehydrogenase (B-protein), which are responsible for azoline formation and azoline oxidation, respectively. Although several studies have focused on the mechanism of azoline formation, many details regarding the role of the dehydrogenase (B-protein) in overall substrate processing remain unknown. In this work, we evaluated the involvement of the dehydrogenase in determining the order of ring formation as well as the promiscuity of the Balh and microcin B17 cyclodehydratases to accept a panel of noncognate dehydrogenases. In support of the observed promiscuity, a fluorescence polarization assay was utilized to measure binding of the dehydrogenase to the cyclodehydratase using the intrinsic fluorescence of the FMN cofactor. Ultimately, the noncognate dehydrogenases were shown to possess cyclodehydratase-independent activity. A previous study identified a conserved Lys-Tyr motif to be important for dehydrogenase activity. Using the tools developed in this study, the Lys-Tyr motif was shown neither to alter complex formation with the cyclodehydratase nor the reduction potential. Taken together with the known crystal structure of a homologue, our data suggest that the Lys-Tyr motif is of catalytic importance. Overall, this study provides a greater level of insight into the complex orchestration of enzymatic activity during TOMM biosynthesis.

  16. Orchestration of Enzymatic Processing by Thiazole/Oxazole-Modified Microcin Dehydrogenases

    PubMed Central

    Melby, Joel O.; Li, Xiangpo; Mitchell, Douglas A.

    2014-01-01

    Thiazole/oxazole-modified microcins (TOMMs) comprise a structurally diverse family of natural products with varied bioactivities linked by the presence of posttranslationally installed thiazol(in)e and oxazol(in)e heterocycles. The detailed investigation of the TOMM biosynthetic enzymes from Bacillus sp. Al Hakam (Balh) has provided significant insight into heterocycle biosynthesis. Thiazoles and oxazoles are installed by the successive action of an ATP-dependent cyclodehydratase (C- and D-protein) and a FMN-dependent dehydrogenase (B-protein), which are responsible for azoline formation and azoline oxidation, respectively. Although several studies have focused on the mechanism of azoline formation, many details regarding the role of the dehydrogenase (B-protein) in overall substrate processing remain unknown. In this work, we evaluated the involvement of the dehydrogenase in determining the order of ring formation, as well as the promiscuity of the Balh and microcin B17 cyclodehydratases to accept a panel of noncognate dehydrogenases. In support of the observed promiscuity, a fluorescence polarization assay was utilized to measure binding of the dehydrogenase to the cyclodehydratase using the intrinsic fluorescence of the FMN cofactor. Ultimately, the noncognate dehydrogenases were shown to possess cyclodehydratase-independent activity. A previous study identified a conserved Lys-Tyr motif to be important for dehydrogenase activity. Using the tools developed in this study, the Lys-Tyr motif was shown to not alter complex formation with the cyclodehydratase nor the reduction potential. Taken with the known crystal structure of a homolog, our data suggest that the Lys-Tyr motif is of catalytic importance. Overall, this study provides a greater level of insight into the complex orchestration of enzymatic activity during TOMM biosynthesis. PMID:24364559

  17. Production of superoxide/hydrogen peroxide by the mitochondrial 2-oxoadipate dehydrogenase complex.

    PubMed

    Goncalves, Renata L S; Bunik, Victoria I; Brand, Martin D

    2016-02-01

    In humans, mutations in dehydrogenase E1 and transketolase domain containing 1 (DHTKD1) are associated with neurological abnormalities and accumulation of 2-oxoadipate, 2-aminoadipate, and reactive oxygen species. The protein encoded by DHTKD1 has sequence and structural similarities to 2-oxoglutarate dehydrogenase, and the 2-oxoglutarate dehydrogenase complex can produce superoxide/H2O2 at high rates. The DHTKD1 enzyme is hypothesized to catalyze the oxidative decarboxylation of 2-oxoadipate, a shared intermediate of the degradative pathways for tryptophan, lysine and hydroxylysine. Here, we show that rat skeletal muscle mitochondria can produce superoxide/H2O2 at high rates when given 2-oxoadipate. We identify the putative mitochondrial 2-oxoadipate dehydrogenase complex as one of the sources and characterize the conditions that favor its superoxide/H2O2 production. Rates increased at higher NAD(P)H/NAD(P)(+) ratios and were higher at each NAD(P)H/NAD(P)(+) ratio when 2-oxoadipate was present, showing that superoxide/H2O2 was produced during the forward reaction from 2-oxoadipate, but not in the reverse reaction from NADH in the absence of 2-oxoadipate. The maximum capacity of the 2-oxoadipate dehydrogenase complex for production of superoxide/H2O2 is comparable to that of site IF of complex I, and seven, four and almost two-fold lower than the capacities of the 2-oxoglutarate, pyruvate and branched-chain 2-oxoacid dehydrogenase complexes, respectively. Regulation by ADP and ATP of H2O2 production driven by 2-oxoadipate was very different from that driven by 2-oxoglutarate, suggesting that site AF of the 2-oxoadipate dehydrogenase complex is a new source of superoxide/H2O2 associated with the NADH isopotential pool in mitochondria.

