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Sample records for 4-hydroxyphenyl pyruvate dioxygenase

  1. Photosystem II-inhibitors play a limited role in sweet corn response to 4-hydroxyphenyl pyruvate dioxygenase-inhibiting herbicides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Postemergence (POST) application of 4-hydroxyphenyl pyruvate dioxygenase (HPPD) inhibitors in combination with a photosystem II (PSII) inhibitor, such as atrazine, is common practice in sweet corn production. Given the sensitivity of sweet corn to HPPD-inhibiting herbicides, the objective of this wo...

  2. Pharmacokinetics and pharmacodynamics of NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione) and mesotrione, inhibitors of 4-hydroxyphenyl pyruvate dioxygenase (HPPD) following a single dose to healthy male volunteers

    PubMed Central

    Hall, Michael G; Wilks, Martin F; Provan, W McLean; Eksborg, Staffan; Lumholtz, Bo

    2001-01-01

    Aims NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione) and mesotrione (2-(4-methylsulphonyl-2-nitrobenzoyl)-1,3-cyclohexanedione) are inhibitors of 4-hydroxyphenyl pyruvate dioxygenase (HPPD). NTBC has been successfully used as a treatment for hereditary tyrosinaemia type 1 (HT-1), while mesotrione has been developed as an herbicide. The pharmacokinetics of the two compounds were investigated in healthy male volunteers following single oral administration. The aim of the NTBC study was to assess the bioequivalence of two different formulations and to determine the extent of the induced tyrosinaemia. The mesotrione study was performed to determine the magnitude and duration of the effect on tyrosine catabolism. Additionally, the urinary excretion of unchanged mesotrione was measured to assess the importance of this route of clearance and to help develop a strategy for monitoring occupational exposure. Methods A total of 28 volunteers participated in two separate studies with the compounds. In the first study, the relative bioavailability of NTBC from liquid and capsule formulations was compared and the effect on plasma tyrosine concentrations measured. In the second study the pharmacokinetics of mesotrione were determined at three doses. Plasma tyrosine concentrations were monitored and the urinary excretion of mesotrione and tyrosine metabolites was measured. Results Both compounds were well tolerated at the dose levels studied. Peak plasma concentrations of NTBC were rapidly attained following a single oral dose of 1 mg kg−1 body weight of either formulation and the half-life in plasma was approximately 54 h. There were no statistical differences in mean (± s.d.) AUC(0,∞) (capsule 602 ± 154 vs solution 602 ± 146 µg ml−1 h) or t½ (capsule 55 ± 13 vs solution 54 ± 8 h) and these parameters supported the bioequivalence of the two formulations. Mesotrione was also rapidly absorbed, with a significant proportion of the dose eliminated unchanged

  3. Estrogenic activity of bis(4-hydroxyphenyl)methanes with cyclic hydrophobic structure.

    PubMed

    Kojima, Tomohiro; Ogawa, Takumi; Kitao, Souichiro; Sato, Manabu; Oda, Akifumi; Ohta, Kiminori; Endo, Yasuyuki

    2015-11-01

    Monoalkylated bis(4-hydroxyphenyl)methanes (e.g., 1) are reported to show weak binding affinity for estrogen receptor (ER). We hypothesized that introduction of appropriately located hydrophobic substituents in these compounds would increase the binding affinity. Indeed, we found that bis(4-hydroxyphenyl)methane bearing a 3,3-dimethylcyclohexyl group (7) shows potent ERα binding affinity, comparable to that of estradiol. Bulkier substituents could be introduced at the 3,3-position without decreasing the affinity. However, the position of the substituents was critical: the 4,4-dimethylcyclohexyl derivative (2) showed very weak binding affinity. The compounds with high ER-binding affinity showed predominantly agonistic activity, together with weak antagonistic activity at high concentration, in cell proliferation assay with human breast cancer cell line MCF-7. Further structure-function studies of these compounds and their derivatives might lead to the development of more selective and potent estrogen receptor modulators. PMID:26462053

  4. Synthesis and crystal structure of 3-ammonium-4-hydroxyphenyl sulfonate hemihydrate

    NASA Astrophysics Data System (ADS)

    Belhouchet, M.; Mhiri, T.

    2013-01-01

    The crystal structure of 3-ammonium-4-hydroxyphenyl sulfonate hemihydrate C6H3(NH3)(OH)SO3 · 0.5H2O is determined by single-crystal X-ray diffraction. The unit cell parameters are as follows: a = 11.2395(3), b = 10.3814(3), c = 13.7509(4) Å, β = 100.326(1)°, V = 1578.49(8) Å3, space group P21/ n, Z = 4. The crystal structure can be described us a succession of infinite corrugated layers parallel to ab plane. These layers consist of rings formed by four sulfonate molecules located around a center of symmetry. The rings are connected to each other and to water molecules via O-H...O hydrogen bonds. The structure is further stabilized by π-π interactions between phenyl rings of organic entities of successive layers.

  5. Synthesis and crystal structure of 3-ammonium-4-hydroxyphenyl sulfonate hemihydrate

    SciTech Connect

    Belhouchet, M. Mhiri, T.

    2013-01-15

    The crystal structure of 3-ammonium-4-hydroxyphenyl sulfonate hemihydrate C{sub 6}H{sub 3}(NH{sub 3})(OH)SO{sub 3} {center_dot} 0.5H{sub 2}O is determined by single-crystal X-ray diffraction. The unit cell parameters are as follows: a = 11.2395(3), b = 10.3814(3), c = 13.7509(4) A, {beta} = 100.326(1) Degree-Sign , V = 1578.49(8) A{sup 3}, space group P2{sub 1}/n, Z = 4. The crystal structure can be described us a succession of infinite corrugated layers parallel to ab plane. These layers consist of rings formed by four sulfonate molecules located around a center of symmetry. The rings are connected to each other and to water molecules via O-H...O hydrogen bonds. The structure is further stabilized by {pi}-{pi} interactions between phenyl rings of organic entities of successive layers.

  6. Solid Phase-Assisted Synthesis and Screening of a Small Library of N-(4-Hydroxyphenyl)retinamide (4-HPR) Analogs

    PubMed Central

    Mershon, Serena M.; Anding, Allyson L.; Chapman, Jason S.; Clagett-Dame, Margaret; Stonerock, Laura A.; Curley, Robert W.

    2007-01-01

    Using solid phase-assisted synthesis and purification, a 49 member library of analogs of the mammary tumor chemopreventive retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) has been prepared. After prescreening for growth inhibitory activity in human mammary tumor cells (MCF-7) in culture, most of those analogs which showed activity (12 of them) were assayed for apoptosis-inducing activity in the MCF-7 cells. At least 3 of the analogs (13, 24, and 28) showed activity approaching that of 4-HPR. PMID:17112722

  7. Synthesis of 8-Phenylphenalenones: 2-Hydroxy-8-(4-hydroxyphenyl)-1H-phenalen-1-one from Eichhornia crassipes.

    PubMed

    Ospina, Felipe; Hidalgo, William; Cano, Marisol; Schneider, Bernd; Otálvaro, Felipe

    2016-02-01

    2-Hydroxy-8-(4-hydroxyphenyl)-1H-phenalen-1-one (1), the first reported 8-phenylphenalenone from the roots of Eichhornia crassipes (water hyacinth), was synthesized starting from 2-methoxynaphthalene in 11 steps and with an overall yield of 2%. A cascade Friedel-Crafts/Michael annulation reaction between acryloyl chloride and 2-methoxynaphthalene afforded 9-methoxyperinaphthanone that, after transformation to 9-methoxy-2-(4-methoxyphenyl)-1H-phenalen-1-one by means of standard Suzuki-Miyaura methodology, was subjected to a reductive carbonyl transposition to afford 8-(4-methoxyphenyl)perinaphthanone. Dehydrogenation, epoxidation, and demethylation of the latter afforded 1. PMID:26741281

  8. Synthesis and crystal structure of 2-(4-hydroxyphenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol

    NASA Astrophysics Data System (ADS)

    Li, S.-J.; Shen, D.; Zhang, C.-Z.

    2015-11-01

    The title compound 2-(4-hydroxyphenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol was synthesized by the reaction of phenol with hexafluoroacetone using mesitylene as solvent and. methanesulfonic acid as catalist. The structure is determined by X-ray structure analysis. Two kinds of strong intermolecular hydrogen bonds O( Alk)-H···O( Ar)and O( Ar)-H···O( Alk) are formed in crystal. These hydrogen bonds connect the molecules into two-dimensional layers. Based on theoretical calculations of the electronic structure of the title compound, its application in fluoro-containing materials is predicted. The title compound may be employed to synthesize many organic fluoro-containing polymers, because alcoholic hydroxyl and phenolic hydroxyl are easily deprotonated.

  9. 3,3-Bis-(4-hydroxyphenyl)-7-methyl-2-indolinone (BHMI), the active metabolite of the laxative sulisatin.

    PubMed

    Moretó, M; Goñalons, E; Mylonakis, N; Giráldez, A; Torralba, A

    1979-01-01

    The disodium salt of the sulphuric diester of 3,3-bis-(4-hydroxyphenyl)-7-methyl-2-indolinone (sodium sulisatin, Laxitex), a synthetic laxative with two phenolic groups esterified with sulfate, has been studied in order to find out if its laxative properties may be attributed to the unaltered compound or to its diphenolic derivative BHMI. We first studied the effect of homogenates of the gastrointestinal tract of rats and of rat cecal content of the hydrolysis of sulfate ester bonds of sulisatin. Results show that sulisatin can be hydrolyzed by cecal content while homogenates of stomach, small intestine and large intestine have no hydrolytic effect. Sulisatin is also a substrate of arylsulfate sulphohydrolase obtained from the snail Helix pomatia. The unaltered drug has no effect on the intestinal motility since it does not change the intestinal transit speed in rats pretreated with neomycin sulfate. Sulisatin (1.5, 3 and 6 mg) is unable to inhibit water absorption in rat colon while small amounts of BHMI (15 and 30 micrograms) may inhibit it significantly. It is concluded that sulisatin passes unaltered through the small intestine and is hydrolyzed in the large intestine by the intestinal microflora to its diphenolic derivative BHMI, which is responsible for the laxative activity of the drug. PMID:583222

  10. Molecular structure and density functional modelling studies of 2-[(E)-2-(4-hydroxyphenyl)ethyliminomethyl]phenol

    NASA Astrophysics Data System (ADS)

    Zeyrek, Celal Tuğrul; Koçak, Selen Bilge; Ünver, Hüseyin; Pektaş, Serhan; Başterzi, Nisan Sevin; Çelik, Ömer

    2015-11-01

    In the present work, the tautomerism in 2-[(E)-2-(4-hydroxyphenyl)ethyliminomethyl]phenol was investigated by experimental (MS, FT-IR, NMR and X-ray diffraction) and computational method [density functional theory (DFT)]. The optimized geometrical structures, atomic charges, molecular electrostatic potential (MEP), natural bond orbital (NBO) and nonlinear optical (NLO) effects of the compound have been investigated by using DFT calculations. The potential energy surface (PES) scans about three important torsion angles are performed by using B3LYP/6-311++G (d,p) level of theoretical approximation for the compound. The experimental (FT-IR) and calculated vibrational frequencies (using DFT) of the title compound have been compared. The 1H and 13C NMR chemical shift assignments have been performed using DFT. To investigate the tautomeric stability, some properties such as total energy, HOMO and LUMO energies of the compound were obtained at B3LYP and B1B95/6-311++G(d,p) level in the gas phase. The calculated results showed that the phenol-imine form of the compound was more favorite than keto-amine form. Moreover, a good correlation between experimental and theoretical data for phenol-imine form of the compound was found.

  11. SYNTHESIS OF THE FULLY PROTECTED PHOSPHORAMIDITE OF THE BENZENE-DNA ADDUCT, N2- (4-HYDROXYPHENYL)-2'-DEOXYGUANOSINE AND INCORPORATION OF THE LATER INTO DNA OLIGOMERS

    SciTech Connect

    Chenna, Ahmed; Gupta, Ramesh C.; Bonala, Radha R.; Johnson, Francis; Huang, Bo

    2008-06-09

    N2-(4-Hydroxyphenyl)-2'-deoxyguanosine-5'-O-DMT-3'-phosphoramidite has been synthesized and used to incorporate the N2-(4-hydroxyphenyl)-2'-dG (N2-4-HOPh-dG) into DNA, using solid-state synthesis technology. The key step to obtaining the xenonucleoside is a palladium (Xantphos-chelated) catalyzed N2-arylation (Buchwald-Hartwig reaction) of a fully protected 2'-deoxyguanosine derivative by 4-isobutyryloxybromobenzene. The reaction proceeded in good yield and the adduct was converted to the required 5'-O-DMT-3'-O-phosphoramidite by standard methods. The latter was used to synthesize oligodeoxynucleotides in which the N2-4-HOPh-dG adduct was incorporated site-specifically. The oligomers were purified by reverse-phase HPLC. Enzymatic hydrolysis and HPLC analysis confirmed the presence of this adduct in the oligomers.

  12. Preparation and properties of polyformals obtained from 2,2-bis(4-hydroxyphenyl)-1,1,1,3,3,3,-hexafluoropropane and dichloromethane

    SciTech Connect

    Nakamura, S.; Suzuki, Y.; Tamura, E.; Kuriki, M.; Saegusa, Y.

    1993-12-31

    Fluorine-containing aromatic polyformal and copolyformals were prepared from 2,2-bis(4-hydroxyphenyl)-1,1,1,3,3,3,-hexafluoropropane (bis-phenol AF) and/or 2,2-bis(4-hydroxyphenyl)-propane (bisphenol A) with dichloromethane. High molecular-weight polyformal and copolyformals were obtained by using NMP as solvent and in the presence of KOH. The thermal stability was lowered with increasing fluorine content. The Tg increased monotonically with fluorine content from 89{degrees}C for bisphenol A polyformal to 17{degrees}C for bisphenol AF polyformal. Bisphenol a polyformal was soluble in limited solvents, whereas bisphenol AF polyformal was soluble in a wide variety of solvents such aprotic polar solvents, aromatic solvents and chlorinated hydrocarbons.

  13. Molecular Genetic and Crystal Structural Analysis of 1-(4-Hydroxyphenyl)-Ethanol Dehydrogenase from 'Aromatoleum aromaticum' EbN1.

    PubMed

    Büsing, Imke; Höffken, H Wolfgang; Breuer, Michael; Wöhlbrand, Lars; Hauer, Bernhard; Rabus, Ralf

    2015-01-01

    The dehydrogenation of 1-(4-hydroxyphenyl)-ethanol to 4-hydroxyacetophenone represents the second reaction step during anaerobic degradation of p-ethylphenol in the denitrifying bacterium 'Aromatoleum aromaticum' EbN1. Previous proteogenomic studies identified two different proteins (ChnA and EbA309) as possible candidates for catalyzing this reaction [Wöhlbrand et al: J Bacteriol 2008;190:5699-5709]. Physiological-molecular characterization of newly generated unmarked in-frame deletion and complementation mutants allowed defining ChnA (renamed here as Hped) as the enzyme responsible for 1-(4-hydroxyphenyl)-ethanol oxidation. Hped [1-(4-hydroxyphenyl)-ethanol dehydrogenase] belongs to the 'classical' family within the short-chain alcohol dehydrogenase/reductase (SDR) superfamily. Hped was overproduced in Escherichia coli, purified and crystallized. The X-ray structures of the apo- and NAD(+)-soaked form were resolved at 1.5 and 1.1 Å, respectively, and revealed Hped as a typical homotetrameric SDR. Modeling of the substrate 4-hydroxyacetophenone (reductive direction of Hped) into the active site revealed the structural determinants of the strict (R)-specificity of Hped (Phe(187)), contrasting the (S)-specificity of previously reported 1-phenylethanol dehydrogenase (Ped; Tyr(93)) from strain EbN1 [Höffken et al: Biochemistry 2006;45:82-93]. PMID:26488297

  14. Benzene-derived N2-(4-hydroxyphenyl)-deoxyguanosine adduct: UvrABC incision and its conformation in DNA

    SciTech Connect

    Hang, Bo; Rodriguez, Ben; Yang, Yanu; Guliaev, Anton B.; Chenna, Ahmed

    2010-06-14

    Benzene, a ubiquitous human carcinogen, forms DNA adducts through its metabolites such as p-benzoquinone (p-BQ) and hydroquinone (HQ). N(2)-(4-Hydroxyphenyl)-2'-deoxyguanosine (N(2)-4-HOPh-dG) is the principal adduct identified in vivo by (32)P-postlabeling in cells or animals treated with p-BQ or HQ. To study its effect on repair specificity and replication fidelity, we recently synthesized defined oligonucleotides containing a site-specific adduct using phosphoramidite chemistry. We here report the repair of this adduct by Escherichia coli UvrABC complex, which performs the initial damage recognition and incision steps in the nucleotide excision repair (NER) pathway. We first showed that the p-BQ-treated plasmid was efficiently cleaved by the complex, indicating the formation of DNA lesions that are substrates for NER. Using a 40-mer substrate, we found that UvrABC incises the DNA strand containing N(2)-4-HOPh-dG in a dose- and time-dependent manner. The specificity of such repair was also compared with that of DNA glycosylases and damage-specific endonucleases of E. coli, both of which were found to have no detectable activity toward N(2)-4-HOPh-dG. To understand why this adduct is specifically recognized and processed by UvrABC, molecular modeling studies were performed. Analysis of molecular dynamics trajectories showed that stable G:C-like hydrogen bonding patterns of all three Watson-Crick hydrogen bonds are present within the N(2)-4-HOPh-G:C base pair, with the hydroxyphenyl ring at an almost planar position. In addition, N(2)-4-HOPh-dG has a tendency to form more stable stacking interactions than a normal G in B-type DNA. These conformational properties may be critical in differential recognition of this adduct by specific repair enzymes.

  15. Conformer-specific vibronic spectroscopy and vibronic coupling in a flexible bichromophore: Bis-(4-hydroxyphenyl)methane

    NASA Astrophysics Data System (ADS)

    Rodrigo, Chirantha P.; Müller, Christian W.; Pillsbury, Nathan R.; James, William H.; Plusquellic, David F.; Zwier, Timothy S.

    2011-04-01

    The vibronic spectroscopy of jet-cooled bis-(4-hydroxyphenyl)methane has been explored using fluorescence excitation, dispersed fluorescence (DFL), UV-UV hole-burning, UV depletion, and fluorescence-dip infrared spectroscopies. Calculations predict the presence of three nearly isoenergetic conformers that differ in the orientations of the two OH groups in the para positions on the two aromatic rings (labeled uu, dd, and ud). In practice, two conformers (labeled A and B) are observed, with S0-S1 origins at 35 184 and 35 209 cm-1, respectively. The two conformers have nearly identical vibronic spectra and hydride stretch infrared spectra. The low-frequency vibronic structure is assigned to bands involving the phenyl torsions (T and bar T), ring-flapping (R and bar R), and butterfly (β) modes. Symmetry arguments lead to a tentative assignment of the two conformers as the C2 symmetric uu and dd conformers. The S0-S2 origins are assigned to bands located 132 cm-1 above the S0-S1 origins of both conformers. DFL spectra from the S2 origin of the two conformers display extensive evidence for vibronic coupling between the two close-lying electronic states. Near-resonant coupling from the S2 origin occurs dominantly to S1 bar R^1 and S1 bar R^1 β ^1 levels, which are located -15 and +31 cm-1 from it. Unusual vibronic activity in the ring-breathing (ν1) and ring-deformation (ν6a) modes is also attributed to vibronic coupling involving these Franck-Condon active modes. A multimode vibronic coupling model is developed based on earlier theoretical descriptions of molecular dimers [Fulton and Gouterman, J. Chem. Phys. 35, 1059 (1961)] and applied here to flexible bichromophores. The model is able to account for the ring-mode activity under conditions in which the S2 origin is strongly mixed (60%/40%) with S1 overline {6a} ^1 and bar 1^1 levels. The direct extension of this model to the T /bar T and R /bar R inter-ring mode pairs is only partially successful and required some

  16. Simultaneous quantification of vortioxetine, carvedilol and its active metabolite 4-hydroxyphenyl carvedilol in rat plasma by UPLC-MS/MS: Application to their pharmacokinetic interaction study.

    PubMed

    Huang, Yi; Zheng, Shuangli; Pan, Yongyang; Li, Tao; Xu, Zhi-Sheng; Shao, Meng-Meng

    2016-09-01

    To establish a rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of vortioxetine, carvedilol and its metabolite 4-hydroxyphenyl carvedilol in rat plasma. The analytes and the internal standard (diazepam) were separated on an Acquity UPLC BEH C18 chromatography column (2.1mm×50mm, 1.7μm) using gradient elution with a mobile phase of acetonitrile and 0.1% formic acid in water at a flow rate of 0.4mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 299.2→150.1 for vortioxetine, m/z 407.2→100.3 for carvedilol, m/z 423.2→100.1 for 4-hydroxyphenyl carvedilol and m/z 285.2→193.1 for diazepam (IS) using a positive electrospray ionization interface. The method was validated over a concentration range of 0.5-100ng/mL for vortioxetine, 0.5-1000ng/mL for carvedilol and 0.1-50ng/mL for 4-hydroxyphenyl carvedilol. Total time for each chromatograph was 3.0min. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels exhibited relative standard deviations (RSD)<11.6% and the accuracy values ranged from -12.2% to 11.3%. The analytical method was successfully applied to a pharmacokinetic interaction study of vortioxetine and carvedilol after oral administration vortioxetine and carvedilol in rats. Results suggested that the co-administration of vortioxetine and carvedilol results in a significant drug interaction in rats. PMID:27262994

  17. Spectroscopic and structural elucidation of merocyanine dye 2,5-[1-metyl-4-[2-(4-hydroxyphenyl)ethenyl)]piridinium]-hexane tetraphenylborate aggregation processes.

    PubMed

    Koleva, Bojidarka B; Stoyanov, Stanimir; Kolev, Tsonko; Petkov, Ivan; Spiteller, Michael

    2008-12-01

    Structural and spectroscopic elucidation of merocyanine dye, 2,5-[1-metyl-4-[2-(4-hydroxyphenyl)ethenyl)]piridinium]-hexane tetraphenylborate, is performed in gas and condense phase by means of solution and solid-state conventional and linear-polarized IR-spectroscopy of oriented colloids in nematic liquid crystal suspension, UV-vis and fluorescence methods, HPLC MS/MS tandem and ESI mass spectrometry, (1)H, (13)C and (1)H-(1)H COSY NMR, TGV and DSC methods. Quantum chemical DFT calculations are performed for structural optimization and spectroscopic properties prediction. PMID:18400554

  18. Spectroscopic and structural elucidation of merocyanine dye 2,5-[1-metyl-4-[2-(4-hydroxyphenyl)ethenyl)]piridinium]-hexane tetraphenylborate. Aggregation processes

    NASA Astrophysics Data System (ADS)

    Koleva, Bojidarka B.; Stoyanov, Stanimir; Kolev, Tsonko; Petkov, Ivan; Spiteller, Michael

    2008-12-01

    Structural and spectroscopic elucidation of merocyanine dye, 2,5-[1-metyl-4-[2-(4-hydroxyphenyl)ethenyl)]piridinium]-hexane tetraphenylborate, is performed in gas and condense phase by means of solution and solid-state conventional and linear-polarized IR-spectroscopy of oriented colloids in nematic liquid crystal suspension, UV-vis and fluorescence methods, HPLC MS/MS tandem and ESI mass spectrometry, 1H, 13C and 1H- 1H COSY NMR, TGV and DSC methods. Quantum chemical DFT calculations are performed for structural optimization and spectroscopic properties prediction.

  19. Erbeta Ligands. Part 5: Synthesis and Structure-Activity Relationships of a Series of 4'-hydroxyphenyl-aryl-carbaldehyde Oxime Derivatives

    SciTech Connect

    Mewshaw,R.; Bowen, S.; Harris, H.; Xu, Z.; Manas, E.; Cohn, S.

    2007-01-01

    A series of 4'-hydroxyphenyl-aryl-carbaldehyde oximes (5b) was prepared and found to have high affinity (4 nM) and modest selectivity (39-fold) for estrogen receptor-{beta} (ER{beta}). Substitution of one of the core rings of the scaffold based around these novel ligands further expanded our knowledge in the quest toward achieving high affinity and selectivity for ER{beta}. An X-ray co-crystal of structure 11 revealed that the oxime moiety was mimicking the C-ring of genistein, as previously predicted by SAR and docking studies.

  20. Fluorescence-based sensor for Pb(II) using tetra-(3-bromo-4-hydroxyphenyl)porphyrin in liquid and immobilized medium

    NASA Astrophysics Data System (ADS)

    Bozkurt, Serap Seyhan; Ayata, Sevda; Kaynak, Ipek

    2009-05-01

    A new optical sensor for sensing of Pb 2+ in immobilized medium (PVC film) and ethanol medium was developed by using 5,10,15,20-tetra-(3-bromo-4-hydroxyphenyl)porphyrin (TBHPP) synthesized. The sensor-based TBHPP showed a linear response towards Pb 2+ in concentration range from 5 × 10 -6 to 4 × 10 -4 mol L -1 in PVC film and 5 × 10 -6 to 3 × 10 -4 mol L -1 in ethanol medium, with a working pH 7. The detection limit was 2 × 10 -8 and 4 × 10 -8 mol L -1 for Pb 2+ in PVC film and ethanol medium respectively. The response time of Pb 2+ was found as 4 min for PVC film and 2 min for ethanol medium. The sensor developed in two different mediums was used for lead determination in standard soil sample with satisfactory results.

  1. Infrared and Raman spectroscopic analyses and theoretical computation of 4-butyl-1-(4-hydroxyphenyl)-2-phenyl-3,5-pyrazolidinedione

    NASA Astrophysics Data System (ADS)

    Binil, P. S.; Mary, Y. Sheena; Varghese, Hema Tresa; Panicker, C. Yohannan; Anoop, M. R.; Manojkumar, T. K.

    Infrared and Raman spectroscopic analyses were carried out on 4-butyl-1-(4-hydroxyphenyl)-2-phenyl-3,5-pyrazolidinedione. The interpretation of the spectra was aided by DFT calculation of the molecule. The vibrational wavenumbers were examined theoretically using the Gaussian03 set of quantum chemistry codes and the normal modes were assigned by potential energy distribution calculations. A computation of the first hyperpolarizability of the compound indicates that the compound may be a good candidate as a NLO material. Optimized geometrical parameters are in agreement with the reported XRD results. The RMS error of the observed Raman bands and IR bands are found to be 35.09 and 39.57 for HF method and 14.31 and 17.17 for DFT method. The predicted infrared intensities and Raman activities are reported.

  2. Experimental and density functional theory and ab initio Hartree-Fock study on the vibrational spectra of 2-(4-fluorobenzylideneamino)-3-(4-hydroxyphenyl) propanoic acid

    NASA Astrophysics Data System (ADS)

    Song, Yuan-Zhi; Ruan, Min; Ye, Yong; Li, Yue-Ying; Xie, Wei; Shen, Jing; Shen, Ai-Guo

    2008-02-01

    2-(4-Fluorobenzylideneamino)-3-(4-hydroxyphenyl) propanoic acid (4-FT) was synthesized through the reaction of 4-fluorobenzaldehyde and L-tyrosine in refluxing EtOH. The structure of 4-FT was verified by measuring 1H NMR, FTIR and Raman. The ground-state geometries were optimized at B3LYP/6-31G**, B3LYP/6-31G*, HF/6-31G** and HF/6-31G* levels without symmetry constrains. The vibrational wavenumbers of 4-FT were calculated at same levels. The scaled spectra using B3LYP methods, which are in a good agreement with the measured spectra, are superior to those calculated using HF methods.

  3. Synthesis, spectroscopic and structural elucidation of 1-butyl-4-[2-(3,5-dimethoxy-4-hydroxyphenyl)ethenyl)]pyridinium chloride tetrahydrate.

    PubMed

    Koleva, B B; Kolev, T; Lamshöft, M; Mayer-Figge, H; Sheldrick, W S; Spiteller, M

    2009-12-01

    The novel chloride salt of 1-butyl-4-[2-(4-hydroxyphenyl)ethenyl)]pyridine (1), has been synthesized as the tetrahydrate and its structure and properties elucidated in detail spectroscopically, thermally and structurally, using single crystal X-ray diffraction, linear-polarized solid-state IR-spectroscopy, UV-spectroscopy and mass spectrometry. Quantum chemical calculations were performed with a view to supporting and explaining the experimental structural and spectroscopic data. The compound (1) crystallizes in triclinic P1 space group and its unit cell contains two independent 1-butyl-4-[2-(3,5-dimethoxy4-hydroxyphenyl)ethenyl)]pyridinium] cations, differing with respect to the butyl chain torsion angle for which values of 80.0(9) degrees and 173.6(3) degrees are observed. The cations and anions are joined into infinite layers, formed by two different dimers and including solvent molecules. Hydrogen bonds OH...OH(2) (2.814 A), HOH...O(CH(3)) (2.960 A), OH...Cl (2.967 A), HOH...Cl(-) (3.034, 3.188, 3.161 and 3.062 A) and HOH...OH(2) (2.772 A) are observed. For first time in the literature, we are reporting the crystal structure of the dye with the syring-fragment in the molecule. The spectroscopic properties of the novel compound are compared and with those of the corresponding quinoide form (2). Both the forms (1) and (2) are characterized by 21 and 140 nm solvatochromic effects depending of the type of the solvent. The UV-spectroscopic data in solution confirm the formation of classical H-aggregates in polar protic solvent mixture. PMID:19833548

  4. Synthesis, spectroscopic and structural elucidation of 1-butyl-4-[2-(3,5-dimethoxy-4-hydroxyphenyl)ethenyl)]pyridinium chloride tetrahydrate

    NASA Astrophysics Data System (ADS)

    Koleva, B. B.; Kolev, T.; Lamshöft, M.; Mayer-Figge, H.; Sheldrick, W. S.; Spiteller, M.

    2009-12-01

    The novel chloride salt of 1-butyl-4-[2-(4-hydroxyphenyl)ethenyl)]pyridine ( 1), has been synthesized as the tetrahydrate and its structure and properties elucidated in detail spectroscopically, thermally and structurally, using single crystal X-ray diffraction, linear-polarized solid-state IR-spectroscopy, UV-spectroscopy and mass spectrometry. Quantum chemical calculations were performed with a view to supporting and explaining the experimental structural and spectroscopic data. The compound ( 1) crystallizes in triclinic P1¯ space group and its unit cell contains two independent 1-butyl-4-[2-(3,5-dimethoxy4-hydroxyphenyl)ethenyl)]pyridinium] cations, differing with respect to the butyl chain torsion angle for which values of 80.0(9)° and 173.6(3)° are observed. The cations and anions are joined into infinite layers, formed by two different dimers and including solvent molecules. Hydrogen bonds OH⋯OH 2 (2.814 Å), HOH⋯O(CH 3) (2.960 Å), OH⋯Cl (2.967 Å), HOH⋯Cl - (3.034, 3.188, 3.161 and 3.062 Å) and HOH⋯OH 2 (2.772 Å) are observed. For first time in the literature, we are reporting the crystal structure of the dye with the syring-fragment in the molecule. The spectroscopic properties of the novel compound are compared and with those of the corresponding quinoide form ( 2). Both the forms ( 1) and ( 2) are characterized by 21 and 140 nm solvatochromic effects depending of the type of the solvent. The UV-spectroscopic data in solution confirm the formation of classical H-aggregates in polar protic solvent mixture.

  5. Pyruvate kinase blood test

    MedlinePlus

    ... break down faster than normal, a condition called hemolytic anemia . This test helps diagnose pyruvate kinase deficiency (PKD) . ... Pa: Elsevier Saunders; 2011:chap 32. Gallagher PG. Hemolytic anemias: red cell membrane and metabolic defects In: Goldman ...

  6. Reactive oxygen species mediate N-(4-hydroxyphenyl)retinamide-induced cell death in malignant T cells and are inhibited by the HTLV-I oncoprotein Tax.

    PubMed

    Darwiche, N; Abou-Lteif, G; Bazarbachi, A

    2007-02-01

    N-(4-hydroxyphenyl)retinamide (HPR) is a synthetic retinoid that inhibits growth of many human tumor cells, including those resistant to natural retinoids. HPR is an effective chemopreventive agent for prostate, cervix, breast, bladder, skin and lung cancers, and has shown promise for the treatment of neuroblastomas. We have previously shown that HPR inhibits proliferation and induces apoptosis of human T-cell lymphotropic virus type I (HTLV-I)-associated adult T-cell leukemia (ATL) and HTLV-I-negative malignant T cells, whereas no effect is observed on normal lymphocytes. In this report, we identified HPR-induced reactive oxygen species (ROS) generation as the key mediator of cell cycle arrest and apoptosis of malignant T cells. HPR treatment of HTLV-I-negative malignant T cells was associated with a rapid and progressive ROS accumulation. Pre-treatment with the antioxidants vitamin C and dithiothreitol inhibited ROS generation, prevented HPR-induced ceramide accumulation, cell cycle arrest, cytochrome c release, caspase-activation and apoptosis. Therefore, anti-oxidants protected malignant T cells from HPR-induced growth inhibition. The expression of the HTLV-I oncoprotein Tax abrogated HPR-induced ROS accumulation in HTLV-I-infected cells, which explains their lower sensitivity to HPR. Defining the mechanism of free radical induction by HPR may support a potential therapeutic role for this synthetic retinoid in ATL and HTLV-I-negative T-cell lymphomas. PMID:17122865

  7. Structural, spectral, thermal and biological studies on (E)-2-(1-(4-hydroxyphenyl)ethylidene)-N-(pyridin-2-yl)hydrazinecarbothioamide and its metal complexes

    NASA Astrophysics Data System (ADS)

    Yousef, T. A.; Abu El-Reash, G. M.; El-Rakhawy, E. R.

    2014-12-01

    Schiff base complexes of Mn(II), Co(II), Ni(II), Cu(II), Cd(II), Hg(II) and U(VI)O2 with (E)-2-(1-(4-hydroxyphenyl)ethylidene)-N-(pyridin-2-yl)hydrazinecarbothioamide (H2PHAT) were synthesized and characterized by different physicochemical methods, elemental analysis, (UV-vis, IR and 1H NMR spectra) and thermal analysis (TG and DTG) techniques. Spectral data showed that H2PHAT behaves as a NS bidentate ligand through both thione sulphur or thiol sulphur and the nitrogen of the pyridine ring or azomethine nitrogen, NSN tridentate ligand through both thione sulphur or thiol sulphur, the nitrogen of the pyridine ring and azomethine nitrogen. ESR spectrum data for Cu(II) solid complex confirms the square planar state is the most fitted one for the coordinated structure. The kinetic parameters were determined for each thermal degradation stage of the complexes using Coats-Redfern and Horowitz-Metzger methods. From modeling studies, the bond length, bond angle, HOMO, LUMO and dipole moment had been calculated to confirm the geometry of the ligand and their investigated complexes. The biological activity was tested against DNA showing that Cd(II), U(VI)O2, Ni(II) and Mn(II) complexes had powerful and complete degradation effect. Also, the ligand and its complexes were screened against Bacillus thuringiensis as Gram-positive bacteria and Pseudomonas aeuroginosa as Gram-negative bacteria using the inhibitory zone diameter.

  8. The Nitrification Inhibitor Methyl 3-(4-Hydroxyphenyl)Propionate Modulates Root Development by Interfering with Auxin Signaling via the NO/ROS Pathway.

    PubMed

    Liu, Yangyang; Wang, Ruling; Zhang, Ping; Chen, Qi; Luo, Qiong; Zhu, Yiyong; Xu, Jin

    2016-07-01

    Methyl 3-(4-hydroxyphenyl)propionate (MHPP) is a root exudate that functions as a nitrification inhibitor and as a modulator of the root system architecture (RSA) by inhibiting primary root (PR) elongation and promoting lateral root formation. However, the mechanism underlying MHPP-mediated modulation of the RSA remains unclear. Here, we report that MHPP inhibits PR elongation in Arabidopsis (Arabidopsis thaliana) by elevating the levels of auxin expression and signaling. MHPP induces an increase in auxin levels by up-regulating auxin biosynthesis, altering the expression of auxin carriers, and promoting the degradation of the auxin/indole-3-acetic acid family of transcriptional repressors. We found that MHPP-induced nitric oxide (NO) production promoted reactive oxygen species (ROS) accumulation in root tips. Suppressing the accumulation of NO or ROS alleviated the inhibitory effect of MHPP on PR elongation by weakening auxin responses and perception and by affecting meristematic cell division potential. Genetic analysis supported the phenotype described above. Taken together, our results indicate that MHPP modulates RSA remodeling via the NO/ROS-mediated auxin response pathway in Arabidopsis. Our study also revealed that MHPP significantly induced the accumulation of glucosinolates in roots, suggesting the diverse functions of MHPP in modulating plant growth, development, and stress tolerance in plants. PMID:27217493

  9. New analgesics synthetically derived from the paracetamol metabolite N-(4-hydroxyphenyl)-(5Z,8Z,11Z,14Z)-icosatetra-5,8,11,14-enamide.

    PubMed

    Sinning, Christian; Watzer, Bernhard; Coste, Ovidiu; Nüsing, Rolf M; Ott, Ingo; Ligresti, Alessia; Di Marzo, Vincenzo; Imming, Peter

    2008-12-25

    N-(4-hydroxyphenyl)-(5Z,8Z,11Z,14Z)-icosatetra-5,8,11,14-enamide (AM404) is a metabolite of the well-known analgesic paracetamol. AM404 inhibits endocannabinoid cellular uptake, binds weakly to CB1 and CB2 cannabinoid receptors, and is formed by fatty acid amide hydrolase (FAAH) in vivo. We prepared three derivatives of this new (endo)cannabinoid using bioisosteric replacement (1), homology (2), and derivatization (3) of the 4-aminophenol moiety in AM404 and tested them against CB1, CB2, and FAAH. We found affinities toward both cannabinoid receptors equal to or greater than that of AM404. Shortening the acyl chain from C20 to C2 led to three new paracetamol analogues: N-(1H-indazol-5-yl)acetamide (5), N-(4-hydroxybenzyl)acetamide (6), and N-(4-hydroxy-3-methoxyphenyl)acetamide (7). Again, 5, 6, and 7 were tested against CB1, CB2, and FAAH without significant activity. However, 5 and 7 behaved like inhibitors of cyclooxygenases in whole blood assays. Finally, 5 (50 mg/kg) and 6 (275 mg/kg) displayed analgesic activities comparable to paracetamol (200 mg/kg) in the mouse formalin test. PMID:19053765

  10. Structural, spectral, thermal and biological studies on (E)-2-(1-(4-hydroxyphenyl)ethylidene)-N-(pyridin-2-yl)hydrazinecarbothioamide and its metal complexes.

    PubMed

    Yousef, T A; Abu El-Reash, G M; El-Rakhawy, E R

    2014-12-10

    Schiff base complexes of Mn(II), Co(II), Ni(II), Cu(II), Cd(II), Hg(II) and U(VI)O2 with (E)-2-(1-(4-hydroxyphenyl)ethylidene)-N-(pyridin-2-yl)hydrazinecarbothioamide (H2PHAT) were synthesized and characterized by different physicochemical methods, elemental analysis, (UV-vis, IR and (1)H NMR spectra) and thermal analysis (TG and DTG) techniques. Spectral data showed that H2PHAT behaves as a NS bidentate ligand through both thione sulphur or thiol sulphur and the nitrogen of the pyridine ring or azomethine nitrogen, NSN tridentate ligand through both thione sulphur or thiol sulphur, the nitrogen of the pyridine ring and azomethine nitrogen. ESR spectrum data for Cu(II) solid complex confirms the square planar state is the most fitted one for the coordinated structure. The kinetic parameters were determined for each thermal degradation stage of the complexes using Coats-Redfern and Horowitz-Metzger methods. From modeling studies, the bond length, bond angle, HOMO, LUMO and dipole moment had been calculated to confirm the geometry of the ligand and their investigated complexes. The biological activity was tested against DNA showing that Cd(II), U(VI)O2, Ni(II) and Mn(II) complexes had powerful and complete degradation effect. Also, the ligand and its complexes were screened against Bacillus thuringiensis as Gram-positive bacteria and Pseudomonas aeuroginosa as Gram-negative bacteria using the inhibitory zone diameter. PMID:24992916

  11. Study of the laxative properties of the disodium salt of the sulfuric diester of 3,3-bis-(4-hydroxyphenyl)-7-methyl-2-indolinone (DAN-603 in the rat.

    PubMed

    Moretó, M; Goñalons, E; Giráldez, A; Torralba, A

    1976-03-01

    The influence of DAN-603 (disodium salt of sulphuric diester of 3,3-bis-(4-hydroxyphenyl)-7-methyl-2-indolinone) on the propulsive motility of the rat digestive tract was studied by means of indicators (charcoal and pyrvinium pamoate) and radioactive tracers (133BaSO4). The results showed that DAN-603 increases selectively the colon motility without modifying the speed of gastric, intestinal (small intestine) and caecal emptying. PMID:1261595

  12. Crystal Structure of Mammalian Cysteine dioxygenase: A Novel Mononuclear Iron Center for Cysteine Thiol Oxidation

    SciTech Connect

    Simmons,C.; Liu, Q.; Huang, Q.; Hao, Q.; Begley, T.; Karplus, P.; Stipanuk, M.

    2006-01-01

    Cysteine dioxygenase is a mononuclear iron-dependent enzyme responsible for the oxidation of cysteine with molecular oxygen to form cysteinesulfinate. This reaction commits cysteine to either catabolism to sulfate and pyruvate or to the taurine biosynthetic pathway. Cysteine dioxygenase is a member of the cupin superfamily of proteins. The crystal structure of recombinant rat cysteine dioxygenase has been determined to 1.5 Angstroms resolution, and these results confirm the canonical cupin {beta}-sandwich fold and the rare cysteinyl-tyrosine intramolecular crosslink (between Cys93 and Tyr157) seen in the recently reported murine cysteine dioxygenase structure. In contrast to the catalytically inactive mononuclear Ni(II) metallocenter present in the murine structure, crystallization of a catalytically competent preparation of rat cysteine dioxygenase revealed a novel tetrahedrally coordinated mononuclear iron center involving three histidines (His86, His88, and His140) and a water molecule. Attempts to acquire a structure with bound ligand using either co-crystallization or soaks with cysteine revealed the formation of a mixed disulfide involving Cys164 near the active site, which may explain previously observed substrate inhibition. This work provides a framework for understanding the molecular mechanisms involved in thiol dioxygenation and sets the stage for exploring the chemistry of both the novel mononuclear iron center and the catalytic role of the cysteinyl-tyrosine linkage.

  13. Synthesis, spectroscopic characterization and thermal behavior of metal complexes formed with N'-(1-(4-hydroxyphenyl) ethylidene)-2-oxo-2-(phenylamino) acetohydrazide (H 3OPAH)

    NASA Astrophysics Data System (ADS)

    Ahmed, Sara F.; El-Gammal, Ola A.; El-Reash, Gaber Abu

    2011-12-01

    Complexes of Co(II), Ni(II), Cu(II), Mn(II), Cd(II), Zn(II), Hg(II) and U(IV)O 22+ with N'-(1-(4-hydroxyphenyl) ethylidene)-2-oxo-2-(phenylamino) acetohydrazide (H 3OPAH) are reported and have been characterized by various spectroscopic techniques like IR, UV-visible, 1H NMR and ESR as well as magnetic and thermal (TG and DTA) measurements. It is found that the ligand behaves as a neutral bidentate, monoanionic tridentate or tetradentate and dianionic tetradentate. An octahedral geometry for [Mn(H 3OPAH) 2Cl 2], [Co 2(H 2OPAH) 2Cl 2(H 2O) 4] and [(UO 2) 2(HOPAH)(OAc) 2(H 2O) 2] complexes, a square planar geometry for [Cu 2(H 2OPAH)Cl 3(H 2O)]H 2O complex, a tetrahedral structure for [Cd(H 3OPAH)Cl 2], [Zn(H 3OPAH)(OAc) 2] and [Hg(H 3OPAH)Cl 2]H 2O complexes. The binuclear [Ni 2(HOPAH)Cl 2(H 2O) 2]H 2O complex contains a mixed geometry of both tetrahedral and square planar structures. The protonation constants of ligand and stepwise stability constants of its complexes at 298, 308 and 318 K as well as the thermodynamic parameters are being calculated. The bond lengths, bond angles, HOMO, LUMO and dipole moments have been calculated to confirm the geometry of the ligand and the investigated complexes. Also, thermal properties and decomposition kinetics of all compounds are investigated. The interpretation, mathematical analysis and evaluation of kinetic parameters ( Ea, A, Δ H, Δ S and Δ G) of all thermal decomposition stages have been evaluated using Coats-Redfern and Horowitz-Metzger methods.

  14. Alkaline Ceramidase 2 (ACER2) and Its Product Dihydrosphingosine Mediate the Cytotoxicity of N-(4-Hydroxyphenyl)retinamide in Tumor Cells*

    PubMed Central

    Mao, Zhehao; Sun, Wei; Xu, Ruijuan; Novgorodov, Sergei; Szulc, Zdzislaw M.; Bielawski, Jacek; Obeid, Lina M.; Mao, Cungui

    2010-01-01

    Increased generation of dihydrosphingosine (DHS), a bioactive sphingolipid, has been implicated in the cytotoxicity of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in tumor cells. However, how 4-HPR increases DHS remains unclear. Here we demonstrate that 4-HPR increases the expression of ACER2, which catalyzes the hydrolysis of dihydroceramides to generate DHS, and that ACER2 up-regulation plays a key role in mediating the 4-HPR-induced generation of DHS as well as the cytotoxicity of 4-HPR in tumor cells. Treatment with 4-HPR induced the accumulation of dihydroceramides (DHCs) in tumor cells by inhibiting dihydroceramide desaturase (DES) activity, which catalyzes the conversion of DHCs to ceramides. Treatment with 4-HPR also increased ACER2 expression through a retinoic acid receptor-independent and caspase-dependent manner. Overexpression of ACER2 augmented the 4-HPR-induced generation of DHS as well as 4-HPR cytotoxicity, and 4-HPR-induced death in tumor cells, whereas knocking down ACER2 had the opposite effects. ACER2 overexpression, along with treatment with GT11, another DES inhibitor, markedly increased cellular DHS, leading to tumor cell death, whereas ACER2 overexpression or GT11 treatment alone failed to do so, suggesting that both ACER2 up-regulation and DES inhibition are necessary and sufficient to mediate 4-HPR-induced DHS accumulation, cytotoxicity, and death in tumor cells. Taken together, these results suggest that up-regulation of the ACER2/DHS pathway mediates the cytotoxicity of 4-HPR in tumor cells and that up-regulating or activating ACER2 may improve the anti-cancer activity of 4-HRR and other DHC-inducing agents. PMID:20628055

  15. Hydroperoxylation by Hydroxyethylphosphonate Dioxygenase

    PubMed Central

    2009-01-01

    Hydroxyethylphosphonate dioxygenase (HEPD) catalyzes the O2-dependent cleavage of the carbon−carbon bond of 2-hydroxyethylphosphonate (2-HEP) to afford hydroxymethylphosphonate (HMP) and formate without input of electrons or use of any organic cofactors. Two mechanisms have been proposed to account for this reaction. One involves initial hydroxylation of substrate to an acetal intermediate and its subsequent attack onto an Fe(IV)-oxo species. The second mechanism features initial hydroperoxylation of substrate followed by a Criegee rearrangement. To distinguish between the two mechanisms, substrate analogues were synthesized and presented to the enzyme. Hydroxymethylphosphonate was converted into phosphate and formate, and 1-hydroxyethylphosphonate was converted to acetylphosphate, which is an inhibitor of the enzyme. These results provide strong support for a Criegee rearrangement with a phosphorus-based migrating group and require that the O−O bond of molecular oxygen is not cleaved prior to substrate activation. (2R)-Hydroxypropylphosphonate partitioned between conversion to 2-oxopropylphosphonate and hydroxymethylphosphonate, with the latter in turn converted to phosphate and formate. Collectively, these results support a mechanism that proceeds by hydroperoxylation followed by a Criegee rearrangement. PMID:19839620

  16. Isolation of mesotrione-degrading bacteria from aquatic environments in Brazil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mesotrione is a benzoylcyclohexane-1,3-dione herbicide that inhibits 4-hydroxyphenyl pyruvate dioxygenase (HPPD) in target plants. Although it has been used since 2000, only a limited number of degrading microorganisms have been reported. Mesotrione-degrading bacteria were selected among strains iso...

  17. Modulation of the insulin-like growth factor-I system by N-(4-hydroxyphenyl)-retinamide in human breast cancer cell lines.

    PubMed Central

    Favoni, R. E.; de Cupis, A.; Bruno, S.; Yee, D.; Ferrera, A.; Pirani, P.; Costa, A.; Decensi, A.

    1998-01-01

    The potent mitogenic activity of insulin-like growth factor I (IGF-I) on breast epithelium is inhibited by retinoic acid in oestrogen receptor-positive (ER+) breast cancer cell lines. We studied and compared the effects of N-(4-hydroxyphenyl)-retinamide (4-HPR) in terms of growth inhibition and modulation of the IGF-I system in ER+ (MCF-7) and oestrogen receptor-negative (ER-) (MDA-MB231) breast cancer cell lines. Treatment with 1-10 microM 4-HPR for up to 96 h induced a dose- and time-dependent inhibition of proliferation in both breast cancer cell lines. Induction of apoptosis was much more evident in MCF-7 than in MDA-MB231 cells (30-40% compared with 0-5% respectively at 5 microM for 48 h). Exogenous human recombinant IGF-I (hr-IGF-I)-stimulated cell proliferation was abolished by 1 microM 4-HPR in MCF-7 cells. Immunoreactive IGF-I-like protein concentration in conditioned medium was reduced by 38% in MCF-7 and by 90% in MDA-MB231 cell lines following treatment for 48 h with 5 microM 4-HPR. Western ligand blot analysis showed a reduction of IGF-binding protein 4 (BP4) and BP5 by 67% and 87%, respectively, in MCF-7, whereas IGF-BP4 and -BP1 were reduced by approximately 20% in MDA-MB231 cells. Exposure to 5 microM 4-HPR for 48 h inhibited [125I]IGF-I binding and Scatchard analysis revealed a decrease of more than 50% in maximum binding capacity (Bmax) and a reduced receptor number/cell in both cancer cell lines. Steady-state type I IGF-receptor mRNA levels were reduced by approximately 30% in both tumour cell lines. We conclude that 4-HPR induces a significant down-regulation of the IGF-I system in both ER+ (MCF-7) and ER- (MDA-MB231) breast cancer cell lines. These findings suggest that, in our model, interference with the ER signalling pathway is not the only mechanism of breast cancer growth inhibition by 4-HPR. Images Figure 6 Figure 8 PMID:9649125

  18. Biomimetic synthesis and antioxidant evaluation of 3,4-DHPEA-EDA [2-(3,4-hydroxyphenyl) ethyl (3S,4E)-4-formyl-3-(2-oxoethyl)hex-4-enoate].

    PubMed

    Nardi, Monica; Bonacci, Sonia; De Luca, Giuseppina; Maiuolo, Jessica; Oliverio, Manuela; Sindona, Giovanni; Procopio, Antonio

    2014-11-01

    Phenolic compounds present in extra virgin olive oil have attracted considerable recent attention. Many of them, show specific anti-inflammatory and anti-tumor activities. In this work we describe the biomimetic synthesis of 3,4-DHPEA-EDA [2-(3,4-hydroxyphenyl) ethyl (3S,4E)-4-formyl-3-(2-oxoethyl)hex-4-enoate], starting from natural demethyloleuropein present in olive tissues. A comparison between 3,4-DHPEA-EDA (6) and oleuropein (1), oleuropein aglycone (4) and hydroxytyrosol ORACFL values was undertaken. PMID:24874361

  19. Genetics Home Reference: pyruvate dehydrogenase deficiency

    MedlinePlus

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

  20. Synthesis and characterization of terbium(III) complexes with the biscoumarin derivative 3,3‧-[(4-hydroxyphenyl)methyl]bis-(4-hydroxy-2H-chromen-2-one)

    NASA Astrophysics Data System (ADS)

    Elenkova, D.; Monov, R.; Tadjer, A.; Manolov, I.; Milanova, M.

    2016-02-01

    Complexes of Tb(III) with the biscoumarin derivative 3,3‧-[(4-hydroxyphenyl)methyl]bis-(4-hydroxy-2H-chromen-2-one) are synthesized by using a solution of tetraethylammonium hydroxide, [(C2H5)4N]OH, in water as deprotonating base. Elemental analysis, IR- and fluorescence spectroscopy are used to characterize the samples obtained. The complexes have good fluorescent properties and show the characteristic emission bands of the Tb(III) ion. The triplet state of the mono-deprotonated ligand H2L- was determined. The optimised geometries of the ligand and the complexes were obtained by means of molecular modelling with first principles (DFT) methods.

  1. Inhibitory Effect of 3-(4-Hydroxyphenyl)-1-(thiophen-2-yl) prop-2-en-1-one, a Chalcone Derivative on MCP-1 Expression in Macrophages via Inhibition of ROS and Akt Signaling

    PubMed Central

    Kim, Mi Jin; Kadayat, Taraman; Um, Yeon Ji; Jeong, Tae Cheon; Lee, Eung-Seok; Park, Pil-Hoon

    2015-01-01

    Chalcones (1,3-diaryl-2-propen-1-ones), a subfamily of flavonoid, are widely known to possess potent anti-inflammatory and anti-oxidant properties. In this study, we investigated the effect of 3-(4-Hydroxyphenyl)-1-(thio3-(4-Hydroxyphenyl phen-2-yl)prop-2-en-1-one (TI-I-175), a synthetic chalcone derivative, on endotoxin-induced expression of monocyte chemoattractant protein-1 (MCP-1), one of the key chemokines that regulates migration and infiltration of immune cells, and its potential mechanisms. TII-175 potently inhibited MCP-1 mRNA expression stimulated by lipopolysaccharide (LPS) in RAW 264.7 macrophages without significant effect on cell viability. Treatment of cells with TI-I-175 markedly prevented LPS-induced transcriptional activation of activator protein-1 (AP-1) as measured by luciferase reporter assay, while nuclear factor-κB (NF-κB) activity was not inhibited by TI-I-175, implying that TI-I-175 suppressed MCP-1 expression probably via regulation of AP-1. In addition, TI-I-175 treatment significantly inhibited LPS-induced Akt phosphorylation and led to a significant decrease in reactive oxygen species (ROS) production by LPS, which act as up-stream signaling events required for AP-1 activation in RAW 264.7 macrophages. Taken together, these results indicate that TI-I-175 suppresses MCP-1 gene expression in LPS-stimulated RAW 264.7 macrophages via suppression of ROS production and Akt activation. PMID:25767679

  2. Pyruvate Metabolism in Sarcina maxima

    PubMed Central

    Kupfer, Dorothy G.; Canale-Parola, E.

    1967-01-01

    The mechanisms of pyruvate cleavage and hydrogen production by Sarcina maxima were studied. It was found that a phosphoroclastic system for pyruvate oxidation, similar to that occurring in saccharolytic clostridia, is present in S. maxima. Cleavage of pyruvate by extracts of the latter organism resulted in the formation of acetyl phosphate, CO2, and electrons which were transferred to ferredoxin. Formate was not an intermediate in this system. Pyruvate oxidation was coupled with ferredoxin-dependent nicotinamide adenine dinucleotide phosphate (NADP) reduction. A hydrogenase, active in particulate extracts of S. maxima, did not accept electrons from reduced ferredoxin. Formate was detected as a fermentation product when S. maxima was grown in media buffered with CaCO3. Whole cells and extracts degraded formate to H2 and CO2. The evidence suggests that electrons generated by ferredoxin-linked pyruvate oxidation by S. maxima are not used for H2 production, but that they serve for the reduction of NADP. Reduced NADP may be utilized by the organisms for synthesis of cell material. Production of H2 by S. maxima may occur through a pyruvate clastic system similar to that present in coliform bacteria. PMID:4383134

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

    PubMed

    Baggetto, L G; Lehninger, A L

    1987-05-29

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

  4. Structural Basis for Small Molecule NDB (N-Benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) Benzamide) as a Selective Antagonist of Farnesoid X Receptor α (FXRα) in Stabilizing the Homodimerization of the Receptor*

    PubMed Central

    Xu, Xing; Xu, Xin; Liu, Peng; Zhu, Zhi-yuan; Chen, Jing; Fu, Hai-an; Chen, Li-li; Hu, Li-hong; Shen, Xu

    2015-01-01

    Farnesoid X receptor α (FXRα) as a bile acid sensor plays potent roles in multiple metabolic processes, and its antagonist has recently revealed special interests in the treatment of metabolic disorders, although the underlying mechanisms still remain unclear. Here, we identified that the small molecule N-benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) benzamide (NDB) functioned as a selective antagonist of human FXRα (hFXRα), and the crystal structure of hFXRα ligand binding domain (hFXRα-LBD) in complex with NDB was analyzed. It was unexpectedly discovered that NDB induced rearrangements of helix 11 (H11) and helix 12 (H12, AF-2) by forming a homodimer of hFXRα-LBD, totally different from the active conformation in monomer state, and the binding details were further supported by the mutation analysis. Moreover, functional studies demonstrated that NDB effectively antagonized the GW4064-stimulated FXR/RXR interaction and FXRα target gene expression in primary mouse hepatocytes, including the small heterodimer partner (SHP) and bile-salt export pump (BSEP); meanwhile, administration of NDB to db/db mice efficiently decreased the gene expressions of phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6-pase), small heterodimer partner, and BSEP. It is expected that our first analyzed crystal structure of hFXRα-LBD·NDB will help expound the antagonistic mechanism of the receptor, and NDB may find its potential as a lead compound in anti-diabetes research. PMID:26100621

  5. Safety assessment for octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)-propionate (CAS Reg. No. 2082-79-3) from use in food contact applications.

    PubMed

    Neal-Kluever, April P; Bailey, Allan B; Hatwell, Karen R

    2015-12-01

    Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate (CAS Reg. No. 2082-79-3), currently marketed as Irganox 1076 (I-76), is a sterically hindered phenolic antioxidant used in a variety of organic substrates, including those used in the manufacture of food contact articles. In 2012, the US Food and Drug Administration (USFDA), Office of Food Additive Safety (OFAS), initiated a post-market re-evaluation of the food contact applications of I-76. This project aimed to ensure that current dietary exposures from the use of I-76 in food contact articles are accurately captured and the safety assessment considered all relevant and available toxicological information. To accomplish these aims, the USFDA reviewed the available toxicological studies and chemistry information on food contact applications of I-76. Based on this in-depth analysis, a NOAEL of 64 mg/kg-bw/d (female rats) from a chronic rat study and a cumulative estimated dietary intake (CEDI) of 4.5 mg/p/d, was used to calculate a margin of exposure (MOE) of ∼850. We concluded that the previous and current exposure levels provide an adequate margin of safety (MOS) and remain protective of human health for the regulated uses. PMID:26482640

  6. Structural Basis for Small Molecule NDB (N-Benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) Benzamide) as a Selective Antagonist of Farnesoid X Receptor α (FXRα) in Stabilizing the Homodimerization of the Receptor.

    PubMed

    Xu, Xing; Xu, Xin; Liu, Peng; Zhu, Zhi-yuan; Chen, Jing; Fu, Hai-an; Chen, Li-li; Hu, Li-hong; Shen, Xu

    2015-08-01

    Farnesoid X receptor α (FXRα) as a bile acid sensor plays potent roles in multiple metabolic processes, and its antagonist has recently revealed special interests in the treatment of metabolic disorders, although the underlying mechanisms still remain unclear. Here, we identified that the small molecule N-benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) benzamide (NDB) functioned as a selective antagonist of human FXRα (hFXRα), and the crystal structure of hFXRα ligand binding domain (hFXRα-LBD) in complex with NDB was analyzed. It was unexpectedly discovered that NDB induced rearrangements of helix 11 (H11) and helix 12 (H12, AF-2) by forming a homodimer of hFXRα-LBD, totally different from the active conformation in monomer state, and the binding details were further supported by the mutation analysis. Moreover, functional studies demonstrated that NDB effectively antagonized the GW4064-stimulated FXR/RXR interaction and FXRα target gene expression in primary mouse hepatocytes, including the small heterodimer partner (SHP) and bile-salt export pump (BSEP); meanwhile, administration of NDB to db/db mice efficiently decreased the gene expressions of phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6-pase), small heterodimer partner, and BSEP. It is expected that our first analyzed crystal structure of hFXRα-LBD·NDB will help expound the antagonistic mechanism of the receptor, and NDB may find its potential as a lead compound in anti-diabetes research. PMID:26100621

  7. Growth profile of Carboxydothermus hydrogenoformans on pyruvate

    PubMed Central

    2013-01-01

    Carboxydothermus hydrogenoformans is a thermophilic anaerobic strain most widely known for its ability to produce hydrogen (H2) when grown on carbon monoxide (CO). Although relatively well studied, growth characterization on pyruvate has never been assessed. The present work fully characterizes growth of the bacterium on pyruvate as a sole carbon source. C. hydrogenoformans demonstrated a growth rate of 0.03 h-1, with pyruvate consumption ranging between 0.21 and 0.48 mol · g-1 volatile suspended solid · d-1. A lag phase was also observed when switching from pyruvate to CO. When grown simultaneously on pyruvate and CO, pyruvate consumption was initiated upon CO depletion. This was attributed to pyruvate oxidation inhibition by CO, and not to a diauxic phenomenom. The strain also showed homoacetogenic activity. PMID:24099169

  8. Spectroscopic Studies of the Catechol Dioxygenases.

    ERIC Educational Resources Information Center

    Que, Lawrence Jr.

    1985-01-01

    The catechol dioxygenases are bacterial iron-containing enzymes that catalyze the oxidative cleavage of catechols. These enzymes serve as a component of nature's mechanisms for degrading aromatic compounds in the environment. The structure and mechanistic aspects of these enzymes are described. (JN)

  9. A thermodynamic study of interaction of Ag+, Mg2+, Ca2+, and K+ cations with 4-hydroxyphenyl-2,5-bis(2-benzofuranyl)pyridine in some binary mixed non-aqueous solvents

    NASA Astrophysics Data System (ADS)

    Khoshnood, Razieh Sanavi; Hatami, Elaheh; Arefi, Donya; Maknoni, Fatemeh Zahra

    2016-02-01

    In the present work the complexation process between Ag+ and Mg2+ cations and 4-hydroxyphenyl-2,5-bis(2-benzofuranyl)pyridine (HBFPY) ligand was studied in pure dimethylformamide (DMF), ethanol (EtOH), acetonitrile (AN) and in (DMF-EtOH), (AN-EtOH) and (DMF-AN) binary mixed solvent solutions at different temperatures using the conductometric method. Also in this work the complexation reaction between Ca2+, K+ cations and HBFPY ligand, was studied in pure dimethylformamide (DMF), propanol (PrOH), 1,4-dioxane (DOX), ethanol (EtOH) and in DMF-PrOH, DMF-DOX and DMF-EtOH binary mixed solvent solutions at different temperatures using the conductometric method. The conductance data show that the stoichiometry of the complexes formed between this ligand and the studied cations is 1 : 1 [ML]. In most cases, addition of HBFPY to solutions of these cations, causes a continuous increase in the molar conductivities which indicates that the mobility of complexed cations is more than the uncomplexed ones. The stability constants of the complexes were obtained from fitting of molar conductivity curves using a computer program, GENPLOT. The stability constant of [Mg(HBFPY)]2+ complex in various neat solvents at 15°C decreases in order: EtOH > DMF > AN and the stability constant of [Ag(HBFPY)]+ complex in various neat solvents at 35°C decreases in order: DMF > EtOH. The values of standard enthalpy changes (Δ H° c ) for complexation reactions were obtained from the slope of the Van't Hoff plots and the changes in standard entropy (Δ S° c ) were calculated from the relationship Δ H° c,295.15= Δ H° c -298.15Δ S° c .

  10. 1-(2,6-Dihydroxy-4-methoxyphenyl)-2-(4-hydroxyphenyl) Ethanone-Induced Cell Cycle Arrest in G1/G0 in HT-29 Cells Human Colon Adenocarcinoma Cells

    PubMed Central

    Lay, Ma Ma; Karsani, Saiful Anuar; Abd Malek, Sri Nurestri

    2014-01-01

    1-(2,6-Dihydroxy-4-methoxyphenyl)-2-(4-hydroxyphenyl) ethanone (DMHE) was isolated from the ethyl acetate fraction of Phaleria macrocarpa (Scheff.) Boerl fruits and the structure confirmed by GC-MS (gas chromatography-mass spectrometry) and NMR (nuclear magnetic resonance) analysis. This compound was tested on the HT-29 human colon adenocarcinoma cell line using MTT (method of transcriptional and translational) cell proliferation assay. The results of MTT assay showed that DMHE exhibited good cytotoxic effect on HT-29 cells in a dose- and time-dependent manner but no cytotoxic effect on the MRC-5 cell line after 72 h incubation. Morphological features of apoptotic cells upon treatment by DMHE, e.g., cell shrinkage and membrane blebbing, were examined by an inverted and phase microscope. Other features, such as chromatin condension and nuclear fragmentation were studied using acridine orange and propidium iodide staining under the fluorescence microscope. Future evidence of apoptosis/necrosis was provided by result fromannexin V-FITC/PI (fluorescein-isothiocyanate/propidium iodide) staining revealed the percentage of early apoptotic, late apoptotic, necrotic and live cells in a dose- and time-dependent manner using flow cytometry. Cell cycle analysis showed G0/G1 arrest in a time-dependent manner. A western blot analysis indicated that cell death might be associated with the up-regulation of the pro-apoptotic proteins Bax PUMA. However, the anit-apotptic proteins Bcl-2, Bcl-xL, and Mcl-1 were also found to increase in a time-dependent manner. The expression of the pro-apoptotic protein Bak was not observed. PMID:24451128

  11. Atmospheric measurements of pyruvic and formic acid

    NASA Technical Reports Server (NTRS)

    Andreae, Meinrat O.; Li, Shao-Meng; Talbot, Robert W.

    1987-01-01

    Pyruvic acid, a product of the atmospheric oxidation of cresols and probably of isoprene, has been determined together with formic acid in atmospheric aerosols and rain as well as in the vapor phase. Both acids are present predominantly as vapor; only about 10-20 percent of the total atmospheric pyruvate and 1-2 percent of the total formate are in the particulate phase. The concentrations of pyruvic and formic acid are highly correlated, with typical formic-to-pyruvic ratios of 10-30 in the gas phase, 20-30 in rain, and 2-10 in aerosols. The gas-phase and rain ratios are comparable to those predicted to result from isoprene oxidation. Pyruvic acid levels were similar in the eastern United States (during summer) and the Amazon Basin, suggesting that natural processes, particularly the photochemical oxidation of isoprene, could account for most of the pyruvic acid present in the atmosphere.

  12. Mitochondrial pyruvate carrier in Trypanosoma brucei.

    PubMed

    Štáfková, Jitka; Mach, Jan; Biran, Marc; Verner, Zdeněk; Bringaud, Frédéric; Tachezy, Jan

    2016-05-01

    Pyruvate is a key product of glycolysis that regulates the energy metabolism of cells. In Trypanosoma brucei, the causative agent of sleeping sickness, the fate of pyruvate varies dramatically during the parasite life cycle. In bloodstream forms, pyruvate is mainly excreted, whereas in tsetse fly forms, pyruvate is metabolized in mitochondria yielding additional ATP molecules. The character of the molecular machinery that mediates pyruvate transport across mitochondrial membrane was elusive until the recent discovery of mitochondrial pyruvate carrier (MPC) in yeast and mammals. Here, we characterized pyruvate import into mitochondrion of T. brucei. We identified mpc1 and mpc2 homologs in the T. brucei genome with attributes of MPC protein family and we demonstrated that both proteins are present in the mitochondrial membrane of the parasite. Investigations of mpc1 or mpc2 gene knock-out cells proved that T. brucei MPC1/2 proteins facilitate mitochondrial pyruvate transport. Interestingly, MPC is expressed not only in procyclic trypanosomes with fully activated mitochondria but also in bloodstream trypanosomes in which most of pyruvate is excreted. Moreover, MPC appears to be essential for bloodstream forms, supporting the recently emerging picture that the functions of mitochondria in bloodstream forms are more diverse than it was originally thought. PMID:26748989

  13. Indoleamine 2,3-dioxygenase vaccination

    PubMed Central

    Andersen, Mads Hald; Svane, Inge Marie

    2015-01-01

    Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme. Remarkably, we discovered IDO-specific T cells that can influence adaptive immune reactions in patients with cancer. Further, a recent phase I clinical trial demonstrated long-lasting disease stabilization without toxicity in patients with non-small-cell lung cancer (NSCLC) who were vaccinated with an IDO-derived HLA-A2-restricted epitope. PMID:25949864

  14. Genetics Home Reference: pyruvate kinase deficiency

    MedlinePlus

    ... National (UK) Information Centre for Metabolic Diseases National Organization for Rare Disorders (NORD): Pyruvate Kinase Deficiency Genetic Testing Registry (1 link) Pyruvate kinase deficiency of red cells Scientific articles on PubMed (1 link) PubMed OMIM (1 link) ...

  15. Thiol Dioxygenases: Unique Families of Cupin Proteins

    PubMed Central

    Simmons, C. R.; Karplus, P. A.; Dominy, J. E.

    2011-01-01

    Proteins in the cupin superfamily have a wide range of biological functions in archaea, bacteria and eukaryotes. Although proteins in the cupin superfamily show very low overall sequence similarity, they all contain two short but partially conserved cupin sequence motifs separated by a less conserved intermotif region that varies both in length and amino acid sequence. Furthermore, these proteins all share a common architecture described as a 6-stranded β-barrel core, and this canonical cupin or “jelly roll” β-barrel is formed with cupin motif 1, the intermotif region, and cupin motif 2 each forming two of the core six β-strands in the folded protein structure. The recently obtained crystal structures of cysteine dioxygenase (CDO), with contains conserved cupin motifs, show that it has the predicted canonical cupin β-barrel fold. Although there had been no reports of CDO activity in prokaryotes, we identified a number of bacterial cupin proteins of unknown function that share low similarity with mammalian CDO and that conserve many residues in the active site pocket of CDO. Putative bacterial CDOs predicted to have CDO activity were shown to have similar substrate specificity and kinetic parameters as eukaryotic CDOs. Information gleaned from crystal structures of mammalian CDO along with sequence information for homologs shown to have CDO activity facilitated the identification of a CDO family fingerprint motif. One key feature of the CDO fingerprint motif is that the canonical metal-binding glutamate residue in cupin motif 1 is replaced by a cysteine (in mammalian CDOs) or by a glycine (bacterial CDOs). The recent report that some putative bacterial CDO homologs are actually 3-mercaptopropionate dioxygenases suggests that the CDO family may include proteins with specificities for other thiol substrates. A paralog of CDO in mammals was also identified and shown to be the other mammalian thiol dioxygenase, cysteamine dioxygenase (ADO). A tentative

  16. Functional response of the isolated, perfused normoxic heart to pyruvate dehydrogenase activation by dichloroacetate and pyruvate

    PubMed Central

    Jaimes, Rafael; Kuzmiak-Glancy, Sarah; Brooks, Daina M.; Swift, Luther M.; Posnack, Nikki G.; Kay, Matthew W.

    2015-01-01

    Dichloroacetate (DCA) and pyruvate activate pyruvate dehydrogenase (PDH), a key enzyme that modulates glucose oxidation and mitochondrial NADH production. Both compounds improve recovery after ischemia in isolated hearts. However, the action of DCA and pyruvate in normoxic myocardium is incompletely understood. We measured the effect of DCA and pyruvate on contraction, mitochondrial redox state, and intracellular calcium cycling in isolated rat hearts during normoxic perfusion. Normalized epicardial NADH fluorescence (nNADH) and left ventricular developed pressure (LVDP) were measured before and after administering DCA (5 mM) or pyruvate (5 mM). Optical mapping of Rhod-2AM was used to measure cytosolic calcium kinetics. DCA maximally activated PDH, increasing the ratio of active to total PDH from 0.48±0.03 to 1.03 ±0.03. Pyruvate sub-maximally activated PDH to a ratio of 0.75±0.02. DCA and pyruvate increased LVDP. When glucose was the only exogenous fuel, pyruvate increased nNADH by 21.4±2.9 % while DCA reduced nNADH by 21.4±6.1 % and elevated the incidence of premature ventricular contractions (PVCs). When lactate, pyruvate, and glucose were provided together as exogenous fuels, nNADH increased with DCA, indicating that PDH activation with glucose as the only exogenous fuel depletes PDH substrate. Calcium transient time-to-peak was shortened by DCA and pyruvate and SR calcium re-uptake was 30 % longer. DCA and pyruvate increased SR calcium load in myocyte monolayers. Overall, during normoxia when glucose is the only exogenous fuel, DCA elevates SR calcium, increases LVDP and contractility, and diminishes mitochondrial NADH. Administering DCA with plasma levels of lactate and pyruvate mitigates the drop in mitochondrial NADH and prevents PVCs. PMID:26142699

  17. Pyruvate kinase and the "high ATP syndrome".

    PubMed Central

    Staal, G E; Jansen, G; Roos, D

    1984-01-01

    The erythrocytes of a patient with the so-called "high ATP syndrome" were characterized by a high ATP content and low 2,3-diphosphoglycerate level. The pyruvate kinase activity was specifically increased (about twice the normal level). After separation of the erythrocytes according to age by discontinuous Percoll density centrifugation, the pyruvate kinase activity was found to be increased in all Percoll fractions. Pyruvate kinase of the patient's cells was characterized by a decreased K0.5 for the substrate phosphoenolpyruvate and no inhibition by ATP. The Michaelis constant (Km) value for ADP, the nucleotide specificity, the thermostability, pH optimum, and immunological specific activity were normal. It is concluded that the high pyruvate kinase activity is due to a shift in the R(elaxed) in equilibrium T(ight) equilibrium to the R(elaxed) form. PMID:6736249

  18. Phosphorylation site on yeast pyruvate dehydrogenase complex

    SciTech Connect

    Uhlinger, D.J.

    1986-01-01

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

  19. The "Gln-Type" Thiol Dioxygenase from Azotobacter vinelandii is a 3-Mercaptopropionic Acid Dioxygenase.

    PubMed

    Pierce, Brad S; Subedi, Bishnu P; Sardar, Sinjinee; Crowell, Joshua K

    2015-12-29

    Cysteine dioxygenase (CDO) is a non-heme iron enzyme that catalyzes the O2-dependent oxidation of l-cysteine to produce cysteinesulfinic acid. Bacterial CDOs have been subdivided as either "Arg-type" or "Gln-type" on the basis of the identity of conserved active site residues. To date, "Gln-type" enzymes remain largely uncharacterized. It was recently noted that the "Gln-type" enzymes are more homologous with another thiol dioxygenase [3-mercaptopropionate dioxygenase (MDO)] identified in Variovorax paradoxus, suggesting that enzymes of the "Gln-type" subclass are in fact MDOs. In this work, a putative "Gln-type" thiol dioxygenase from Azotobacter vinelandii (Av) was purified to homogeneity and characterized. Steady-state assays were performed using three substrates [3-mercaptopropionic acid (3mpa), l-cysteine (cys), and cysteamine (ca)]. Despite comparable maximal velocities, the "Gln-type" Av enzyme exhibited a specificity for 3mpa (kcat/KM = 72000 M(-1) s(-1)) nearly 2 orders of magnitude greater than those for cys (110 M(-1) s(-1)) and ca (11 M(-1) s(-1)). Supporting X-band electron paramagnetic resonance (EPR) studies were performed using nitric oxide (NO) as a surrogate for O2 binding to confirm obligate-ordered addition of substrate prior to NO. Stoichimetric addition of NO to solutions of 3mpa-bound enzyme quantitatively yields an iron-nitrosyl species (Av ES-NO) with EPR features consistent with a mononuclear (S = (3)/2) {FeNO}(7) site. Conversely, two distinct substrate-bound conformations were observed in Av ES-NO samples prepared with cys and ca, suggesting heterogeneous binding within the enzymatic active site. Analytical EPR simulations are provided to establish the relative binding affinity for each substrate (3map > cys > ca). Both kinetic and spectroscopic results presented here are consistent with 3mpa being the preferred substrate for this enzyme. PMID:26624219

  20. Molecular characterization of the gallate dioxygenase from Pseudomonas putida KT2440. The prototype of a new subgroup of extradiol dioxygenases.

    PubMed

    Nogales, Juan; Canales, Angeles; Jiménez-Barbero, Jesús; García, José Luis; Díaz, Eduardo

    2005-10-21

    In this work we have characterized the galA gene product from Pseudomonas putida KT2440, a ring-cleavage dioxygenase that acts specifically on gallate to produce 4-oxalomesaconate. The protein is a trimer composed by three identical subunits of 47.6 kDa (419 amino acids) that uses Fe2+ as the main cofactor. The gallate dioxygenase showed maximum activity at pH 7.0, and the Km and Vmax values for gallate were 144 microM and 53.2 micromol/min/mg of protein, respectively. A phylogenetic study suggests that the gallate dioxygenase from P. putida KT2440 is the prototype of a new subgroup of type II extradiol dioxygenases that share a common ancestor with protocatechuate 4,5-dioxygenases and whose two-domain architecture might have evolved from the fusion of the large and small subunits of the latter. A three-dimensional model for the N-terminal domain (residues 1-281) and C-terminal domain (residues 294-420) of the gallate dioxygenase from P. putida KT2440 was generated by comparison with the crystal structures of the large (LigB) and small (LigA) subunits of the protocatechuate 4,5-dioxygenase from Sphingomonas paucimobilis SYK-6. The expression of the galA gene was specifically induced when P. putida KT2440 cells grew in the presence of gallate. A P. putida KT2440 galA mutant strain was unable to use gallate as the sole carbon source and it did not show gallate dioxygenase activity, suggesting that the GalA protein is the only dioxygenase involved in gallate cleavage in this bacterium. This work points to the existence of a new pathway that is devoted to the catabolism of gallic acid and that remained unknown in the paradigmatic P. putida KT2440 strain. PMID:16030014

  1. Engineering dioxygenases for efficient degradation of environmental pollutants.

    PubMed

    Furukawa, K

    2000-06-01

    Dioxygenases have recently been engineered to improve their capabilities for environmental pollutant degradation. The techniques used to achieve this include in vitro DNA shuffling and subunit or domain exchanges between dioxygenases of different bacterial origins. Such evolved enzymes acquire novel and enhanced degradation capabilities of xenobiotic compounds, such as polychlorinated biphenyls, trichloroethylene and a variety of aromatic compounds. Hybrid strains in which the evolved genes are integrated into the chromosomal operons exhibit efficient degradation of xenobiotic chlorinated compounds. PMID:10851151

  2. Production of pyruvate from mannitol by mannitol-assimilating pyruvate decarboxylase-negative Saccharomyces cerevisiae.

    PubMed

    Yoshida, Shiori; Tanaka, Hideki; Hirayama, Makoto; Murata, Kousaku; Kawai, Shigeyuki

    2015-01-01

    Mannitol is contained in brown macroalgae up to 33% (w/w, dry weight), and thus is a promising carbon source for white biotechnology. However, Saccharomyces cerevisiae, a key cell factory, is generally regarded to be unable to assimilate mannitol for growth. We have recently succeeded in producing S. cerevisiae that can assimilate mannitol through spontaneous mutations of Tup1-Cyc8, each of which constitutes a general corepressor complex. In this study, we demonstrate production of pyruvate from mannitol using this mannitol-assimilating S. cerevisiae through deletions of all 3 pyruvate decarboxylase genes. The resultant mannitol-assimilating pyruvate decarboxylase-negative strain produced 0.86 g/L pyruvate without use of acetate after cultivation for 4 days, with an overall yield of 0.77 g of pyruvate per g of mannitol (the theoretical yield was 79%). Although acetate was not needed for growth of this strain in mannitol-containing medium, addition of acetate had a significant beneficial effect on production of pyruvate. This is the first report of production of a valuable compound (other than ethanol) from mannitol using S. cerevisiae, and is an initial platform from which the productivity of pyruvate from mannitol can be improved. PMID:26588105

  3. Microcolony formation by the opportunistic pathogen Pseudomonas aeruginosa requires pyruvate and pyruvate fermentation

    PubMed Central

    Petrova, Olga E.; Schurr, Jill R.; Schurr, Michael J.; Sauer, Karin

    2012-01-01

    SUMMARY A hallmark of the biofilm architecture is the presence of microcolonies. However, little is known about the underlying mechanisms governing microcolony formation. In the pathogen Pseudomonas aeruginosa, microcolony formation is dependent on the two-component regulator MifR, with mifR mutant biofilms exhibiting an overall thin structure lacking microcolonies, and overexpression of mifR resulting in hyper-microcolony formation. Using global transcriptomic and proteomic approaches, we demonstrate that microcolony formation is associated with stressful, oxygen-limiting but electron-rich conditions, as indicated by the activation of stress response mechanisms and anaerobic and fermentative processes, in particular pyruvate fermentation. Inactivation of genes involved in pyruvate utilization including uspK, acnA and ldhA abrogated microcolony formation in a manner similar to mifR inactivation. Moreover, depletion of pyruvate from the growth medium impaired biofilm and microcolony formation, while addition of pyruvate significantly increased microcolony formation. Addition of pyruvate to or expression of mifR in lactate dehydrogenase (ldhA) mutant biofilms did not restore microcolony formation while addition of pyruvate partly restored microcolony formation in mifR mutant biofilms. In contrast, expression of ldhA in mifR::Mar fully restored microcolony formation by this mutant strain. Our findings indicate the fermentative utilization of pyruvate to be a microcolony-specific adaptation of the P. aeruginosa biofilm environment. PMID:22931250

  4. Induction of bphA, Encoding Biphenyl Dioxygenase, in Two Polychlorinated Biphenyl-Degrading Bacteria, Psychrotolerant Pseudomonas Strain Cam-1 and Mesophilic Burkholderia Strain LB400

    PubMed Central

    Master, Emma R.; Mohn, William W.

    2001-01-01

    We investigated induction of biphenyl dioxygenase in the psychrotolerant polychlorinated biphenyl (PCB) degrader Pseudomonas strain Cam-1 and in the mesophilic PCB degrader Burkholderia strain LB400. Using a counterselectable gene replacement vector, we inserted a lacZ-Gmr fusion cassette between chromosomal genes encoding the large subunit (bphA) and small subunit (bphE) of biphenyl dioxygenase in Cam-1 and LB400, generating Cam-10 and LB400-1, respectively. Potential inducers of bphA were added to cell suspensions of Cam-10 and LB400-1 incubated at 30°C, and then beta-galactosidase activity was measured. Biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately six times greater than the basal level in cells incubated with pyruvate. In contrast, the beta-galactosidase activities in LB400-1 incubated with biphenyl and in LB400-1 incubated with pyruvate were indistinguishable. At a concentration of 1 mM, most of the 40 potential inducers tested were inhibitory to induction by biphenyl of beta-galactosidase activity in Cam-10. The exceptions were naphthalene, salicylate, 2-chlorobiphenyl, and 4-chlorobiphenyl, which induced beta-galactosidase activity in Cam-10, although at levels that were no more than 30% of the levels induced by biphenyl. After incubation for 24 h at 7°C, biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately four times greater than the basal level in cells incubated with pyruvate. The constitutive level of beta-galactosidase activity in LB400-1 grown at 15°C was approximately five times less than the level in LB400-1 grown at 30°C. Thus, there are substantial differences in the effects of physical and chemical environmental conditions on genetic regulation of PCB degradation in different bacteria. PMID:11375179

  5. The Pyruvate-Tricarboxylic Acid Cycle Node

    PubMed Central

    Bücker, René; Heroven, Ann Kathrin; Becker, Judith; Dersch, Petra; Wittmann, Christoph

    2014-01-01

    Despite our increasing knowledge of the specific pathogenicity factors in bacteria, the contribution of metabolic processes to virulence is largely unknown. Here, we elucidate a tight connection between pathogenicity and core metabolism in the enteric pathogen Yersinia pseudotuberculosis by integrated transcriptome and [13C]fluxome analysis of the wild type and virulence-regulator mutants. During aerobic growth on glucose, Y. pseudotuberculosis reveals an unusual flux distribution with a high level of secreted pyruvate. The absence of the transcriptional and post-transcriptional regulators RovA, CsrA, and Crp strongly perturbs the fluxes of carbon core metabolism at the level of pyruvate metabolism and the tricarboxylic acid (TCA) cycle, and these perturbations are accompanied by transcriptional changes in the corresponding enzymes. Knock-outs of regulators of this metabolic branch point and of its central enzyme, pyruvate kinase (ΔpykF), result in mutants with significantly reduced virulence in an oral mouse infection model. In summary, our work identifies the pyruvate-TCA cycle node as a focal point for controlling the host colonization and virulence of Yersinia. PMID:25164818

  6. A mimic of the pyruvate dehydrogenase complex.

    PubMed

    Zhao, Huanyu; Breslow, Ronald

    2010-10-15

    Pyruvic acid undergo decarboxylation catalyzed by a hydrophobic thiazolium salt and reacts with a hydrophobic analog of lipoic acid to form a hydrophobic acylthioester that reacts with aniline to form acetanilide in water, but only in the presence of a hydrophobically modified polyaziridine that acts to gather the reactants just as the enzyme complex does. PMID:20826089

  7. BACTERIAL EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF ARABIDOPSIS THALIANA PYRUVATE DEHYDROGENASE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pyruvate dehydrogenase complex (PDC) is a very large multi-component structure that catalyzes decarboxylation of pyruvate, yielding CO2, NADH, and acetyl-CoA as products. The decarboxylation reaction is catalyzed by pyruvate dehydrogenase (E1). The PDC occupies a key position in intermediary met...

  8. Ring-Cleaving Dioxygenases with a Cupin Fold

    PubMed Central

    2012-01-01

    Ring-cleaving dioxygenases catalyze key reactions in the aerobic microbial degradation of aromatic compounds. Many pathways converge to catecholic intermediates, which are subject to ortho or meta cleavage by intradiol or extradiol dioxygenases, respectively. However, a number of degradation pathways proceed via noncatecholic hydroxy-substituted aromatic carboxylic acids like gentisate, salicylate, 1-hydroxy-2-naphthoate, or aminohydroxybenzoates. The ring-cleaving dioxygenases active toward these compounds belong to the cupin superfamily, which is characterized by a six-stranded β-barrel fold and conserved amino acid motifs that provide the 3His or 2- or 3His-1Glu ligand environment of a divalent metal ion. Most cupin-type ring cleavage dioxygenases use an FeII center for catalysis, and the proposed mechanism is very similar to that of the canonical (type I) extradiol dioxygenases. The metal ion is presumed to act as an electron conduit for single electron transfer from the metal-bound substrate anion to O2, resulting in activation of both substrates to radical species. The family of cupin-type dioxygenases also involves quercetinase (flavonol 2,4-dioxygenase), which opens up two C-C bonds of the heterocyclic ring of quercetin, a wide-spread plant flavonol. Remarkably, bacterial quercetinases are capable of using different divalent metal ions for catalysis, suggesting that the redox properties of the metal are relatively unimportant for the catalytic reaction. The major role of the active-site metal ion could be to correctly position the substrate and to stabilize transition states and intermediates rather than to mediate electron transfer. The tentative hypothesis that quercetinase catalysis involves direct electron transfer from metal-bound flavonolate to O2 is supported by model chemistry. PMID:22287012

  9. Hemoglobin: A Nitric-Oxide Dioxygenase

    PubMed Central

    Gardner, Paul R.

    2012-01-01

    Members of the hemoglobin superfamily efficiently catalyze nitric-oxide dioxygenation, and when paired with native electron donors, function as NO dioxygenases (NODs). Indeed, the NOD function has emerged as a more common and ancient function than the well-known role in O2 transport-storage. Novel hemoglobins possessing a NOD function continue to be discovered in diverse life forms. Unique hemoglobin structures evolved, in part, for catalysis with different electron donors. The mechanism of NOD catalysis by representative single domain hemoglobins and multidomain flavohemoglobin occurs through a multistep mechanism involving O2 migration to the heme pocket, O2 binding-reduction, NO migration, radical-radical coupling, O-atom rearrangement, nitrate release, and heme iron re-reduction. Unraveling the physiological functions of multiple NODs with varying expression in organisms and the complexity of NO as both a poison and signaling molecule remain grand challenges for the NO field. NOD knockout organisms and cells expressing recombinant NODs are helping to advance our understanding of NO actions in microbial infection, plant senescence, cancer, mitochondrial function, iron metabolism, and tissue O2 homeostasis. NOD inhibitors are being pursued for therapeutic applications as antibiotics and antitumor agents. Transgenic NOD-expressing plants, fish, algae, and microbes are being developed for agriculture, aquaculture, and industry. PMID:24278729

  10. Substrate Oxidation by Indoleamine 2,3-Dioxygenase

    PubMed Central

    Booth, Elizabeth S.; Basran, Jaswir; Lee, Michael; Handa, Sandeep; Raven, Emma L.

    2015-01-01

    The kynurenine pathway is the major route of l-tryptophan (l-Trp) catabolism in biology, leading ultimately to the formation of NAD+. The initial and rate-limiting step of the kynurenine pathway involves oxidation of l-Trp to N-formylkynurenine. This is an O2-dependent process and catalyzed by indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase. More than 60 years after these dioxygenase enzymes were first isolated (Kotake, Y., and Masayama, I. (1936) Z. Physiol. Chem. 243, 237–244), the mechanism of the reaction is not established. We examined the mechanism of substrate oxidation for a series of substituted tryptophan analogues by indoleamine 2,3-dioxygenase. We observed formation of a transient intermediate, assigned as a Compound II (ferryl) species, during oxidation of l-Trp, 1-methyl-l-Trp, and a number of other substrate analogues. The data are consistent with a common reaction mechanism for indoleamine 2,3-dioxygenase-catalyzed oxidation of tryptophan and other tryptophan analogues. PMID:26511316

  11. Measurement of Cysteine Dioxygenase Activity and Protein Abundance

    PubMed Central

    Stipanuk, Martha H.; Dominy, John E.; Ueki, Iori; Hirschberger, Lawrence L.