  18. Tandem orientation of duplicated xanthine dehydrogenase genes from Arabidopsis thaliana: differential gene expression and enzyme activities.

    PubMed

    Hesberg, Christine; Hänsch, Robert; Mendel, Ralf R; Bittner, Florian

    2004-04-02

    Xanthine dehydrogenase from the plant Arabidopsis thaliana was analyzed on molecular and biochemical levels. Whereas most other organisms appear to own only one gene for xanthine dehydrogenase A. thaliana possesses two genes in tandem orientation spaced by 704 base pairs. The cDNAs as well as the proteins AtXDH1 and AtXDH2 share an overall identity of 93% and show high homologies to xanthine dehydrogenases from other organisms. Whereas AtXDH2 mRNA is expressed constitutively, alterations of AtXDH1 transcript levels were observed at various stresses like drought, salinity, cold, and natural senescence, but also after abscisic acid treatment. Transcript alteration did not mandatorily result in changes of xanthine dehydrogenase activities. Whereas salt treatment had no effect on xanthine dehydrogenase activities, cold stress caused a decrease, but desiccation and senescence caused a strong increase of activities in leaves. Because AtXDH1 presumably is the more important isoenzyme in A. thaliana it was expressed in Pichia pastoris, purified, and used for biochemical studies. AtXDH1 protein is a homodimer of about 300 kDa consisting of identical subunits of 150 kDa. Like xanthine dehydrogenases from other organisms AtXDH1 uses hypoxanthine and xanthine as main substrates and is strongly inhibited by allopurinol. AtXDH1 could be activated by the purified molybdenum cofactor sulfurase ABA3 that converts inactive desulfo-into active sulfoenzymes. Finally it was found that AtXDH1 is a strict dehydrogenase and not an oxidase, but is able to produce superoxide radicals indicating that besides purine catabolism it might also be involved in response to various stresses that require reactive oxygen species.

  19. Mechanism of citrinin-induced dysfunction of mitochondria. V. Effect on the homeostasis of the reactive oxygen species.

    PubMed

    Ribeiro, S M; Chagas, G M; Campello, A P; Klüppel, M L

    1997-09-01

    The effects of citrinin in the maintenance of the homeostasis of the reactive oxygen species in rat liver cells were evaluated. Citrinin (CTN) modifies the antioxidant enzymatic defences of cells through the inhibition of GSSG-reductase and transhydrogenase. No effect was observed on GSH-peroxidase, catalase, glucose 6-phosphate and 6 phosphogluconate dehydrogenases, and superoxide dismutase. The mycotoxin increased the generation of reactive oxygen species, stimulating the production of the superoxide anion in the respiratory chain. The results suggest that oxidative stress is an important mechanism, side by side with other effects previously shown, in the establishment of the cytotoxicity and cellular death provoked by CTN in several tissues.

  20. Xanthine oxidase and xanthine dehydrogenase from an estivating land snail.

    PubMed

    Hermes-Lima, M; Storey, K B

    1995-01-01

    During arousal from estivation in land snails, Otala lactea, active metabolic functions are restored within minutes and oxygen consumption increases dramatically. During the transition from the hypoxic conditions of estivation to normoxia it is possible that xanthine oxidase (XO) in hepatopancreas contributes to the observed lipid peroxidation. Using a fluorometric assay that is based on the oxidation of pterin, the activities and some properties of XO and XO+XDH (sum of XO and xanthine dehydrogenase activities) were measured in hepatopancreas extracts. Km values for pterin for XO and XO+XDH were 9 and 6 microM, respectively, and the Km of XDH for methylene blue was 5 microM. Both XO+XDH and XO activities were inhibited by allopurinol (I50 = 2 microM), pre-incubation at 40 degrees C, and by 5 min H2O2 pre-exposure. Inclusion of azide in the reaction promoted a rise of approximately 70-fold in the inactivation power of H2O2 due to inhibition of high endogenous catalase activity. The I50 for H2O2 of XO+XDH and XO activities in the presence of azide was 0.04 and 0.11 mM, respectively. Unlike the situation for mammalian XO, a previous reduction of O. lactea XO (by pterin) was not necessary to make the enzyme susceptible to H2O2 effects. Interestingly, methylene blue partially prevented both heat- and H2O2-induced inactivation of XO+XDH activity. These data indicate that the formation of an enzyme-methylene blue complex induces protection against heat and oxidative damage at the FAD-active site. Both XO and XO+XDH activites were significantly higher in snails after 35 days of estivation compared with active snails 24 h after arousal from dormancy. The ratio of XO/(XO+XDH) activities was also slightly increased in estivating O. lactea (from 0.07 to 0.09; P < 0.025). XO activity was 0.03 nmol.min-1.mg protein-1 in estivating snails. Compared with hepatopancreas catalase, XO activity is probably too low to contribute significantly to the net generation of oxyradicals, and