    2009-01-01

    Cysteine dioxygenase is an iron (Fe2+)-dependent thiol dioxygenase that uses molecular oxygen to oxidize the sulfhydryl group of cysteine to generate 3-sulfinoalanine (commonly called cysteinesulfinic acid). Cysteine dioxygenase activity is routinely assayed by measuring cysteinesulfinate formation from substrate L-cysteine at pH 6.1 in the presence of ferrous ions to saturate the enzyme with metal cofactor, a copper chelator to diminish substrate oxidation, and hydroxylamine to inhibit pyridoxal 5′-phosphate-dependent degradation of product. The amount of cysteine dioxygenase may be measured by immunoblotting. Upon SDS-PAGE, cysteine dioxygenase can be separated into two major bands, with the upper band representing the 23-kDa protein and the lower band representing the mature enzyme that has undergone formation of an internal thioether cross link in the active site. Formation of this cross link is dependent upon the catalytic turnover of substrate and produces an enzyme with a higher catalytic efficiency and catalytic half-life. PMID:19885389

  12. Mitochondrial pyruvate transport: a historical perspective and future research directions

    PubMed Central

    McCommis, Kyle S.; Finck, Brian N.

    2015-01-01

    Pyruvate is the end-product of glycolysis, a major substrate for oxidative metabolism, and a branching point for glucose, lactate, fatty acid and amino acid synthesis. The mitochondrial enzymes that metabolize pyruvate are physically separated from cytosolic pyruvate pools and rely on a membrane transport system to shuttle pyruvate across the impermeable inner mitochondrial membrane (IMM). Despite long-standing acceptance that transport of pyruvate into the mitochondrial matrix by a carrier-mediated process is required for the bulk of its metabolism, it has taken almost 40 years to determine the molecular identity of an IMM pyruvate carrier. Our current understanding is that two proteins, mitochondrial pyruvate carriers MPC1 and MPC2, form a hetero-oligomeric complex in the IMM to facilitate pyruvate transport. This step is required for mitochondrial pyruvate oxidation and carboxylation – critical reactions in intermediary metabolism that are dysregulated in several common diseases. The identification of these transporter constituents opens the door to the identification of novel compounds that modulate MPC activity, with potential utility for treating diabetes, cardiovascular disease, cancer, neurodegenerative diseases, and other common causes of morbidity and mortality. The purpose of the present review is to detail the historical, current and future research investigations concerning mitochondrial pyruvate transport, and discuss the possible consequences of altered pyruvate transport in various metabolic tissues. PMID:25748677

  13. Gentisate 1,2-dioxygenase from Haloferax sp. D1227.

    PubMed

    Fu, W; Oriel, P

    1998-11-01

    Gentisate 1,2-dioxygenase from the extreme halophile Haloferax sp. D1227 (Hf. D1227) was purified using a three-step procedure. The enzyme was found to be a homotetramer of 42,000 +/- 1,000 Da subunits, with a native molecular weight of 174,000 +/- 6,000 Da. The optimal salt concentration, temperature, and pH for enzyme activity were 2 M KCl or NaCl, 45 degrees C, and pH 7.2, respectively. The gene encoding Hf. D1227 gentisate 1,2-dioxygenase was cloned, sequenced, and expressed in Haloferax volcanii. The deduced amino acid sequence exhibited a 9.2% excess acidic over basic amino acids typical of halophilic enzymes. Four novel histidine clusters and a possible extradiol dioxygenase fingerprint region were identified. PMID:9827334

  14. Structure, function and regulation of pyruvate carboxylase.

    PubMed Central

    Jitrapakdee, S; Wallace, J C

    1999-01-01

    Pyruvate carboxylase (PC; EC 6.4.1.1), a member of the biotin-dependent enzyme family, catalyses the ATP-dependent carboxylation of pyruvate to oxaloacetate. PC has been found in a wide variety of prokaryotes and eukaryotes. In mammals, PC plays a crucial role in gluconeogenesis and lipogenesis, in the biosynthesis of neurotransmitter substances, and in glucose-induced insulin secretion by pancreatic islets. The reaction catalysed by PC and the physical properties of the enzyme have been studied extensively. Although no high-resolution three-dimensional structure has yet been determined by X-ray crystallography, structural studies of PC have been conducted by electron microscopy, by limited proteolysis, and by cloning and sequencing of genes and cDNA encoding the enzyme. Most well characterized forms of active PC consist of four identical subunits arranged in a tetrahedron-like structure. Each subunit contains three functional domains: the biotin carboxylation domain, the transcarboxylation domain and the biotin carboxyl carrier domain. Different physiological conditions, including diabetes, hyperthyroidism, genetic obesity and postnatal development, increase the level of PC expression through transcriptional and translational mechanisms, whereas insulin inhibits PC expression. Glucocorticoids, glucagon and catecholamines cause an increase in PC activity or in the rate of pyruvate carboxylation in the short term. Molecular defects of PC in humans have recently been associated with four point mutations within the structural region of the PC gene, namely Val145-->Ala, Arg451-->Cys, Ala610-->Thr and Met743-->Thr. PMID:10229653

  15. [Pyruvate dehydrogenase deficiency and cerebral malformations].

    PubMed

    Eirís, J; Alvarez-Moreno, A; Briones, P; Alonso-Alonso, C; Castro-Gago, M

    1996-10-01

    Pyruvate dehydrogenase (PDH) deficiency is a major cause of primary lactic acidosis and severe global developmental delay. A deficiency of PDH E1 alpha, a subunit of the PDH complex is a prominent cause of congenital lactic acidosis. The E1 alpha cDNA and corresponding genomic DNA have been located in the short arm of the X-chromosome (Xp22-1). A isolated 'cerebral' lactic acidosis with cerebral dysgenesis is a recognized pattern of presentation of PDH deficiency. Here, we report clinical features, magnetic resonance, and biochemical studies of two females aged 6 months (case 1) and 26 months (case 2). Both had severe development delay, minor dysmorphic features, microcephaly, severe hypoplasia of the corpus callosum, cerebral atrophy, ventricular dilatation and increase in serum lactate levels without systemic acidosis. Urinary organic acid profile was compatible with PDH deficiency. Increased CSF lactate and pyruvate levels and reduced total PDH and PDH E1 activities in muscle and fibroblasts were observed in case 1. Otherwise, decreased total PDH activity in muscle but not in fibroblasts was seen in case 2. The PDH E1á gene was sequenced in the case 1 and a deletion in exon 7 was demonstrated. Dysmorphism with severe cerebral malformations in female patients merits a metabolic evaluation, including determination of lactate and pyruvate levels in CSF. PMID:8983728

  16. Transport of pyruvate into mitochondria is involved in methylmercury toxicity

    PubMed Central

    Lee, Jin-Yong; Ishida, Yosuke; Takahashi, Tsutomu; Naganuma, Akira; Hwang, Gi-Wook

    2016-01-01

    We have previously demonstrated that the overexpression of enzymes involved in the production of pyruvate, enolase 2 (Eno2) and D-lactate dehydrogenase (Dld3) renders yeast highly sensitive to methylmercury and that the promotion of intracellular pyruvate synthesis may be involved in intensifying the toxicity of methylmercury. In the present study, we showed that the addition of pyruvate to culture media in non-toxic concentrations significantly enhanced the sensitivity of yeast and human neuroblastoma cells to methylmercury. The results also suggested that methylmercury promoted the transport of pyruvate into mitochondria and that the increased pyruvate concentrations in mitochondria were involved in intensifying the toxicity of methylmercury without pyruvate being converted to acetyl-CoA. Furthermore, in human neuroblastoma cells, methylmercury treatment alone decreased the mitochondrial membrane potential, and the addition of pyruvate led to a further significant decrease. In addition, treatment with N-acetylcysteine (an antioxidant) significantly alleviated the toxicity of methylmercury and significantly inhibited the intensification of methylmercury toxicity by pyruvate. Based on these data, we hypothesize that methylmercury exerts its toxicity by raising the level of pyruvate in mitochondria and that mitochondrial dysfunction and increased levels of reactive oxygen species are involved in the action of pyruvate. PMID:26899208

  17. Mercaptosuccinate Dioxygenase, a Cysteine Dioxygenase Homologue, from Variovorax paradoxus Strain B4 Is the Key Enzyme of Mercaptosuccinate Degradation

    PubMed Central

    Brandt, Ulrike; Schürmann, Marc; Steinbüchel, Alexander

    2014-01-01

    The versatile thiol mercaptosuccinate has a wide range of applications, e.g. in quantum dot research or in bioimaging. Its metabolism is investigated in Variovorax paradoxus strain B4, which can utilize this compound as the sole source of carbon and sulfur. Proteomic studies of strain B4 resulted in the identification of a putative mercaptosuccinate dioxygenase, a cysteine dioxygenase homologue, possibly representing the key enzyme in the degradation of mercaptosuccinate. Therefore, the putative mercaptosuccinate dioxygenase was heterologously expressed, purified, and characterized in this study. The results clearly demonstrated that the enzyme utilizes mercaptosuccinate with concomitant consumption of oxygen. Thus, the enzyme is designated as mercaptosuccinate dioxygenase. Succinate and sulfite were verified as the final reaction products. The enzyme showed an apparent Km of 0.4 mm, and a specific activity (Vmax) of 20.0 μmol min−1 mg−1 corresponding to a kcat of 7.7 s−1. Furthermore, the enzyme was highly specific for mercaptosuccinate, no activity was observed with cysteine, dithiothreitol, 2-mercaptoethanol, and 3-mercaptopropionate. These structurally related thiols did not have an inhibitory effect either. Fe(II) could clearly be identified as metal cofactor of the mercaptosuccinate dioxygenase with a content of 0.6 mol of Fe(II)/mol of enzyme. The recently proposed hypothesis for the degradation pathway of mercaptosuccinate based on proteome analyses could be strengthened in the present study. (i) Mercaptosuccinate is first converted to sulfinosuccinate by this mercaptosuccinate dioxygenase; (ii) sulfinosuccinate is spontaneously desulfinated to succinate and sulfite; and (iii) whereas succinate enters the central metabolism, sulfite is detoxified by the previously identified putative molybdopterin oxidoreductase. PMID:25228698

  18. Substrate Stereo-specificity in Tryptophan dioxygenase and Indoleamine 2,3- dioxygenase

    PubMed Central

    Capece, L.; Arrar, M.; Roitberg, A. E.; Yeh, Syun-Ru; Marti, M. A.; Estrin, D. A.

    2010-01-01

    The first and rate-limiting step of the kynurenine pathway, in which tryptophan (Trp) is converted to N-formylkynurenine is catalyzed by two heme-containing proteins, Indoleamine 2,3-dioxygenase (IDO) and Tryptophan 2,3-dioxygenase (TDO). In mammals, TDO is found exclusively in liver tissue, IDO is found ubiquitously in all tissues. IDO has become increasingly popular in pharmaceutical research as it was found to be involved in many physiological situations, including immune escape of cancer. More importantly, small-molecule inhibitors of IDO are currently utilized in cancer therapy. One of the main concerns for the design of human IDO (hIDO) inhibitors is that they should be selective enough to avoid inhibition of TDO. In this work we have used a combination of classical molecular dynamics (MD) and hybrid quantum-classical (QM/MM) methodologies to establish the structural basis that determine the differences in a) the interactions of TDO and IDO with small ligands (CO/O2) and b) the substrate stereo-specificity in hIDO and TDO. Our results indicate that the differences in small ligand bound structures of IDO and TDO arise from slight differences in the structure of the bound substrate complex. The results also show that substrate stereo-specificity of TDO is achieved by the perfect fit of L-Trp, but not D-Trp, which exhibits weaker interactions with the protein matrix-. For hIDO, the presence of multiple stable binding conformations for L/D-Trp reveal the presence of a large and dynamic active site. Taken together, our data allow determination of key interactions useful for the future design of more potent hIDO-selective inhibitors. PMID:20715188

  19. Microbial Dioxygenase Gene Population Shifts during Polycyclic Aromatic Hydrocarbon Biodegradation

    PubMed Central

    Ní Chadhain, Sinéad M.; Norman, R. Sean; Pesce, Karen V.; Kukor, Jerome J.; Zylstra, Gerben J.

    2006-01-01

    The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely studied. While many pure cultures have been isolated and characterized for their ability to grow on PAHs, limited information is available on the diversity of microbes involved in PAH degradation in the environment. We have designed generic PCR primers targeting the gene fragment encoding the Rieske iron sulfur center common to all PAH dioxygenase enzymes. These Rieske primers were employed to track dioxygenase gene population shifts in soil enrichment cultures following exposure to naphthalene, phenanthrene, or pyrene. PAH degradation was monitored by gas chromatograph with flame ionization detection. DNA was extracted from the enrichment cultures following PAH degradation. 16S rRNA and Rieske gene fragments were PCR amplified from DNA extracted from each enrichment culture and an unamended treatment. The PCR products were cloned and sequenced. Molecular monitoring of the enrichment cultures before and after PAH degradation using denaturing gradient gel electrophoresis and 16S rRNA gene libraries suggests that specific phylotypes of bacteria were associated with the degradation of each PAH. Sequencing of the cloned Rieske gene fragments showed that different suites of genes were present in soil microbe populations under each enrichment culture condition. Many of the Rieske gene fragment sequences fell into clades which are distinct from the reference dioxygenase gene sequences used to design the PCR primers. The ability to profile not only the bacterial community but also the dioxygenases which they encode provides a powerful tool for both assessing bioremediation potential in the environment and for the discovery of novel dioxygenase genes. PMID:16751518

  20. Engineering nitric oxide synthase chimeras to function as NO dioxygenases.

    PubMed

    Wang, Zhi-Qiang; Haque, Mohammad Mahfuzul; Binder, Katherine; Sharma, Manisha; Wei, Chin-Chuan; Stuehr, Dennis J

    2016-05-01

    Nitric oxide synthases (NOSs) catalyze a two-step oxidation of l-arginine to form nitric oxide (NO) and l-citrulline. NOS contains a N-terminal oxygenase domain (NOSoxy) that is the site of NO synthesis, and a C-terminal reductase domain (NOSred) that binds nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN) and provides electrons to the NOSoxy heme during catalysis. The three NOS isoforms in mammals inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS) share high structural similarity but differ in NO release rates and catalytic properties due to differences in enzyme kinetic parameters. These parameters must be balanced for NOS enzymes to release NO, rather than consume it in a competing, inherent NO dioxygenase reaction. To improve understanding, we drew on a global catalytic model and previous findings to design three NOS chimeras that may predominantly function as NO dioxygenases: iNOSoxy/nNOSred (Wild type (WT) chimera), V346I iNOSoxy/nNOSred (V346I chimera) and iNOSoxy/S1412D nNOSred (S1412D chimera). The WT and S1412D chimeras had higher NO release than the parent iNOS, while the V346I chimera exhibited much lower NO release, consistent with expectations. Measurements indicated that a greater NO dioxygenase activity was achieved, particularly in the V346I chimera, which dioxygenated an estimated two to four NO per NO that it released, while the other chimeras had nearly equivalent NO dioxygenase and NO release activities. Computer simulations of the global catalytic model using the measured kinetic parameters produced results that mimicked the measured outcomes, and this provided further insights on the catalytic behaviors of the chimeras and basis of their increased NO dioxygenase activities. PMID:27013266

  1. Experimental evidence of phosphoenolpyruvate resynthesis from pyruvate in illuminated leaves.

    PubMed

    Tcherkez, Guillaume; Mahé, Aline; Boex-Fontvieille, Edouard; Gout, Elisabeth; Guérard, Florence; Bligny, Richard

    2011-09-01

    Day respiration is the cornerstone of nitrogen assimilation since it provides carbon skeletons to primary metabolism for glutamate (Glu) and glutamine synthesis. However, recent studies have suggested that the tricarboxylic acid pathway is rate limiting and mitochondrial pyruvate dehydrogenation is partly inhibited in the light. Pyruvate may serve as a carbon source for amino acid (e.g. alanine) or fatty acid synthesis, but pyruvate metabolism is not well documented, and neither is the possible resynthesis of phosphoenolpyruvate (PEP). Here, we examined the capacity of pyruvate to convert back to PEP using (13)C and (2)H labeling in illuminated cocklebur (Xanthium strumarium) leaves. We show that the intramolecular labeling pattern in Glu, 2-oxoglutarate, and malate after (13)C-3-pyruvate feeding was consistent with (13)C redistribution from PEP via the PEP-carboxylase reaction. Furthermore, the deuterium loss in Glu after (2)H(3)-(13)C-3-pyruvate feeding suggests that conversion to PEP and back to pyruvate washed out (2)H atoms to the solvent. Our results demonstrate that in cocklebur leaves, PEP resynthesis occurred as a flux from pyruvate, approximately 0.5‰ of the net CO(2) assimilation rate. This is likely to involve pyruvate inorganic phosphate dikinase and the fundamental importance of this flux for PEP and inorganic phosphate homeostasis is discussed. PMID:21730197

  2. 21 CFR 862.1650 - Pyruvate kinase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862... to pyruvate kinase deficiency or of acute leukemias. (b) Classification. Class I (general...

  3. 21 CFR 862.1650 - Pyruvate kinase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862... to pyruvate kinase deficiency or of acute leukemias. (b) Classification. Class I (general...

  4. 21 CFR 862.1650 - Pyruvate kinase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862... to pyruvate kinase deficiency or of acute leukemias. (b) Classification. Class I (general...

  5. 21 CFR 862.1650 - Pyruvate kinase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862... to pyruvate kinase deficiency or of acute leukemias. (b) Classification. Class I (general...

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

    PubMed Central

    Williams, Michael; Randall, Douglas D.

    1979-01-01

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

  7. Liver specific pyruvate kinase in pheasants Phasianus colchicus.

    PubMed

    Guderley, H; Jacques, L

    1987-01-01

    While in chickens, the existence of a liver form of pyruvate kinase is controversial, the liver form of pyruvate kinase in pheasants, murres and puffins is electrophoretically distinct from that in muscle, brain, kidney, lung and small intestine. Although the forms in lungs, muscle, heart, brain and small intestine could not be reliably separated by electrophoresis, the functional characteristics of the lung and muscle forms of pyruvate kinase in the pheasant are distinct and can be classified as K and M isozymes respectively. Our data suggest that these birds possess at least three distinct isozymes of pyruvate kinase. PMID:3609446

  8. Plant cytosolic pyruvate kinase: a kinetic study.

    PubMed

    Podestá, F E; Plaxton, W C

    1992-11-20

    The kinetic properties of cytosolic pyruvate kinase (PKc) from germinating castor oil seeds (COS) have been investigated. From experiments in which the free Mg2+ concentration was varied at constant levels of either the complexed or free forms of the substrates it was determined that the true substrates are the free forms of both phosphoenolpyruvate (PEP) and ADP. This conclusion is corroborated by the quenching of intrinsic PKC tryptophan fluorescence by free PEP and ADP. Mg2+ is bound as the free bivalent cation but is likely released as MgATP. The fluorescence data, substrate interaction kinetics, and pattern of inhibition by products and substrate analogues (adenosine 5'-O-(2-thiodiphosphate) for ADP and phenyl phosphate for PEP) are compatible with a sequential, compulsory-ordered, Tri-Bi type kinetic reaction mechanism. PEP is the leading substrate, and pyruvate the last product to abandon the enzyme. The dissociation constant and limiting Km for free PEP (8.2 to 22 and 38 microM, respectively) and the limiting Km for free ADP (2.9 microM) are considerably lower than those reported for the non-plant enzyme. The results indicate that COS PKc exists naturally in an activated state, similar to the fructose 1,6-bisphosphate-activated yeast enzyme. This deduction is consistent with a previous study (F.E. Podestá and W.C. Plaxton (1991) Biochem. J. 279, 495-501) that failed to identify any allosteric activators for the COS PKc, but which proposed a regulatory mechanism based upon ATP levels and pH-dependent alterations in the enzyme's response to various metabolite inhibitors. As plant phosphofructokinases display potent inhibition by PEP, the overall rate of glycolytic flux from hexose 6-phosphate to pyruvate in the plant cytosol will ultimately depend upon variations in PEP levels brought about by the regulation of PKc. PMID:1445948

  9. Pyruvate kinase M2 at a glance

    PubMed Central

    Yang, Weiwei; Lu, Zhimin

    2015-01-01

    Reprogrammed metabolism is a key feature of cancer cells. The pyruvate kinase M2 (PKM2) isoform, which is commonly upregulated in many human cancers, has been recently shown to play a crucial role in metabolism reprogramming, gene transcription and cell cycle progression. In this Cell Science at a glance article and accompanying poster, we provide a brief overview of recent advances in understanding the mechanisms underlying the regulation of PKM2 expression, enzymatic activity, metabolic functions and subcellular location. We highlight the instrumental role of the non-metabolic functions of PKM2 in tumorigenesis and evaluate the potential to target PKM2 for cancer treatment. PMID:25770102

  10. Redirection of pyruvate flux toward desired metabolic pathways through substrate channeling between pyruvate kinase and pyruvate-converting enzymes in Saccharomyces cerevisiae

    PubMed Central

    Kim, Sujin; Bae, Sang-Jeong; Hahn, Ji-Sook

    2016-01-01

    Spatial organization of metabolic enzymes allows substrate channeling, which accelerates processing of intermediates. Here, we investigated the effect of substrate channeling on the flux partitioning at a metabolic branch point, focusing on pyruvate metabolism in Saccharomyces cerevisiae. As a platform strain for the channeling of pyruvate flux, PYK1-Coh-Myc strain was constructed in which PYK1 gene encoding pyruvate kinase is tagged with cohesin domain. By using high-affinity cohesin-dockerin interaction, the pyruvate-forming enzyme Pyk1 was tethered to heterologous pyruvate-converting enzymes, lactate dehydrogenase and α-acetolactate synthase, to produce lactic acid and 2,3-butanediol, respectively. Pyruvate flux was successfully redirected toward desired pathways, with a concomitant decrease in ethanol production even without genetic attenuation of the ethanol-producing pathway. This pyruvate channeling strategy led to an improvement of 2,3-butanediol production by 38%, while showing a limitation in improving lactic acid production due to a reduced activity of lactate dehydrogenase by dockerin tagging. PMID:27052099

  11. Salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans: crystal structure of a peculiar ring-cleaving dioxygenase.

    PubMed

    Matera, Irene; Ferraroni, Marta; Bürger, Sibylle; Scozzafava, Andrea; Stolz, Andreas; Briganti, Fabrizio

    2008-07-25

    The crystallographic structure of salicylate 1,2-dioxygenase (SDO), a new ring fission dioxygenase from the naphthalenesulfonate-degrading strain Pseudaminobacter salicylatoxidans BN12, which oxidizes salicylate to 2-oxohepta-3,5-dienedioic acid by a novel ring fission mechanism, has been solved by molecular replacement techniques and refined at 2.9 A resolution (R(free) 26.1%; R-factor 19.3%). SDO is a homo-tetramer member of type III extradiol-type dioxygenases with a subunit topology characteristic of the bicupin beta-barrel folds. The catalytic center contains a mononuclear iron(II) ion coordinated to three histidine residues (His119, His121, and His160), located within the N-terminal domain in a solvent-accessible pocket. SDO is markedly different from the known gentisate 1,2-dioxygenases (GDO) or 1-hydroxy-2-naphthoate dioxygenase because of its unique ability to oxidatively cleave numerous salicylates, gentisates and 1-hydroxy-2-naphthoate with high catalytic efficiency. The comparison of the structure and substrate specificity for a series of different substrates with the corresponding data for several GDOs and the docking of salicylates/gentisates in the active site of SDO, allowed the identification of several active site residues responsible for differences of substrate specificity. In particular, a more defined electron density of the N-terminal region allowed the discovery of a novel structure fragment in SDO previously unobserved in GDO. This region contributes several residues to the active site that influence substrate specificity for both of these enzymes. Implications on the catalytic mechanism are discussed. PMID:18572191

  12. Ethyl pyruvate protects colonic anastomosis from ischemia-reperfusion injury.

    PubMed

    Unal, B; Karabeyoglu, M; Huner, T; Canbay, E; Eroglu, A; Yildirim, O; Dolapci, M; Bilgihan, A; Cengiz, O

    2009-03-01

    Ethyl pyruvate is a simple derivative in Ca(+2)- and K(+)-containing balanced salt solution of pyruvate to avoid the problems associated with the instability of pyruvate in solution. It has been shown to ameliorate the effects of ischemia-reperfusion (I/R) injury in many organs. It has also been shown that I/R injury delays the healing of colonic anastomosis. In this study, the effect of ethyl pyruvate on the healing of colon anastomosis and anastomotic strength after I/R injury was investigated. Anastomosis of the colon was performed in 32 adult male Wistar albino rats divided into 4 groups of 8 individuals: (1) sham-operated control group (group 1); (2) 30 minutes of intestinal I/R by superior mesenteric artery occlusion (group 2); (3) I/R+ ethyl pyruvate (group 3), ethyl pyruvate was administered as a 50-mg/kg/d single dose; and (4) I/R+ ethyl pyruvate (group 4), ethyl pyruvate administration was repeatedly (every 6 hours) at the same dose (50 mg/kg). On the fifth postoperative day, animals were killed. Perianastomotic tissue hydroxyproline contents and anastomotic bursting pressures were measured in all groups. When the anastomotic bursting pressures and tissue hydroxyproline contents were compared, it was found that they were decreased in group 2 when compared with groups 1, 3, and 4 (P < .05). Both anastomotic bursting pressure (P = .005) and hydroxyproline content (P < .001) levels were found to be significantly increased with ethyl pyruvate administration when compared with group 2. When ethyl pyruvate administration doses were compared, a significant difference was not observed (P > .05). Ethyl pyruvate significantly prevents the delaying effect of I/R injury on anastomotic strength and healing independent from doses of administration. PMID:19064591

  13. Phosphorylation-dephosphorylation of yeast pyruvate dehydrogenase

    SciTech Connect

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

    1986-05-01

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

  14. Postischemic hyperoxia reduces hippocampal pyruvate dehydrogenase activity

    PubMed Central

    Richards, Erica M.; Rosenthal, Robert E.; Kristian, Tibor; Fiskum, Gary

    2008-01-01

    The pyruvate dehydrogenase complex (PDHC) is a mitochondrial matrix enzyme that catalyzes the oxidative decarboxylation of pyruvate and represents the sole bridge between anaerobic and aerobic cerebral energy metabolism. Previous studies demonstrating loss of PDHC enzyme activity and immunoreactivity during reperfusion after cerebral ischemia suggest that oxidative modifications are involved. This study tested the hypothesis that hyperoxic reperfusion exacerbates loss of PDHC enzyme activity, possibly due to tyrosine nitration or S-nitrosation. We used a clinically relevant canine ventricular fibrillation cardiac arrest model in which, after resuscitation and ventilation on either 100% O2 (hyperoxic) or 21–30% O2 (normoxic), animals were sacrificed at 2 h reperfusion and the brains removed for enzyme activity and immunoreactivity measurements. Animals resuscitated under hyperoxic conditions exhibited decreased PDHC activity and elevated 3-nitrotyrosine immunoreactivity in the hippocampus but not the cortex, compared to nonischemic controls. These measures were unchanged in normoxic animals. In vitro exposure of purified PDHC to peroxynitrite resulted in a dose-dependent loss of activity and increased nitrotyrosine immunoreactivity. These results support the hypothesis that oxidative stress contributes to loss of hippocampal PDHC activity during cerebral ischemia and reperfusion and suggest that PDHC is a target of peroxynitrite. PMID:16716897

  15. A monomeric form of pyruvate kinase in human pyruvate kinase deficiency.

    PubMed Central

    Adachi, K; Ghory, P K; Asakura, T; Schwartz, E

    1977-01-01

    A mutant pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from human erythrocytes which is easily separated into monomers and dimers by gel chromatography is described. Tht mutant enzyme shows almost the same pH optimum and thermostability as normal enzyme, but has a decreased stability on shaking with air, a decreased Km for phosphoenolpyruvate and a loss of allosteric properties. The apparent Km values for phosphoenolpyruvate of tetramers and monomers were the same. The tetrameric enzyme was slightly activated by fructose-1,6-diphosphate but the monomeric form was not. The tetrameric enzyme was found to dissociate spontaneously to dimeric and monomeric forms. PMID:15247

  16. Probes of the Catalytic Site of Cysteine Dioxygenase

    SciTech Connect

    Chai,S.; Bruyere, J.; Maroney, M.

    2006-01-01

    The first major step of cysteine catabolism, the oxidation of cysteine to cysteine sulfinic acid, is catalyzed by cysteine dioxygenase (CDO). In the present work, we utilize recombinant rat liver CDO and cysteine derivatives to elucidate structural parameters involved in substrate recognition and x-ray absorption spectroscopy to probe the interaction of the active site iron center with cysteine. Kinetic studies using cysteine structural analogs show that most are inhibitors and that a terminal functional group bearing a negative charge (e.g. a carboxylate) is required for binding. The substrate-binding site has no stringent restrictions with respect to the size of the amino acid. Lack of the amino or carboxyl groups at the a-carbon does not prevent the molecules from interacting with the active site. In fact, cysteamine is shown to be a potent activator of the enzyme without being a substrate. CDO was also rendered inactive upon complexation with the metal-binding inhibitors azide and cyanide. Unlike many non-heme iron dioxygenases that employ a-keto acids as cofactors, CDO was shown to be the only dioxygenase known to be inhibited by {alpha}-ketoglutarate.

  17. 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.

  18. Conformational Dynamics and Allostery in Pyruvate Kinase.

    PubMed

    Donovan, Katherine A; Zhu, Shaolong; Liuni, Peter; Peng, Fen; Kessans, Sarah A; Wilson, Derek J; Dobson, Renwick C J

    2016-04-22

    Pyruvate kinase catalyzes the final step in glycolysis and is allosterically regulated to control flux through the pathway. Two models are proposed to explain how Escherichia coli pyruvate kinase type 1 is allosterically regulated: the "domain rotation model" suggests that both the domains within the monomer and the monomers within the tetramer reorient with respect to one another; the "rigid body reorientation model" proposes only a reorientation of the monomers within the tetramer causing rigidification of the active site. To test these hypotheses and elucidate the conformational and dynamic changes that drive allostery, we performed time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange studies followed by mutagenic analysis to test the activation mechanism. Global exchange experiments, supported by thermostability studies, demonstrate that fructose 1,6-bisphosphate binding to the allosteric domain causes a shift toward a globally more dynamic ensemble of conformations. Mapping deuterium exchange to peptides within the enzyme highlight site-specific regions with altered conformational dynamics, many of which increase in conformational flexibility. Based upon these and mutagenic studies, we propose an allosteric mechanism whereby the binding of fructose 1,6-bisphosphate destabilizes an α-helix that bridges the allosteric and active site domains within the monomeric unit. This destabilizes the β-strands within the (β/α)8-barrel domain and the linked active site loops that are responsible for substrate binding. Our data are consistent with the domain rotation model but inconsistent with the rigid body reorientation model given the increased flexibility at the interdomain interface, and we can for the first time explain how fructose 1,6-bisphosphate affects the active site. PMID:26879751

  19. Studies on the isopropylbenzene 2,3-dioxygenase and the 3-isopropylcatechol 2,3-dioxygenase genes encoded by the linear plasmid of Rhodococcus erythropolis BD2.

    PubMed

    Kesseler, M; Dabbs, E R; Averhoff, B; Gottschalk, G

    1996-11-01

    The enzymes responsible for the degradation of isopropylbenzene (IPB) and co-oxidation of trichloroethene (TCE) by Rhodococcus erythropolis BD2 are encoded by the linear plasmid pBD2. Fragments containing IPB catabolic genes were cloned from pBD2 and the nucleotide sequence was determined. By means of database searches and expression of the cloned genes in recombinant strains, we identified five clustered genes, ipbA1A2A3A4C, which encode the three components of the IPB 2,3-dioxygenase system, reductaseIPB (ipbA4), ferredoxinIPB (ipbA3) and the two subunits of the terminal dioxygenase (ipbA1A2), as well as the 3-isopropylcatechol (IPC) 2,3-dioxygenase (ipbC). The protein sequences deduced from the ipbA1A2A3A4C gene cluster exhibited significant homology with the corresponding proteins of analogous degradative pathways in Gram-negative and Gram-positive bacteria, but the gene order differed from most of them. IPB 2,3-dioxygenase and 3-IPC 2,3-dioxygenase could both be expressed in Escherichia coli, but the IPB 2,3-dioxygenase activities were too low to be detected by polarographic and TCE degradative means. However, inhibitor studies with the R. erythropolis BD2 wild-type are in accordance with the involvement of the IPB 2,3-dioxygenase in TCE oxidation. PMID:8969521

  20. Catechol dioxygenases from Escherichia coli (MhpB) and Alcaligenes eutrophus (MpcI): sequence analysis and biochemical properties of a third family of extradiol dioxygenases.

    PubMed Central

    Spence, E L; Kawamukai, M; Sanvoisin, J; Braven, H; Bugg, T D

    1996-01-01

    The nucleotide sequence of the Escherichia coli mhpB gene, encoding 2,3-dihydroxyphenylpropionate 1,2-dioxygenase, was determined by sequencing of a 3.1-kb fragment of DNA from Kohara phage 139. The inferred amino acid sequence showed 58% sequence identity with the sequence of an extradiol dioxygenase, MpcI, from Alcaligenes eutrophus and 10 to 20% sequence identity with protocatechuate 4,5-dioxygenase from Pseudomonas paucimobilis, with 3,4-dihydroxyphenylacetate 2,3-dioxygenase from E. coli, and with human 3-hydroxyanthranilate dioxygenase. Sequence similarity between the N- and C-terminal halves of this new family of dioxygenases was detected, with conserved histidine residues in the N-terminal domain. A model is proposed to account for the relationship between this family of enzymes and other extradiol dioxygenases. The A. eutrophus MpcI enzyme was expressed in E. coli, purified, and characterized as a protein with a subunit size of 33.8 kDa. Purified MhpB and MpcI showed similar substrate specificities for a range of 3-substituted catechols, and evidence for essential histidine and cysteine residues in both enzymes was obtained. PMID:8752345

  1. Biochemical Characterization of Inducible 'Reductase' Component of Benzoate Dioxygenase and Phthalate Isomer Dioxygenases from Pseudomonas aeruginosa strain PP4.

    PubMed

    Karandikar, Rohini; Badri, Abinaya; Phale, Prashant S

    2015-09-01

    The first step involved in the degradation of phthalate isomers (phthalate, isophthalate and terephthalate) is the double hydroxylation by respective aromatic-ring hydroxylating dioxygenases. These are two component enzymes consisting of 'oxygenase' and 'reductase' components. Soil isolate Pseudomonas aeruginosa strain PP4 degrades phthalate isomers via protocatechuate and benzoate via catechol 'ortho' ring cleavage pathway. Metabolic studies suggest that strain PP4 has carbon source-specific inducible phthalate isomer dioxygenase and benzoate dioxygenase. Thus, it was of interest to study the properties of reductase components of these enzymes. Reductase activity from phthalate isomer-grown cells was 3-5-folds higher than benzoate grown cells. In-gel activity staining profile showed a reductase activity band of R f 0.56 for phthalate isomer-grown cells as compared to R f 0.73 from benzoate-grown cells. Partially purified reductase components from phthalate isomer grown cells showed K m in the range of 30-40 μM and V max = 34-48 μmol min(-1) mg(-1). However, reductase from benzoate grown cells showed K m = 49 μM and V max = 10 μmol min(-1) mg(-1). Strikingly similar molecular and kinetic properties of reductase component from phthalate isomer-grown cells suggest that probably the same reductase component is employed in three phthalate isomer dioxygenases. However, reductase component is different, with respect to kinetic properties and zymogram analysis, from benzoate-grown cells when compared to that from phthalate isomer grown cells of PP4. PMID:26201480

  2. Neuronal pyruvate carboxylation supports formation of transmitter glutamate.

    PubMed

    Hassel, B; Brâthe, A

    2000-02-15

    Release of transmitter glutamate implies a drain of alpha-ketoglutarate from neurons, because glutamate, which is formed from alpha-ketoglutarate, is taken up by astrocytes. It is generally believed that this drain is compensated by uptake of glutamine from astrocytes, because neurons are considered incapable of de novo synthesis of tricarboxylic acid cycle intermediates, which requires pyruvate carboxylation. Here we show that cultured cerebellar granule neurons form releasable [(14)C]glutamate from H(14)CO(3)(-) and [1-(14)C]pyruvate via pyruvate carboxylation, probably mediated by malic enzyme. The activity of pyruvate carboxylation was calculated to be approximately one-third of the pyruvate dehydrogenase activity in neurons. Furthermore, intrastriatal injection of NaH(14)CO(3) or [1-(14)C]pyruvate labeled glutamate better than glutamine, showing that pyruvate carboxylation occurs in neurons in vivo. This means that neurons themselves to a large extent may support their release of glutamate, and thus entails a revision of the current view of glial-neuronal interactions and the importance of the glutamine cycle. PMID:10662824

  3. Activation of Cytosolic Pyruvate Kinase by Polyethylene Glycol.

    PubMed Central

    Podesta, F. E.; Plaxton, W. C.

    1993-01-01

    Homogeneous cytosolic pyruvate kinase from endosperm of germinating castor oil (Ricinus communis L. cv Hale) seeds was potently activated by polyethylene glycol. The addition of 5% (w/v) polyethylene glycol to the pyruvate kinase reaction mixture caused a 2.6-fold increase in maximal velocity and 12.5- and 2-fold reductions in Km values for phosphoenolpyruvate and ADP, respectively. Glycerol, ethylene glycol, and bovine serum albumin also enhanced pyruvate kinase activity, albeit to a lesser extent than polyethylene glycol. The addition of 5% (w/v) polyethylene glycol to the elution buffer during high-performance gel filtration chromatography of purified cytosolic pyruvate kinase helped to stabilize the active heterotetrameric native structure of the enzyme. A higher degree of inhibition by MgATP, but lower sensitivity to the inhibitors 3-phosphoglycerate and fructose- 1,6-bisphosphate, was also observed in the presence of 5% (w/v) polyethylene glycol. It is concluded that (a) plant cytosolic pyruvate kinase activity and regulation, like that of other regulatory pyruvate kinases, is modified by extreme dilution in the assay medium, probably as a result of deaggregation of the native tetrameric enzyme, and (b) ATP is probably the major metabolic effector of germinating castor endosperm cytosolic pyruvate kinase in vivo. PMID:12231936

  4. Activation of Cytosolic Pyruvate Kinase by Polyethylene Glycol.

    PubMed

    Podesta, F. E.; Plaxton, W. C.

    1993-09-01

    Homogeneous cytosolic pyruvate kinase from endosperm of germinating castor oil (Ricinus communis L. cv Hale) seeds was potently activated by polyethylene glycol. The addition of 5% (w/v) polyethylene glycol to the pyruvate kinase reaction mixture caused a 2.6-fold increase in maximal velocity and 12.5- and 2-fold reductions in Km values for phosphoenolpyruvate and ADP, respectively. Glycerol, ethylene glycol, and bovine serum albumin also enhanced pyruvate kinase activity, albeit to a lesser extent than polyethylene glycol. The addition of 5% (w/v) polyethylene glycol to the elution buffer during high-performance gel filtration chromatography of purified cytosolic pyruvate kinase helped to stabilize the active heterotetrameric native structure of the enzyme. A higher degree of inhibition by MgATP, but lower sensitivity to the inhibitors 3-phosphoglycerate and fructose- 1,6-bisphosphate, was also observed in the presence of 5% (w/v) polyethylene glycol. It is concluded that (a) plant cytosolic pyruvate kinase activity and regulation, like that of other regulatory pyruvate kinases, is modified by extreme dilution in the assay medium, probably as a result of deaggregation of the native tetrameric enzyme, and (b) ATP is probably the major metabolic effector of germinating castor endosperm cytosolic pyruvate kinase in vivo. PMID:12231936

  5. Decarboxylation of Pyruvate to Acetaldehyde for Ethanol Production by Hyperthermophiles

    PubMed Central

    Eram, Mohammad S.; Ma, Kesen

    2013-01-01

    Pyruvate decarboxylase (PDC encoded by pdc) is a thiamine pyrophosphate (TPP)-containing enzyme responsible for the conversion of pyruvate to acetaldehyde in many mesophilic organisms. However, no pdc/PDC homolog has yet been found in fully sequenced genomes and proteomes of hyper/thermophiles. The only PDC activity reported in hyperthermophiles was a bifunctional, TPP- and CoA-dependent pyruvate ferredoxin oxidoreductase (POR)/PDC enzyme from the hyperthermophilic archaeon Pyrococcus furiosus. Another enzyme known to be involved in catalysis of acetaldehyde production from pyruvate is CoA-acetylating acetaldehyde dehydrogenase (AcDH encoded by mhpF and adhE). Pyruvate is oxidized into acetyl-CoA by either POR or pyruvate formate lyase (PFL), and AcDH catalyzes the reduction of acetyl-CoA to acetaldehyde in mesophilic organisms. AcDH is present in some mesophilic (such as clostridia) and thermophilic bacteria (e.g., Geobacillus and Thermoanaerobacter). However, no AcDH gene or protein homologs could be found in the released genomes and proteomes of hyperthermophiles. Moreover, no such activity was detectable from the cell-free extracts of different hyperthermophiles under different assay conditions. In conclusion, no commonly-known PDCs was found in hyperthermophiles. Instead of the commonly-known PDC, it appears that at least one multifunctional enzyme is responsible for catalyzing the non-oxidative decarboxylation of pyruvate to acetaldehyde in hyperthermophiles. PMID:24970182

  6. Contributions of tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase to the conversion of D-tryptophan to nicotinamide analyzed by using tryptophan 2,3-dioxygenase-knockout mice.

    PubMed

    Maeta, Akihiro; Sano, Mitsue; Fukuwatari, Tsutomu; Funakoshi, Hiroshi; Nakamura, Toshikazu; Shibata, Katsumi

    2014-01-01

    We investigated the contribution percentage of tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) to the conversion of D-tryptophan to nicotinamide in TDO-knockout mice. The calculated percentage conversions indicated that TDO and IDO oxidized 70 and 30%, respectively, of the dietary L-tryptophan. These results indicate that both TDO and IDO biosynthesize nicotinamide from D-tryptophan and L-tryptophan in mice. PMID:25035993

  7. Purification and characterization of protocatechuate 2,3-dioxygenase from Bacillus macerans: a new extradiol catecholic dioxygenase.

    PubMed Central

    Wolgel, S A; Dege, J E; Perkins-Olson, P E; Jaurez-Garcia, C H; Crawford, R L; Münck, E; Lipscomb, J D

    1993-01-01

    Protocatechuate 2,3-dioxygenase (2,3-PCD) from Bacillus macerans JJ1b has been purified to homogeneity for the first time. The enzyme catalyzes proximal extradiol ring cleavage of protocatechuate (PCA) with the attendant incorporation of both atoms of oxygen from O2. The holoenzyme has a mass of 143 +/- 7 kDa as determined by ultracentrifugation and other techniques. It is composed of four apparently identical subunits with M(r)s of 35,500, each containing one iron atom. Mössbauer spectroscopy of 57Fe-enriched enzyme showed that the irons are indistinguishable and are high spin (S = 2) Fe2+ in both the uncomplexed and substrate-bound enzyme. However, the quadrupole splitting, delta EQ, and isomer shift, delta, of the Mössbauer spectrum changed from delta EQ = 2.57 mm/s and delta = 1.29 mm/s to delta EQ = 2.73 mm/s and delta = 1.19 mm/s upon PCA binding to the enzyme, showing that the iron environment is altered when substrate is present. The enzyme was also found to bind variable and substoichiometric amounts of Mn2+, but this metal could be removed without loss of activity or stability. The inherently electron paramagnetic resonance (EPR)-silent Fe2+ of the enzyme reversibly bound nitric oxide to produce an EPR-active species (g = 4.11, 3.95; S = 3/2). The specific activity of the enzyme was found to be correlated with the amount of the S = 3/2 species formed, showing that activity is dependent on Fe2+. Anaerobic addition of substrates to the enzyme-nitric oxide complex significantly altered the EPR spectrum, suggesting that substrates bind to or near the iron. The enzyme was inactivated by reagents that oxidize the Fe2+, such as H2O2 and K3FE(CN)6; full activity was restored after reduction of the iron by ascorbate. Steady-state kinetic data were found to be consistent with an ordered bi-uni mechanism in which the organic substrate must add to 2,3-PCD before O2. The enzyme has the broadest substrate range of any of the well-studied catecholic dioxygenases. All

  8. A Novel Angular Dioxygenase Gene Cluster Encoding 3-Phenoxybenzoate 1′,2′-Dioxygenase in Sphingobium wenxiniae JZ-1

    PubMed Central

    Wang, Chenghong; Chen, Qing; Wang, Rui; Shi, Chao; Yan, Xin; Li, Shunpeng

    2014-01-01

    Sphingobium wenxiniae JZ-1 utilizes a wide range of pyrethroids and their metabolic product, 3-phenoxybenzoate, as sources of carbon and energy. A mutant JZ-1 strain, MJZ-1, defective in the degradation of 3-phenoxybenzoate was obtained by successive streaking on LB agar. Comparison of the draft genomes of strains JZ-1 and MJZ-1 revealed that a 29,366-bp DNA fragment containing a putative angular dioxygenase gene cluster (pbaA1A2B) is missing in strain MJZ-1. PbaA1, PbaA2, and PbaB share 65%, 52%, and 10% identity with the corresponding α and β subunits and the ferredoxin component of dioxin dioxygenase from Sphingomonas wittichii RW1, respectively. Complementation of pbaA1A2B in strain MJZ-1 resulted in the active 3-phenoxybenzoate 1′,2′-dioxygenase, but the enzyme activity in Escherichia coli was achieved only through the coexpression of pbaA1A2B and a glutathione reductase (GR)-type reductase gene, pbaC, indicating that the 3-phenoxybenzoate 1′,2′-dioxygenase belongs to a type IV Rieske non-heme iron aromatic ring-hydroxylating oxygenase system consisting of a hetero-oligomeric oxygenase, a [2Fe-2S]-type ferredoxin, and a GR-type reductase. The pbaC gene is not located in the immediate vicinity of pbaA1A2B. 3-Phenoxybenzoate 1′,2′-dioxygenase catalyzes the hydroxylation in the 1′ and 2′ positions of the benzene moiety of 3-phenoxybenzoate, yielding 3-hydroxybenzoate and catechol. Transcription of pbaA1A2B and pbaC was induced by 3-phenoxybenzoate, but the transcriptional level of pbaC was far less than that of pbaA1A2B, implying the possibility that PbaC may not be the only reductase that can physiologically transfer electrons to PbaA1A2B in strain JZ-1. Some GR-type reductases from other sphingomonad strains could also transfer electrons to PbaA1A2B, suggesting that PbaA1A2B has a low specificity for reductase. PMID:24747891

  9. Physiological roles of pyruvate ferredoxin oxidoreductase and pyruvate formate-lyase in Thermoanaerobacterium saccharolyticum JW/SL-YS485

    SciTech Connect

    Zhou, Jilai; Olson, Daniel G.; Lanahan, Anthony A.; Tian, Liang; Murphy, Sean Jean-Loup; Lo, Jonathan; Lynd, Lee R.

    2015-09-15

    We report that Thermoanaerobacter saccharolyticum is a thermophilic microorganism that has been engineered to produce ethanol at high titer (30–70 g/L) and greater than 90 % theoretical yield. However, few genes involved in pyruvate to ethanol production pathway have been unambiguously identified. In T. saccharolyticum, the products of six putative pfor gene clusters and one pfl gene may be responsible for the conversion of pyruvate to acetyl-CoA. To gain insights into the physiological roles of PFOR and PFL, we studied the effect of deletions of several genes thought to encode these activities. We found that that pyruvate ferredoxin oxidoreductase enzyme (PFOR) is encoded by the pforA gene and plays a key role in pyruvate dissimilation. We further demonstrated that pyruvate formate-lyase activity (PFL) is encoded by the pfl gene. Although the pfl gene is normally expressed at low levels, it is crucial for biosynthesis in T. saccharolyticum. In pforA deletion strains, pfl expression increased and was able to partially compensate for the loss of PFOR activity. Deletion of both pforA and pfl resulted in a strain that required acetate and formate for growth and produced lactate as the primary fermentation product, achieving 88 % theoretical lactate yield. PFOR encoded by Tsac_0046 and PFL encoded by Tsac_0628 are only two routes for converting pyruvate to acetyl-CoA in T. saccharolyticum. The physiological role of PFOR is pyruvate dissimilation, whereas that of PFL is supplying C1 units for biosynthesis.

  10. Photosolvolysis of bulky (4-hydroxyphenyl)naphthalene derivatives.

    PubMed

    Skalamera, Dani; Mlinarić-Majerski, Kata; Uzelac, Lidija; Kralj, Marijeta; Wan, Peter; Basarić, Nikola

    2013-11-01

    Six new naphthylphenols , bearing bulky hydroxymethyl substituents on the naphthalene, were synthesized and their photoreactivity was investigated by preparative irradiation, fluorescence measurements, and laser flash photolysis. All derivatives (in S1) undergo deprotonation of the phenolic OH in the aqueous solution. Also, fluorescence quenching with HClO4 in the pH range 2-4 indicates that can be protonated in S1. Formation of QMs most probably takes place sequentially, triggered by the phenol deprotonation. However, with the present data, a mechanism that involves simultaneous deprotonation and the loss of OH(-) cannot be ruled out. Photodehydration takes place only for , , and , delivering the corresponding QMs which react with nucleophilic solvents giving the corresponding photosolvolysis products. The other less likely option for the formation of the observed solvolysis products from , , and may involve some radical species. Photodehydration of and was not observed which may be due to the anticipated high energy of the corresponding sterically-congested and . The most efficient photosolvolyses were observed for the 2,6-substituted naphthalenes. PMID:24057421

  11. 21 CFR 862.1655 - Pyruvic acid test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... measure pyruvic acid (an intermediate compound in the metabolism of carbohydrate) in plasma. Measurements obtained by this device are used in the evaluation of electrolyte metabolism and in the diagnosis...

  12. 21 CFR 862.1655 - Pyruvic acid test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... measure pyruvic acid (an intermediate compound in the metabolism of carbohydrate) in plasma. Measurements obtained by this device are used in the evaluation of electrolyte metabolism and in the diagnosis...

  13. 21 CFR 862.1655 - Pyruvic acid test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... measure pyruvic acid (an intermediate compound in the metabolism of carbohydrate) in plasma. Measurements obtained by this device are used in the evaluation of electrolyte metabolism and in the diagnosis...

  14. 21 CFR 862.1655 - Pyruvic acid test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... measure pyruvic acid (an intermediate compound in the metabolism of carbohydrate) in plasma. Measurements obtained by this device are used in the evaluation of electrolyte metabolism and in the diagnosis...

  15. 21 CFR 862.1655 - Pyruvic acid test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... measure pyruvic acid (an intermediate compound in the metabolism of carbohydrate) in plasma. Measurements obtained by this device are used in the evaluation of electrolyte metabolism and in the diagnosis...

  16. Genetic analysis of the pyruvate decarboxylase reaction in yeast glycolysis.

    PubMed Central

    Schmitt, H D; Zimmermann, F K

    1982-01-01

    Six different pyruvate decarboxylase mutants of Saccharomyces cerevisiae were isolated. They belong to two unlinked complementation groups. Evidence is presented that one group is affected in a structural gene. The fact that five of the six mutants had residual pyruvate decarboxylase activity provided the opportunity for an intensive physiological characterization. It was shown that the loss of enzyme activity in vitro is reflected in a lower fermentation rate, an increased pyruvate secretion, and slower growth on a 2% glucose medium. The different effects of antimycin A on leaky mutants grown on ethanol versus the same mutants grown on glucose support the view that glucose induces some of the glycolytic enzymes, especially pyruvate decarboxylase. PMID:7050079

  17. Pyruvate Protects Pathogenic Spirochetes from H2O2 Killing

    PubMed Central

    Troxell, Bryan; Zhang, Jun-Jie; Bourret, Travis J.; Zeng, Melody Yue; Blum, Janice; Gherardini, Frank; Hassan, Hosni M.; Yang, X. Frank

    2014-01-01

    Pathogenic spirochetes cause clinically relevant diseases in humans and animals, such as Lyme disease and leptospirosis. The causative agent of Lyme disease, Borrelia burgdorferi, and the causative agent of leptospirosis, Leptospria interrogans, encounter reactive oxygen species (ROS) during their enzootic cycles. This report demonstrated that physiologically relevant concentrations of pyruvate, a potent H2O2 scavenger, and provided passive protection to B. burgdorferi and L. interrogans against H2O2. When extracellular pyruvate was absent, both spirochetes were sensitive to a low dose of H2O2 (≈0.6 µM per h) generated by glucose oxidase (GOX). Despite encoding a functional catalase, L. interrogans was more sensitive than B. burgdorferi to H2O2 generated by GOX, which may be due to the inherent resistance of B. burgdorferi because of the virtual absence of intracellular iron. In B. burgdorferi, the nucleotide excision repair (NER) and the DNA mismatch repair (MMR) pathways were important for survival during H2O2 challenge since deletion of the uvrB or the mutS genes enhanced its sensitivity to H2O2 killing; however, the presence of pyruvate fully protected ΔuvrB and ΔmutS from H2O2 killing further demonstrating the importance of pyruvate in protection. These findings demonstrated that pyruvate, in addition to its classical role in central carbon metabolism, serves as an important H2O2 scavenger for pathogenic spirochetes. Furthermore, pyruvate reduced ROS generated by human neutrophils in response to the Toll-like receptor 2 (TLR2) agonist zymosan. In addition, pyruvate reduced neutrophil-derived ROS in response to B. burgdorferi, which also activates host expression through TLR2 signaling. Thus, pathogenic spirochetes may exploit the metabolite pyruvate, present in blood and tissues, to survive H2O2 generated by the host antibacterial response generated during infection. PMID:24392147

  18. Pyruvate kinase: function, regulation and role in cancer

    PubMed Central

    Israelsen, William J.; Vander Heiden, Matthew G.

    2015-01-01

    Pyruvate kinase is an enzyme that catalyzes the conversion of phosphoenolpyruvate and ADP to pyruvate and ATP in glycolysis and plays a role in regulating cell metabolism. There are four mammalian pyruvate kinase isoforms with unique tissue expression patterns and regulatory properties. The M2 isoform of pyruvate kinase (PKM2) supports anabolic metabolism and is expressed both in cancer and normal tissue. The enzymatic activity of PKM2 is allosterically regulated by both intracellular signaling pathways and metabolites; PKM2 thus integrates signaling and metabolic inputs to modulate glucose metabolism according to the needs of the cell. Recent advances have increased our understanding of metabolic regulation by pyruvate kinase, raised new questions, and suggested the possibility of non-canonical PKM2 functions to regulate gene expression and cell cycle progression via protein-protein interactions and protein kinase activity. Here we review the structure, function, and regulation of pyruvate kinase and discuss how these properties enable regulation of PKM2 for cell proliferation and tumor growth. PMID:26277545

  19. An energy-conserving pyruvate-to-acetate pathway in Entamoeba histolytica. Pyruvate synthase and a new acetate thiokinase.

    PubMed

    Reeves, R E; Warren, L G; Susskind, B; Lo, H S

    1977-01-25

    Under anaerobic conditions, cells of Entamoeba histolytica grown with bacteria produce H2 and acetate while cells grown axenically produce neither. Aerobically, acetate is produced and O2 is consumed by amebae from either type of cells. Centrifuged extracts, 2.4 x 106 x g x min, from both types of cells contain pyruvate synthase (EC 1.2.7.1) and an acetate thiokinase which, together, form a system capable of converting pyruvate to acetate. Pyruvate synthase catalyzes the reaction: pyruvate + CoA leads to CO2 + acetyl-CoA + 2E. Electron acceptors which function with this enzyme are FAD, FMN, riboflavin, ferredoxin, and methyl viologen, but not NAD or NADP. The amebal acetate thiokinase catalyzes the reaction acetyl-CoA + ADP + Pi leads to acetate + ATP + CoA. For this apparently new enzyme we suggest the trivial name acetyl-CoA-synthetase (ADP-forming). Extracts from axenic amebae do not contain hydrogenase, but extracts from cells grown with bacteria do. It is postulated that in bacteria-grown amebae electrons generated at the pyruvate synthase step are utilized anaerobically to produce H2 via the hydrogenase and that the acetyl-CoA is converted to acetate in an energy-conserving step catalyzed by amebal acetyl-CoA synthetase. Aerobically, cells grown under either regimen may utilize the energy-conserving pyruvate-to-acetate pathway since O2 then serves as the ultimate electron acceptor. PMID:13076

  20. Pyruvate Dehydrogenase and Pyruvate Dehydrogenase Kinase Expression in Non Small Cell Lung Cancer and Tumor-Associated Stroma1

    PubMed Central

    Koukourakis, Michael I; Giatromanolaki, Alexandra; Sivridis, Efthimios; Gatter, Kevin C; Harris, Adrian L; “Tumor and Angiogenesis Research Group”

    2005-01-01

    Abstract Pyruvate dehydrogenase (PDH) catalyzes the conversion of pyruvate to acetyl-coenzyme A, which enters into the Krebs cycle, providing adenosine triphosphate (ATP) to the cell. PDH activity is under the control of pyruvate dehydrogenase kinases (PDKs). Under hypoxic conditions, conversion of pyruvate to lactate occurs, a reaction catalyzed by lactate dehydrogenase 5 (LDH5). In cancer cells, however, pyruvate is transformed to lactate occurs, regardless of the presence of oxygen (aerobic glycolysis/Warburg effect). Although hypoxic intratumoral conditions account for HIF1α stabilization and induction of anaerobic metabolism, recent data suggest that high pyruvate concentrations also result in HIF1α stabilization independently of hypoxia. In the present immunohistochemical study, we provide evidence that the PDH/PDK pathway is repressed in 73% of non small cell lung carcinomas, which may be a key reason for HIF1α stabilization and “aerobic glycolysis.” However, about half of PDH-deficient carcinomas are not able to switch on the HIF pathway, and patients harboring these tumors have an excellent postoperative outcome. A small subgroup of clinically aggressive tumors maintains a coherent PDH and HIF/LDH5 expression. In contrast to cancer cells, fibroblasts in the tumor-supporting stroma exhibit an intense PDH but reduced PDK1 expression favoring maximum PDH activity. This means that stroma may use lactic acid produced by tumor cells, preventing the creation of an intolerable intratumoral acidic environment at the same time. PMID:15736311

  1. Oxidative Transformation of Aminodinitrotoluene Isomers by Multicomponent Dioxygenases

    PubMed Central

    Johnson, Glenn R.; Smets, Barth F.; Spain, Jim C.

    2001-01-01

    The electron-withdrawing nitro substituents of 2,4,6-trinitrotoluene (TNT) make the aromatic ring highly resistant to oxidative transformation. The typical biological transformation of TNT involves reduction of one or more of the nitro groups of the ring to produce the corresponding amine. Reduction of a single nitro substituent of TNT to an amino substituent increases the electron density of the aromatic nucleus considerably. The comparatively electron-dense nuclei of the aminodinitrotoluene (ADNT) isomers would be expected to be more susceptible to oxygenase attack than TNT. The hypothesis was tested by evaluating three nitroarene dioxygenases for the ability to hydroxylate the ADNT isomers. The predominant reaction was dioxygenation of the ring to yield nitrite and the corresponding aminomethylnitrocatechol. A secondary reaction was benzylic monooxygenation to form aminodinitrobenzyl alcohol. The substrate preferences and catalytic specificities of the three enzymes differed considerably. The discovery that the ADNT isomers are substrates for the nitroarene dioxygenases reveals the potential for extensive bacterial transformation of TNT under aerobic conditions. PMID:11722893

  2. Physiological roles of pyruvate ferredoxin oxidoreductase and pyruvate formate-lyase in Thermoanaerobacterium saccharolyticum JW/SL-YS485

    DOE PAGESBeta

    Zhou, Jilai; Olson, Daniel G.; Lanahan, Anthony A.; Tian, Liang; Murphy, Sean Jean-Loup; Lo, Jonathan; Lynd, Lee R.

    2015-09-15

    We report that Thermoanaerobacter saccharolyticum is a thermophilic microorganism that has been engineered to produce ethanol at high titer (30–70 g/L) and greater than 90 % theoretical yield. However, few genes involved in pyruvate to ethanol production pathway have been unambiguously identified. In T. saccharolyticum, the products of six putative pfor gene clusters and one pfl gene may be responsible for the conversion of pyruvate to acetyl-CoA. To gain insights into the physiological roles of PFOR and PFL, we studied the effect of deletions of several genes thought to encode these activities. We found that that pyruvate ferredoxin oxidoreductase enzymemore » (PFOR) is encoded by the pforA gene and plays a key role in pyruvate dissimilation. We further demonstrated that pyruvate formate-lyase activity (PFL) is encoded by the pfl gene. Although the pfl gene is normally expressed at low levels, it is crucial for biosynthesis in T. saccharolyticum. In pforA deletion strains, pfl expression increased and was able to partially compensate for the loss of PFOR activity. Deletion of both pforA and pfl resulted in a strain that required acetate and formate for growth and produced lactate as the primary fermentation product, achieving 88 % theoretical lactate yield. PFOR encoded by Tsac_0046 and PFL encoded by Tsac_0628 are only two routes for converting pyruvate to acetyl-CoA in T. saccharolyticum. The physiological role of PFOR is pyruvate dissimilation, whereas that of PFL is supplying C1 units for biosynthesis.« less

  3. Multiphase Chemistry of Pyruvic Acid Under Atmospherically Relevant Conditions

    NASA Astrophysics Data System (ADS)

    Vaida, V.; Monod, A.; Doussin, J. F.; Reed Harris, A. E.; Griffith, E. C.; Kroll, J. A.; Rapf, R.

    2014-12-01

    Chemistry in the natural environment proceeds in multiple phases and is subject to effects from atmospheric constituents and conditions. This presentation will use pyruvic acid as a case study to demonstrate the complexity of atmospheric multiphase chemistry. The photophysics and photochemistry of pyruvic acid proceeds on different potential energy surfaces with different reaction mechanisms, rates, and products in gas versus the aqueous phase. While the gas phase reaction generally decreases the complexity of products, the aqueous chemistry creates higher molecular weight, surface-active compounds. The studies presented involve a combination of laboratory studies that focus on the photochemistry of pyruvic acid in both the gas and aqueous phases. Further, experiments in an environmental simulation chamber (CESAM) that follow the photochemistry chemistry of pyruvic acid under atmospherically relevant conditions will be presented to highlight the effect of pressure, oxygen, relative humidity, and phase on the photochemistry of pyruvic acid. The results provide new input for atmospheric chemistry models that is required to better describe the behavior of α-keto acids in the environment.

  4. Reactivity landscape of pyruvate under simulated hydrothermal vent conditions

    PubMed Central

    Novikov, Yehor; Copley, Shelley D.

    2013-01-01

    Pyruvate is an important “hub” metabolite that is a precursor for amino acids, sugars, cofactors, and lipids in extant metabolic networks. Pyruvate has been produced under simulated hydrothermal vent conditions from alkyl thiols and carbon monoxide in the presence of transition metal sulfides at 250 °C [Cody GD et al. (2000) Science 289(5483):1337–1340], so it is plausible that pyruvate was formed in hydrothermal systems on the early earth. We report here that pyruvate reacts readily in the presence of transition metal sulfide minerals under simulated hydrothermal vent fluids at more moderate temperatures (25–110 °C) that are more conducive to survival of biogenic molecules. We found that pyruvate partitions among five reaction pathways at rates that depend upon the nature of the mineral present; the concentrations of H2S, H2, and NH4Cl; and the temperature. In most cases, high yields of one or two primary products are found due to preferential acceleration of certain pathways. Reactions observed include reduction of ketones to alcohols and aldol condensation, both reactions that are common in extant metabolic networks. We also observed reductive amination to form alanine and reduction to form propionic acid. Amino acids and fatty acids formed by analogous processes may have been important components of a protometabolic network that allowed the emergence of life. PMID:23872841

  5. Reactivity landscape of pyruvate under simulated hydrothermal vent conditions.

    PubMed

    Novikov, Yehor; Copley, Shelley D

    2013-08-13

    Pyruvate is an important "hub" metabolite that is a precursor for amino acids, sugars, cofactors, and lipids in extant metabolic networks. Pyruvate has been produced under simulated hydrothermal vent conditions from alkyl thiols and carbon monoxide in the presence of transition metal sulfides at 250 °C [Cody GD et al. (2000) Science 289(5483):1337-1340], so it is plausible that pyruvate was formed in hydrothermal systems on the early earth. We report here that pyruvate reacts readily in the presence of transition metal sulfide minerals under simulated hydrothermal vent fluids at more moderate temperatures (25-110 °C) that are more conducive to survival of biogenic molecules. We found that pyruvate partitions among five reaction pathways at rates that depend upon the nature of the mineral present; the concentrations of H2S, H2, and NH4Cl; and the temperature. In most cases, high yields of one or two primary products are found due to preferential acceleration of certain pathways. Reactions observed include reduction of ketones to alcohols and aldol condensation, both reactions that are common in extant metabolic networks. We also observed reductive amination to form alanine and reduction to form propionic acid. Amino acids and fatty acids formed by analogous processes may have been important components of a protometabolic network that allowed the emergence of life. PMID:23872841

  6. Ethyl Pyruvate Emerges as a Safe and Fast Acting Agent against Trypanosoma brucei by Targeting Pyruvate Kinase Activity

    PubMed Central

    Worku, Netsanet; Stich, August; Daugschies, Arwid; Wenzel, Iris; Kurz, Randy; Thieme, Rene; Kurz, Susanne; Birkenmeier, Gerd

    2015-01-01

    Background Human African Trypanosomiasis (HAT) also called sleeping sickness is an infectious disease in humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. Currently available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to similarities in cell metabolism between cancerous tumors and trypanosoma cells, some of the current registered drugs against HAT have also been tested in cancer chemotherapy. Here we demonstrate for the first time that the simple ester, ethyl pyruvate, comprises such properties. Results The current study covers the efficacy and corresponding target evaluation of ethyl pyruvate on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, phasecontrast microscopic video imaging and ex vivo toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki = 3.0±0.29 mM). The potential of ethyl pyruvate as a trypanocidal compound is also strengthened by its fast acting property, killing cells within three hours post exposure. This has been demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, ethyl pyruvate produces minimal side effects in human red cells and is known to easily cross the blood-brain-barrier. This makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug-resistance tests indicate irreversible cell death and a low incidence of resistance development under experimental conditions. Conclusion Our results present ethyl pyruvate as a safe and fast acting trypanocidal compound and show that it inhibits the enzyme pyruvate kinase. Competitive inhibition of this enzyme was found to cause ATP depletion and cell death. Due to its ability to

  7. Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase

    SciTech Connect

    Catalanotti, C.; Dubini, A.; Subramanian, V.; Yang, W. Q.; Magneschi, L.; Mus, F.; Seibert, M.; Posewitz, M. C.; Grossman, A. R.

    2012-02-01

    Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H2 production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.

  8. /sup 14/CO/sub 2/ ratios method for detecting pyruvate carboxylation

    SciTech Connect

    Kelleher, J.K.; Bryan, B.M. III

    1985-11-15

    The pattern of oxidative metabolism of pyruvate may be assessed by comparing the steady-state /sup 14/CO/sub 2/ production from four isotopes in identical samples. The assay requires measuring the ratios of steady-state /sup 14/CO/sub 2/ production from two isotope pairs, (2-/sup 14/C)pyruvate:(3-/sup 14/C)pyruvate and (1-/sup 14/C)acetate:(2-/sup 14/C)acetate. These ratios are defined as the ''pyruvate /sup 14/CO/sub 2/ ratio'' and the ''acetate /sup 14/CO/sub 2/ ratio,'' respectively. If pyruvate is metabolized exclusively via pyruvate dehydrogenase (PDH), the two ratios will be identical. Alternatively, if any pyruvate enters the tricarboxylic acid (TCA) cycle via pyruvate carboxylation (PC), the pyruvate /sup 14/CO/sub 2/ ratio will be less than the acetate /sup 14/CO/sub 2/ ratio. If pyruvate enters the TCA cycle only through PC (with oxaloacetate and fumarate in equilibrium) the pyruvate /sup 14/CO/sub 2/ ratio will approach a value of 1.0. An equation is presented for the quantitative evaluation of pyruvate oxidation by these two pathways. We have used this method to detect relative changes in the pattern of pyruvate metabolism in rat liver mitochondria produced by exposure to 1 mM octanoyl carnitine, a compound known to alter the PC:PDH activity ratio. The major advantages of the method are (i) that it provides a sensitive method for detecting pyruvate carboxylation at physiological pyruvate concentrations and (ii) that it provides a method for distinguishing between effects on pyruvate transport and effects on pyruvate oxidation.

  9. Antitumour agents as inhibitors of tryptophan 2,3-dioxygenase.

    PubMed

    Pantouris, Georgios; Mowat, Christopher G

    2014-01-01

    The involvement of tryptophan 2,3-dioxygenase (TDO) in cancer biology has recently been described, with the enzyme playing an immunomodulatory role, suppressing antitumour immune responses and promoting tumour cell survival and proliferation. This finding reinforces the need for specific inhibitors of TDO that may potentially be developed for therapeutic use. In this work we have screened ~2800 compounds from the library of the National Cancer Institute USA and identified seven potent inhibitors of TDO with inhibition constants in the nanomolar or low micromolar range. All seven have antitumour properties, killing various cancer cell lines. For comparison, the inhibition potencies of these compounds were tested against IDO and their inhibition constants are reported. Interestingly, this work reveals that NSC 36398 (dihydroquercetin, taxifolin), with an in vitro inhibition constant of ~16 μM, is the first TDO-selective inhibitor reported. PMID:24269239

  10. NADH Oxidase Activity of Indoleamine 2,3-Dioxygenase*

    PubMed Central

    Rosell, Federico I.; Kuo, Hsin H.; Mauk, A. Grant

    2011-01-01

    The heme enzyme indoleamine 2,3-dioxygenase (IDO) was found to oxidize NADH under aerobic conditions in the absence of other enzymes or reactants. This reaction led to the formation of the dioxygen adduct of IDO and supported the oxidation of Trp to N-formylkynurenine. Formation of the dioxygen adduct and oxidation of Trp were accelerated by the addition of small amounts of hydrogen peroxide, and both processes were inhibited in the presence of either superoxide dismutase or catalase. Anaerobic reaction of IDO with NADH proceeded only in the presence of a mediator (e.g. methylene blue) and resulted in formation of the ferrous form of the enzyme. We propose that trace amounts of peroxide previously proposed to occur in NADH solutions as well as solid NADH activate IDO and lead to aerobic formation of superoxide and the reactive dioxygen adduct of the enzyme. PMID:21690092

  11. Crystallization and preliminary crystallographic studies of human indoleamine 2,3-dioxygenase

    SciTech Connect

    Oda, Shun-ichiro; Sugimoto, Hiroshi; Yoshida, Tadashi; Shiro, Yoshitsugu

    2006-03-01

    Human indoleamine 2,3-dioxygenase, a haem-containing dioxygenase, was crystallized. The crystal diffracted to 2.3 Å resolution. Indoleamine 2,3-dioxygenase (IDO) is a haem-containing dioxygenase that catalyzes the oxidative cleavage of the pyrrole ring of indoleamines by the insertion of molecular oxygen. This reaction is the first and the rate-limiting step in the kynurenine pathway, the major Trp catabolic pathway in mammals. Recombinant human IDO was crystallized by the vapour-diffusion technique. The addition of 4-phenylimidazole as a haem ligand was essential for crystallization. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 86.1, b = 98.0, c = 131.0 Å. Diffraction data were collected to 2.3 Å resolution.

  12. INHIBITION OF INDOLEAMINE 2,3-DIOXYGENASE DOES NOT IMPEDE ORAL TOLERANCE

    EPA Science Inventory

    Rationale: Indoleamine 2,3-dioxygenase (IDO), a tryptophan catabolizing enzyme, regulates immune tolerance through inhibition of T-cell proliferation. Pharmacologic inhibition of IDO, which causes fetal rejection and increased tumor resistance in mice, may prove useful in cancer...

  13. Molecular basis for the substrate stereoselectivity in Tryptophan Dioxygenase

    PubMed Central

    Capece, Luciana; Lewis-Ballester, Ariel; Marti, Marcelo A.; Estrin, Dario A.; Yeh, Syun-Ru

    2011-01-01

    Tryptophan dioxygenase (TDO) and Indoleamine 2,3 dioxygenase (IDO) are the only two heme-proteins that catalyze the oxidation reaction of tryptophan (Trp) to N-formylkynurenine (NFK). While human IDO (hIDO) is able to oxidize both L and D-Trp, human TDO (hTDO) displays a major specificity towards L-Trp. In this work we aim to interrogate the molecular basis for the substrate stereoselectivity of hTDO. Our previous molecular dynamics simulation studies of Xanthomonas campestris TDO (xcTDO) showed that an H-bond between T254 (T342 in hTDO) and the ammonium group of the substrate is present in the L-Trp-bound enzyme, but not in the D-Trp bound enzyme. The fact that this is the only notable structural alteration induced by the change in the stereo structure of the substrate prompted us to produce and characterize the T342A mutant of hTDO to evaluate the structural role of T342 in controlling the substrate stereoselectivity of the enzyme. The experimental results indicate that the mutation only slightly perturbs the global structural properties of the enzyme, but it totally abolishes the substrate stereoselectivity. Molecular Dynamics simulations of xcTDO show that T254 controls the substrate stereoselectivity of the enzyme by (i) modulating the H-bonding interaction between the NH3+ group and epoxide oxygen of the ferryl/indole 2,3-epoxide intermediate of the enzyme, and (ii) regulating the dynamics of two active site loops, loop250–260 and loop117–130, critical for substrate-binding. PMID:22082147

  14. Pyruvate protects against experimental stroke via an anti-inflammatory mechanism

    PubMed Central

    Wang, Qing; van Hoecke, Michael; Tang, Xiannan; Lee, Hokyou; Zheng, Zheng; Swanson, Raymond A.; Yenari, Midori A.

    2009-01-01

    Pyruvate, a key intermediate in glucose metabolism, was explored as a potential treatment in models of experimental stroke and inflammation. Pyruvate was administered to rodents after the onset of middle cerebral artery occlusion (MCAO). Since the extent of inflammation is often proportional to the size of the infarct, we also studied a group of animals given lipopolysaccharide (LPS) to cause brain inflammation without cell death. Following MCAO, pyruvate did not affect physiological parameters but significantly reduced infarct volume, improved behavioral tests and reduced numbers of neutrophils, microglial and NF-kB activation. Animals given LPS showed increased microglial and NF-kB activation which was almost completely abolished by pyruvate. Lactate, a major metabolite of pyruvate, was increased after pyruvate administration. However, administration of lactate itself did not have any anti-inflammatory effects. Pyruvate protects against ischemia possibly by blocking inflammation, but lactate itself does not appear to explain pyruvate's anti-inflammatory properties. PMID:19635562

  15. Intestinal anti-inflammatory activity of calcium pyruvate in the TNBS model of rat colitis: Comparison with ethyl pyruvate.

    PubMed

    Algieri, F; Rodriguez-Nogales, A; Garrido-Mesa, J; Camuesco, D; Vezza, T; Garrido-Mesa, N; Utrilla, P; Rodriguez-Cabezas, M E; Pischel, I; Galvez, J

    2016-03-01

    Pyruvate is a key intermediate of the carbohydrate metabolism with endogenous scavenger properties. However, it cannot be used in clinics due to its instability. Ethyl pyruvate (EP) has shown better stability as well as an antioxidant and anti-inflammatory activity. Calcium pyruvate monohydrate (CPM) is another stable pyruvate derivative that could also provide the benefits from calcium, fundamental for bone health. Considering everything, we propose CPM as a therapeutic strategy to treat diseases with an immune component in which there is also a significant dysregulation of the skeletal homeostasis. This could be applicable to inflammatory bowel disease, which is characterized by over-production of pro-inflammatory mediators, including cytokines and reactive oxygen and nitrogen metabolites that induces intestinal mucosal damage and chronic inflammation, and extra-intestinal symptoms like osteopenia and osteoporosis. The effects of CPM and EP (20, 40 and 100mg/kg) were evaluated on the trinitrobenzenesulfonic acid (TNBS) model of colitis in rats, after a 7-day oral treatment, with main focus on colonic histology and inflammatory mediators. Both pyruvates showed intestinal anti-inflammatory effects in the TNBS-induced colitis. They were evident both histologically, with a recovery of the mucosal cytoarchitecture and a reduction of the neutrophil infiltration, and through the profile of inflammatory mediators (IL-1, IL-6, IL-17, IL-23, iNOS). However, CPM appeared to be more effective than ethyl pyruvate. In conclusion, CPM exerts intestinal anti-inflammatory effect on the TNBS-induced colitis in rats, although further experiments are needed to explore its beneficial effects on bone health and osteoporosis. PMID:26774455

  16. Indoleamine 2,3-dioxygenase-2; a new enzyme in the kynurenine pathway.

    PubMed

    Ball, Helen J; Yuasa, Hajime J; Austin, Christopher J D; Weiser, Silvia; Hunt, Nicholas H

    2009-03-01

    The kynurenine pathway of tryptophan metabolism converts the amino acid tryptophan into a number of biologically active metabolites. The first and rate-limiting step in this pathway is the conversion of tryptophan to N-formylkynurenine and until recently this reaction was thought to be performed by either of two enzymes, tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase. A third enzyme, indoleamine 2,3-dioxygenase-2, indoleamine 2,3-dioxygenase-like protein or proto-indoleamine 2,3-dioxygenase (IDO2, IDO-2, INDOL1 or proto-IDO), with this activity recently has been described. The gene encoding IDO2 is adjacent and structurally similar to the indoleamine 2,3-dioxygenase gene and both mouse genes use multiple promoters to express transcripts with alternate 5' exons. The IDO2 protein is expressed in the murine kidney, liver, male and female reproductive system. The two IDO enzymes can utilise a similar range of substrates, however they differ in their selectivity for some inhibitors. The selective inhibition of IDO2 by 1-methyl-D-tryptophan suggests that IDO2 activity may have a role in the inhibition of immune responses to tumours. PMID:18282734

  17. Structures of the multicomponent Rieske non-heme iron toluene 2, 3-dioxygenase enzyme system

    SciTech Connect

    Friemann, Rosmarie; Lee, Kyoung; Brown, Eric N.; Gibson, David T.; Eklund, Hans; Ramaswamy, S.

    2009-01-01

    The crystal structures of the three-component toluene 2, 3-dioxygenase system provide a model for electron transfer among bacterial Rieske non-heme iron dioxygenases. Bacterial Rieske non-heme iron oxygenases catalyze the initial hydroxylation of aromatic hydrocarbon substrates. The structures of all three components of one such system, the toluene 2, 3-dioxygenase system, have now been determined. This system consists of a reductase, a ferredoxin and a terminal dioxygenase. The dioxygenase, which was cocrystallized with toluene, is a heterohexamer containing a catalytic and a structural subunit. The catalytic subunit contains a Rieske [2Fe–2S] cluster and mononuclear iron at the active site. This iron is not strongly bound and is easily removed during enzyme purification. The structures of the enzyme with and without mononuclear iron demonstrate that part of the structure is flexible in the absence of iron. The orientation of the toluene substrate in the active site is consistent with the regiospecificity of oxygen incorporation seen in the product formed. The ferredoxin is Rieske type and contains a [2Fe–2S] cluster close to the protein surface. The reductase belongs to the glutathione reductase family of flavoenzymes and consists of three domains: an FAD-binding domain, an NADH-binding domain and a C-terminal domain. A model for electron transfer from NADH via FAD in the reductase and the ferredoxin to the terminal active-site mononuclear iron of the dioxygenase is proposed.

  18. Crystal structure of pyruvate decarboxylase from Zymobacter palmae

    PubMed Central

    Buddrus, Lisa; Andrews, Emma S. V.; Leak, David J.; Danson, Michael J.; Arcus, Vickery L.; Crennell, Susan J.

    2016-01-01

    Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a thiamine pyrophosphate- and Mg2+ ion-dependent enzyme that catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. It is rare in bacteria, but is a key enzyme in homofermentative metabolism, where ethanol is the major product. Here, the previously unreported crystal structure of the bacterial pyruvate decarboxylase from Zymobacter palmae is presented. The crystals were shown to diffract to 2.15 Å resolution. They belonged to space group P21, with unit-cell parameters a = 204.56, b = 177.39, c = 244.55 Å and R r.i.m. = 0.175 (0.714 in the highest resolution bin). The structure was solved by molecular replacement using PDB entry 2vbi as a model and the final R values were R work = 0.186 (0.271 in the highest resolution bin) and R free = 0.220 (0.300 in the highest resolution bin). Each of the six tetramers is a dimer of dimers, with each monomer sharing its thiamine pyrophosphate across the dimer interface, and some contain ethylene glycol mimicking the substrate pyruvate in the active site. Comparison with other bacterial PDCs shows a correlation of higher thermostability with greater tetramer interface area and number of interactions. PMID:27599861

  19. Crystal structure of pyruvate decarboxylase from Zymobacter palmae.

    PubMed

    Buddrus, Lisa; Andrews, Emma S V; Leak, David J; Danson, Michael J; Arcus, Vickery L; Crennell, Susan J

    2016-09-01

    Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a thiamine pyrophosphate- and Mg(2+) ion-dependent enzyme that catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. It is rare in bacteria, but is a key enzyme in homofermentative metabolism, where ethanol is the major product. Here, the previously unreported crystal structure of the bacterial pyruvate decarboxylase from Zymobacter palmae is presented. The crystals were shown to diffract to 2.15 Å resolution. They belonged to space group P21, with unit-cell parameters a = 204.56, b = 177.39, c = 244.55 Å and Rr.i.m. = 0.175 (0.714 in the highest resolution bin). The structure was solved by molecular replacement using PDB entry 2vbi as a model and the final R values were Rwork = 0.186 (0.271 in the highest resolution bin) and Rfree = 0.220 (0.300 in the highest resolution bin). Each of the six tetramers is a dimer of dimers, with each monomer sharing its thiamine pyrophosphate across the dimer interface, and some contain ethylene glycol mimicking the substrate pyruvate in the active site. Comparison with other bacterial PDCs shows a correlation of higher thermostability with greater tetramer interface area and number of interactions. PMID:27599861

  20. Evolution of the functional properties of pyruvate kinase isozymes: pyruvate kinase L from Rana pipiens.

    PubMed

    Fournier, P; Guderley, H

    1986-01-01

    The regulatory properties of type L pyruvate kinase from Rana pipiens are intermediate between those of the mammalian K and L isozymes. As with mammalian type L, the levels of the frog isozyme are affected by the animal's nutritional state. The mammalian and amphibian isozymes show similar sensitivities to fructose 1,6-bisphosphate activation and amino acid inhibition. By contrast, the frog L isozyme shares several properties of the K class: i.e. irreversible inactivation by oxidized glutathione and lack of response to a cyclic AMP stimulated phosphorylation. Furthermore, as for some mammalian K isozymes, frog type L shows a high PEP affinity and a low cooperativity of PEP binding. Insofar as the properties of this present day enzyme reflect those of its counterpart in the amphibian ancestor of higher vertebrates, our results suggest that at its first expression, the type L resembled the type K. Many important regulatory properties of the L isozyme, especially the sensitivity to phosphorylation, were acquired more recently perhaps in association with an increased importance of constant blood glucose. PMID:3489743

  1. IDH1 Mutation Induces Reprogramming of Pyruvate Metabolism.

    PubMed

    Izquierdo-Garcia, Jose L; Viswanath, Pavithra; Eriksson, Pia; Cai, Larry; Radoul, Marina; Chaumeil, Myriam M; Blough, Michael; Luchman, H Artee; Weiss, Samuel; Cairncross, J Gregory; Phillips, Joanna J; Pieper, Russell O; Ronen, Sabrina M

    2015-08-01

    Mutant isocitrate dehydrogenase 1 (IDH1) catalyzes the production of 2-hydroxyglutarate but also elicits additional metabolic changes. Levels of both glutamate and pyruvate dehydrogenase (PDH) activity have been shown to be affected in U87 glioblastoma cells or normal human astrocyte (NHA) cells expressing mutant IDH1, as compared with cells expressing wild-type IDH1. In this study, we show how these phenomena are linked through the effects of IDH1 mutation, which also reprograms pyruvate metabolism. Reduced PDH activity in U87 glioblastoma and NHA IDH1 mutant cells was associated with relative increases in PDH inhibitory phosphorylation, expression of pyruvate dehydrogenase kinase-3, and levels of hypoxia inducible factor-1α. PDH activity was monitored in these cells by hyperpolarized (13)C-magnetic resonance spectroscopy ((13)C-MRS), which revealed a reduction in metabolism of hyperpolarized 2-(13)C-pyruvate to 5-(13)C-glutamate, relative to cells expressing wild-type IDH1. (13)C-MRS also revealed a reduction in glucose flux to glutamate in IDH1 mutant cells. Notably, pharmacological activation of PDH by cell exposure to dichloroacetate (DCA) increased production of hyperpolarized 5-(13)C-glutamate in IDH1 mutant cells. Furthermore, DCA treatment also abrogated the clonogenic advantage conferred by IDH1 mutation. Using patient-derived mutant IDH1 neurosphere models, we showed that PDH activity was essential for cell proliferation. Taken together, our results established that the IDH1 mutation induces an MRS-detectable reprogramming of pyruvate metabolism, which is essential for cell proliferation and clonogenicity, with immediate therapeutic implications. PMID:26045167

  2. Activation and inhibition of pyruvate carboxylase from Rhizobium etli.

    PubMed

    Zeczycki, Tonya N; Menefee, Ann L; Jitrapakdee, Sarawut; Wallace, John C; Attwood, Paul V; St Maurice, Martin; Cleland, W Wallace

    2011-11-15

    While crystallographic structures of the R. etli pyruvate carboxylase (PC) holoenzyme revealed the location and probable positioning of the essential activator, Mg(2+), and nonessential activator, acetyl-CoA, an understanding of how they affect catalysis remains unclear. The current steady-state kinetic investigation indicates that both acetyl-CoA and Mg(2+) assist in coupling the MgATP-dependent carboxylation of biotin in the biotin carboxylase (BC) domain with pyruvate carboxylation in the carboxyl transferase (CT) domain. Initial velocity plots of free Mg(2+) vs pyruvate were nonlinear at low concentrations of Mg(2+) and a nearly complete loss of coupling between the BC and CT domain reactions was observed in the absence of acetyl-CoA. Increasing concentrations of free Mg(2+) also resulted in a decrease in the K(a) for acetyl-CoA. Acetyl phosphate was determined to be a suitable phosphoryl donor for the catalytic phosphorylation of MgADP, while phosphonoacetate inhibited both the phosphorylation of MgADP by carbamoyl phosphate (K(i) = 0.026 mM) and pyruvate carboxylation (K(i) = 2.5 mM). In conjunction with crystal structures of T882A R. etli PC mutant cocrystallized with phosphonoacetate and MgADP, computational docking studies suggest that phosphonoacetate could coordinate to one of two Mg(2+) metal centers in the BC domain active site. Based on the pH profiles, inhibition studies, and initial velocity patterns, possible mechanisms for the activation, regulation, and coordination of catalysis between the two spatially distinct active sites in pyruvate carboxylase from R. etli by acetyl-CoA and Mg(2+) are described. PMID:21958066

  3. Propionate stimulates pyruvate oxidation in the presence of acetate

    PubMed Central

    Purmal, Colin; Kucejova, Blanka; Sherry, A. Dean; Burgess, Shawn C.; Malloy, Craig. R.

    2014-01-01

    Flux through pyruvate dehydrogenase (PDH) in the heart may be reduced by various forms of injury to the myocardium, or by oxidation of alternative substrates in normal heart tissue. It is important to distinguish these two mechanisms because imaging of flux through PDH based on the appearance of hyperpolarized (HP) [13C]bicarbonate derived from HP [1-13C]pyruvate has been proposed as a method for identifying viable myocardium. The efficacy of propionate for increasing PDH flux in the setting of PDH inhibition by an alternative substrate was studied using isotopomer analysis paired with exams using HP [1-13C]pyruvate. Hearts from C57/bl6 mice were supplied with acetate (2 mM) and glucose (8.25 mM). 13C NMR spectra were acquired in a cryogenically cooled probe at 14.1 Tesla. After addition of hyperpolarized [1-13C]pyruvate, 13C NMR signals from lactate, alanine, malate, and aspartate were easily detected, in addition to small signals from bicarbonate and CO2. The addition of propionate (2 mM) increased appearance of HP [13C]bicarbonate >30-fold without change in O2 consumption. Isotopomer analysis of extracts from the freeze-clamped hearts indicated that acetate was the preferred substrate for energy production, glucose contribution to energy production was minimal, and anaplerosis was stimulated in the presence of propionate. Under conditions where production of acetyl-CoA is dominated by the availability of an alternative substrate, acetate, propionate markedly stimulated PDH flux as detected by the appearance of hyperpolarized [13C]bicarbonate from metabolism of hyperpolarized [1-13C]pyruvate. PMID:25320331

  4. In vivo13C spectroscopy in the rat brain using hyperpolarized [1- 13C]pyruvate and [2- 13C]pyruvate

    NASA Astrophysics Data System (ADS)

    Marjańska, Małgorzata; Iltis, Isabelle; Shestov, Alexander A.; Deelchand, Dinesh K.; Nelson, Christopher; Uğurbil, Kâmil; Henry, Pierre-Gilles

    2010-10-01

    The low sensitivity of 13C spectroscopy can be enhanced using dynamic nuclear polarization. Detection of hyperpolarized [1- 13C]pyruvate and its metabolic products has been reported in kidney, liver, and muscle. In this work, the feasibility of measuring 13C signals of hyperpolarized 13C metabolic products in the rat brain in vivo following the injection of hyperpolarized [1- 13C]pyruvate and [2- 13C]pyruvate is investigated. Injection of [2- 13C]pyruvate led to the detection of [2- 13C]lactate, but no other downstream metabolites such as TCA cycle intermediates were detected. Injection of [1- 13C]pyruvate enabled the detection of both [1- 13C]lactate and [ 13C]bicarbonate. A metabolic model was used to fit the hyperpolarized 13C time courses obtained during infusion of [1- 13C]pyruvate and to determine the values of VPDH and VLDH.

  5. Molecular and biochemical characterization of bifunctional pyruvate decarboxylases and pyruvate ferredoxin oxidoreductases from Thermotoga maritima and Thermotoga hypogea.

    PubMed

    Eram, Mohammad S; Wong, Alton; Oduaran, Erica; Ma, Kesen

    2015-12-01

    Hyperthermophilic bacteria Thermotoga maritima and Thermotoga hypogea produce ethanol as a metabolic end product, which is resulted from acetaldehyde reduction catalysed by an alcohol dehydrogenase (ADH). However, the enzyme that is involved in the production of acetaldehyde from pyruvate is not well characterized. An oxygen sensitive and coenzyme A-dependent pyruvate decarboxylase (PDC) activity was found to be present in cell free extracts of T. maritima and T. hypogea. Both enzymes were purified and found to have pyruvate ferredoxin oxidoreductase (POR) activity, indicating their bifunctionality. Both PDC and POR activities from each of the purified enzymes were characterized in regards to their optimal assay conditions including pH dependency, oxygen sensitivity, thermal stability, temperature dependency and kinetic parameters. The close relatedness of the PORs that was shown by sequence analysis could be an indication of the presence of such bifunctionality in other hyperthermophilic bacteria. This is the first report of a bifunctional PDC/POR enzyme in hyperthermophilic bacteria. The PDC and the previously reported ADHs are most likely the key enzymes catalysing the production of ethanol from pyruvate in bacterial hyperthermophiles. PMID:26032540

  6. Metabolism of hyperpolarized [1-(13) C]pyruvate through alternate pathways in rat liver.

    PubMed

    Jin, Eunsook S; Moreno, Karlos X; Wang, Jian-Xiong; Fidelino, Leila; Merritt, Matthew E; Sherry, A Dean; Malloy, Craig R

    2016-04-01

    The source of hyperpolarized (HP) [(13) C]bicarbonate in the liver during metabolism of HP [1-(13) C]pyruvate is uncertain and likely changes with physiology. Multiple processes including decarboxylation through pyruvate dehydrogenase or pyruvate carboxylase followed by subsequent decarboxylation via phosphoenolpyruvate carboxykinase (gluconeogenesis) could play a role. Here we tested which metabolic fate of pyruvate contributed to the appearance of HP [(13) C]bicarbonate during metabolism of HP [1-(13) C]pyruvate by the liver in rats after 21 h of fasting compared to rats with free access to food. The (13) C NMR of HP [(13) C]bicarbonate was observed in the liver of fed rats, but not in fasted rats where pyruvate carboxylation and gluconeogenesis was active. To further explore the relative fluxes through pyruvate carboxylase versus pyruvate dehydrogenase in the liver under typical conditions of hyperpolarization studies, separate parallel experiments were performed with rats given non-hyperpolarized [2,3-(13) C]pyruvate. (13) C NMR analysis of glutamate isolated from the liver of rats revealed that flux from injected pyruvate through pyruvate dehydrogenase was dominant under fed conditions whereas flux through pyruvate carboxylase dominated under fasted conditions. The NMR signal of HP [(13) C]bicarbonate does not parallel pyruvate carboxylase activity followed by subsequent decarboxylation reaction leading to glucose production. In the liver of healthy well-fed rats, the appearance of HP [(13) C]bicarbonate exclusively reflects decarboxylation of HP [1-(13) C]pyruvate via pyruvate dehydrogenase. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd. PMID:26836042

  7. Molecular evolution of bacterial indoleamine 2,3-dioxygenase.

    PubMed

    Yuasa, Hajime J; Ushigoe, Akiko; Ball, Helen J

    2011-10-01

    Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that catalyze the first step in L-Trp catabolism via the kynurenine pathway. In mammals, TDO is mainly expressed in the liver and primarily supplies nicotinamide adenine dinucleotide (NAD(+)). TDO is widely distributed from mammals to bacteria. Active IDO enzymes have been reported only in vertebrates and fungi. In mammals, IDO activity plays a significant role in the immune system while in fungal species, IDO is constitutively expressed and supplies NAD(+), like mammalian TDO. A search of genomic databases reveals that some bacterial species also have a putative IDO gene. A phylogenetic analysis clustered bacterial IDOs into two groups, group I or group II bacterial IDOs. The catalytic efficiencies of group I bacterial IDOs were very low and they are suspected not to contribute significantly to L-Trp metabolism. The bacterial species bearing the group I bacterial IDO are scattered across a few phyla and no phylogenetically close relationship is observed between them. This suggests that the group I bacterial IDOs might be acquired by horizontal gene transmission that occurred in each lineage independently. In contrast, group II bacterial IDOs showed rather high catalytic efficiency. Particularly, the enzymatic characteristics (K(m), V(max) and inhibitor selectivity) of the Gemmatimonas aurantiaca IDO are comparable to those of mammalian IDO1, although comparison of the IDO sequences does not suggest a close evolutionary relationship. In several bacteria, TDO and the kynureninase gene (kynU) are clustered on their chromosome suggesting that these genes could be transcribed in an operon. Interestingly, G. aurantiaca has no TDO, and the IDO is clustered with kynU on its chromosome. Although the G. aurantiaca also has NadA and NadB to synthesize a quinolinic acid (a precursor of NAD(+)) via the aspartate pathway, the high activity of the G. aurantiaca IDO flanking

  8. Pro-inflammatory Macrophages Sustain Pyruvate Oxidation through Pyruvate Dehydrogenase for the Synthesis of Itaconate and to Enable Cytokine Expression.

    PubMed

    Meiser, Johannes; Krämer, Lisa; Sapcariu, Sean C; Battello, Nadia; Ghelfi, Jenny; D'Herouel, Aymeric Fouquier; Skupin, Alexander; Hiller, Karsten

    2016-02-19

    Upon stimulation with Th1 cytokines or bacterial lipopolysaccharides, resting macrophages shift their phenotype toward a pro-inflammatory state as part of the innate immune response. LPS-activated macrophages undergo profound metabolic changes to adapt to these new physiological requirements. One key step to mediate this metabolic adaptation is the stabilization of HIF1α, which leads to increased glycolysis and lactate release, as well as decreased oxygen consumption. HIF1 abundance can result in the induction of the gene encoding pyruvate dehydrogenase kinase 1 (PDK1), which inhibits pyruvate dehydrogenase (PDH) via phosphorylation. Therefore, it has been speculated that pyruvate oxidation through PDH is decreased in pro-inflammatory macrophages. However, to answer this open question, an in-depth analysis of this metabolic branching point was so far lacking. In this work, we applied stable isotope-assisted metabolomics techniques and demonstrate that pyruvate oxidation is maintained in mature pro-inflammatory macrophages. Glucose-derived pyruvate is oxidized via PDH to generate citrate in the mitochondria. Citrate is used for the synthesis of the antimicrobial metabolite itaconate and for lipogenesis. An increased demand for these metabolites decreases citrate oxidation through the tricarboxylic acid cycle, whereas increased glutamine uptake serves to replenish the TCA cycle. Furthermore, we found that the PDH flux is maintained by unchanged PDK1 abundance, despite the presence of HIF1. By pharmacological intervention, we demonstrate that the PDH flux is an important node for M(LPS) macrophage activation. Therefore, PDH represents a metabolic intervention point that might become a research target for translational medicine to treat chronic inflammatory diseases. PMID:26679997

  9. Pro-inflammatory Macrophages Sustain Pyruvate Oxidation through Pyruvate Dehydrogenase for the Synthesis of Itaconate and to Enable Cytokine Expression*

    PubMed Central

    Meiser, Johannes; Krämer, Lisa; Sapcariu, Sean C.; Battello, Nadia; Ghelfi, Jenny; D'Herouel, Aymeric Fouquier; Skupin, Alexander; Hiller, Karsten

    2016-01-01

    Upon stimulation with Th1 cytokines or bacterial lipopolysaccharides, resting macrophages shift their phenotype toward a pro-inflammatory state as part of the innate immune response. LPS-activated macrophages undergo profound metabolic changes to adapt to these new physiological requirements. One key step to mediate this metabolic adaptation is the stabilization of HIF1α, which leads to increased glycolysis and lactate release, as well as decreased oxygen consumption. HIF1 abundance can result in the induction of the gene encoding pyruvate dehydrogenase kinase 1 (PDK1), which inhibits pyruvate dehydrogenase (PDH) via phosphorylation. Therefore, it has been speculated that pyruvate oxidation through PDH is decreased in pro-inflammatory macrophages. However, to answer this open question, an in-depth analysis of this metabolic branching point was so far lacking. In this work, we applied stable isotope-assisted metabolomics techniques and demonstrate that pyruvate oxidation is maintained in mature pro-inflammatory macrophages. Glucose-derived pyruvate is oxidized via PDH to generate citrate in the mitochondria. Citrate is used for the synthesis of the antimicrobial metabolite itaconate and for lipogenesis. An increased demand for these metabolites decreases citrate oxidation through the tricarboxylic acid cycle, whereas increased glutamine uptake serves to replenish the TCA cycle. Furthermore, we found that the PDH flux is maintained by unchanged PDK1 abundance, despite the presence of HIF1. By pharmacological intervention, we demonstrate that the PDH flux is an important node for M(LPS) macrophage activation. Therefore, PDH represents a metabolic intervention point that might become a research target for translational medicine to treat chronic inflammatory diseases. PMID:26679997

  10. Mechanism and Substrate Recognition of 2-Hydroxyethylphosphonate Dioxygenase

    SciTech Connect

    Peck, Spencer C.; Cooke, Heather A.; Cicchillo, Robert M.; Malova, Petra; Hammerschmidt, Friedrich; Nair, Satish K.; van der Donk, Wilfred A.

    2011-09-20

    HEPD belongs to the superfamily of 2-His-1-carboxylate non-heme iron-dependent dioxygenases. It converts 2-hydroxyethylphosphonate (2-HEP) to hydroxymethylphosphonate (HMP) and formate. Previously postulated mechanisms for the reaction catalyzed by HEPD cannot explain its conversion of 1-HEP to acetylphosphate. Alternative mechanisms that involve either phosphite or methylphosphonate as intermediates, which potentially explain all experimental studies including isotope labeling experiments and use of substrate analogues, were investigated. The results of these studies reveal that these alternative mechanisms are not correct. Site-directed mutagenesis studies of Lys16, Arg90, and Tyr98 support roles of these residues in binding of 2-HEP. Mutation of Lys16 to Ala resulted in an inactive enzyme, whereas mutation of Arg90 to Ala or Tyr98 to Phe greatly decreased k{sub cat}/K{sub m,2-HEP}. Furthermore, the latter mutants could not be saturated in O{sub 2}. These results suggest that proper binding of 2-HEP is important for O{sub 2} activation and that the enzyme uses a compulsory binding order with 2-HEP binding before O{sub 2}. The Y98F mutant produces methylphosphonate as a minor side product providing indirect support for the proposal that the last step during catalysis involves a ferric hydroxide reacting with a methylphosphonate radical.

  11. p-Hydroxyphenylpyruvate dioxygenase inhibitor-resistant plants.

    PubMed

    Matringe, Michel; Sailland, Alain; Pelissier, Bernard; Rolland, Anne; Zink, Olivier

    2005-03-01

    The enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the formation of homogentisic acid, the aromatic precursor of plastoquinone and vitamin E. HPPD is the specific target of several herbicide families: isoxazoles, triketones and pyroxazoles. Its inhibition results in the depletion of the plant plastoquinone and vitamin E pools, leading to bleaching symptoms. These herbicides are very potent for the selective pre- and in some cases post-emergence control of a wide range of broadleaf and grass weeds in maize and rice. Their herbicidal potential raised interest in the development of highly resistant transgenic crops. This goal was first achieved by over-expression of a bacterial HPPD in crop plants, and an increased level of resistance was obtained by using a mutant enzyme. A second strategy based on bypassing HPPD in the production of homogentisate was then developed. Recently, a third strategy of resistance based on the increase of p-hydroxyphenylpyruvate substrate flux has been developed. This was achieved by the introduction of the yeast prephenate dehydrogenase gene (PDH) into transgenic plants already overexpressing HPPD. In addition to a high level of herbicide resistance, a massive accumulation of vitamin E, mainly tocotrienols, was observed in leaves of the transgenic HPPD-PDH plants. PMID:15633191

  12. Antitumour agents as inhibitors of tryptophan 2,3-dioxygenase

    SciTech Connect

    Pantouris, Georgios; Mowat, Christopher G.

    2014-01-03

    Highlights: •∼2800 National Cancer Institute USA compounds have been screened as potential inhibitors of TDO and/or IDO. •Seven compounds with anti-tumour properties have been identified as potent inhibitors. •NSC 36398 (taxifolin, dihydroquercetin) is selective for TDO with a K{sub i} of 16 M. •This may help further our understanding of the role of TDO in cancer. -- Abstract: The involvement of tryptophan 2,3-dioxygenase (TDO) in cancer biology has recently been described, with the enzyme playing an immunomodulatory role, suppressing antitumour immune responses and promoting tumour cell survival and proliferation. This finding reinforces the need for specific inhibitors of TDO that may potentially be developed for therapeutic use. In this work we have screened ∼2800 compounds from the library of the National Cancer Institute USA and identified seven potent inhibitors of TDO with inhibition constants in the nanomolar or low micromolar range. All seven have antitumour properties, killing various cancer cell lines. For comparison, the inhibition potencies of these compounds were tested against IDO and their inhibition constants are reported. Interestingly, this work reveals that NSC 36398 (dihydroquercetin, taxifolin), with an in vitro inhibition constant of ∼16 μM, is the first TDO-selective inhibitor reported.

  13. Isoenzymes of Pyruvate Kinase in Etioplasts and Chloroplasts 1

    PubMed Central

    Ireland, Robert J.; Deluca, Vincenzo; Dennis, David T.

    1979-01-01

    Isoenzymes of pyruvate kinase from green leaves of castor bean and etiolated leaves of pea plants have been separated by ion filtration chromatography. One of the isoenzymes is localized in the plastid, whereas the other is in the cytosol. The cytosolic enzyme has a pH optimum from pH 7 to pH 9, and is able to utilize nucleotides other than ADP as the phosphoryl acceptor. The plastid enzyme has a much sharper optimum at pH 8, and is less efficient at using alternative nucleotides. The plastic pyruvate kinase, unlike the cytosolic enzyme, requires the presence of dithiothreitol or 2-mercaptoethanol during isolation and storage to stabilize the activity. PMID:16660835

  14. Ethyl pyruvate protects against sepsis by regulating energy metabolism

    PubMed Central

    Kang, Hongjun; Mao, Zhi; Zhao, Yan; Yin, Ting; Song, Qing; Pan, Liang; Hu, Xin; Hu, Jie; Zhou, Feihu

    2016-01-01

    Background Ethyl pyruvate (EP) is a derivative of pyruvic acid that has been demonstrated to be a potential scavenger of reactive oxygen species as well as an anti-inflammatory agent. In this study, we investigated the protective effects of EP and its role in regulating the energy metabolism in the livers of cecal-ligation-and-puncture-induced septic mice. Methods The animals were treated intraperitoneally with 0.2 mL of Ringer’s lactate solution or an equivalent volume of Ringer’s lactate solution containing EP immediately after cecal ligation and puncture. Each mouse in the Sham group was only subjected to a laparotomy. At 30-, 60-, 180-, and 360-minute time points, we measured the histopathological alterations of the intestines, and the plasma levels of interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor-α, and the total antioxidative capacity, malondialdehyde content, and lactate and lactate/pyruvate levels in livers. Furthermore, we detected the levels of adenosine triphosphate, total adenylate, and energy charge in the livers. Results Our results demonstrated that the administration of EP significantly improved the survival rate and reduced intestinal histological alterations. EP inhibited the plasma levels of IL-1β, IL-6, and tumor necrosis factor-α and increased the IL-10 level. EP significantly inhibited the elevation of the malondialdehyde, lactate, and lactate/pyruvate levels and enhanced the total antioxidative capacity levels in the liver tissues. The downregulation of the adenosine triphosphate, total adenylate, and energy charge levels in the liver tissues was reversed in the septic mice treated with EP. Conclusion The results suggest that EP administration effectively modulates the energy metabolism, which may be an important component in treatment of sepsis. PMID:26966369

  15. Siblings with severe pyruvate kinase deficiency and a complex genotype.

    PubMed

    Christensen, Robert D; Yaish, Hassan M; Nussenzveig, Roberto H; Agarwal, Archana M

    2016-09-01

    Siblings presented as neonates with severe jaundice and transfusion-dependent hemolytic anemia. Next-generation sequencing revealed both to have three heterozygous mutations in the gene encoding erythrocyte pyruvate kinase (PKLR), plus a heterozygous splice mutation in the beta-spectrin gene (SPTB). In addition, both have a different 5th mutation in a gene encoding other erythrocyte membrane proteins. The asymptomatic parents and all three asymptomatic siblings have different sets of these mutations. © 2016 Wiley Periodicals, Inc. PMID:27354418

  16. Development of catechol 2,3-dioxygenase-specific primers for monitoring bioremediation by competitive quantitative PCR

    SciTech Connect

    Mesarch, M.B.; Nakatsu, C.H.; Nies, L.

    2000-02-01

    Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primes that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 10{sup 2} to 10{sup 3} gene copies, which was lowered to 10{sup 0} to 10{sup 1} gene copies of hybridization. Using the dioxygenase-specific primers, an increase in catechol 2,3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR and a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2,3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivation-based molecular techniques for detecting and enumerating microbial pollutant-biodegrading genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.

  17. Impact of Perturbed Pyruvate Metabolism on Adipocyte Triglyceride Accumulation

    PubMed Central

    Si, Yaguang; Shi, Hai; Lee, Kyongbum

    2009-01-01

    This study aimed to test the hypothesis that adipocyte TG accumulation could be altered by specifically perturbing pyruvate metabolism. We treated cultured 3T3-L1 adipocytes with chemical inhibitors of lactate dehydrogenase (LDH) and pyruvate carboxylase (PC), and characterized their global effects on intermediary metabolism using metabolic flux and isotopomer analysis. Inhibiting the enzymes over several days did not alter the adipocyte differentiation program as assessed by the expression levels of peroxisome proliferator-activated receptor-γ and glycerol-3-phosphate dehydrogenase. The main metabolic effects were to up-regulate intracellular lipolysis and decrease TG accumulation. Inhibiting PC also up-regulated glycolysis. Flux estimates indicated that the reduction in TG was due to decreased de novo fatty acid synthesis. Exogenous addition of free fatty acids dose-dependently increased the cellular TG level in the inhibitor-treated adipocytes, but not in untreated control cells. The results of this study support our hypothesis regarding the critical role of pyruvate reactions in TG synthesis. PMID:19683593

  18. Phosphoenolpyruvate carboxykinase and pyruvate carboxylase in developing rat liver

    PubMed Central

    Ballard, F. J.; Hanson, R. W.

    1967-01-01

    1. Phosphoenolpyruvate carboxykinase and pyruvate carboxylase were measured in foetal, newborn and adult rat liver extracts by a radiochemical assay involving the fixation of [14C]bicarbonate. 2. Pyruvate-carboxylase activity in both foetal and adult liver occurs mainly in mitochondrial and nuclear fractions, with about 10% of the activity in the cytoplasm. 3. Similar studies of the intracellular distribution of phosphoenolpyruvate carboxykinase show that more than 90% of the activity is in the cytoplasm. However, in the 17-day foetal liver about 90% of the activity is in mitochondria and nuclei. 4. Pyruvate-carboxylase activity in both particulate and soluble fractions is very low in the 17-day foetal liver and increases to near adult levels before birth. 5. Phosphoenolpyruvate-carboxykinase activity in the soluble cell fraction increases 25-fold in the first 2 days after birth. This same enzyme in the mitochondria has considerable activity in the foetal and adult liver and is lower in the newborn. 6. Kinetic and other studies on the properties of phosphoenolpyruvate carboxykinase have shown no differences between the soluble and mitochondrial enzymes. 7. It is suggested that the appearance of the soluble phosphoenolpyruvate carboxykinase at birth initiates the rapid increase in overall gluconeogenesis at this stage. PMID:6049928

  19. Biosynthesis of pyruvic acid from glucose by Blastobotrys adeninivorans.

    PubMed

    Kamzolova, Svetlana V; Morgunov, Igor G

    2016-09-01

    The ability of taxonomically different yeasts to synthesize pyruvic acid (PA) from glucose was studied. The study showed that many yeasts are able to produce PA from glucose under the condition of growth limitation by thiamine. This ability was found in the yeast Blastobotrys adeninivorans for the first time. The production (oversynthesis) of PA in this yeast can be explained by disturbance in the function of thiamine-dependent pyruvate dehydrogenase. Namely, the partial inhibition of this enzyme brings about the excretion of PA from the yeast cells. Due to incomplete inhibition of pyruvate dehydrogenase, the formation of acetyl-CoA continues, although at a lower level, maintaining the synthesis of α-ketoglutaric acid (KGA) in the tricarboxylic acid (TCA) cycle. KGA is no longer oxidized in the TCA cycle, because thiamine limitation inhibits α-ketoglutarate dehydrogenase. As a result, KGA is excreted from the yeast cells as a byproduct of PA oversynthesis. Furthermore, the increased level of KGA in the yeast cells inhibits NAD-dependent isocitrate dehydrogenase in the TCA cycle and enhances the production and excretion of citric acid, another byproduct of PA oversynthesis. During cultivation in a fermentor, the strain Blastobotrys adeninivorans VKM Y-2677 produced 43.2 g l(-1) PA from glucose with a product yield (YPA) of 0.77 g PA/g glucose. The proportion of PA to byproducts was 18:1 for KGA and 8:1 for citric acid. PMID:27221290

  20. Identification of the Elusive Pyruvate Reductase of Chlamydomonas reinhardtii Chloroplasts.

    PubMed

    Burgess, Steven J; Taha, Hussein; Yeoman, Justin A; Iamshanova, Oksana; Chan, Kher Xing; Boehm, Marko; Behrends, Volker; Bundy, Jacob G; Bialek, Wojciech; Murray, James W; Nixon, Peter J

    2016-01-01

    Under anoxic conditions the green alga Chlamydomonas reinhardtii activates various fermentation pathways leading to the creation of formate, acetate, ethanol and small amounts of other metabolites including d-lactate and hydrogen. Progress has been made in identifying the enzymes involved in these pathways and their subcellular locations; however, the identity of the enzyme involved in reducing pyruvate to d-lactate has remained unclear. Based on sequence comparisons, enzyme activity measurements, X-ray crystallography, biochemical fractionation and analysis of knock-down mutants, we conclude that pyruvate reduction in the chloroplast is catalyzed by a tetrameric NAD(+)-dependent d-lactate dehydrogenase encoded by Cre07.g324550. Its expression during aerobic growth supports a possible function as a 'lactate valve' for the export of lactate to the mitochondrion for oxidation by cytochrome-dependent d-lactate dehydrogenases and by glycolate dehydrogenase. We also present a revised spatial model of fermentation based on our immunochemical detection of the likely pyruvate decarboxylase, PDC3, in the cytoplasm. PMID:26574578

  1. Pyruvate dehydrogenase deficiency: molecular basis for intrafamilial heterogeneity.

    PubMed

    Fujii, T; Van Coster, R N; Old, S E; Medori, R; Winter, S; Gubits, R M; Matthews, P M; Brown, R M; Brown, G K; Dahl, H H

    1994-07-01

    Two half-brothers and their mother had symptomatic pyruvate dehydrogenase complex deficiency. The infants had severe congenital lactic acidosis, seizures, and apneic spells and died at the ages 3 and 4 months. The mother was less symptomatic with mental retardation, truncal ataxia, and dysarthria. The residual pyruvate dehydrogenase activities in cultured skin fibroblasts from the 2 infants and their mother were 7, 15, and 10% of control values. Immunoblot analysis showed negligible amounts of E1 alpha and E1 beta subunits of the complex. Northern blot analysis for the E1 alpha subunit showed normal results. In the 2 sons, complementary DNA sequence analysis revealed a cytosine to thymine mutation in exon 4, resulting in a change of arginine 127 to tryptophan in the E1 alpha subunit. Restriction enzyme analysis of the polymerase chain reaction product representing exon 4 of the E1 alpha gene revealed that the mother was a heterozygotes. Complementary DNA restriction analysis and methylation analysis of the X chromosome DXS255 loci revealed skewed activation of the mutant allele, consistent with the deficient pyruvate dehydrogenase activity in the mother's fibroblasts. The milder maternal phenotype is consistent with variable X-inactivation patterns in different organs of female heterozygotes. PMID:8024267

  2. Identification of the Elusive Pyruvate Reductase of Chlamydomonas reinhardtii Chloroplasts

    PubMed Central

    Burgess, Steven J.; Taha, Hussein; Yeoman, Justin A.; Iamshanova, Oksana; Chan, Kher Xing; Boehm, Marko; Behrends, Volker; Bundy, Jacob G.; Bialek, Wojciech; Murray, James W.; Nixon, Peter J.

    2016-01-01

    Under anoxic conditions the green alga Chlamydomonas reinhardtii activates various fermentation pathways leading to the creation of formate, acetate, ethanol and small amounts of other metabolites including d-lactate and hydrogen. Progress has been made in identifying the enzymes involved in these pathways and their subcellular locations; however, the identity of the enzyme involved in reducing pyruvate to d-lactate has remained unclear. Based on sequence comparisons, enzyme activity measurements, X-ray crystallography, biochemical fractionation and analysis of knock-down mutants, we conclude that pyruvate reduction in the chloroplast is catalyzed by a tetrameric NAD+-dependent d-lactate dehydrogenase encoded by Cre07.g324550. Its expression during aerobic growth supports a possible function as a ‘lactate valve’ for the export of lactate to the mitochondrion for oxidation by cytochrome-dependent d-lactate dehydrogenases and by glycolate dehydrogenase. We also present a revised spatial model of fermentation based on our immunochemical detection of the likely pyruvate decarboxylase, PDC3, in the cytoplasm. PMID:26574578

  3. Spinach pyruvate kinase isoforms: partial purification and regulatory properties

    SciTech Connect

    Baysdorfer, C.; Bassham, J.A.

    1984-02-01

    Pyruvate kinase from spinach (Spinacea oleracea L.) leaves consists of two isoforms, separable by blue agarose chromatography. Both isoforms share similar pH profiles and substrate and alternate nucleotide K/sub m/ values. In addition, both isoforms are inhibited by oxalate and ATP and activated by AMP. The isoforms differ in their response to three key metabolites; citrate, aspartate, and glutamate. The first isoform is similar to previously reported plant pyruvate kinases in its sensitivity to citrate inhibition. The K/sub i/ for this inhibition is 1.2 millimolar citrate. The second isoform is not affected by citrate but is regulated by aspartate and glutamate. Aspartate is an activator with a K/sub a/ of 0.05 millimolar, and glutamate is an inhibitor with a K/sub i/ of 0.68 millimolar. A pyruvate kinase with these properties has not been previously reported. Based on these considerations, the authors suggest that the activity of the first isoform is regulated by respiratory metabolism. The second isoform, in contrast, may be regulated by the demand for carbon skeletons for use in ammonia assimilation.

  4. [Different properties of pyruvate kinase from rabbit and hare muscles].

    PubMed

    Strumilo, S; Tylicki, A

    2015-01-01

    Some catalytic and kinetic properties of pyruvate kinase (PK, EC 2.7.1.40) isolated from the heart and skeletal muscles of rabbits and hares with a 9-16-fold purification were studied. The initial specific activity of the enzyme in hare heart homogenates was 66% and in skeletal muscles 25% as high as in respective rabbit tissues. Temperature optimums and thermostability of PK from hare tissues were higher as compared with those in rabbits. From the comparison of K(M) (S0.5) values it follows that hare skeletal muscle PK exhibits a highest affinity to phosphoenol pyruvate, but lowest to ADP, as compared with rabbit skeletal muscle PK. Moreover, PK from both hare tissues exhibits a positive kinetic cooperativity (Hill coefficient > 1.35) of the phosphoenol pyruvate and ADP binding sites. In contrast to PK from rabbit tissues, the enzyme from the hare heart and muscles PK is presented by its allosteric isoform which might by advantageous under extreme conditions of the hare's habitation. PMID:26027383

  5. Developmental changes in the pyruvate kinase isozymes of coho salmon.

    PubMed

    Guderley, H E; Cardenas, J M

    1979-04-01

    Pyruvate kinase exists as two major isozymes in coho salmon. As in mammals and birds, one form is present in the early embryo and maintains a wide tissue distribution in adults. This salmonid type K shows anodal migration during electrophoresis at pH 7.5. The appearence of functional musculature in the developing embryos. In adult animals this second form is the only pyruvate kinase in muscle. Brain, kidney, liver and gill contain primarily the type K pyruvate kinase while heart contains both major forms along with three intermediate forms which presumably constitute a hybrid set. Since there is no additional isozyme restricted to gluconeogenic tissues, we conclude that a type L isozyme has not developed in these animals. The two major isozymes are immunologically distincy. Both forms are dubject to fructose 1,6-bisphosphate activation of phosphoenolpyruvate binding, but the magnitude of the effect is small. The affinities for phosphoenolpyruvate are similar, but salmon type K has hyperbolic saturation curves with this substrate and type M has sigmoidal saturation curves. While the immunological data indicates considerable divergence in structure, the kinetic parameters of the two forms have remained relatively similar. PMID:469475

  6. Loss of Mitochondrial Pyruvate Carrier 2 in the Liver Leads to Defects in Gluconeogenesis and Compensation via Pyruvate-Alanine Cycling.

    PubMed

    McCommis, Kyle S; Chen, Zhouji; Fu, Xiaorong; McDonald, William G; Colca, Jerry R; Kletzien, Rolf F; Burgess, Shawn C; Finck, Brian N

    2015-10-01

    Pyruvate transport across the inner mitochondrial membrane is believed to be a prerequisite for gluconeogenesis in hepatocytes, which is important for the maintenance of normoglycemia during prolonged food deprivation but also contributes to hyperglycemia in diabetes. To determine the requirement for mitochondrial pyruvate import in gluconeogenesis, mice with liver-specific deletion of mitochondrial pyruvate carrier 2 (LS-Mpc2(-/-)) were generated. Loss of MPC2 impaired, but did not completely abolish, hepatocyte conversion of labeled pyruvate to TCA cycle intermediates and glucose. Unbiased metabolomic analyses of livers from fasted LS-Mpc2(-/-) mice suggested that alterations in amino acid metabolism, including pyruvate-alanine cycling, might compensate for the loss of MPC2. Indeed, inhibition of pyruvate-alanine transamination further reduced mitochondrial pyruvate metabolism and glucose production by LS-Mpc2(-/-) hepatocytes. These data demonstrate an important role for MPC2 in controlling hepatic gluconeogenesis and illuminate a compensatory mechanism for circumventing a block in mitochondrial pyruvate import. PMID:26344101

  7. 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...

  8. Pyruvate ingestion for 7 days does not improve aerobic performance in well-trained individuals

    NASA Technical Reports Server (NTRS)

    Morrison, M. A.; Spriet, L. L.; Dyck, D. J.

    2000-01-01

    The purposes of the present studies were to test the hypotheses that lower dosages of oral pyruvate ingestion would increase blood pyruvate concentration and that the ingestion of a commonly recommended dosage of pyruvate (7 g) for 7 days would enhance performance during intense aerobic exercise in well-trained individuals. Nine recreationally active subjects (8 women, 1 man) consumed 7, 15, and 25 g of pyruvate and were monitored for a 4-h period to determine whether blood metabolites were altered. Pyruvate consumption failed to significantly elevate blood pyruvate, and it had no effect on indexes of carbohydrate (blood glucose, lactate) or lipid metabolism (blood glycerol, plasma free fatty acids). As a follow-up, we administered 7 g/day of either placebo or pyruvate, for a 1-wk period to seven, well-trained male cyclists (maximal oxygen consumption, 62.3 +/- 3.0 ml. kg(-1). min(-1)) in a randomized, double-blind, crossover trial. Subjects cycled at 74-80% of their maximal oxygen consumption until exhaustion. There was no difference in performance times between the two trials (placebo, 91 +/- 9 min; pyruvate, 88 +/- 8 min). Measured blood parameters (insulin, peptide C, glucose, lactate, glycerol, free fatty acids) were also unaffected. Our results indicate that oral pyruvate supplementation does not increase blood pyruvate content and does not enhance performance during intense exercise in well-trained cyclists.

  9. Characterization of a Novel Angular Dioxygenase from Fluorene-Degrading Sphingomonas sp. Strain LB126▿

    PubMed Central

    Schuler, Luc; Ní Chadhain, Sinéad M.; Jouanneau, Yves; Meyer, Christine; Zylstra, Gerben J.; Hols, Pascal; Agathos, Spiros N.

    2008-01-01

    In this study, the genes involved in the initial attack on fluorene by Sphingomonas sp. strain LB126 were investigated. The α and β subunits of a dioxygenase complex (FlnA1-FlnA2), showing 63 and 51% sequence identity, respectively, to the subunits of an angular dioxygenase from the gram-positive dibenzofuran degrader Terrabacter sp. strain DBF63, were identified. When overexpressed in Escherichia coli, FlnA1-FlnA2 was responsible for the angular oxidation of fluorene, 9-hydroxyfluorene, 9-fluorenone, dibenzofuran, and dibenzo-p-dioxin. Moreover, FlnA1-FlnA2 was able to oxidize polycyclic aromatic hydrocarbons and heteroaromatics, some of which were not oxidized by the dioxygenase from Terrabacter sp. strain DBF63. The quantification of resulting oxidation products showed that fluorene and phenanthrene were the preferred substrates of FlnA1-FlnA2. PMID:18156320

  10. Pyruvate modifies metabolic flux and nutrient sensing during extracorporeal membrane oxygenation in an immature swine model

    PubMed Central

    Ledee, Dolena R.; Kajimoto, Masaki; O'Kelly Priddy, Colleen M.; Olson, Aaron K.; Isern, Nancy; Robillard-Frayne, Isabelle; Des Rosiers, Christine

    2015-01-01

    Extracorporeal membrane oxygenation (ECMO) provides mechanical circulatory support for infants and children with postoperative cardiopulmonary failure. Nutritional support is mandatory during ECMO although specific actions for substrates on the heart have not been delineated. Prior work shows that enhancing pyruvate oxidation promotes successful weaning from ECMO. Accordingly, we tested the hypothesis that prolonged systemic pyruvate supplementation activates pyruvate oxidation in an immature swine model in vivo. Twelve male mixed-breed Yorkshire piglets (age 30–49 days) received systemic infusion of either normal saline (group C) or pyruvate (group P) during the final 6 h of 8 h of ECMO. Over the final hour, piglets received [2-13C] pyruvate, as a reference substrate for oxidation, and [13C6]-l-leucine, as an indicator for amino acid oxidation and protein synthesis. A significant increase in lactate and pyruvate concentrations occurred, along with an increase in the absolute concentration of the citric acid cycle intermediates. An increase in anaplerotic flux through pyruvate carboxylation in group P occurred compared with no change in pyruvate oxidation. Additionally, pyruvate promoted an increase in the phosphorylation state of several nutrient-sensitive enzymes, like AMP-activated protein kinase and acetyl CoA carboxylase, suggesting activation for fatty acid oxidation. Pyruvate also promoted O-GlcNAcylation through the hexosamine biosynthetic pathway. In conclusion, although prolonged pyruvate supplementation did not alter pyruvate oxidation, it did elicit changes in nutrient- and energy-sensitive pathways. Therefore, the observed results support the further study of pyruvate and its downstream effect on cardiac function. PMID:25910802

  11. A hydrogenosome with pyruvate formate-lyase: anaerobic chytrid fungi use an alternative route for pyruvate catabolism.

    PubMed

    Akhmanova, A; Voncken, F G; Hosea, K M; Harhangi, H; Keltjens, J T; op den Camp, H J; Vogels, G D; Hackstein, J H

    1999-06-01

    The chytrid fungi Piromyces sp. E2 and Neocallimastix sp. L2 are obligatory amitochondriate anaerobes that possess hydrogenosomes. Hydrogenosomes are highly specialized organelles engaged in anaerobic carbon metabolism; they generate molecular hydrogen and ATP. Here, we show for the first time that chytrid hydrogenosomes use pyruvate formate-lyase (PFL) and not pyruvate:ferredoxin oxidoreductase (PFO) for pyruvate catabolism, unlike all other hydrogenosomes studied to date. Chytrid PFLs are encoded by a multigene family and are abundantly expressed in Piromyces sp. E2 and Neocallimastix sp. L2. Western blotting after cellular fractionation, proteinase K protection assays and determinations of enzyme activities reveal that PFL is present in the hydrogenosomes of Piromyces sp. E2. The main route of the hydrogenosomal carbon metabolism involves PFL; the formation of equimolar amounts of formate and acetate by isolated hydrogenosomes excludes a significant contribution by PFO. Our data support the assumption that chytrid hydrogenosomes are unique and argue for a polyphyletic origin of these organelles. PMID:10361311

  12. Molecular Cloning, Nucleotide Sequence, and Expression of Genes Encoding a Polycyclic Aromatic Ring Dioxygenase from Mycobacterium sp. Strain PYR-1

    PubMed Central

    Khan, Ashraf A.; Wang, Rong-Fu; Cao, Wei-Wen; Doerge, Daniel R.; Wennerstrom, David; Cerniglia, Carl E.

    2001-01-01

    Mycobacterium sp. strain PYR-1 degrades high-molecular-weight polycyclic hydrocarbons (PAHs) primarily through the introduction of both atoms of molecular oxygen by a dioxygenase. To clone the dioxygenase genes involved in PAH degradation, two-dimensional (2D) gel electrophoresis of PAH-induced proteins from cultures of Mycobacterium sp. strain PYR-1 was used to detect proteins that increased after phenanthrene, dibenzothiophene, and pyrene exposure. Comparison of proteins from induced and uninduced cultures on 2D gels indicated that at least six major proteins were expressed (105, 81, 52, 50, 43, and 13 kDa). The N-terminal sequence of the 50-kDa protein was similar to those of other dioxygenases. A digoxigenin-labeled oligonucleotide probe designed from this protein sequence was used to screen dioxygenase-positive clones from a genomic library of Mycobacterium sp. strain PYR-1. Three clones, each containing a 5,288-bp DNA insert with three genes of the dioxygenase system, were obtained. The genes in the DNA insert, from the 5′ to the 3′ direction, were a dehydrogenase, the dioxygenase small (β)-subunit, and the dioxygenase large (α)-subunit genes, arranged in a sequence different from those of genes encoding other bacterial dioxygenase systems. Phylogenetic analysis showed that the large α subunit did not cluster with most of the known α-subunit sequences but rather with three newly described α subunits of dioxygenases from Rhodococcus spp. and Nocardioides spp. The genes from Mycobacterium sp. strain PYR-1 were subcloned and overexpressed in Escherichia coli with the pBAD/ThioFusion system. The functionality of the genes for PAH degradation was confirmed in a phagemid clone containing all three genes, as well as in plasmid subclones containing the two genes encoding the dioxygenase subunits. PMID:11472934

  13. Cloning and functional characterization of carotenoid cleavage dioxygenase 4 genes.

    PubMed

    Huang, Fong-Chin; Molnár, Péter; Schwab, Wilfried

    2009-01-01

    Although a number of plant carotenoid cleavage dioxygenase (CCD) genes have been functionally characterized in different plant species, little is known about the biochemical role and enzymatic activities of members of the subclass 4 (CCD4). To gain insight into their biological function, CCD4 genes were isolated from apple (Malus x domestica, MdCCD4), chrysanthemum (Chrysanthemum x morifolium, CmCCD4a), rose (Rosa x damascena, RdCCD4), and osmanthus (Osmanthus fragrans, OfCCD4), and were expressed, together with AtCCD4, in Escherichia coli. In vivo assays showed that CmCCD4a and MdCCD4 cleaved beta-carotene well to yield beta-ionone, while OfCCD4, RdCCD4, and AtCCD4 were almost inactive towards this substrate. No cleavage products were found for any of the five CCD4 genes when they were co-expressed in E. coli strains that accumulated cis-zeta-carotene and lycopene. In vitro assays, however, demonstrated the breakdown of 8'-apo-beta-caroten-8'-al by AtCCD4 and RdCCD4 to beta-ionone, while this apocarotenal was almost not degraded by OfCCD4, CmCCD4a, and MdCCD4. Sequence analysis of genomic clones of CCD4 genes revealed that RdCCD4, like AtCCD4, contains no intron, while MdCCD, OfCCD4, and CmCCD4a contain introns. These results indicate that plants produce at least two different forms of CCD4 proteins. Although CCD4 enzymes cleave their substrates at the same position (9,10 and 9',10'), they might have different biochemical functions as they accept different (apo)-carotenoid substrates, show various expression patterns, and are genomically differently organized. PMID:19436048

  14. Metal-Dependent Function of a Mammalian Acireductone Dioxygenase.

    PubMed

    Deshpande, Aditi R; Wagenpfeil, Karina; Pochapsky, Thomas C; Petsko, Gregory A; Ringe, Dagmar

    2016-03-01

    The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella oxytoca are the only known pair of naturally occurring metalloenzymes with distinct chemical and physical properties determined solely by the identity of the divalent transition metal ion (Fe(2+) or Ni(2+)) in the active site. We now show that this dual chemistry can also occur in mammals. ARD from Mus musculus (MmARD) was studied to relate the metal ion identity and three-dimensional structure to enzyme function. The iron-containing isozyme catalyzes the cleavage of 1,2-dihydroxy-3-keto-5-(thiomethyl)pent-1-ene (acireductone) by O2 to formate and the ketoacid precursor of methionine, which is the penultimate step in methionine salvage. The nickel-bound form of ARD catalyzes an off-pathway reaction resulting in formate, carbon monoxide (CO), and 3-(thiomethyl) propionate. Recombinant MmARD was expressed and purified to obtain a homogeneous enzyme with a single transition metal ion bound. The Fe(2+)-bound protein, which shows about 10-fold higher activity than that of others, catalyzes on-pathway chemistry, whereas the Ni(2+), Co(2+), or Mn(2+) forms exhibit off-pathway chemistry, as has been seen with ARD from Klebsiella. Thermal stability of the isozymes is strongly affected by the metal ion identity, with Ni(2+)-bound MmARD being the most stable, followed by Co(2+) and Fe(2+), and Mn(2+)-bound ARD being the least stable. Ni(2+)- and Co(2+)-bound MmARD were crystallized, and the structures of the two proteins found to be similar. Enzyme-ligand complexes provide insight into substrate binding, metal coordination, and the catalytic mechanism. PMID:26858196

  15. Superior Cardiac Function Via Anaplerotic Pyruvate in the Immature Swine Heart After Cardiopulmonary Bypass and Reperfusion

    SciTech Connect

    Olson, Aaron; Hyyti, Outi M.; Cohen, Gordon A.; Ning, Xue-Han; Sadilek, Martin; Isern, Nancy G.; Portman, Michael A.

    2008-12-01

    Pyruvate produces inotropic responses in the adult reperfused heart. Pyruvate oxidation and anaplerotic entry into the citric acid cycle (CAC) via carboxylation are linked to stimulation of contractile function. The goals of this study were to determine if these metabolic pathways operate and are maintained in the developing myocardium after reperfusion. Immature male swine (age 10-18 days) were subjected to cardiopulmonary bypass (CPB). Intracoronary infusion of [2]-13C-pyruvate (to achieve a final concentration of 8 mM) was given for 35 minutes starting either during weaning (Group I), after discontinuation (Group II) or without (Control) CPB. Hemodynamic data was collected. 13C NMR spectroscopy was used to determine the fraction of pyruvate entering the CAC via pyruvate carboxylation (PC) to total CAC entry (PC plus decarboxlyation via pyruvate dehydrogenase). Liquid chromatography-mass spectrometry was used to determine total glutamate enrichment.

  16. High levels of expression of the Iron-Sulfur Proteins Phthalate Dioxygenase and Phthalate Dioxygenase Reductase in Escherichia coli

    PubMed Central

    Jaganaman, Sunil; Pinto, Alex; Tarasev, Michael; Ballou, David P.

    2007-01-01

    Phthalate dioxygenase (PDO), a hexamer with one Rieske-type [2Fe-2S] and one Fe (II) - mononuclear center per monomer, and its reductase (PDR), which contains flavin mononucleotide and a plant-type ferredoxin [2Fe-2S] center, are expressed by Burkholderia cepacia at ∼30 mg of crude PDO and ∼1 mg of crude PDR per liter of cell culture when grown with phthalate as the main carbon source. A high level expression system in Escherichia coli was developed for PDO and PDR. Optimization relative to Escherichia coli cell line, growth parameters, time of induction, media composition, and iron-sulfur additives resulted in yields of about 1 g/L for PDO and about 0.2 g/L for PDR. Protein expression was correlated to the increase in pH of the cell culture and exhibited a pronounced (variable from 5 to 20 hours) lag after the induction. The specific activity of purified PDO did not depend on the pH of the cell culture when harvested. However, when the pH of the culture reached 8.5-9, a large fraction of the PDR that was expressed lacked its ferredoxin domain, presumably because of proteolysis. Termination of growth while the pH of the cell culture was < 8 decreased the fraction of proteolyzed enzyme, whereas yields of the unclipped PDR were only marginally lower. Overall, changes in pH of the cell culture were found to be an excellent indicator of the overall level of native protein expression. Its monitoring allowed the real time tracking of the protein expression and made it possible to tailor the expression times to achieve a combination of high quality and high yield of protein. PMID:17049880

  17. Dealing with the sulfur part of cysteine: four enzymatic steps degrade l-cysteine to pyruvate and thiosulfate in Arabidopsis mitochondria.

    PubMed

    Höfler, Saskia; Lorenz, Christin; Busch, Tjorven; Brinkkötter, Mascha; Tohge, Takayuki; Fernie, Alisdair R; Braun, Hans-Peter; Hildebrandt, Tatjana M

    2016-07-01

    Amino acid catabolism is essential for adjusting pool sizes of free amino acids and takes part in energy production as well as nutrient remobilization. The carbon skeletons are generally converted to precursors or intermediates of the tricarboxylic acid cycle. In the case of cysteine, the reduced sulfur derived from the thiol group also has to be oxidized in order to prevent accumulation to toxic concentrations. Here we present a mitochondrial sulfur catabolic pathway catalyzing the complete oxidation of l-cysteine to pyruvate and thiosulfate. After transamination to 3-mercaptopyruvate, the sulfhydryl group from l-cysteine is transferred to glutathione by sulfurtransferase 1 and oxidized to sulfite by the sulfur dioxygenase ETHE1. Sulfite is then converted to thiosulfate by addition of a second persulfide group by sulfurtransferase 1. This pathway is most relevant during early embryo development and for vegetative growth under light-limiting conditions. Characterization of a double mutant produced from Arabidopsis thaliana T-DNA insertion lines for ETHE1 and sulfurtransferase 1 revealed that an intermediate of the ETHE1 dependent pathway, most likely a persulfide, interferes with amino acid catabolism and induces early senescence. PMID:27105581

  18. The first step of the dioxygenation reaction carried out by tryptophan dioxygenase and indoleamine 2,3-dioxygenase as revealed by quantum mechanical/molecular mechanical studies

    PubMed Central

    Capece, Luciana; Lewis-Ballester, Ariel; Batabyal, Dipanwita; Di Russo, Natali; Estrin, Dario A.

    2015-01-01

    Tryptophan dioxygenase (TDO) and indole-amine 2,3-dioxygenase (IDO) are two heme-containing enzymes which catalyze the conversion of L-tryptophan to N-formylkynurenine (NFK). In mammals, TDO is mostly expressed in liver and is involved in controlling homeostatic serum tryptophan concentrations, whereas IDO is ubiquitous and is involved in modulating immune responses. Previous studies suggested that the first step of the dioxygenase reaction involves the deprotonation of the indoleamine group of the substrate by an evolutionarily conserved distal histidine residue in TDO and the heme-bound dioxygen in IDO. Here, we used classical molecular dynamics and hybrid quantum mechanical/molecular mechanical methods to evaluate the base-catalyzed mechanism. Our data suggest that the deprotonation of the indoleamine group of the substrate by either histidine in TDO or heme-bound dioxygen in IDO is not energetically favorable. Instead, the dioxygenase reaction can be initiated by a direct attack of heme-bound dioxygen on the C2=C3 bond of the indole ring, leading to a protein-stabilized 2,3-alkylperoxide transition state and a ferryl epoxide intermediate, which subsequently recombine to generate NFK. The novel sequential two-step oxygen addition mechanism is fully supported by our recent resonance Raman data that allowed identification of the ferryl intermediate (Lewis-Ballester et al. in Proc Natl Acad Sci USA 106:17371–17376, 2009). The results reveal the subtle differences between the TDO and IDO reactions and highlight the importance of protein matrix in modulating stereoelectronic factors for oxygen activation and the stabilization of both transition and intermediate states. PMID:20361220

  19. The pyruvate dehydrogenase complex of Corynebacterium glutamicum: an attractive target for metabolic engineering.

    PubMed

    Eikmanns, Bernhard J; Blombach, Bastian

    2014-12-20

    The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative thiamine pyrophosphate-dependent decarboxylation of pyruvate to acetyl-CoA and CO2. Since pyruvate is a key metabolite of the central metabolism and also the precursor for several relevant biotechnological products, metabolic engineering of this multienzyme complex is a promising strategy to improve microbial production processes. This review summarizes the current knowledge and achievements on metabolic engineering approaches to tailor the PDHC of Corynebacterium glutamicum for the bio-based production of l-valine, 2-ketosiovalerate, pyruvate, succinate and isobutanol and to improve l-lysine production. PMID:24486441

  20. 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

  1. Molecular cloning, expression and characterization of pyridoxamine–pyruvate aminotransferase

    PubMed Central

    Yoshikane, Yu; Yokochi, Nana; Ohnishi, Kouhei; Hayashi, Hideyuki; Yagi, Toshiharu

    2006-01-01

    Pyridoxamine–pyruvate aminotransferase is a PLP (pyridoxal 5′-phosphate) (a coenzyme form of vitamin B6)-independent aminotransferase which catalyses a reversible transamination reaction between pyridoxamine and pyruvate to form pyridoxal and L-alanine. The gene encoding the enzyme has been identified, cloned and overexpressed for the first time. The mlr6806 gene on the chromosome of a symbiotic nitrogen-fixing bacterium, Mesorhizobium loti, encoded the enzyme, which consists of 393 amino acid residues. The primary sequence was identical with those of archaeal aspartate aminotransferase and rat serine–pyruvate aminotransferase, which are PLP-dependent aminotransferases. The results of fold-type analysis and the consensus amino acid residues found around the active-site lysine residue identified in the present study showed that the enzyme could be classified into class V aminotransferases of fold type I or the AT IV subfamily of the α family of the PLP-dependent enzymes. Analyses of the absorption and CD spectra of the wild-type and point-mutated enzymes showed that Lys197 was essential for the enzyme activity, and was the active-site lysine residue that corresponded to that found in the PLP-dependent aminotransferases, as had been suggested previously [Hodsdon, Kolb, Snell and Cole (1978) Biochem. J. 169, 429–432]. The Kd value for pyridoxal determined by means of CD was 100-fold lower than the Km value for it, suggesting that Schiff base formation between pyridoxal and the active-site lysine residue is partially rate determining in the catalysis of pyridoxal. The active-site structure and evolutionary aspects of the enzyme are discussed. PMID:16545075

  2. Regulation of Blood Glucose by Hypothalamic Pyruvate Metabolism

    NASA Astrophysics Data System (ADS)

    Lam, Tony K. T.; Gutierrez-Juarez, Roger; Pocai, Alessandro; Rossetti, Luciano

    2005-08-01

    The brain keenly depends on glucose for energy, and mammalians have redundant systems to control glucose production. An increase in circulating glucose inhibits glucose production in the liver, but this negative feedback is impaired in type 2 diabetes. Here we report that a primary increase in hypothalamic glucose levels lowers blood glucose through inhibition of glucose production in rats. The effect of glucose requires its conversion to lactate followed by stimulation of pyruvate metabolism, which leads to activation of adenosine triphosphate (ATP)-sensitive potassium channels. Thus, interventions designed to enhance the hypothalamic sensing of glucose may improve glucose homeostasis in diabetes.

  3. The Metabolic and Developmental Roles of Carotenoid Cleavage Dioxygenase 4 from Potato (Solanum tuberosum L)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The factors that regulate storage organ carotenoid content remain to be fully elucidated despite the nutritional and economic importance of this class of compound. Recent findings suggest that carotenoid pool size is determined at least in part, by the activity of carotenoid cleavage dioxygenases. T...

  4. Structures of the Multicomponent Rieske Non-Heme Iron Toluene 2,3-Dioxygenase Enzyme System

    SciTech Connect

    Friemann, R.; Lee, K; Brown, E; Gibson, D; Eklund, H; Ramaswamy, S

    2009-01-01

    Bacterial Rieske non-heme iron oxygenases catalyze the initial hydroxylation of aromatic hydrocarbon substrates. The structures of all three components of one such system, the toluene 2,3-dioxygenase system, have now been determined. This system consists of a reductase, a ferredoxin and a terminal dioxygenase. The dioxygenase, which was cocrystallized with toluene, is a heterohexamer containing a catalytic and a structural subunit. The catalytic subunit contains a Rieske [2Fe-2S] cluster and mononuclear iron at the active site. This iron is not strongly bound and is easily removed during enzyme purification. The structures of the enzyme with and without mononuclear iron demonstrate that part of the structure is flexible in the absence of iron. The orientation of the toluene substrate in the active site is consistent with the regiospecificity of oxygen incorporation seen in the product formed. The ferredoxin is Rieske type and contains a [2Fe-2S] cluster close to the protein surface. The reductase belongs to the glutathione reductase family of flavoenzymes and consists of three domains: an FAD-binding domain, an NADH-binding domain and a C-terminal domain. A model for electron transfer from NADH via FAD in the reductase and the ferredoxin to the terminal active-site mononuclear iron of the dioxygenase is proposed.

  5. Novel bacterial bioassay for a high-throughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is the molecular target of a range of synthetic ß-triketone herbicides that are currently used commercially. Their mode of action is based on an irreversible inhibition of HPPD. Therefore, this inhibitory capacity was used to develop a whole cell colo...

  6. Benzoate degradation by Rhodococcus opacus 1CP after dormancy: Characterization of dioxygenases involved in the process.

    PubMed

    Solyanikova, Inna P; Emelyanova, Elena V; Borzova, Oksana V; Golovleva, Ludmila A

    2016-01-01

    The process of benzoate degradation by strain Rhodococcus opacus 1CP after a five-year dormancy was investigated and its peculiarities were revealed. The strain was shown to be capable of growth on benzoate at a concentration of up to 10 g L(-1). The substrate specificity of benzoate dioxygenase (BDO) during the culture growth on a medium with a low (200-250 mg L(-1)) and high (4 g L(-1)) concentration of benzoate was assessed. BDO of R. opacus 1CP was shown to be an extremely narrow specificity enzyme. Out of 31 substituted benzoates, only with one, 3-chlorobenzoate, its activity was higher than 9% of that of benzoate. Two dioxygenases, catechol 1,2-dioxygenase (Cat 1,2-DO) and protocatechuate 3,4-dioxygenase (PCA 3,4-DO), were identified in a cell-free extract, purified and characterized. The substrate specificity of Cat 1,2-DO isolated from cells of strain 1CP after the dormancy was found to differ significantly from that of Cat 1,2-DO isolated earlier from cells of this strain grown on benzoate. By its substrate specificity, the described Cat 1,2-DO was close to the Cat 1,2-DO from strain 1CP grown on 4-methylbenzoate. Neither activity nor inhibition by protocatechuate was observed during the reaction of Cat 1,2-DO with catechol, and catechol had no inhibitory effect on the reaction of PCA 3,4-DO with protocatechuate. PMID:26669259

  7. CLONING IN PSEUDOMONAS CEPACIA: EXPRESSION AND REGULATION OF THE PROTOCATECHUATE 3,4-DIOXYGENASE GENES

    EPA Science Inventory

    Genes for the a and B subunits of the enzyme protocatechuate 3,4-dioxygenase were cloned from the Pseudomonas cepacia DB01 chromosome on a 9.5 kilobase pair PstI fragment into the broad-host-range cloning vector pR023l7. he resultant clone was able to complement protocatechuate 3...

  8. Relationship between indoleamine 2,3-dioxygenase activity and lymphatic invasion propensity of colorectal carcinoma

    PubMed Central

    Engin, Atilla; Gonul, Ipek Isik; Engin, Ayse Basak; Karamercan, Ahmet; Sepici Dincel, Aylin; Dursun, Ayse

    2016-01-01

    AIM: To evaluate whether serum and tumor indoleamine 2,3-dioxygenase activities can predict lymphatic invasion (LI) or lymph node metastasis in colorectal carcinoma. METHODS: The study group consisted of 44 colorectal carcinoma patients. The patients were re-grouped according to the presence or absence of LI and lymph node metastasis. Forty-three cancer-free subjects without any metabolic disturbances were included into the control group. Serum neopterin was measured by enzyme linked immunosorbent assay. Urinary neopterin and biopterin, serum tryptophan (Trp) and kynurenine (Kyn) concentrations of all patients were determined by high performance liquid chromatography. Kyn/Trp was calculated and its correlation with serum neopterin was determined to estimate the serum indoleamine 2,3-dioxygenase activity. Tissue sections from the studied tumors were re-examined histopathologically and were stained by immunohistochemistry with indoleamine-2,3-dioxygenase antibodies. RESULTS: Neither serum nor urinary neopterin was significantly different between the patient and control groups (both P > 0.05). However, colorectal carcinoma patients showed a significant positive correlation between the serum neopterin levels and Kyn/Trp (r = 0.450, P < 0.01). Urinary biopterin was significantly higher in cancer cases (P < 0.05). Serum Kyn/Trp was significantly higher in colorectal carcinoma patients (P < 0.01). Lymphatic invasion was present in 23 of 44 patients, of which only 12 patients had lymph node metastasis. Eleven patients with LI had no lymph node metastasis. Indoleamine-2,3-dioxygenase intensity score was significantly higher in LI positive cancer group (44.56% ± 6.11%) than negative colorectal cancer patients (24.04% ± 6.90%), (P < 0.05). Indoleamine 2,3-dioxygenase expression correlated both with the presence of LI and lymph node metastasis (P < 0.01 and P < 0.05, respectively). A significant difference between the accuracy of diagnosis by using either total indoleamine-2

  9. Crystal structures of alkylperoxo and anhydride intermediates in an intradiol ring-cleaving dioxygenase

    PubMed Central

    Knoot, Cory J.; Purpero, Vincent M.; Lipscomb, John D.

    2015-01-01

    Intradiol aromatic ring-cleaving dioxygenases use an active site, nonheme Fe3+ to activate O2 and catecholic substrates for reaction. The inability of Fe3+ to directly bind O2 presents a mechanistic conundrum. The reaction mechanism of protocatechuate 3,4-dioxygenase is investigated here using the alternative substrate 4-fluorocatechol. This substrate is found to slow the reaction at several steps throughout the mechanistic cycle, allowing the intermediates to be detected in solution studies. When the reaction was initiated in an enzyme crystal, it was found to halt at one of two intermediates depending on the pH of the surrounding solution. The X-ray crystal structure of the intermediate at pH 6.5 revealed the key alkylperoxo-Fe3+ species, and the anhydride-Fe3+ intermediate was found for a crystal reacted at pH 8.5. Intermediates of these types have not been structurally characterized for intradiol dioxygenases, and they validate four decades of spectroscopic, kinetic, and computational studies. In contrast to our similar in crystallo crystallographic studies of an Fe2+-containing extradiol dioxygenase, no evidence for a superoxo or peroxo intermediate preceding the alkylperoxo was found. This observation and the lack of spectroscopic evidence for an Fe2+ intermediate that could bind O2 are consistent with concerted formation of the alkylperoxo followed by Criegee rearrangement to yield the anhydride and ultimately ring-opened product. Structural comparison of the alkylperoxo intermediates from the intra- and extradiol dioxygenases provides a rationale for site specificity of ring cleavage. PMID:25548185

  10. Pyruvate kinase M2 is a phosphotyrosine-binding protein

    SciTech Connect

    Christofk, H.R.; Vander Heiden, M.G.; Wu, N.; Asara, J.M.; Cantley, L.C.

    2008-06-03

    Growth factors stimulate cells to take up excess nutrients and to use them for anabolic processes. The biochemical mechanism by which this is accomplished is not fully understood but it is initiated by phosphorylation of signalling proteins on tyrosine residues. Using a novel proteomic screen for phosphotyrosine-binding proteins, we have made the observation that an enzyme involved in glycolysis, the human M2 (fetal) isoform of pyruvate kinase (PKM2), binds directly and selectively to tyrosine-phosphorylated peptides. We show that binding of phosphotyrosine peptides to PKM2 results in release of the allosteric activator fructose-1,6-bisphosphate, leading to inhibition of PKM2 enzymatic activity. We also provide evidence that this regulation of PKM2 by phosphotyrosine signalling diverts glucose metabolites from energy production to anabolic processes when cells are stimulated by certain growth factors. Collectively, our results indicate that expression of this phosphotyrosine-binding form of pyruvate kinase is critical for rapid growth in cancer cells.

  11. 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

  12. Protective effects of ethyl pyruvate in cisplatin-induced nephrotoxicity

    PubMed Central

    Kelle, Ilker; Akkoc, Hasan; Tunik, Selcuk; Nergiz, Yusuf; Erdinc, Meral; Erdinc, Levent

    2014-01-01

    This study was performed to investigate the effect of ethyl pyruvate on changes in renal functions and oxidative stress related renal injury caused by cisplatin (cis-dichlorodiammine platinum-II; CDDP). Male Wistar albino rats were divided into four groups (n = 8): (1) control group (1 ml Ringer's lactate solution i.p.); (2) ethyl pyruvate (EP) group (50 mg/kg Ringer's EP solution (REPS) i.p.); (3) cisplatin group (a single dose of cisplatin (5 mg/kg, i.p.); and (4) cisplatin + EP group (a single dose of cisplatin (5 mg/kg, i.p.) + REPS 50 mg/kg/day, i.p.) for five days. At the sixth day, kidneys of rats were mounted to a Langendorff apparatus. Renal perfusion pressures were recorded. Blood samples were taken for serum urea, creatinine, total oxidant status (TOS), total antioxidant status (TAS) and oxidative stres index (OSI) evaluations. Kidney tissues were obtained for malondialdehyde (MDA) analyses and histopathological examination. Perfusion pressures, serum urea, creatinine, TOS, OSI and tissue MDA levels were found significantly higher, whereas TAS was notably lower in cisplatin group. Histopathological examination showed apparent renal paranchymal injury in cisplatin group. In cisplatin + REPS group, perfusion pressures, serum urea, creatinine and tissue MDA levels were decreased. Moreover, EP co-administration provided less inflammatory cell infiltration, tubular dilatation, whereas TOS, TAS and OSI improved significantly versus cisplatin group. These findings show that EP has protective effects against cisplatin nephrotoxicity. PMID:26019553

  13. Comparison of fumerate-pyruvate media and beef extract media for aerobically culturing Campylobacter species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Media supplemented with fumarate, pyruvate, and a vitamin-mineral solution or with beef extract were compared for the ability to support aerobic growth of Campylobacter. Basal broth composed of tryptose, yeast extract, bicarbonate, and agar was supplemented with 30 mM fumarate, 100 mM pyruvate, and ...

  14. Pyruvate orthophosphate dikinase: intracellular site of synthesis in maize leaf cells

    SciTech Connect

    Gee, S.L.; Ruzin, S.; Bassham, J.A.

    1984-01-01

    Pyruvate orthophosphate dikinase is synthesized in non-green leaf cells of the maize mutant iojap. Since iojap plastids lack ribosomes, it is concluded that the site of the synthesis of pyruvate orthophosphate dikinase in maize leaf cells is on ribosomes in the cytoplasm.

  15. Effect of bicarbonate concentration on aerobic growth of campylobacter in a fumarate-pyruvate medium

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The purpose of the present study was to examine the effect of sodium bicarbonate (NaHCO3) concentration on aerobic growth of Campylobacter in a fumarate-pyruvate medium. Fumarate-pyruvate broth medium was supplemented with 0.00 to 0.10% NaHCO3 and inoculated with Campylobacter coli 33559, Campyloba...

  16. Metabolic networks to generate pyruvate, PEP and ATP from glycerol in Pseudomonas fluorescens.

    PubMed

    Alhasawi, Azhar; Thomas, Sean C; Appanna, Vasu D

    2016-04-01

    Glycerol is a major by-product of the biodiesel industry. In this study we report on the metabolic networks involved in its transformation into pyruvate, phosphoenolpyruvate (PEP) and ATP. When the nutritionally-versatile Pseudomonas fluorescens was exposed to hydrogen peroxide (H2O2) in a mineral medium with glycerol as the sole carbon source, the microbe reconfigured its metabolism to generate adenosine triphosphate (ATP) primarily via substrate-level phosphorylation (SLP). This alternative ATP-producing stratagem resulted in the synthesis of copious amounts of PEP and pyruvate. The production of these metabolites was mediated via the enhanced activities of such enzymes as pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC). The high energy PEP was subsequently converted into ATP with the aid of pyruvate phosphate dikinase (PPDK), phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK) with the concomitant formation of pyruvate. The participation of the phospho-transfer enzymes like adenylate kinase (AK) and acetate kinase (ACK) ensured the efficiency of this O2-independent energy-generating machinery. The increased activity of glycerol dehydrogenase (GDH) in the stressed bacteria provided the necessary precursors to fuel this process. This H2O2-induced anaerobic life-style fortuitously evokes metabolic networks to an effective pathway that can be harnessed into the synthesis of ATP, PEP and pyruvate. The bioconversion of glycerol to pyruvate will offer interesting economic benefit. PMID:26920481

  17. Regulation of synthesis of pyruvate carboxylase in the photosynthetic bacterium Rhodobacter capsulatus.

    PubMed Central

    Yakunin, A F; Hallenbeck, P C

    1997-01-01

    The synthesis of pyruvate carboxylase (PC) was studied by using quantitative immunoblot analysis with an antibody raised against PC purified from Rhodobacter capsulatus and was found to vary 20-fold depending on the growth conditions. The PC content was high in cells grown on pyruvate or on carbon substrates metabolized via pyruvate (lactate, D-malate, glucose, or fructose) and low in cells grown on tricarboxylic acid (TCA) cycle intermediates or substrates metabolized without intermediate formation of pyruvate (acetate or glutamate). Under dark aerobic growth conditions with lactate as a carbon source, the PC content was approximately twofold higher than that found under light anaerobic growth conditions. The results of incubation experiments demonstrate that PC synthesis is induced by pyruvate and repressed by TCA cycle intermediates, with negative control dominating over positive control. The content of PC in R. capsulatus cells was also directly related to the growth rate in continuous cultures. The analysis of intracellular levels of pyruvate and TCA cycle intermediates in cells grown under different conditions demonstrated that the content of PC is directly proportional to the ratio between pyruvate and C4 dicarboxylates. These results suggest that the regulation of PC synthesis by oxygen and its direct correlation with growth rate may reflect effects on the balance of intracellular pyruvate and C4 dicarboxylates. Thus, this important enzyme is potentially regulated both allosterically and at the level of synthesis. PMID:9045800

  18. Differential resuscitative effect of pyruvate and its analogues on VBNC (viable but non-culturable) Salmonella.

    PubMed

    Morishige, Yuta; Fujimori, Ko; Amano, Fumio

    2013-01-01

    An environmental isolate of Salmonella Enteritidis (SE), grown to the logarithmic phase, rapidly lost culturability by the addition of 3 mM H2O2 to cultures grown in Luria-Bertani (LB) medium; however, some H2O2-treated bacteria regained their culturability in M9 minimal medium, if sodium pyruvate was present at at least 0.3 mM. In addition, most pyruvate analogues, such as bromopyruvate or phenylpyruvate, did not show restoration activity similar to that of pyruvate, except in the case of α-ketobutyrate. Further analysis of the mechanism underlying the resuscitation by pyruvate revealed that although many of the bacteria showed respiratory activity on CTC (5-cyano-2,3-di-(p-tolyl) tetrazolium chloride) reduction with or without pyruvate, the biosynthesis of DNA and protein synthesis were quite different in the presence or absence of pyruvate, i.e., pyruvate endowed the cells with the ability to incorporate much more radio-label into precursors during the resuscitation process. These results suggest that pyruvate is one of the key molecules working in the resuscitation process by taking bacteria from the non-culturable state to the growing and colony-forming state by triggering the synthesis of macromolecules such as DNA and protein. PMID:23595023

  19. Knockout of the murine cysteine dioxygenase gene results in severe impairment in ability to synthesize taurine and an increased catabolism of cysteine to hydrogen sulfide

    PubMed Central

    Ueki, Iori; Roman, Heather B.; Valli, Alessandro; Fieselmann, Krista; Lam, Jimmy; Peters, Rachel; Hirschberger, Lawrence L.

    2011-01-01

    Cysteine homeostasis is dependent on the regulation of cysteine dioxygenase (CDO) in response to changes in sulfur amino acid intake. CDO oxidizes cysteine to cysteinesulfinate, which is further metabolized to either taurine or to pyruvate plus sulfate. To gain insight into the physiological function of CDO and the consequence of a loss of CDO activity, mice carrying a null CDO allele (CDO+/− mice) were crossed to generate CDO−/−, CDO+/−, and CDO+/+ mice. CDO−/− mice exhibited postnatal mortality, growth deficit, and connective tissue pathology. CDO−/− mice had extremely low taurine levels and somewhat elevated cysteine levels, consistent with the lack of flux through CDO-dependent catabolic pathways. However, plasma sulfate levels were slightly higher in CDO−/− mice than in CDO+/− or CDO+/+ mice, and tissue levels of acid-labile sulfide were elevated, indicating an increase in cysteine catabolism by cysteine desulfhydration pathways. Null mice had lower hepatic cytochrome c oxidase levels, suggesting impaired electron transport capacity. Supplementation of mice with taurine improved survival of male pups but otherwise had little effect on the phenotype of the CDO−/− mice. H2S has been identified as an important gaseous signaling molecule as well as a toxicant, and pathology may be due to dysregulation of H2S production. Control of cysteine levels by regulation of CDO may be necessary to maintain low H2S/sulfane sulfur levels and facilitate the use of H2S as a signaling molecule. PMID:21693692

  20. Improvement of isobutanol production in Saccharomyces cerevisiae by increasing mitochondrial import of pyruvate through mitochondrial pyruvate carrier.

    PubMed

    Park, Seong-Hee; Kim, Sujin; Hahn, Ji-Sook

    2016-09-01

    Subcellular compartmentalization of the biosynthetic enzymes is one of the limiting factors for isobutanol production in Saccharomyces cerevisiae. Previously, it has been shown that mitochondrial compartmentalization of the biosynthetic pathway through re-locating cytosolic Ehrlich pathway enzymes into the mitochondria can increase isobutanol production. In this study, we improved mitochondrial isobutanol production by increasing mitochondrial pool of pyruvate, a key substrate for isobutanol production. Mitochondrial isobutanol biosynthetic pathway was introduced into bat1Δald6Δlpd1Δ strain, where genes involved in competing pathways were deleted, and MPC1, MPC2, and MPC3 genes encoding the subunits of mitochondrial pyruvate carrier (MPC) hetero-oligomeric complex were overexpressed with different combinations. Overexpression of Mpc1 and Mpc3 forming high-affinity MPCOX was more effective in improving isobutanol production than overexpression of Mpc1 and Mpc2 forming low-affinity MPCFERM. The final engineered strain overexpressing MPCOX produced 330.9 mg/L isobutanol from 20 g/L glucose, exhibiting about 22-fold increase in production compared to wild type. PMID:27225475

  1. Crystal engineering using bisphenols and trisphenols. Complexes with 1,10-phenanthroline: hydrogen-bonded chains in adducts with 4,4'-biphenol (1/1) and 4,4'-sulfonyldiphenol (2/3), pi-pi stacked chains in the (1/2) adduct with 4,4'-thiodiphenol, and pairwise-interwoven nets in 1,1,1-tris(4-hydroxyphenyl)ethane-1,10-phenanthroline-methanol (1/1/1).

    PubMed

    Ferguson; Glidewell; Lavender

    1999-08-01

    In 4,4'-biphenol-1,10-phenanthroline (1/1) [systematic name: 4,4'-biphenyldiol-1,10-phenanthroline (1/1)] the diphenol molecules lie across centres of inversion and the phenanthroline molecules lie across twofold rotation axes; the phenanthroline molecules act as chain-building units and the molecular components are linked into steeply zigzag C(16) chains parallel to [101] by means of O-H.N hydrogen bonds. In the structure of 4,4'-thiodiphenol-1,10-phenanthroline (1/2) the phenanthroline molecules act as chain-terminating units; the supramolecular aggregation is finite, with the bisphenol linked to each phenanthroline molecule by means of a single O-H.N hydrogen bond. pi-pi stacking interactions between the phenanthroline molecules in neighbouring hydrogen-bonded aggregates serve to link these aggregates into a continuous two-dimensional array. The phenanthroline molecules in 4,4'-sulfonyldiphenol-1,10-phenanthroline (2/3) play two roles: molecules in general positions act as chain-terminating units and are linked to the sulfonyldiphenol molecules by means of three-centre O-H.(N)(2) hydrogen bonds, while those lying across twofold rotation axes act as chain builders and are linked to two different sulfonyldiphenol molecules by means of a two-centre O-H.N hydrogen bond in each case; the resulting U-shaped five-component aggregates are further linked by C-H.O=S hydrogen bonds into a C(3)(3)(17)[R(2)(2)(12)] 'chain of rings' along [001]. In 1,1,1-tris(4-hydroxyphenyl)ethane-1,10-phenanthroline-methanol (1/1/1) [systematic name: 4,4',4"-ethylidynetriphenol-1,10-phenanthroline-methanol (1/1/1)] the phenanthroline molecules again act as chain-terminating units: the trisphenol molecules and the methanol molecules are linked by O-H.O hydrogen bonds into two-dimensional nets built from R(6)(6)(42) rings, and pairs of these nets are interwoven. The formation of each net utilizes two hydroxyl groups per trisphenol molecule as hydrogen-bond donors and the remaining hydroxyl

  2. Isolation of mesotrione-degrading bacteria from aquatic environments in Brazil.

    PubMed

    Pileggi, Marcos; Pileggi, Sônia Alvim Veiga; Olchanheski, Luiz Ricardo; da Silva, Paulo Augusto Garbugio; Munoz Gonzalez, Ana M; Koskinen, Willian C; Barber, Brian; Sadowsky, Michael Jay

    2012-03-01

    Mesotrione is a benzoylcyclohexane-1,3-dione herbicide that inhibits 4-hydroxyphenyl pyruvate dioxygenase in target plants. Although it has been used since 2000, only a limited number of degrading microorganisms have been reported. Mesotrione-degrading bacteria were selected among strains isolated from Brazilian aquatic environments, located near corn fields treated with this herbicide. Pantoea ananatis was found to rapidly and completely degrade mesotrione. Mesotrione did not serve as a sole C, N, or S source for growth of P. ananatis, and mesotrione catabolism required glucose supplementation to minimal media. LC-MS/MS analyses indicated that mesotrione degradation produced intermediates other than 2-amino-4-methylsulfonyl benzoic acid or 4-methylsulfonyl-2-nitrobenzoic acid, two metabolites previously identified in a mesotrione-degrading Bacillus strain. Since P. ananatis rapidly degraded mesotrione, this strain might be useful for bioremediation purposes. PMID:22245060

  3. Carbohydrate metabolism during prolonged exercise and recovery: interactions between pyruvate dehydrogenase, fatty acids, and amino acids.

    PubMed

    Mourtzakis, Marina; Saltin, Bengt; Graham, Terry; Pilegaard, Henriette

    2006-06-01

    During prolonged exercise, carbohydrate oxidation may result from decreased pyruvate production and increased fatty acid supply and ultimately lead to reduced pyruvate dehydrogenase (PDH) activity. Pyruvate also interacts with the amino acids alanine, glutamine, and glutamate, whereby the decline in pyruvate production could affect tricarboxycylic acid cycle flux as well as gluconeogenesis. To enhance our understanding of these interactions, we studied the time course of changes in substrate utilization in six men who cycled at 44+/-1% peak oxygen consumption (mean+/-SE) until exhaustion (exhaustion at 3 h 23 min+/-11 min). Femoral arterial and venous blood, blood flow measurements, and muscle samples were obtained hourly during exercise and recovery (3 h). Carbohydrate oxidation peaked at 30 min of exercise and subsequently decreased for the remainder of the exercise bout (P<0.05). PDH activity peaked at 2 h of exercise, whereas pyruvate production peaked at 1 h of exercise and was reduced (approximately 30%) thereafter, suggesting that pyruvate availability primarily accounted for reduced carbohydrate oxidation. Increased free fatty acid uptake (P<0.05) was also associated with decreasing PDH activity (P<0.05) and increased PDH kinase 4 mRNA (P<0.05) during exercise and recovery. At 1 h of exercise, pyruvate production was greatest and was closely linked to glutamate, which was the predominant amino acid taken up during exercise and recovery. Alanine and glutamine were also associated with pyruvate metabolism, and they comprised approximately 68% of total amino-acid release during exercise and recovery. Thus reduced pyruvate production was primarily associated with reduced carbohydrate oxidation, whereas the greatest production of pyruvate was related to glutamate, glutamine, and alanine metabolism in early exercise. PMID:16424076

  4. Pyruvate modifies metabolic flux and nutrient sensing during extracorporeal membrane oxygenation in an immature swine model

    SciTech Connect

    Ledee, Dolena R.; Kajimoto, Masaki; O'Kelly-Priddy, Colleen M.; Olson, Aaron; Isern, Nancy G.; Robillard Frayne, Isabelle; Des Rosiers, Christine; Portman, Michael A.

    2015-07-01

    Extracorporeal membrane oxygenation (ECMO) provides mechanical circulatory support for infants and children with postoperative cardiopulmonary failure. Nutritional support is mandatory during ECMO, although specific actions for substrates on the heart have not been delineated. Prior work shows that enhancing pyruvate oxidation promotes successful weaning from ECMO. Accordingly, we closely examined the role of prolonged systemic pyruvate supplementation in modifying metabolic parameters during the unique conditions of ventricular unloading provided by ECMO. Twelve male mixed breed Yorkshire piglets (age 30-49 days) received systemic infusion of either normal saline (Group C) or pyruvate (Group P) during ECMO for 8 hours. Over the final hour piglets received [2-13C] pyruvate, and [13C6]-L-leucine, as an indicator for oxidation and protein synthesis. A significant increase in lactate and pyruvate concentrations occurred, along with an increase in the absolute concentration of all measured CAC intermediates. Group P showed greater anaplerotic flux through pyruvate carboxylation although pyruvate oxidation relative to citrate synthase flux was similar to Group C. The groups demonstrated similar leucine fractional contributions to acetyl-CoA and fractional protein synthesis rates. Pyruvate also promoted an increase in the phosphorylation state of several nutrient sensitive enzymes, such as AMPK and ACC, and promoted O-GlcNAcylation through the hexosamine biosynthetic pathway (HBP). In conclusion, prolonged pyruvate supplementation during ECMO modified anaplerotic pyruvate flux and elicited changes in important nutrient and energy sensitive pathways, while preserving protein synthesis. Therefore, the observed results support the further study of nutritional supplementation and its downstream effects on cardiac adaptation during ventricular unloading.

  5. Validation of the in vivo assessment of pyruvate dehydrogenase activity using hyperpolarised 13C MRS.

    PubMed

    Atherton, Helen J; Schroeder, Marie A; Dodd, Michael S; Heather, Lisa C; Carter, Emma E; Cochlin, Lowri E; Nagel, Simon; Sibson, Nicola R; Radda, George K; Clarke, Kieran; Tyler, Damian J

    2011-02-01

    Many diseases of the heart are characterised by changes in substrate utilisation, which is regulated in part by the activity of the enzyme pyruvate dehydrogenase (PDH). Consequently, there is much interest in the in vivo evaluation of PDH activity in a range of physiological and pathological states to obtain information on the metabolic mechanisms of cardiac diseases. Hyperpolarised [1-(13)C]pyruvate, detected using MRS, is a novel technique for the noninvasive evaluation of PDH flux. PDH flux has been assumed to directly reflect in vivo PDH activity, although to date this assumption remains unproven. Control animals and animals undergoing interventions known to modulate PDH activity, namely high fat feeding and dichloroacetate infusion, were used to investigate the relationship between in vivo hyperpolarised MRS measurements of PDH flux and ex vivo measurements of PDH enzyme activity (PDH(a)). Further, the plasma concentrations of pyruvate and other important metabolites were evaluated following pyruvate infusion to assess the metabolic consequences of pyruvate infusion during hyperpolarised MRS experiments. Hyperpolarised MRS measurements of PDH flux correlated significantly with ex vivo measurements of PDH(a), confirming that PDH activity influences directly the in vivo flux of hyperpolarised pyruvate through cardiac PDH. The maximum plasma concentration of pyruvate reached during hyperpolarised MRS experiments was approximately 250 µM, equivalent to physiological pyruvate concentrations reached during exercise or with dietary interventions. The concentrations of other metabolites, including lactate, glucose and β-hydroxybutyrate, did not vary during the 60 s following pyruvate infusion. Hence, during the 60-s data acquisition period, metabolism was minimally affected by pyruvate infusion. PMID:20799252

  6. A Novel Aromatic-Ring-Hydroxylating Dioxygenase from the Diterpenoid-Degrading Bacterium Pseudomonas abietaniphila BKME-9

    PubMed Central

    Martin, Vincent J. J.; Mohn, William W.

    1999-01-01

    Pseudomonas abietaniphila BKME-9 is able to degrade dehydroabietic acid (DhA) via ring hydroxylation by a novel dioxygenase. The ditA1, ditA2, and ditA3 genes, which encode the α and β subunits of the oxygenase and the ferredoxin of the diterpenoid dioxygenase, respectively, were isolated and sequenced. The ferredoxin gene is 9.2 kb upstream of the oxygenase genes and 872 bp upstream of a putative meta ring cleavage dioxygenase gene, ditC. A Tn5 insertion in the α subunit gene, ditA1, resulted in the accumulation by the mutant strain BKME-941 of the pathway intermediate, 7-oxoDhA. Disruption of the ferredoxin gene, ditA3, in wild-type BKME-9 by mutant-allele exchange resulted in a strain (BKME-91) with a phenotype identical to that of the mutant strain BKME-941. Sequence analysis of the putative ferredoxin indicated that it is likely to be a [4Fe-4S]- or [3Fe-4S]-type ferredoxin and not a [2Fe-2S]-type ferredoxin, as found in all previously described ring-hydroxylating dioxygenases. Expression in Escherichia coli of ditA1A2A3, encoding the diterpenoid dioxygenase without its putative reductase component, resulted in a functional enzyme. The diterpenoid dioxygenase attacks 7-oxoDhA, and not DhA, at C-11 and C-12, producing 7-oxo-11,12-dihydroxy-8,13-abietadien acid, which was identified by 1H nuclear magnetic resonance, UV-visible light, and high-resolution mass spectrometry. The organization of the genes encoding the various components of the diterpenoid dioxygenase, the phylogenetic distinctiveness of both the α subunit and the ferredoxin component, and the unusual Fe-S cluster of the ferredoxin all suggest that this enzyme belongs to a new class of aromatic ring-hydroxylating dioxygenases. PMID:10217753

  7. Similarities between the antABC-Encoded Anthranilate Dioxygenase and the benABC-Encoded Benzoate Dioxygenase of Acinetobacter sp. Strain ADP1

    PubMed Central

    Bundy, Becky M.; Campbell, Alan L.; Neidle, Ellen L.

    1998-01-01

    Acinetobacter sp. strain ADP1 can use benzoate or anthranilate as a sole carbon source. These structurally similar compounds are independently converted to catechol, allowing further degradation to proceed via the β-ketoadipate pathway. In this study, the first step in anthranilate catabolism was characterized. A mutant unable to grow on anthranilate, ACN26, was selected. The sequence of a wild-type DNA fragment that restored growth revealed the antABC genes, encoding 54-, 19-, and 39-kDa proteins, respectively. The deduced AntABC sequences were homologous to those of class IB multicomponent aromatic ring-dihydroxylating enzymes, including the dioxygenase that initiates benzoate catabolism. Expression of antABC in Escherichia coli, a bacterium that normally does not degrade anthranilate, enabled the conversion of anthranilate to catechol. Unlike benzoate dioxygenase (BenABC), anthranilate dioxygenase (AntABC) catalyzed catechol formation without requiring a dehydrogenase. In Acinetobacter mutants, benC substituted for antC during growth on anthranilate, suggesting relatively broad substrate specificity of the BenC reductase, which transfers electrons from NADH to the terminal oxygenase. In contrast, the benAB genes did not substitute for antAB. An antA point mutation in ACN26 prevented anthranilate degradation, and this mutation was independent of a mucK mutation in the same strain that prevented exogenous muconate degradation. Anthranilate induced expression of antA, although no associated transcriptional regulators were identified. Disruption of three open reading frames in the immediate vicinity of antABC did not prevent the use of anthranilate as a sole carbon source. The antABC genes were mapped on the ADP1 chromosome and were not linked to the two known supraoperonic gene clusters involved in aromatic compound degradation. PMID:9721284

  8. A biochemical and genetic study of Leishmania donovani pyruvate kinase.

    PubMed

    Sandoval, Will; Isea, Raúl; Rodriguez, Evelyn; Ramirez, Jose Luis

    2008-11-15

    Here we present a biochemical and molecular biology study of the enzyme pyruvate kinase (PYK) from the parasitic protozoa Leishmania donovani. The PYK gene was cloned, mutagenised and over expressed and its kinetic parameters determined. Like in other kinetoplastids, L. donovani PYK is allosterically stimulated by the effector fructose 2,6 biphosphate and not by fructose 1,6 biphosphate. When the putative effector binding site of L. donovani PYK was mutagenised, we obtained two mutants with extreme kinetic behavior: Lys453Leu, which retained a sigmoidal kinetics and was little affected by the effector; and His480Gln, which deployed a hyperbolic kinetics that was not changed by the addition of the effector. Molecular Dynamics (MD) studies revealed that the mutations not only altered the effector binding site of L. donovani PYK but also changed the folding of its domain C. PMID:18725273

  9. Parkin Regulates the Activity of Pyruvate Kinase M2*

    PubMed Central

    Liu, Kun; Li, Fanzhou; Han, Haichao; Chen, Yue; Mao, Zebin; Luo, Jianyuan; Zhao, Yingming; Zheng, Bin; Gu, Wei; Zhao, Wenhui

    2016-01-01

    Parkin, a ubiquitin E3 ligase, is mutated in most cases of autosomal recessive early onset Parkinson disease. It was discovered that Parkin is also mutated in glioblastoma and other human malignancies and that it inhibits tumor cell growth. Here, we identified pyruvate kinase M2 (PKM2) as a unique substrate for parkin through biochemical purification. We found that parkin interacts with PKM2 both in vitro and in vivo, and this interaction dramatically increases during glucose starvation. Ubiquitylation of PKM2 by parkin does not affect its stability but decreases its enzymatic activity. Parkin regulates the glycolysis pathway and affects the cell metabolism. Our studies revealed the novel important roles of parkin in tumor cell metabolism and provided new insight for therapy of Parkinson disease. PMID:26975375

  10. Parkin Regulates the Activity of Pyruvate Kinase M2.

    PubMed

    Liu, Kun; Li, Fanzhou; Han, Haichao; Chen, Yue; Mao, Zebin; Luo, Jianyuan; Zhao, Yingming; Zheng, Bin; Gu, Wei; Zhao, Wenhui

    2016-05-01

    Parkin, a ubiquitin E3 ligase, is mutated in most cases of autosomal recessive early onset Parkinson disease. It was discovered that Parkin is also mutated in glioblastoma and other human malignancies and that it inhibits tumor cell growth. Here, we identified pyruvate kinase M2 (PKM2) as a unique substrate for parkin through biochemical purification. We found that parkin interacts with PKM2 both in vitro and in vivo, and this interaction dramatically increases during glucose starvation. Ubiquitylation of PKM2 by parkin does not affect its stability but decreases its enzymatic activity. Parkin regulates the glycolysis pathway and affects the cell metabolism. Our studies revealed the novel important roles of parkin in tumor cell metabolism and provided new insight for therapy of Parkinson disease. PMID:26975375

  11. Transformation linked decrease of pyruvate dehydrogenase complex in human epidermis.

    PubMed

    Eboli, M L; Pasquini, A

    1994-10-14

    Epidermis exhibits glycolytic features peculiar to cancer cells. The activity of pyruvate dehydrogenase complex, both active (PDHa) and total (PDHt) forms, has been investigated and compared in epidermis and epidermal carcinomas from human source. Low or undetectable PDHa is found in either normal and neoplastic tissue. PDHt is unchanged in human epidermis between the second and seventh decades of life but is dramatically decreased following neoplastic transformation (0.107 and 0.026 units/g fresh tissue for epidermis and epidermal carcinoma, respectively). As PDH plays a key role in mitochondrial carbohydrate metabolism, the decrease of total enzymic capacity found in tumors suggest that different mechanisms regulate PDH expression and, in turn, glycolytic mechanisms of epidermis and cancer cells. PMID:7954343

  12. Light regulation of pyruvate allocation into primary and secondary carbon metabolism in plants

    NASA Astrophysics Data System (ADS)

    Jardine, K.; Werner, C.; Wegener, F.; Meyers, K.; Abrell, L.

    2012-12-01

    While plant metabolic processes are known to exert a large influence on climate and air quality through the emission of CO2 and volatile organic compounds (VOCs), controls over the allocation of assimilated carbon to these important components of the global carbon cycle are poorly understood. In plants, pyruvate lies at the heart of carbon metabolism by acting as a key product of photosynthesis and glycolysis and as a substrate used in respiratory and secondary biosynthetic pathways (e.g. VOCs). It is now well recognized that light has a strong inhibitory effect on mitochondrial respiration and recent studies have shown this contributes to an accumulation of pyruvate. However, little is known about the impact(s) this has on biosynthetic processes including VOCs and how the different carbon atoms within pyruvate are utilized. In this study, we quantified diurnal VOC and CO2 fluxes from intact branches of a Mediterranean shrub (Halimium halimifolium) under controlled light conditions. In addition, we utilized positionally specific 13C-labeled pyruvate branch feeding together with stable isotope analysis to trace the partitioning of C1, C2, and C3 carbon atoms of pyruvate into VOCs and CO2 emissions in the light and in the dark. In the light, we found high emission rates of a large array of VOC including volatile isoprenoids, oxygenated VOCs, green leaf volatiles, aromatics, sulfides, and nitrogen containing VOCs. In addition, elevated 13C-VOC emissions were stimulated by pyruvate-2-13C and pyruvate-2,3-13C but not pyruvate-1-13C while the opposite was the case for 13CO2 emissions (respiration). Moreover, we found that in the dark, 13C-VOC emissions dramatically declined while 13CO2 emissions were strongly stimulated. Our observations suggest that in the light, H. halimifolium dedicates a high pyruvate flux through secondary biosynthetic pathways including the pyruvate dehydrogenase bypass, mevalonic acid, MEP/DOXP, shikimic acid, and fatty acid pathways, which are

  13. Purification and characterization of cytosolic pyruvate kinase from banana fruit.

    PubMed Central

    Turner, W L; Plaxton, W C

    2000-01-01

    Cytosolic pyruvate kinase (PK(c)) from ripened banana (Musa cavendishii L.) fruits has been purified 543-fold to electrophoretic homogeneity and a final specific activity of 59.7 micromol of pyruvate produced/min per mg of protein. SDS/PAGE and gel-filtration FPLC of the final preparation indicated that this enzyme exists as a 240 kDa homotetramer composed of subunits of 57 kDa. Although the enzyme displayed a pH optimum of 6.9, optimal efficiency in substrate utilization [in terms of V(max)/K(m) for phosphoenolpyruvate (PEP) or ADP] was equivalent at pH 6.9 and 7.5. PK(c) activity was absolutely dependent upon the presence of a bivalent and a univalent cation, with Mg(2+) and K(+) respectively fulfilling this requirement. Hyperbolic saturation kinetics were observed for the binding of PEP, ADP, Mg(2+) and K(+) (K(m) values of 0.098, 0.12, 0.27 and 0.91 mM respectively). Although the enzyme utilized UDP, IDP, GDP and CDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate and MgATP were the most effective inhibitors, whereas L-aspartate functioned as an activator by reversing the inhibition of PK(c) by L-glutamate. The allosteric features of banana PK(c) are compared with those of banana PEP carboxylase [Law and Plaxton (1995) Biochem. J. 307, 807-816]. A model is presented which highlights the roles of cytosolic pH, MgATP, L-glutamate and L-aspartate in the co-ordinate control of the PEP branchpoint in ripening bananas. PMID:11104698

  14. The Crystal Structure of Toxoplasma gondii Pyruvate Kinase 1

    SciTech Connect

    Bakszt, R.; Wernimont, A; Allali-Hassani, A; Mok, M; Hills, T; Hui, R; Pizarro, J

    2010-01-01

    Pyruvate kinase (PK), which catalyzes the final step in glycolysis converting phosphoenolpyruvate to pyruvate, is a central metabolic regulator in most organisms. Consequently PK represents an attractive therapeutic target in cancer and human pathogens, like Apicomplexans. The phylum Aplicomplexa, a group of exclusively parasitic organisms, includes the genera Plasmodium, Cryptosporidium and Toxoplasma, the etiological agents of malaria, cryptosporidiosis and toxoplasmosis respectively. Toxoplasma gondii infection causes a mild illness and is a very common infection affecting nearly one third of the world's population. We have determined the crystal structure of the PK1 enzyme from T. gondii, with the B domain in the open and closed conformations. We have also characterized its enzymatic activity and confirmed glucose-6-phosphate as its allosteric activator. This is the first description of a PK enzyme in a closed inactive conformation without any bound substrate. Comparison of the two tetrameric TgPK1 structures indicates a reorientation of the monomers with a concomitant change in the buried surface among adjacent monomers. The change in the buried surface was associated with significant B domain movements in one of the interacting monomers. We hypothesize that a loop in the interface between the A and B domains plays an important role linking the position of the B domain to the buried surface among monomers through two {alpha}-helices. The proposed model links the catalytic cycle of the enzyme with its domain movements and highlights the contribution of the interface between adjacent subunits. In addition, an unusual ordered conformation was observed in one of the allosteric binding domains and it is related to a specific apicomplexan insertion. The sequence and structural particularity would explain the atypical activation by a mono-phosphorylated sugar. The sum of peculiarities raises this enzyme as an emerging target for drug discovery.

  15. Specific inhibition by synthetic analogs of pyruvate reveals that the pyruvate dehydrogenase reaction is essential for metabolism and viability of glioblastoma cells.

    PubMed

    Bunik, Victoria I; Artiukhov, Artem; Kazantsev, Alexey; Goncalves, Renata; Daloso, Danilo; Oppermann, Henry; Kulakovskaya, Elena; Lukashev, Nikolay; Fernie, Alisdair; Brand, Martin; Gaunitz, Frank

    2015-11-24

    The pyruvate dehydrogenase complex (PDHC) and its phosphorylation are considered essential for oncotransformation, but it is unclear whether cancer cells require PDHC to be functional or silenced. We used specific inhibition of PDHC by synthetic structural analogs of pyruvate to resolve this question. With isolated and intramitochondrial PDHC, acetyl phosphinate (AcPH, KiAcPH = 0.1 μM) was a much more potent competitive inhibitor than the methyl ester of acetyl phosphonate (AcPMe, KiAcPMe = 40 μM). When preincubated with the complex, AcPH also irreversibly inactivated PDHC. Pyruvate prevented, but did not reverse the inactivation. The pyruvate analogs did not significantly inhibit other 2-oxo acid dehydrogenases. Different cell lines were exposed to the inhibitors and a membrane-permeable precursor of AcPMe, dimethyl acetyl phosphonate, which did not inhibit isolated PDHC. Using an ATP-based assay, dependence of cellular viability on the concentration of the pyruvate analogs was followed. The highest toxicity of the membrane-permeable precursor suggested that the cellular action of charged AcPH and AcPMe requires monocarboxylate transporters. The relevant cell-specific transcripts extracted from Gene Expression Omnibus database indicated that cell lines with higher expression of monocarboxylate transporters and PDHC components were more sensitive to the PDHC inhibitors. Prior to a detectable antiproliferative action, AcPH significantly changed metabolic profiles of the investigated glioblastoma cell lines. We conclude that catalytic transformation of pyruvate by pyruvate dehydrogenase is essential for the metabolism and viability of glioblastoma cell lines, although metabolic heterogeneity causes different cellular sensitivities and/or abilities to cope with PDHC inhibition. PMID:26503465

  16. Specific inhibition by synthetic analogs of pyruvate reveals that the pyruvate dehydrogenase reaction is essential for metabolism and viability of glioblastoma cells

    PubMed Central

    Bunik, Victoria I.; Artiukhov, Artem; Kazantsev, Alexey; Goncalves, Renata; Daloso, Danilo; Oppermann, Henry; Kulakovskaya, Elena; Lukashev, Nikolay; Fernie, Alisdair; Brand, Martin; Gaunitz, Frank

    2015-01-01

    The pyruvate dehydrogenase complex (PDHC) and its phosphorylation are considered essential for oncotransformation, but it is unclear whether cancer cells require PDHC to be functional or silenced. We used specific inhibition of PDHC by synthetic structural analogs of pyruvate to resolve this question. With isolated and intramitochondrial PDHC, acetyl phosphinate (AcPH, KiAcPH = 0.1 μM) was a much more potent competitive inhibitor than the methyl ester of acetyl phosphonate (AcPMe, KiAcPMe = 40 μM). When preincubated with the complex, AcPH also irreversibly inactivated PDHC. Pyruvate prevented, but did not reverse the inactivation. The pyruvate analogs did not significantly inhibit other 2-oxo acid dehydrogenases. Different cell lines were exposed to the inhibitors and a membrane-permeable precursor of AcPMe, dimethyl acetyl phosphonate, which did not inhibit isolated PDHC. Using an ATP-based assay, dependence of cellular viability on the concentration of the pyruvate analogs was followed. The highest toxicity of the membrane-permeable precursor suggested that the cellular action of charged AcPH and AcPMe requires monocarboxylate transporters. The relevant cell-specific transcripts extracted from Gene Expression Omnibus database indicated that cell lines with higher expression of monocarboxylate transporters and PDHC components were more sensitive to the PDHC inhibitors. Prior to a detectable antiproliferative action, AcPH significantly changed metabolic profiles of the investigated glioblastoma cell lines. We conclude that catalytic transformation of pyruvate by pyruvate dehydrogenase is essential for the metabolism and viability of glioblastoma cell lines, although metabolic heterogeneity causes different cellular sensitivities and/or abilities to cope with PDHC inhibition. PMID:26503465

  17. A two-electron shell game: Intermediates of the extradiol-cleaving catechol dioxygenases

    PubMed Central

    Fielding, Andrew J.

    2014-01-01

    Extradiol catechol ring-cleaving dioxygenases function by binding both the organic substrate and O2 at a divalent metal center in the active site. They have proven to be a particularly versatile group of enzymes with which to study the O2 activation process. Here, recent studies of homoprotocatechuate 2,3-dioxygenase (HPCD) are summarized with the objective of showing how Nature can utilize the enzyme structure and the properties of the metal and the substrate to select among many possible chemical paths to achieve both specificity and efficiency. Possible intermediates in the mechanism have been trapped by swapping active site metals, introducing active site amino acid substituted variants, and using substrates with different electron donating capacities. While each of these intermediates could form part of a viable reaction pathway, kinetic measurements significantly limit the likely candidates. Structural, kinetic, spectroscopic and computational analysis of the various intermediates shed light on how catalytic efficiency can be achieved. PMID:24615282

  18. Crystal structure of the TK2203 protein from Thermococcus kodakarensis, a putative extradiol dioxygenase.

    PubMed

    Nishitani, Yuichi; Simons, Jan Robert; Kanai, Tamotsu; Atomi, Haruyuki; Miki, Kunio

    2016-06-01

    The TK2203 protein from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (262 residues, 29 kDa) is a putative extradiol dioxygenase catalyzing the cleavage of C-C bonds in catechol derivatives. It contains three metal-binding residues, but has no significant sequence similarity to proteins for which structures have been determined. Here, the first crystal structure of the TK2203 protein was determined at 1.41 Å resolution to investigate its functional role. Structure analysis reveals that this protein shares the same fold and catalytic residues as other extradiol dioxygenases, strongly suggesting the same enzymatic activity. Furthermore, the important region contributing to substrate selectivity is discussed. PMID:27303894

  19. Oxidation of nitrotoluenes by toluene dioxygenase: Evidence for a monooxygenase reaction

    SciTech Connect

    Robertson, J.B.; Spain, J.C. ); Haddock, J.D.; Gibson, D.T. )

    1992-08-01

    Pseudomonas putida F1 and Pseudomonas sp. strain JS150 initiate toluene degradation by incorporating molecular oxygen into the aromatic nucleus to form cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene. When toluene-grown cells were incubated with 2- and 3-nitrotoluene, the major products identified were 2- and 3-nitrobenzyl alcohol, respectively. The same cells oxidized 4-nitrotoluene to 2-methyl-5-nitrophenol and 3-methyl-6-nitrocatechol. Escherichia coli JM109(pDTG601), which contains the toluene dioxygenase genes from P. putida F1 under the control of the tac promoter, oxidized the isomeric nitrotoluenes to the same metabolites as those formed by P. putida F1 and Pseudomonas sp. strain JS150. These results extend the range of substrates known to be oxidized by this versatile enzyme and demonstrate for the first time that toluene dioxygenase can oxidize an aromatic methyl substituent.

  20. Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes

    SciTech Connect

    Zylstra, G.J.; Gibson, D.T. ); Wackett, L.P. )

    1989-12-01

    Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene. This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter. The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene. A linear rate of trichloroethylene degradation was observed with the E. coli recombinant strain. In contrast, P. putida F39/D, a mutant strain of P. putida F1 that does not contain cis-toluene dihydrodiol dehydrogenase, showed a much faster initial rate of trichloroethylene degradation which decreased over time.

  1. A two-electron-shell game: intermediates of the extradiol-cleaving catechol dioxygenases.

    PubMed

    Fielding, Andrew J; Lipscomb, John D; Que, Lawrence

    2014-06-01

    Extradiol-cleaving catechol dioxygenases function by binding both the organic substrate and O2 at a divalent metal center in the active site. They have proven to be a particularly versatile group of enzymes with which to study the O2 activation process. Here, recent studies of homoprotocatechuate 2,3-dioxygenase are summarized, showing how nature can utilize the enzyme structure and the properties of the metal and the substrate to select among many possible chemical paths to achieve both specificity and efficiency. Possible intermediates in the mechanism have been trapped by swapping active-site metals, introducing active-site amino acid substituted variants, and using substrates with different electron-donating capacities. Although each of these intermediates could form part of a viable reaction pathway, kinetic measurements significantly limit the likely candidates. Structural, kinetic, spectroscopic, and computational analyses of the various intermediates shed light on how catalytic efficiency can be achieved. PMID:24615282

  2. Interrogating the Druggability of the 2-Oxoglutarate-Dependent Dioxygenase Target Class by Chemical Proteomics.

    PubMed

    Joberty, Gérard; Boesche, Markus; Brown, Jack A; Eberhard, Dirk; Garton, Neil S; Humphreys, Philip G; Mathieson, Toby; Muelbaier, Marcel; Ramsden, Nigel G; Reader, Valérie; Rueger, Anne; Sheppard, Robert J; Westaway, Susan M; Bantscheff, Marcus; Lee, Kevin; Wilson, David M; Prinjha, Rab K; Drewes, Gerard

    2016-07-15

    The 2-oxoglutarate-dependent dioxygenase target class comprises around 60 enzymes including several subfamilies with relevance to human disease, such as the prolyl hydroxylases and the Jumonji-type lysine demethylases. Current drug discovery approaches are largely based on small molecule inhibitors targeting the iron/2-oxoglutarate cofactor binding site. We have devised a chemoproteomics approach based on a combination of unselective active-site ligands tethered to beads, enabling affinity capturing of around 40 different dioxygenase enzymes from human cells. Mass-spectrometry-based quantification of bead-bound enzymes using a free-ligand competition-binding format enabled the comprehensive determination of affinities for the cosubstrate 2-oxoglutarate and for oncometabolites such as 2-hydroxyglutarate. We also profiled a set of representative drug-like inhibitor compounds. The results indicate that intracellular competition by endogenous cofactors and high active site similarity present substantial challenges for drug discovery for this target class. PMID:27197014

  3. Mechanisms by which dichloroacetate lowers lactic acid levels: the kinetic interrelationships between lactate, pyruvate, alanine, and glucose.

    PubMed

    Jahoor, F; Zhang, X J; Frazer, E

    1994-01-01

    Dichloroacetate (DCA) is gaining use as an alternative to bicarbonate therapy in the treatment of lactic acidosis. To determine the mechanism(s) by which DCA lowers blood lactate levels, we studied its effect on the kinetic interrelationships between pyruvate, lactate, alanine, and glucose in the hindlimb of dogs during hormonal stimulation of pyruvate production (Ra) and its conversion to lactate. Three groups of dogs (n = 6) were infused with 1-13C-pyruvate to measure whole body pyruvate Ra, and pyruvate Ra and utilization (Rd) across the hindlimb during either a 4-hr infusion of saline (controls), or somatostatin, glucagon, and epinephrine (SGE), or SGE plus dichloroacetate (SGE + DCA). Pyruvate Ra was used as an index of rate of glycolysis and Rd as an index of pyruvate oxidation. In the controls, all kinetic parameters were constant during the saline infusion. Hindlimb pyruvate Ra and Rd were almost equal, and lactate release negligible. Compared to controls, SGE administration significantly increased (P < 0.05) wholebody pyruvate Ra (48.5 +/- 6.2 vs 33.6 +/- 2.4 mumol/kg/min) and blood lactate levels (P < 0.05). Hindlimb pyruvate Ra increased by approximately 150%, but Rd remained unchanged resulting in marked increases in lactate and alanine effluxes. Adding DCA to the SGE infusion significantly reduced wholebody pyruvate Ra (P < 0.05) and blood lactate levels (P < 0.01). In the hindlimb, however, there was no decrease in lactate output, despite a 91% increase in pyruvate utilization because pyruvate Ra also increased. These results suggest that during stimulation of rate of glycolysis, DCA lowers lactate levels by reducing the overall availability of pyruvate for lactate synthesis. This is accomplished by suppressing the rate of glycolysis in tissues other than skeletal muscle and stimulating pyruvate oxidation. PMID:7906882

  4. Pyruvate metabolism: A therapeutic opportunity in radiation-induced skin injury

    SciTech Connect

    Yoo, Hyun; Kang, Jeong Wook; Lee, Dong Won; Oh, Sang Ho; Lee, Yun-Sil; Lee, Eun-Jung; Cho, Jaeho

    2015-05-08

    Ionizing radiation is used to treat a range of cancers. Despite recent technological progress, radiation therapy can damage the skin at the administration site. The specific molecular mechanisms involved in this effect have not been fully characterized. In this study, the effects of pyruvate, on radiation-induced skin injury were investigated, including the role of the pyruvate dehydrogenase kinase 2 (PDK2) signaling pathway. Next generation sequencing (NGS) identified a wide range of gene expression differences between the control and irradiated mice, including reduced expression of PDK2. This was confirmed using Q-PCR. Cell culture studies demonstrated that PDK2 overexpression and a high cellular pyruvate concentration inhibited radiation-induced cytokine expression. Immunohistochemical studies demonstrated radiation-induced skin thickening and gene expression changes. Oral pyruvate treatment markedly downregulated radiation-induced changes in skin thickness and inflammatory cytokine expression. These findings indicated that regulation of the pyruvate metabolic pathway could provide an effective approach to the control of radiation-induced skin damage. - Highlights: • The effects of radiation on skin thickness in mice. • Next generation sequencing revealed that radiation inhibited pyruvate dehydrogenase kinase 2 expression. • PDK2 inhibited irradiation-induced cytokine gene expression. • Oral pyruvate treatment markedly downregulated radiation-induced changes in skin thickness.

  5. 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.

  6. Pyruvic acid levels in serum and saliva: A new course for oral cancer screening?

    PubMed Central

    Bhat, Manohara A; Prasad, KVV; Trivedi, Dheeraj; Rajeev, BR; Battur, Hemanth

    2016-01-01

    Objective: Cancerous cells show increased glycolysis rate. This will increase overall levels of pyruvate as it is one of the end products of glycolysis. The present on-going study is to estimate the levels of pyruvate in saliva and serum among healthy and oral cancer subjects. Settings and Design: Hospital-based cross-sectional comparative study. Methodology: A total of 50 subjects among healthy and oral cancer subjects were selected based on clinical and histological criteria. Saliva and serum samples were collected and subjected to pyruvate level estimation using biochemical analysis. Statistical Analysis: Descriptive analysis and Mann-Whitney test were used to find the statistical difference between the two independent groups. Results: Serum pyruvic acid levels of the healthy group were 1.09 ± 0.14 and for oral cancer, it was 2.95 ± 0.59 and salivary level were 3.49 ± 0.47 and 1.32 ± 0.10 respectively. Mann-Whitney test showed statistically significant difference in serum and salivary pyruvate level in between two groups (P < 0.000 respectively). Conclusion: The present study showed noticeable variation in the level of pyruvic acid among healthy and oral cancer subjects. This generates the hypothesis that estimation of the pyruvic acid can be a new tool to screening of the cancer. PMID:27194870

  7. Targeting Pyruvate Carboxylase Reduces Gluconeogenesis and Adiposity and Improves Insulin Resistance

    PubMed Central

    Kumashiro, Naoki; Beddow, Sara A.; Vatner, Daniel F.; Majumdar, Sachin K.; Cantley, Jennifer L.; Guebre-Egziabher, Fitsum; Fat, Ioana; Guigni, Blas; Jurczak, Michael J.; Birkenfeld, Andreas L.; Kahn, Mario; Perler, Bryce K.; Puchowicz, Michelle A.; Manchem, Vara Prasad; Bhanot, Sanjay; Still, Christopher D.; Gerhard, Glenn S.; Petersen, Kitt Falk; Cline, Gary W.; Shulman, Gerald I.; Samuel, Varman T.

    2013-01-01

    We measured the mRNA and protein expression of the key gluconeogenic enzymes in human liver biopsy specimens and found that only hepatic pyruvate carboxylase protein levels related strongly with glycemia. We assessed the role of pyruvate carboxylase in regulating glucose and lipid metabolism in rats through a loss-of-function approach using a specific antisense oligonucleotide (ASO) to decrease expression predominantly in liver and adipose tissue. Pyruvate carboxylase ASO reduced plasma glucose concentrations and the rate of endogenous glucose production in vivo. Interestingly, pyruvate carboxylase ASO also reduced adiposity, plasma lipid concentrations, and hepatic steatosis in high fat–fed rats and improved hepatic insulin sensitivity. Pyruvate carboxylase ASO had similar effects in Zucker Diabetic Fatty rats. Pyruvate carboxylase ASO did not alter de novo fatty acid synthesis, lipolysis, or hepatocyte fatty acid oxidation. In contrast, the lipid phenotype was attributed to a decrease in hepatic and adipose glycerol synthesis, which is important for fatty acid esterification when dietary fat is in excess. Tissue-specific inhibition of pyruvate carboxylase is a potential therapeutic approach for nonalcoholic fatty liver disease, hepatic insulin resistance, and type 2 diabetes. PMID:23423574

  8. Decomposition of Pyruvic Acid on the Ground-State Potential Energy Surface.

    PubMed

    da Silva, Gabriel

    2016-01-21

    A potential energy surface is reported for isomerization and decomposition of gas-phase pyruvic acid (CH3C(O)C(O)OH) in its ground electronic state. Consistent with previous works, the lowest energy pathway for pyruvic acid decomposition is identified as decarboxylation to produce hydroxymethylcarbene (CH3COH), with overall barrier of 43 kcal mol(-1). This study discovers that pyruvic acid can also isomerize to the α-lactone form with a barrier of only 36 kcal mol(-1), from which CO elimination can occur at 49 kcal mol(-1) above pyruvic acid. An additional novel channel is identified for the tautomerisation of pyruvic acid to the enol form, via a double H-shift mechanism. The barrier for this process is 51 kcal mol(-1), which is around 20 kcal mol(-1) lower than the barrier for conventional keto-enol tautomerization via a 1,3-H shift transition state. Rate coefficients are calculated for pyruvic acid decomposition through RRKM theory/master equation simulations at 800-2000 K and 1 atm, showing good agreement with the available experimental data. The dissociation of vibrationally excited pyruvic acid produced through photoexcitation and subsequent internal conversion to the ground state is also modeled under tropospheric conditions and is seen to produce appreciable quantities of CO (∼1-4%) in addition to CH3COH via the dominant CO2 loss channel. PMID:26587666

  9. Induction of indoleamine 2,3-dioxygenase: a mechanism of the antitumor activity of interferon. gamma

    SciTech Connect

    Ozaki, Y.; Edelstein, M.P.; Duch, D.S.

    1988-02-01

    The antiproliferative effects of interferon ..cap alpha.. (IFN-..cap alpha..) and interferon ..gamma.. (IFN-..gamma..) were found to be cell-dependent. Among the human cell lines examined, IFN-..gamma.. had a greater antiproliferative effect against cell lines that exhibited induction of indoleamine 2,3-dioxygenase, such as the KB oral carcinoma or WiDr colon adenocarcinoma, than against those that lacked the enzyme activity, such as the SW480 colon adenocarcinoma of NCI-H128 small-cell lung carcinoma. Induction of this dioxygenase showed a clear temporal relationship with increased metabolism of L-tryptophan and the depletion of this amino acid in the culture medium. While 70-80% of L-tryptophan remained in the medium of IFN-..cap alpha..- or vehicle-treated cells, virtually all of this amino acid was depleted in the medium of the IFN-..gamma..-treated group following 2-3 days of culture. Supplementing the growth medium with additional L-tryptophan reversed the antiproliferative effect of IFN-..gamma.. against KB cells in a dose- and time-dependent manner. The antiproliferative effects of IFN-..cap alpha.. and IFN-..gamma.. on SW480 and NCI-H128 cells, which are independent of the dioxygenase activity, and the inability of added L-tryptophan to reverse the effects of IFN-..gamma.. in WiDr cells suggest multiple mechanisms of action of the IFNs. The data show that the antiproliferative effect of IFN-..gamma.. through induction of indoleamine 2,3-dioxygenase, with a consequent L-tryptophan deprivation, is an effective means of regulating cell growth.

  10. Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis

    PubMed Central

    Frusciante, Sarah; Diretto, Gianfranco; Bruno, Mark; Ferrante, Paola; Pietrella, Marco; Prado-Cabrero, Alfonso; Rubio-Moraga, Angela; Beyer, Peter; Gomez-Gomez, Lourdes; Al-Babili, Salim; Giuliano, Giovanni

    2014-01-01

    Crocus sativus stigmas are the source of the saffron spice and accumulate the apocarotenoids crocetin, crocins, picrocrocin, and safranal, responsible for its color, taste, and aroma. Through deep transcriptome sequencing, we identified a novel dioxygenase, carotenoid cleavage dioxygenase 2 (CCD2), expressed early during stigma development and closely related to, but distinct from, the CCD1 dioxygenase family. CCD2 is the only identified member of a novel CCD clade, presents the structural features of a bona fide CCD, and is able to cleave zeaxanthin, the presumed precursor of saffron apocarotenoids, both in Escherichia coli and in maize endosperm. The cleavage products, identified through high-resolution mass spectrometry and comigration with authentic standards, are crocetin dialdehyde and crocetin, respectively. In vitro assays show that CCD2 cleaves sequentially the 7,8 and 7′,8′ double bonds adjacent to a 3-OH-β-ionone ring and that the conversion of zeaxanthin to crocetin dialdehyde proceeds via the C30 intermediate 3-OH-β-apo-8′-carotenal. In contrast, zeaxanthin cleavage dioxygenase (ZCD), an enzyme previously claimed to mediate crocetin formation, did not cleave zeaxanthin or 3-OH-β-apo-8′-carotenal in the test systems used. Sequence comparison and structure prediction suggest that ZCD is an N-truncated CCD4 form, lacking one blade of the β-propeller structure conserved in all CCDs. These results constitute strong evidence that CCD2 catalyzes the first dedicated step in crocin biosynthesis. Similar to CCD1, CCD2 has a cytoplasmic localization, suggesting that it may cleave carotenoids localized in the chromoplast outer envelope. PMID:25097262

  11. Novel flavonol 2-oxoglutarate dependent dioxygenase: affinity purification, characterization, and kinetic properties.

    PubMed

    Anzellotti, D; Ibrahim, R K

    2000-10-15

    A 2-oxoglutarate-dependent dioxygenase [EC 1.14.11-] that catalyzes the 6-hydroxylation of partially methylated flavonols has been purified to near homogeneity from Chrysosplenium americanum. Enzyme purification was achieved by fast protein liquid chromatography on Superose 12 and Mono Q columns as well as by affinity chromatography on 2-oxoglutarate-Sepharose and immunoaffinity columns. The specific activity of the 6-hydroxylase eluted from Mono Q (97.1 pkat/mg) was enriched 538-fold, with a 0.63% recovery. Both affinity chromatography steps resulted in the elimination of most contaminating proteins, but not without loss of enzyme activity and stability. The molecular mass of both the native and denatured enzyme was found to be 42 and 45 kDa, respectively, suggesting a monomeric protein. The enzyme exhibits strict specificity for position 6 of partially methylated flavonols possessing a 7-methoxyl group, indicating its involvement in the biosynthesis of polymethylated flavonols in this plant. The cofactor dependence of the enzyme is similar to that of other plant dioxygenases, particularly its dependence on ferrous ions for catalytic activity and reactivation. Internal amino acid sequence information indicated its relatedness to other plant flavonoid dioxygenases. The results of substrate interaction kinetics and product inhibition studies suggest an ordered, sequential reaction mechanism (TerTer), where 2-oxoglutarate is the first substrate to bind, followed by O2 and the flavonol substrate. Product release occurs in the reverse order where the hydroxylated flavonol is the first to be released, followed by CO2 and succinate. To our knowledge, this is the first reported 2-oxoglutarate-dependent dioxygenase that catalyzes the aromatic hydroxylation of a flavonoid compound. PMID:11068865

  12. Factors Altering Pyruvate Excretion in a Glycogen Storage Mutant of the Cyanobacterium, Synechococcus PCC7942.

    PubMed

    Benson, Phoebe J; Purcell-Meyerink, Diane; Hocart, Charles H; Truong, Thy T; James, Gabriel O; Rourke, Loraine; Djordjevic, Michael A; Blackburn, Susan I; Price, G D

    2016-01-01

    Interest in the production of carbon commodities from photosynthetically fixed CO2 has focused attention on cyanobacteria as a target for metabolic engineering and pathway investigation. We investigated the redirection of carbon flux in the model cyanobacterial species, Synechococcus elongatus PCC 7942, under nitrogen deprivation, for optimized production of the industrially desirable compound, pyruvate. Under nitrogen limited conditions, excess carbon is naturally stored as the multi-branched polysaccharide, glycogen, but a block in glycogen synthesis, via knockout mutation in the gene encoding ADP-glucose pyrophosphorylase (glgC), results in the accumulation of the organic acids, pyruvate and 2-oxoglutarate, as overflow excretions into the extracellular media. The ΔglgC strain, under 48 h of N-deprivation was shown to excrete pyruvate for the first time in this strain. Additionally, by increasing culture pH, to pH 10, it was possible to substantially elevate excretion of pyruvate, suggesting the involvement of an unknown substrate/proton symporter for export. The ΔglgC mutant was also engineered to express foreign transporters for glucose and sucrose, and then grown photomixotrophically with exogenous organic carbon supply, as added 5 mM glucose or sucrose during N- deprivation. Under these conditions we observed a fourfold increase in extracellular pyruvate excretion when glucose was added, and a smaller increase with added sucrose. Although the magnitude of pyruvate excretion did not correlate with the capacity of the ΔglgC strain for bicarbonate-dependent photosynthetic O2 evolution, or with light intensity, there was, however, a positive correlation observed between the density of the starter culture prior to N-deprivation and the final extracellular pyruvate concentration. The factors that contribute to enhancement of pyruvate excretion are discussed, as well as consideration of whether the source of carbon for pyruvate excretion might be derived from

  13. Factors Altering Pyruvate Excretion in a Glycogen Storage Mutant of the Cyanobacterium, Synechococcus PCC7942

    PubMed Central

    Benson, Phoebe J.; Purcell-Meyerink, Diane; Hocart, Charles H.; Truong, Thy T.; James, Gabriel O.; Rourke, Loraine; Djordjevic, Michael A.; Blackburn, Susan I.; Price, G. D.

    2016-01-01

    Interest in the production of carbon commodities from photosynthetically fixed CO2 has focused attention on cyanobacteria as a target for metabolic engineering and pathway investigation. We investigated the redirection of carbon flux in the model cyanobacterial species, Synechococcus elongatus PCC 7942, under nitrogen deprivation, for optimized production of the industrially desirable compound, pyruvate. Under nitrogen limited conditions, excess carbon is naturally stored as the multi-branched polysaccharide, glycogen, but a block in glycogen synthesis, via knockout mutation in the gene encoding ADP-glucose pyrophosphorylase (glgC), results in the accumulation of the organic acids, pyruvate and 2-oxoglutarate, as overflow excretions into the extracellular media. The ΔglgC strain, under 48 h of N-deprivation was shown to excrete pyruvate for the first time in this strain. Additionally, by increasing culture pH, to pH 10, it was possible to substantially elevate excretion of pyruvate, suggesting the involvement of an unknown substrate/proton symporter for export. The ΔglgC mutant was also engineered to express foreign transporters for glucose and sucrose, and then grown photomixotrophically with exogenous organic carbon supply, as added 5 mM glucose or sucrose during N- deprivation. Under these conditions we observed a fourfold increase in extracellular pyruvate excretion when glucose was added, and a smaller increase with added sucrose. Although the magnitude of pyruvate excretion did not correlate with the capacity of the ΔglgC strain for bicarbonate-dependent photosynthetic O2 evolution, or with light intensity, there was, however, a positive correlation observed between the density of the starter culture prior to N-deprivation and the final extracellular pyruvate concentration. The factors that contribute to enhancement of pyruvate excretion are discussed, as well as consideration of whether the source of carbon for pyruvate excretion might be derived from

  14. Induction of pyruvate carboxylase apoenzyme and holoenzyme in 3T3-L1 cells during differentiation

    PubMed Central

    Freytag, Svend O.; Utter, Merton F.

    1980-01-01

    The specific activity of pyruvate carboxylase [pyruvate:carbon-dioxide ligase (ADP-forming); EC 6.4.1.1] in 3T3-L1 cells increases approximately 20-fold when these cells differentiate to an adipocyte-like form [Mackall, J. C. & Lane, M. D. (1977) Biochem. Biophys. Res. Commun. 79, 720-725]. A specific antibody to the purified rat liver enzyme quantitatively precipitated pyruvate carboxylase from 3T3-L1 crude homogenates. Use of this immunological technique permitted us to demonstrate that the increase in pyruvate carboxylase activity is due to an increase in the intracellular concentration of the enzyme. The content of pyruvate carboxylase in differentiated 3T3-L1 cells is sufficiently high (1-2% of total protein) that the increase in this large protein (subunit Mr = 130,000) can be visualized when 3T3-L1 crude extracts are subjected to electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. When 3T3-L1 cells differentiated in the presence of avidin, they contained less than 5% of the pyruvate carboxylase activity of cells that differentiated in the absence of avidin. However, the immunoprecipitable pyruvate carboxylase content of the avidin-treated cells was essentially the same as that of cells that differentiated without avidin. Full activity of the enzyme was rapidly restored in the avidin-treated cells upon the addition of excess biotin. The recovery of activity was closely correlated with the incorporation of [14C]biotin into immunoprecipitable pyruvate carboxylase. The rapidity with which the activity was restored and the insensitivity of the process to inhibitors of protein synthesis strongly suggest that the apoenzyme of pyruvate carboxylase accumulates during differentiation in the presence of avidin. Images PMID:6929488

  15. Genetic, Genomic, and Transcriptomic Studies of Pyruvate Metabolism in Methanosarcina barkeri Fusaro

    PubMed Central

    López Muñoz, Madeline M.; Schönheit, Peter

    2015-01-01

    ABSTRACT Pyruvate, a central intermediate in the carbon fixation pathway of methanogenic archaea, is rarely used as an energy source by these organisms. The sole exception to this rule is a genetically uncharacterized Methanosarcina barkeri mutant capable of using pyruvate as a sole energy and carbon source (the Pyr+ phenotype). Here, we provide evidence that suggests that the Pyr+ mutant is able to metabolize pyruvate by overexpressing pyruvate ferredoxin oxidoreductase (por) and mutating genes involved in central carbon metabolism. Genomic analysis showed that the Pyr+ strain has two mutations localized to Mbar_A1588, the biotin protein ligase subunit of the pyruvate carboxylase (pyc) operon, and Mbar_A2165, a putative transcriptional regulator. Mutants expressing the Mbar_A1588 mutation showed no growth defect compared to the wild type (WT), yet the strains lacked pyc activity. Recreation of the Mbar_A2165 mutation resulted in a 2-fold increase of Por activity and gene expression, suggesting a role in por transcriptional regulation. Further transcriptomic analysis revealed that Pyr+ strains also overexpress the gene encoding phosphoenolpyruvate carboxylase, indicating the presence of a previously uncharacterized route for synthesizing oxaloacetate in M. barkeri and explaining the unimpaired growth in the absence of Pyc. Surprisingly, stringent repression of the por operon was lethal, even when the media were supplemented with pyruvate and/or Casamino Acids, suggesting that por plays an unidentified essential function in M. barkeri. IMPORTANCE The work presented here reveals a complex interaction between anabolic and catabolic pathways involving pyruvate metabolism in Methanosarcina barkeri Fusaro. Among the unexpected findings were an essential role for the enzyme pyruvate-ferredoxin oxidoreductase and an alternate pathway for synthesis of oxaloacetate. These results clarify the mechanism of methanogenic catabolism of pyruvate and expand our understanding of

  16. Characterization and evolution of vertebrate indoleamine 2, 3-dioxygenases IDOs from monotremes and marsupials.

    PubMed

    Yuasa, Hajime J; Ball, Helen J; Ho, Yuen Fern; Austin, Christopher J D; Whittington, Camilla M; Belov, Katherine; Maghzal, Ghassan J; Jermiin, Lars S; Hunt, Nicholas H

    2009-06-01

    Indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that catalyze the first step in tryptophan catabolism via the kynurenine pathway. TDO is widely distributed in both eukaryotes and bacteria. In contrast, IDO has been found only in mammals and yeast. In 2007, a third enzyme, indoleamine 2,3-dioxygenase-2 (IDO2), was discovered. IDO2 is found not only in mammals but also in lower vertebrates. Interestingly, the Km value of IDO2 for L-Trp was 500-1000 fold higher than that of IDO1. In this study, we isolated both IDO1 and IDO2 cDNA from a monotreme, the platypus (Ornithorhynchus anatinus), and a marsupial, the gray short-tailed opossum (Monodelphis domestica). We characterized the recombinant proteins and those of other known IDO1/IDO2 in intact cells and a cell-free system. It was found that methylene blue may not be suitable reductant for IDO2, hence resulting in an underestimation of recombinant IDO2 activity. In intact cells, the Km value of IDO2 for L-Trp was estimated to be much higher than that of IDO1 and this high Km value appears to have been conserved during the evolution of IDO2. The protein encoded by the ancestor gene of IDO1 and IDO2 is likely to have had properties more similar to present day IDO2 than to IDO1. PMID:19402226

  17. Sll0254 (CrtLdiox) Is a Bifunctional Lycopene Cyclase/Dioxygenase in Cyanobacteria Producing Myxoxanthophyll

    PubMed Central

    Mohamed, Hatem E.; Vermaas, Wim F. J.

    2006-01-01

    Upon depletion of Sll0254 in Synechocystis sp. strain PCC 6803, cyclized carotenoids were replaced by linear, relatively hydrophilic carotenoids, and the amount of the two photosystems decreased greatly. Full segregants of the sll0254 deletion in Synechocystis were not obtained, implying that this gene is essential for survival, most likely to allow normal cell division. The N-terminal half of Sll0254 has limited similarity to the family of lycopene cyclases, has an additional dehydrogenase motif near the N terminus, and is followed by a Rieske 2Fe-2S center sequence signature. To test whether Sll0254 serves as a lycopene cyclase in Synechocystis, the corresponding gene was expressed in Escherichia coli strains that can produce lycopene or neurosporene. In the presence of Sll0254 these linear carotenoids were converted into cyclized, relatively hydrophilic pigments, with masses consistent with the introduction of two hydroxyl groups and with spectra indicative of only small changes in the number of conjugated double bonds. This suggests that Sll0254 catalyzes formation of oxygenated, cyclized carotenoids. We interpret the appearance of the hydroxyl groups in the carotenoids to be due to dioxygenase activity involving the Rieske 2Fe-2S center and the additional dehydrogenase domain. This dioxygenase activity is required in the myxoxanthophyll biosynthesis pathway, after or concomitant with cyclization on the other end of the molecule. We interpret Sll0254 to be a dual-function enzyme with both lycopene cyclase and dioxygenase activity and have named it CrtLdiox. PMID:16621828

  18. Crystal structure of 2-nitropropane dioxygenase complexed with FMN and substrate. Identification of the catalytic base.

    PubMed

    Ha, Jun Yong; Min, Ji Young; Lee, Su Kyung; Kim, Hyoun Sook; Kim, Do Jin; Kim, Kyoung Hoon; Lee, Hyung Ho; Kim, Hye Kyung; Yoon, Hye-Jin; Suh, Se Won

    2006-07-01

    Nitroalkane compounds are widely used in chemical industry and are also produced by microorganisms and plants. Some nitroalkanes have been demonstrated to be carcinogenic, and enzymatic oxidation of nitroalkanes is of considerable interest. 2-Nitropropane dioxygenases from Neurospora crassa and Williopsis mrakii (Hansenula mrakii), members of one family of the nitroalkane-oxidizing enzymes, contain FMN and FAD, respectively. The enzymatic oxidation of nitroalkanes by 2-nitropropane dioxygenase operates by an oxidase-style catalytic mechanism, which was recently shown to involve the formation of an anionic flavin semiquinone. This represents a unique case in which an anionic flavin semiquinone has been experimentally observed in the catalytic pathway for oxidation catalyzed by a flavin-dependent enzyme. Here we report the first crystal structure of 2-nitropropane dioxygenase from Pseudomonas aeruginosa in two forms: a binary complex with FMN and a ternary complex with both FMN and 2-nitropropane. The structure identifies His(152) as the proposed catalytic base, thus providing a structural framework for a better understanding of the catalytic mechanism. PMID:16682407

  19. Biochemical characteristics and inhibitor selectivity of mouse indoleamine 2,3-dioxygenase-2.

    PubMed

    Austin, Christopher Jonathan Daraius; Mailu, B M; Maghzal, G J; Sanchez-Perez, A; Rahlfs, S; Zocher, K; Yuasa, H J; Arthur, J W; Becker, K; Stocker, R; Hunt, N H; Ball, H J

    2010-07-01

    The first step in the kynurenine pathway of tryptophan catabolism is the cleavage of the 2,3-double bond of the indole ring of tryptophan. In mammals, this reaction is performed independently by indoleamine 2,3-dioxygenase-1 (IDO1), tryptophan 2,3-dioxygenase (TDO) and the recently discovered indoleamine 2,3-dioxygenase-2 (IDO2). Here we describe characteristics of a purified recombinant mouse IDO2 enzyme, including its pH stability, thermal stability and structural features. An improved assay system for future studies of recombinant/isolated IDO2 has been developed using cytochrome b (5) as an electron donor. This, the first description of the interaction between IDO2 and cytochrome b (5), provides further evidence of the presence of a physiological electron carrier necessary for activity of enzymes in the "IDO family". Using this assay, the kinetic activity and substrate range of IDO2 were shown to be different to those of IDO1. 1-Methyl-D-tryptophan, a current lead IDO inhibitor used in clinical trials, was a poor inhibitor of both IDO1 and IDO2 activity. This suggests that its immunosuppressive effect may be independent of pharmacological inhibition of IDO enzymes, in the mouse at least. The different biochemical characteristics of the mouse IDO proteins suggest that they have evolved to have distinct biological roles. PMID:20140689

  20. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs

    PubMed Central

    Driggers, Camden M; Hartman, Steven J; Karplus, P Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ∼15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases. PMID:25307852

  1. Crystal Structure of the Terminal Oxygenase Component of Cumene Dioxygenase from Pseudomonas fluorescens IP01†

    PubMed Central

    Dong, Xuesong; Fushinobu, Shinya; Fukuda, Eriko; Terada, Tohru; Nakamura, Shugo; Shimizu, Kentaro; Nojiri, Hideaki; Omori, Toshio; Shoun, Hirofumi; Wakagi, Takayoshi

    2005-01-01

    The crystal structure of the terminal component of the cumene dioxygenase multicomponent enzyme system of Pseudomonas fluorescens IP01 (CumDO) was determined at a resolution of 2.2 Å by means of molecular replacement by using the crystal structure of the terminal oxygenase component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4 (NphDO). The ligation of the two catalytic centers of CumDO (i.e., the nonheme iron and Rieske [2Fe-2S] centers) and the bridging between them in neighboring catalytic subunits by hydrogen bonds through a single amino acid residue, Asp231, are similar to those of NphDO. An unidentified external ligand, possibly dioxygen, was bound at the active site nonheme iron. The entrance to the active site of CumDO is different from the entrance to the active site of NphDO, as the two loops forming the lid exhibit great deviation. On the basis of the complex structure of NphDO, a biphenyl substrate was modeled in the substrate-binding pocket of CumDO. The residues surrounding the modeled biphenyl molecule include residues that have already been shown to be important for its substrate specificity by a number of engineering studies of biphenyl dioxygenases. PMID:15774891

  2. Identification of metabolites from phenanthrene oxidation by phenoloxidases and dioxygenases of Polyporus sp. S133.

    PubMed

    Hadibarata, Tony; Tachibana, Sanro; Askari, Muhamad

    2011-03-01

    Phenanthrene degradation by Polyporus sp. S133, a new phenanthrene-degrading strain, was investigated in this work. The analysis of degradation was performed by calculation of the remaining phenanthrene by gas chromatography-mass spectrometry. When cells were grown in phenanthrene culture after 92 h, all but 200 and 250 mg/l of the phenanthrene had been degraded. New metabolic pathways of phenanthrene and a better understanding of the phenoloxidases and dioxygenase mechanism involved in degradation of phenanthrene were explored in this research. The mechanism of degradation was determined through identification of the several metabolites; 9,10-phenanthrenequinone, 2,2'-diphenic acid, salicylic acid, and catechol. 9,10-Oxidation and ring cleavage to give 9,10-phenanthrenequinone is the major fate of phenanthrene in ligninolytic Polyporus sp. S133. The identification of 2,2'-diphenic acid in culture extracts indicates that phenanthrene was initially attacked through dioxigenation at C9 and C10 to give cis-9,10-dihydrodiol. Dehydrogenation of phenanthrene-cis-9,10-dihydrodiol to produce the corresponding diol, followed by ortho-cleavage of the oxygenated ring, produced 2,2'-diphenic acid. Several enzymes (manganese peroxidase, lignin peroxidase, laccase, 1,2-dioxygenase, and 2,3-dioxygenase) produced by Polyporus sp. S133 was detected during the incubation. The highest level of activity was shown at 92 h of culture. PMID:21464602

  3. Isolation and characterization of two novel halotolerant Catechol 2, 3-dioxygenases from a halophilic bacterial consortium

    NASA Astrophysics Data System (ADS)

    Guo, Guang; Fang, Tingting; Wang, Chongyang; Huang, Yong; Tian, Fang; Cui, Qijia; Wang, Hui

    2015-12-01

    Study of enzymes in halophiles will help to understand the mechanism of aromatic hydrocarbons degradation in saline environment. In this study, two novel catechol 2,3-dioxygenases (C23O1 and C23O2) were cloned and overexpressed from a halophilic bacterial consortium enriched from an oil-contaminated saline soil. Phylogenetic analysis indicated that the novel C23Os and their relatives formed a new branch in subfamily I.2.A of extradiol dioxygenases and the sequence differences were further analyzed by amino acid sequence alignment. Two enzymes with the halotolerant feature were active over a range of 0-30% salinity and they performed more stable at high salinity than in the absence of salt. Surface electrostatic potential and amino acids composition calculation suggested high acidic residues content, accounting for their tolerance to high salinity. Moreover, two enzymes were further characterized. The enzymes activity both increased in the presence of Fe3+, Fe2+, Cu2+ and Al3+ and showed no significant inhibition by other tested metal ions. The optimal temperatures for the C23Os were 40 °C and 60 °C and their best substrates were catechol and 4-methylcatechol respectively. As the firstly isolated and characterized catechol dioxygenases from halophiles, the two halotolerant C23Os presented novel characteristics suggesting their potential application in aromatic hydrocarbons biodegradation.

  4. Isolation and characterization of two novel halotolerant Catechol 2, 3-dioxygenases from a halophilic bacterial consortium.

    PubMed

    Guo, Guang; Fang, Tingting; Wang, Chongyang; Huang, Yong; Tian, Fang; Cui, Qijia; Wang, Hui

    2015-01-01

    Study of enzymes in halophiles will help to understand the mechanism of aromatic hydrocarbons degradation in saline environment. In this study, two novel catechol 2,3-dioxygenases (C23O1 and C23O2) were cloned and overexpressed from a halophilic bacterial consortium enriched from an oil-contaminated saline soil. Phylogenetic analysis indicated that the novel C23Os and their relatives formed a new branch in subfamily I.2.A of extradiol dioxygenases and the sequence differences were further analyzed by amino acid sequence alignment. Two enzymes with the halotolerant feature were active over a range of 0-30% salinity and they performed more stable at high salinity than in the absence of salt. Surface electrostatic potential and amino acids composition calculation suggested high acidic residues content, accounting for their tolerance to high salinity. Moreover, two enzymes were further characterized. The enzymes activity both increased in the presence of Fe(3+), Fe(2+), Cu(2+) and Al(3+) and showed no significant inhibition by other tested metal ions. The optimal temperatures for the C23Os were 40 °C and 60 °C and their best substrates were catechol and 4-methylcatechol respectively. As the firstly isolated and characterized catechol dioxygenases from halophiles, the two halotolerant C23Os presented novel characteristics suggesting their potential application in aromatic hydrocarbons biodegradation. PMID:26621792

  5. Purification and characterization of hydroquinone dioxygenase from Sphingomonas sp. strain TTNP3

    PubMed Central

    2011-01-01

    Hydroquinone-1,2-dioxygenase, an enzyme involved in the degradation of alkylphenols in Sphingomonas sp. strain TTNP3 was purified to apparent homogeneity. The extradiol dioxygenase catalyzed the ring fission of hydroquinone to 4-hydroxymuconic semialdehyde and the degradation of chlorinated and several alkylated hydroquinones. The activity of 1 mg of the purified enzyme with unsubstituted hydroquinone was 6.1 μmol per minute, the apparent Km 2.2 μM. ICP-MS analysis revealed an iron content of 1.4 moles per mole enzyme. The enzyme lost activity upon exposure to oxygen, but could be reactivated by Fe(II) in presence of ascorbate. SDS-PAGE analysis of the purified enzyme yielded two bands of an apparent size of 38 kDa and 19 kDa, respectively. Data from MALDI-TOF analyses of peptides of the respective bands matched with the deduced amino acid sequences of two neighboring open reading frames found in genomic DNA of Sphingomonas sp strain TTNP3. The deduced amino acid sequences showed 62% and 47% identity to the large and small subunit of hydroquinone dioxygenase from Pseudomonas fluorescens strain ACB, respectively. This heterotetrameric enzyme is the first of its kind found in a strain of the genus Sphingomonas sensu latu. PMID:21906340

  6. Isolation and characterization of two novel halotolerant Catechol 2, 3-dioxygenases from a halophilic bacterial consortium

    PubMed Central

    Guo, Guang; Fang, Tingting; Wang, Chongyang; Huang, Yong; Tian, Fang; Cui, Qijia; Wang, Hui

    2015-01-01

    Study of enzymes in halophiles will help to understand the mechanism of aromatic hydrocarbons degradation in saline environment. In this study, two novel catechol 2,3-dioxygenases (C23O1 and C23O2) were cloned and overexpressed from a halophilic bacterial consortium enriched from an oil-contaminated saline soil. Phylogenetic analysis indicated that the novel C23Os and their relatives formed a new branch in subfamily I.2.A of extradiol dioxygenases and the sequence differences were further analyzed by amino acid sequence alignment. Two enzymes with the halotolerant feature were active over a range of 0–30% salinity and they performed more stable at high salinity than in the absence of salt. Surface electrostatic potential and amino acids composition calculation suggested high acidic residues content, accounting for their tolerance to high salinity. Moreover, two enzymes were further characterized. The enzymes activity both increased in the presence of Fe3+, Fe2+, Cu2+ and Al3+ and showed no significant inhibition by other tested metal ions. The optimal temperatures for the C23Os were 40 °C and 60 °C and their best substrates were catechol and 4-methylcatechol respectively. As the firstly isolated and characterized catechol dioxygenases from halophiles, the two halotolerant C23Os presented novel characteristics suggesting their potential application in aromatic hydrocarbons biodegradation. PMID:26621792

  7. Oxidation of aliphatic olefins by toluene dioxygenase: enzyme rates and product identification.

    PubMed Central

    Lange, C C; Wackett, L P

    1997-01-01

    Toluene dioxygenase from Pseudomonas putida F1 has been studied extensively with aromatic substrates. The present work examined the toluene dioxygenase-catalyzed oxidation of various halogenated ethenes, propenes, butenes and nonhalogenated cis-2-pentene, an isomeric mix of 2-hexenes, cis-2-heptene, and cis-2-octene as substrates for toluene dioxygenase. Enzyme specific activities were determined for the more water-soluble C2 to C5 compounds and ranged from <4 to 52 nmol per min per mg of protein. Trichloroethene was oxidized at a rate of 33 nmol per min per mg of protein. Products from enzyme reactions were identified by gas chromatography-mass spectrometry. Proton and carbon nuclear magnetic resonance spectroscopy of compounds from whole-cell incubation confirmed the identity of products. Substrates lacking a halogen substituent on sp2 carbon atoms were dioxygenated, while those with halogen and one or more unsubstituted allylic methyl groups were monooxygenated to yield allylic alcohols. 2,3-Dichloro-1-propene, containing both a halogenated double bond and a halogenated allylic methyl group, underwent monooxygenation with allylic rearrangement to yield an isomeric mixture of cis- and trans-2,3-dichloro-2-propene-1-ol. PMID:9190800

  8. Expression Pattern and Clinicopathological Relevance of the Indoleamine 2,3-Dioxygenase 1/Tryptophan 2,3-Dioxygenase Protein in Colorectal Cancer.

    PubMed

    Chen, I-Chien; Lee, Kuen-Haur; Hsu, Ying-Hua; Wang, Wei-Ran; Chen, Chuan-Mu; Cheng, Ya-Wen

    2016-01-01

    Aims. Cancer cells use the indoleamine 2,3-dioxygenase 1 (IDO1) pathway to suppress the host's immune response in order to facilitate survival, growth, invasion, and metastasis of malignant cells. Higher IDO1 expression was shown to be involved in colorectal cancer (CRC) progression and to be correlated with impaired clinical outcome. However, the potential correlation between the expression of IDO1 in a CRC population with a low mutation rate of the APC gene remains unknown. Material and Methods. Tissues and blood samples were collected from 192 CRC patients. The expressions of IDO1, tryptophan 2,3-dioxygenase (TDO2), and beta-catenin proteins were analyzed by immunohistochemistry. Microsatellite instability (MSI) was determined by PCR amplification of microsatellite loci. Results. The results showed that high IDO1 or TDO2 protein expression was associated with characteristics of more aggressive phenotypes of CRC. For the first time, they also revealed a positive correlation between the abnormal expression of beta-catenin and IDO1 or TDO2 proteins in a CRC population with a low mutation rate of APC. Conclusion. We concluded that an IDO1-regulated molecular pathway led to abnormal expression of beta-catenin in the nucleus/cytoplasm of CRC patients with low mutation rate of APC, making IDO1 an interesting target for immunotherapy in CRC. PMID:27578919

  9. Expression Pattern and Clinicopathological Relevance of the Indoleamine 2,3-Dioxygenase 1/Tryptophan 2,3-Dioxygenase Protein in Colorectal Cancer

    PubMed Central

    Wang, Wei-Ran

    2016-01-01

    Aims. Cancer cells use the indoleamine 2,3-dioxygenase 1 (IDO1) pathway to suppress the host's immune response in order to facilitate survival, growth, invasion, and metastasis of malignant cells. Higher IDO1 expression was shown to be involved in colorectal cancer (CRC) progression and to be correlated with impaired clinical outcome. However, the potential correlation between the expression of IDO1 in a CRC population with a low mutation rate of the APC gene remains unknown. Material and Methods. Tissues and blood samples were collected from 192 CRC patients. The expressions of IDO1, tryptophan 2,3-dioxygenase (TDO2), and beta-catenin proteins were analyzed by immunohistochemistry. Microsatellite instability (MSI) was determined by PCR amplification of microsatellite loci. Results. The results showed that high IDO1 or TDO2 protein expression was associated with characteristics of more aggressive phenotypes of CRC. For the first time, they also revealed a positive correlation between the abnormal expression of beta-catenin and IDO1 or TDO2 proteins in a CRC population with a low mutation rate of APC. Conclusion. We concluded that an IDO1-regulated molecular pathway led to abnormal expression of beta-catenin in the nucleus/cytoplasm of CRC patients with low mutation rate of APC, making IDO1 an interesting target for immunotherapy in CRC. PMID:27578919

  10. Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase[W

    PubMed Central

    Catalanotti, Claudia; Dubini, Alexandra; Subramanian, Venkataramanan; Yang, Wenqiang; Magneschi, Leonardo; Mus, Florence; Seibert, Michael; Posewitz, Matthew C.; Grossman, Arthur R.

    2012-01-01

    Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H2 production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism. PMID:22353371

  11. A Novel Phenanthrene Dioxygenase from Nocardioides sp. Strain KP7: Expression in Escherichia coli

    PubMed Central

    Saito, Atsushi; Iwabuchi, Tokuro; Harayama, Shigeaki

    2000-01-01

    Nocardioides sp. strain KP7 grows on phenanthrene but not on naphthalene. This organism degrades phenanthrene via 1-hydroxy-2-naphthoate, o-phthalate, and protocatechuate. The genes responsible for the degradation of phenanthrene to o-phthalate (phd) were found by Southern hybridization to reside on the chromosome. A 10.6-kb DNA fragment containing eight phd genes was cloned and sequenced. The phdA, phdB, phdC, and phdD genes, which encode the α and β subunits of the oxygenase component, a ferredoxin, and a ferredoxin reductase, respectively, of phenanthrene dioxygenase were identified. The gene cluster, phdAB, was located 8.3 kb downstream of the previously characterized phdK gene, which encodes 2-carboxybenzaldehyde dehydrogenase. The phdCD gene cluster was located 2.9 kb downstream of the phdB gene. PhdA and PhdB exhibited moderate (less than 60%) sequence identity to the α and β subunits of other ring-hydroxylating dioxygenases. The PhdC sequence showed features of a [3Fe-4S] or [4Fe-4S] type of ferredoxin, not of the [2Fe-2S] type of ferredoxin that has been found in most of the reported ring-hydroxylating dioxygenases. PhdD also showed moderate (less than 40%) sequence identity to known reductases. The phdABCD genes were expressed poorly in Escherichia coli, even when placed under the control of strong promoters. The introduction of a Shine-Dalgarno sequence upstream of each initiation codon of the phdABCD genes improved their expression in E. coli. E. coli cells carrying phdBCD or phdACD exhibited no phenanthrene-degrading activity, and those carrying phdABD or phdABC exhibited phenanthrene-degrading activity which was significantly less than that in cells carrying the phdABCD genes. It was thus concluded that all of the phdABCD genes are necessary for the efficient expression of phenanthrene-degrading activity. The genetic organization of the phd genes, the phylogenetically diverged positions of these genes, and an unusual type of ferredoxin component

  12. Crystallization and initial X-ray diffraction analysis of human pyruvate dehydrogenase

    NASA Technical Reports Server (NTRS)

    Ciszak, E.; Korotchkina, L. G.; Hong, Y. S.; Joachimiak, A.; Patel, M. S.

    2001-01-01

    Human pyruvate dehydrogenase (E1) is a component enzyme of the pyruvate dehydrogenase complex. The enzyme catalyzes the irreversible decarboxylation of pyruvic acid and the rate-limiting reductive acetylation of the lipoyl moiety linked to the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex. E1 is an alpha(2)beta(2) tetramer ( approximately 154 kDa). Crystals of this recombinant enzyme have been grown in polyethylene glycol 3350 using a vapor-diffusion method at 295 K. The crystals are characterized as orthorhombic, space group P2(1)2(1)2(1), with unit-cell parameters a = 64.2, b = 126.9, c = 190.2 A. Crystals diffracted to a minimum d spacing of 2.5 A. The asymmetric unit contains one alpha(2)beta(2) tetrameric E1 assembly; self-rotation function analysis showed a pseudo-twofold symmetry relating the two alphabeta dimers.

  13. Ethyl Pyruvate Combats Human Leukemia Cells but Spares Normal Blood Cells.

    PubMed

    Birkenmeier, Gerd; Hemdan, Nasr Y A; Kurz, Susanne; Bigl, Marina; Pieroh, Philipp; Debebe, Tewodros; Buchold, Martin; Thieme, Rene; Wichmann, Gunnar; Dehghani, Faramarz

    2016-01-01

    Ethyl pyruvate, a known ROS scavenger and anti-inflammatory drug was found to combat leukemia cells. Tumor cell killing was achieved by concerted action of necrosis/apoptosis induction, ATP depletion, and inhibition of glycolytic and para-glycolytic enzymes. Ethyl lactate was less harmful to leukemia cells but was found to arrest cell cycle in the G0/G1 phase. Both, ethyl pyruvate and ethyl lactate were identified as new inhibitors of GSK-3β. Despite the strong effect of ethyl pyruvate on leukemia cells, human cognate blood cells were only marginally affected. The data were compiled by immune blotting, flow cytometry, enzyme activity assay and gene array analysis. Our results inform new mechanisms of ethyl pyruvate-induced cell death, offering thereby a new treatment regime with a high therapeutic window for leukemic tumors. PMID:27579985

  14. 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.

  15. Molecular structure of the pyruvate dehydrogenase complex from Escherichia coli K-12.

    PubMed

    Vogel, O; Hoehn, B; Henning, U

    1972-06-01

    The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex that can be obtained with a unique polypeptide chain composition, has a molecular weight of 3.75 x 10(6). All results obtained agree with the following numerology. The core complex consists of 48 polypeptide chains. There are 16 chains (molecular weight = 100,000) of the pyruvate dehydrogenase component, 16 chains (molecular weight = 80,000) of the dihydrolipoamide dehydrogenase component, and 16 chains (molecular weight = 56,000) of the dihydrolipoamide dehydrogenase component. Usually, but not always, pyruvate dehydrogenase complex is produced in vivo containing at least 2-3 mol more of dimers of the pyruvate dehydrogenase component than the stoichiometric ratio with respect to the core complex. This "excess" component is bound differently than are the eight dimers in the core complex. PMID:4556465

  16. Molecular Structure of the Pyruvate Dehydrogenase Complex from Escherichia coli K-12

    PubMed Central

    Vogel, Otto; Hoehn, Barbara; Henning, Ulf

    1972-01-01

    The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex that can be obtained with a unique polypeptide chain composition, has a molecular weight of 3.75 × 106. All results obtained agree with the following numerology. The core complex consists of 48 polypeptide chains. There are 16 chains (molecular weight = 100,000) of the pyruvate dehydrogenase component, 16 chains (molecular weight = 80,000) of the dihydrolipoamide dehydrogenase component, and 16 chains (molecular weight = 56,000) of the dihydrolipoamide dehydrogenase component. Usually, but not always, pyruvate dehydrogenase complex is produced in vivo containing at least 2-3 mol more of dimers of the pyruvate dehydrogenase component than the stoichiometric ratio with respect to the core complex. This “excess” component is bound differently than are the eight dimers in the core complex. Images PMID:4556465

  17. A Simple Experiment Demonstrating the Allosteric Regulation of Yeast Pyruvate Kinase.

    ERIC Educational Resources Information Center

    Taber, Richard L.; Campbell, Angela; Spencer, Scott

    1998-01-01

    Explains the procedures used to determine the regulatory properties of yeast pyruvate kinase. Involves a partial purification using PEG precipitation that can be done in one laboratory period with simple equipment. (DDR)

  18. Pyruvate Occupancy in the Carboxyl Transferase Domain of Pyruvate Carboxylase Facilitates Product Release from the Biotin Carboxylase Domain through an Intermolecular Mechanism.

    PubMed

    Westerhold, Lauren E; Adams, Stephanie L; Bergman, Hanna L; Zeczycki, Tonya N

    2016-06-21

    Protein structure, ligand binding, and catalytic turnover contributes to the governance of catalytic events occurring at spatially distinct domains in multifunctional enzymes. Coordination of these catalytic events partially rests on the ability of spatially discrete active sites to communicate with other allosteric and active sites on the same polypeptide chain (intramolecular) or on different polypeptide chains (intermolecular) within the holoenzyme. Often, communication results in long-range effects on substrate binding or product release. For example, pyruvate binding to the carboxyl transferase (CT) domain of pyruvate carboxylase (PC) increases the rate of product release in the biotin carboxylase (BC) domain. In order to address how CT domain ligand occupancy is "sensed" by other domains, we generated functional, mixed hybrid tetramers using the E218A (inactive BC domain) and T882S (low pyruvate binding, low activity) mutant forms of PC. The apparent Ka pyruvate for the pyruvate-stimulated release of Pi catalyzed by the T882S:E218A[1:1] hybrid tetramer was comparable to the wild-type enzyme and nearly 10-fold lower than that for the T882S homotetramer. In addition, the ratio of the rates of oxaloacetate formation to Pi release for the WT:T882S[1:1] and E218A:T882S[1:1] hybrid tetramer-catalyzed reactions was 0.5 and 0.6, respectively, while the T882S homotetramer exhibited a near 1:1 coupling of the two domains, suggesting that the mechanisms coordinating catalytic events is more complicated that we initially assumed. The results presented here are consistent with an intermolecular communication mechanism, where pyruvate binding to the CT domain is "sensed" by domains on a different polypeptide chain within the tetramer. PMID:27254467

  19. Robust hyperpolarized (13)C metabolic imaging with selective non-excitation of pyruvate (SNEP).

    PubMed

    Chen, Way Cherng; Teo, Xing Qi; Lee, Man Ying; Radda, George K; Lee, Philip

    2015-08-01

    In vivo metabolic imaging using hyperpolarized [1-(13)C]pyruvate provides localized biochemical information and is particularly useful in detecting early disease changes, as well as monitoring disease progression and treatment response. However, a major limitation of hyperpolarized magnetization is its unrecoverable decay, due not only to T1 relaxation but also to radio-frequency (RF) excitation. RF excitation schemes used in metabolic imaging must therefore be able to utilize available hyperpolarized magnetization efficiently and robustly for the optimal detection of substrate and metabolite activities. In this work, a novel RF excitation scheme called selective non-excitation of pyruvate (SNEP) is presented. This excitation scheme involves the use of a spectral selective RF pulse to specifically exclude the excitation of [1-(13)C]pyruvate, while uniformly exciting the key metabolites of interest (namely [1-(13)C]lactate and [1-(13)C]alanine) and [1-(13)C]pyruvate-hydrate. By eliminating the loss of hyperpolarized [1-(13)C]pyruvate magnetization due to RF excitation, the signal from downstream metabolite pools is increased together with enhanced dynamic range. Simulation results, together with phantom measurements and in vivo experiments, demonstrated the improvement in signal-to-noise ratio (SNR) and the extension of the lifetime of the [1-(13)C]lactate and [1-(13)C]alanine pools when compared with conventional non-spectral selective (NS) excitation. SNEP has also been shown to perform comparably well with multi-band (MB) excitation, yet SNEP possesses distinct advantages, including ease of implementation, less stringent demands on gradient performance, increased robustness to frequency drifts and B0 inhomogeneity as well as easier quantification involving the use of [1-(13)C]pyruvate-hydrate as a proxy for the actual [1-(13)C] pyruvate signal. SNEP is therefore a promising alternative for robust hyperpolarized [1-(13)C]pyruvate metabolic imaging with high

  20. Isoenzyme of Pyruvate Kinase in Proplastids from Developing Castor Bean Endosperm 1

    PubMed Central

    De Luca, Vincenzo; Dennis, David T.

    1978-01-01

    Proplastids from developing castor bean (Ricinus communis) endosperm have a pyruvate kinase activity which is extremely unstable on isolation from the organelle. It can be stabilized by 20 mm 2-mercaptoethanol in 20% ethylene glycol. In contrast the soluble pyruvate kinase is stable at 60 C for 10 minutes. The two activities have different pH optima. The soluble and the proplastid activities are eluted from a diethylaminoethyl-Sephadex A-25 sievorptive column at different ionic strengths. PMID:16660412

  1. Association of phosphoenolpyruvate phosphatase activity with the cytosolic pyruvate kinase of germinating mung beans.

    PubMed

    Podestá, F E; Plaxton, W C

    1991-12-01

    The procedure of Malhotra and Kayastha ([1990] Plant Physiology 93: 194-200) for the purification to homogeneity of a phosphoenolpyruvate-specific alkaline phosphatase (PEP phosphatase) from germinating mung beans (Vigna radiata) was followed. Although a higher specific activity of 1.4 micromoles pyruvate produced per minute per milligram protein was obtained, the final preparation was less than 10% pure as judged by polyacrylamide gel electrophoresis. Attempts to further purify the enzyme resulted in loss of activity. The partially purified enzyme contained significant pyruvate kinase activity (0.13 micromole pyruvate produced per minute per milligram protein) when assayed at pH 7.2, but not at pH 8.5. The PEP phosphatase activity of the final preparation exhibited hysteresis; a lag time of 5 to 6 minutes was required before a steady-state reaction rate was attained. A western blot of the final preparation revealed an immunoreactive 57 kilodalton polypeptide when probed with monospecific rabbit polyclonal antibodies prepared against germinating castor bean cytosolic pyruvate kinase. No antigenic cross-reaction of the final preparation was observed with antibodies against castor bean leucoplast pyruvate kinase, or black mustard PEP-specific acid phosphatase. Nondenaturing polyacrylamide gel electrophoresis of the final preparation resulted in a single PEP phosphatase activity band; when this band was excised and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting, a 57 kilodalton silver-staining polypeptide was obtained that strongly cross-reacted with the anti-(cytosolic pyruvate kinase) immunoglobulin G. It is suggested that mung bean PEP-specific alkaline phosphatase activity is due to cytosolic pyruvate kinase, in which pyruvate and ortho-phosphate are formed in the absence of ADP. PMID:16668551

  2. Association of Phosphoenolpyruvate Phosphatase Activity with the Cytosolic Pyruvate Kinase of Germinating Mung Beans 1

    PubMed Central

    Podestá, Florencio E.; Plaxton, William C.

    1991-01-01

    The procedure of Malhotra and Kayastha ([1990] Plant Physiology 93: 194-200) for the purification to homogeneity of a phosphoenolpyruvate-specific alkaline phosphatase (PEP phosphatase) from germinating mung beans (Vigna radiata) was followed. Although a higher specific activity of 1.4 micromoles pyruvate produced per minute per milligram protein was obtained, the final preparation was less than 10% pure as judged by polyacrylamide gel electrophoresis. Attempts to further purify the enzyme resulted in loss of activity. The partially purified enzyme contained significant pyruvate kinase activity (0.13 micromole pyruvate produced per minute per milligram protein) when assayed at pH 7.2, but not at pH 8.5. The PEP phosphatase activity of the final preparation exhibited hysteresis; a lag time of 5 to 6 minutes was required before a steady-state reaction rate was attained. A western blot of the final preparation revealed an immunoreactive 57 kilodalton polypeptide when probed with monospecific rabbit polyclonal antibodies prepared against germinating castor bean cytosolic pyruvate kinase. No antigenic cross-reaction of the final preparation was observed with antibodies against castor bean leucoplast pyruvate kinase, or black mustard PEP-specific acid phosphatase. Nondenaturing polyacrylamide gel electrophoresis of the final preparation resulted in a single PEP phosphatase activity band; when this band was excised and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting, a 57 kilodalton silver-staining polypeptide was obtained that strongly cross-reacted with the anti-(cytosolic pyruvate kinase) immunoglobulin G. It is suggested that mung bean PEP-specific alkaline phosphatase activity is due to cytosolic pyruvate kinase, in which pyruvate and ortho-phosphate are formed in the absence of ADP. ImagesFigure 1Figure 2 PMID:16668551

  3. Slow-binding inhibition of the Escherichia coli pyruvate dehydrogenase multienzyme complex by acetylphosphinate.

    PubMed

    Schönbrunn-Hanebeck, E; Laber, B; Amrhein, N

    1990-05-22

    The pyruvate analogue acetylphosphinate (CH3-CO-PO2H2) inhibits the pyruvate dehydrogenase component (E1) of the Escherichia coli pyruvate dehydrogenase multienzyme complex in a time-dependent process with biphasic reaction kinetics. The formation of an initial, rapidly reversible enzyme-inhibitor complex (EI) with an apparent Ki of 0.12 +/- 0.025 microM is followed by the conversion to a tighter complex (EI) at a maximal rate of k3 = 0.87 +/- 0.34 min-1. The inhibition is reversible (dissociation rate constant k4 = 0.038 +/- 0.002 min-1), requires the presence of the cofactors thiamin pyrophosphate and Mg2+, and is competitive with regard to pyruvate. The microscopic rate constants give a value of 5 nM for the overall dissociation constant [Ki = [E] [I]/[( EI] + [EI]) = Kik4/(k3 + k4)] compared with values of 10 and 3.5 nM obtained by steady-state methods. Thus acetylphosphinate binds by 5 orders of magnitude more tightly to pyruvate dehydrogenase than does pyruvate (Km = 0.35 mM). Acetylphosphinate also affects the pyruvate dehydrogenase complex fluorescence when excited at 290 nm in a time-dependent manner with a maximal rate constant of 0.99 min-1, suggesting a conformational change in the enzyme complex as the slow step in conversion of EI to EI (k3). All these features taken together suggest that the interaction of the pyruvate dehydrogenase with acetylphosphinate involves the formation of a thiamin pyrophosphate-acetylphosphinate adduct that resembles the normal reaction intermediate, 2-(1-carboxy-1-hydroxyethyl)thiamin pyrophosphate (alpha-lactylthiamin pyrophosphate). PMID:2194562

  4. Genetic Analysis of Dioxin Dioxygenase of Sphingomonas sp. Strain RW1: Catabolic Genes Dispersed on the Genome

    PubMed Central

    Armengaud, Jean; Happe, Birgitta; Timmis, Kenneth N.

    1998-01-01

    The dioxin dioxygenase of Sphingomonas sp. strain RW1 activates dibenzo-p-dioxin and dibenzofuran for further metabolism by introducing two atoms of oxygen at a pair of vicinal carbon atoms, one of which is involved in one of the bridges between the two aromatic rings, i.e., an angular dioxygenation. The dxnA1 and dxnA2 cistrons encoding this dioxygenase have been cloned and shown to be located just upstream of a hydrolase gene which specifies an enzyme involved in the subsequent step of the dibenzofuran biodegradative pathway. Genes encoding the electron supply system of the dioxygenase are not clustered with the dioxygenase gene but rather are located on two other distinct and separate genome segments. Moreover, whereas expression of dxnA1A2 is modulated according to the available carbon source, expression of the dbfB gene encoding the ring cleavage enzyme of the dibenzofuran pathway, which is located in the neighborhood of dxnA1A2 but oriented in the opposite direction, is constitutive. The scattering of genes for the component proteins of dioxin dioxygenase system around the genome of Sphingomonas sp. strain RW1, and the differential expression of dioxin pathway genes, is unusual and contrasts with the typical genetic organization of catabolic pathways where component cistrons tend to be clustered in multicistronic transcriptional units. The sequences of the α and β subunits of the dioxin dioxygenase exhibit only weak similarity to other three component dioxygenases, but some motifs such as the Fe(II) binding site and the [2Fe-2S] cluster ligands are conserved. Dioxin dioxygenase activity in Escherichia coli cells containing the cloned dxnA1A2 gene was achieved only through coexpression of the cognate electron supply system from RW1. Under these conditions, exclusively angular dioxygenation of dibenzofuran and dibenzo-p-dioxin was obtained. The dioxin dioxygenase was not active in E. coli cells coexpressing a class IIB electron supply system. In the course of

  5. Pyruvate dehydrogenase kinase regulates hepatitis C virus replication.

    PubMed

    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

  6. 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

  7. Human pyruvate kinase M2: a multifunctional protein.

    PubMed

    Gupta, Vibhor; Bamezai, Rameshwar N K

    2010-11-01

    Glycolysis, a central metabolic pathway, harbors evolutionary conserved enzymes that modulate and potentially shift the cellular metabolism on requirement. Pyruvate kinase, which catalyzes the last but rate-limiting step of glycolysis, is expressed in four isozymic forms, depending on the tissue requirement. M2 isoform (PKM2) is exclusively expressed in embryonic and adult dividing/tumor cells. This tetrameric allosterically regulated isoform is intrinsically designed to downregulate its activity by subunit dissociation (into dimer), which results in partial inhibition of glycolysis at the last step. This accumulates all upstream glycolytic intermediates as an anabolic feed for synthesis of lipids and nucleic acids, whereas reassociation of PKM2 into active tetramer replenishes the normal catabolism as a feedback after cell division. In addition, involvement of this enzyme in a variety of pathways, protein-protein interactions, and nuclear transport suggests its potential to perform multiple nonglycolytic functions with diverse implications, although multidimensional role of this protein is as yet not fully explored. This review aims to provide an overview of the involvement of PKM2 in various physiological pathways with possible functional implications. PMID:20857498

  8. Pyruvate Kinase M2: A Potential Target for Regulating Inflammation

    PubMed Central

    Alves-Filho, Jose C.; Pålsson-McDermott, Eva M.

    2016-01-01

    Pyruvate kinase (PK) is the enzyme responsible for catalyzing the last step of glycolysis. Of the four PK isoforms expressed in mammalian cells, PKM2 has generated the most interest due to its impact on changes in cellular metabolism observed in cancer as well as in activated immune cells. As our understanding of dysregulated metabolism in cancer develops, and in light of the growing field of immunometabolism, intense efforts are in place to define the mechanism by which PKM2 regulates the metabolic profile of cancer as well as of immune cells. The enzymatic activity of PKM2 is heavily regulated by endogenous allosteric effectors as well as by intracellular signaling pathways, affecting both the enzymatic activity of PKM2 as a PK and the regulation of the recently described non-canonical nuclear functions of PKM2. We here review the current literature on PKM2 and its regulation, and discuss the potential for this protein as a therapeutic target in inflammatory disorders. PMID:27148264

  9. Ethanolic fermentation in transgenic tobacco expressing Zymomonas mobilis pyruvate decarboxylase.

    PubMed Central

    Bucher, M; Brändle, R; Kuhlemeier, C

    1994-01-01

    During oxygen limitation in higher plants, energy metabolism switches from respiration to fermentation. As part of this anaerobic response the expression of genes encoding pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) is strongly induced. In addition there is ample evidence for post-translational regulation. In order to understand this multi-level regulation of the anaerobic response, we provided tobacco with the constitutive capacity of ethanolic fermentation by expressing a PDC gene derived from the obligate anaerobe Zymomonas mobilis. The protein accumulated to high levels and was active in an in vitro assay. During the first 2-4 h of anoxia, acetaldehyde accumulated to 10- to 35-fold and ethanol to 8- to 20-fold higher levels than in wild-type. Under normoxic conditions no accumulation of acetaldehyde and ethanol could be measured. Instead, the two products may be immediately re-metabolized in tobacco leaf tissue. We show that aerobic fermentation takes place when the respiratory system is inhibited. Although these conditions enhance ethanolic fermentation under normoxia, they fail to increase ADH transcript levels. These results indicate that anaerobic transcription is triggered not by the metabolic consequences of oxygen limitation, but directly through an oxygen-sensing system. Images PMID:8026460

  10. Phylogeny congruence analysis and isozyme classification: the pyruvate kinase system.

    PubMed

    Guderley, H; Fournier, P; Auclair, J C

    1989-09-22

    As the isozymes of pyruvate kinase (PK) are best known in rats, the characteristics of the rat isozymes are generally used to classify the PK isozymes in other species. Given the discrepancies generated by this classification by analogy, we evaluated a classification using a phylogeny congruence analysis of the compositional relatedness of vertebrate PK's. While our phylogenetic analysis confirmed the well established separation of the L and R isozymes from the K and M isozymes, its power became most evident in the identification of non-orthologous (or variant) forms of PK. Our analysis emphasized the uniqueness of chicken liver PK which cannot be classified either as a K or an L isozyme, confirmed that tumors express a variety of forms of PK, and indicated that lungs systematically express PK's which are not orthologous with PK's from other tissues. The determination of orthology by the phylogeny congruence analysis assumes that the structural data from different sources are subject to similar methodological error. However, we cannot reject the possibility that an apparent lack of orthology be due to artifacts during purification and analysis. PMID:2615396

  11. Structural Basis for Glycyl Radical Formation By Pyruvate Formate-Lyase Activating Enzyme

    SciTech Connect

    Vey, J.L.; Yang, J.; Li, M.; Broderick, W.E.; Broderick, J.B.; Drennan, C.L.

    2009-05-26

    Pyruvate formate-lyase activating enzyme generates a stable and catalytically essential glycyl radical on G{sup 734} of pyruvate formate-lyase via the direct, stereospecific abstraction of a hydrogen atom from pyruvate formate-lyase. The activase performs this remarkable feat by using an iron-sulfur cluster and S-adenosylmethionine (AdoMet), thus placing it among the AdoMet radical superfamily of enzymes. We report here structures of the substrate-free and substrate-bound forms of pyruvate formate-lyase-activating enzyme, the first structures of an AdoMet radical activase. To obtain the substrate-bound structure, we have used a peptide substrate, the 7-mer RVSGYAV, which contains the sequence surrounding G{sup 734}. Our structures provide fundamental insights into the interactions between the activase and the G{sup 734} loop of pyruvate formate-lyase and provide a structural basis for direct and stereospecific H atom abstraction from the buried G{sup 734}4 of pyruvate formate-lyase.

  12. Pyruvate kinases of salmon: purification and comparison with the isozymes from birds and mammals.

    PubMed

    Guderley, H; Cardenas, J M

    1980-02-01

    Pyruvate kinase occurs as two major forms in coho salmon; the type M isozyme occurs primarily in muscle and heart, but type K has a more generalized tissue distribution, in parallel with the type K isozyme in other vertebrate systems. In order to assess the evolutionary relationships among the fish, avian, and mammalian isozymes of pyruvate kinase, we have purified the two isozymes from fish, have examined some of their physical properties, and have studied their immunological relationships to the avian and mammalian isozymes. Salmon type K is at least partially inactivated by antibody to bivine type L pyruvate kinase as well as by antibodies produced against chicken, bovine, and salmon type M isozymes. Salmon type M pyruvate kinase, on the other hand, is not significantly corss-reactive with the bovine type L isozyme, but is at least partially inactivated by antibodies produced against bovine or chicken type M isozymes. Mammalian type L pyruvate kinase is immunologically distinct from either mammalian type K or type M, but salmon type K has some structural features in common with all three mammalian isozymes. Thus, salmon fish type K pyruvate kinase could be similar to a primordial form that was antecedent to the three major differentiated isozymes of higher vertebrates. PMID:7373271

  13. Characterization and evolution of vertebrate indoleamine 2, 3-dioxygenases IDOs from monotremes and marsupials.

    PubMed

    Yuasa, Hajime J; Ball, Helen J; Ho, Yuen Fern; Austin, Christopher J D; Whittington, Camilla M; Belov, Katherine; Maghzal, Ghassan J; Jermiin, Lars S; Hunt, Nicholas H

    2009-06-01

    Indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that catalyze the first step in tryptophan catabolism via the kynurenine pathway. TDO is widely distributed in both eukaryotes and bacteria. In contrast, IDO has been found only in mammals and yeast. In 2007, a third enzyme, indoleamine 2,3-dioxygenase-2 (IDO2), was discovered. IDO2 is found not only in mammals but also in lower vertebrates. Interestingly, the K(m) value of IDO2 for L-Trp was 500-1000 fold higher than that of IDO1. In this study, we isolated both IDO1 and IDO2 cDNA from a monotreme, the platypus (Ornithorhynchus anatinus), and a marsupial, the gray short-tailed opossum (Monodelphis domestica). We characterized the recombinant proteins and those of other known IDO1/IDO2 in intact cells and a cell-free system. It was found that methylene blue may not be suitable reductant for IDO2, hence resulting in an underestimation of recombinant IDO2 activity. In intact cells, the K(m) value of IDO2 for L-Trp was estimated to be much higher than that of IDO1 and this high K(m) value appears to have been conserved during the evolution of IDO2. The protein encoded by the ancestor gene of IDO1 and IDO2 is likely to have had properties more similar to present day IDO2 than to IDO1. PMID:19416693

  14. Structure of the 2,4′-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP

    PubMed Central

    Keegan, R.; Lebedev, A.; Erskine, P.; Guo, J.; Wood, S. P.; Hopper, D. J.; Rigby, S. E. J.; Cooper, J. B.

    2014-01-01

    The enzyme 2,4′-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2,4′-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C—C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits, each containing nonhaem iron, and its sequence suggests that it belongs to the cupin family of dioxygenases. In this paper, the first X-ray structure of a DAD enzyme from the Gram-negative bacterium Alcaligenes sp. 4HAP is reported, at a resolution of 2.2 Å. The structure establishes that the enzyme adopts a cupin fold, forming dimers with a pronounced hydrophobic interface between the monomers. The catalytic iron is coordinated by three histidine residues (76, 78 and 114) within a buried active-site cavity. The iron also appears to be tightly coordinated by an additional ligand which was putatively assigned as a carbonate dianion since this fits the electron density optimally, although it might also be the product formate. The modelled carbonate is located in a position which is highly likely to be occupied by the α-hydroxyketone group of the bound substrate during catalysis. Modelling of a substrate molecule in this position indicates that it will interact with many conserved amino acids in the predominantly hydrophobic active-site pocket where it undergoes peroxide radical-mediated heterolysis. PMID:25195757

  15. Exploring De Novo metabolic pathways from pyruvate to propionic acid.

    PubMed

    Stine, Andrew; Zhang, Miaomin; Ro, Soo; Clendennen, Stephanie; Shelton, Michael C; Tyo, Keith E J; Broadbelt, Linda J

    2016-03-01

    Industrial biotechnology provides an efficient, sustainable solution for chemical production. However, designing biochemical pathways based solely on known reactions does not exploit its full potential. Enzymes are known to accept non-native substrates, which may allow novel, advantageous reactions. We have previously developed a computational program named Biological Network Integrated Computational Explorer (BNICE) to predict promiscuous enzyme activities and design synthetic pathways, using generalized reaction rules curated from biochemical reaction databases. Here, we use BNICE to design pathways synthesizing propionic acid from pyruvate. The currently known natural pathways produce undesirable by-products lactic acid and succinic acid, reducing their economic viability. BNICE predicted seven pathways containing four reaction steps or less, five of which avoid these by-products. Among the 16 biochemical reactions comprising these pathways, 44% were validated by literature references. More than 28% of these known reactions were not in the BNICE training dataset, showing that BNICE was able to predict novel enzyme substrates. Most of the pathways included the intermediate acrylic acid. As acrylic acid bioproduction has been well advanced, we focused on the critical step of reducing acrylic acid to propionic acid. We experimentally validated that Oye2p from Saccharomyces cerevisiae can catalyze this reaction at a slow turnover rate (10(-3) s(-1) ), which was unknown to occur with this enzyme, and is an important finding for further propionic acid metabolic engineering. These results validate BNICE as a pathway-searching tool that can predict previously unknown promiscuous enzyme activities and show that computational methods can elucidate novel biochemical pathways for industrial applications. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:303-311, 2016. PMID:26821575

  16. Structure of the 2, 4′-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP

    SciTech Connect

    Keegan, R.; Lebedev, A.; Erskine, P.; Guo, J.; Wood, S. P.; Hopper, D. J.; Rigby, S. E. J.; Cooper, J. B.

    2014-09-01

    The first X-ray structure of a 2, 4′-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP at a resolution of 2.2 Å is reported. This structure establishes that the enzyme adopts the cupin-fold, forming compact dimers with a pronounced hydrophobic interface between the monomers. Each monomer possesses a catalytic ferrous iron that is coordinated by three histidines (76, 78 and 114) and an additional ligand which has been putatively assigned as a carbonate, although formate and acetate are possibilities. The enzyme 2, 4′-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2, 4′-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C—C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits, each containing nonhaem iron, and its sequence suggests that it belongs to the cupin family of dioxygenases. In this paper, the first X-ray structure of a DAD enzyme from the Gram-negative bacterium Alcaligenes sp. 4HAP is reported, at a resolution of 2.2 Å. The structure establishes that the enzyme adopts a cupin fold, forming dimers with a pronounced hydrophobic interface between the monomers. The catalytic iron is coordinated by three histidine residues (76, 78 and 114) within a buried active-site cavity. The iron also appears to be tightly coordinated by an additional ligand which was putatively assigned as a carbonate dianion since this fits the electron density optimally, although it might also be the product formate. The modelled carbonate is located in a position which is highly likely to be occupied by the α-hydroxyketone group of the bound substrate during catalysis. Modelling of a substrate molecule in this position indicates that it will interact with many conserved amino acids in

  17. Metabolism of Doubly para-Substituted Hydroxychlorobiphenyls by Bacterial Biphenyl Dioxygenases

    PubMed Central

    Pham, Thi Thanh My; Sondossi, Mohammad

    2015-01-01

    In this work, we examined the profile of metabolites produced from the doubly para-substituted biphenyl analogs 4,4′-dihydroxybiphenyl, 4-hydroxy-4′-chlorobiphenyl, 3-hydroxy-4,4′-dichlorobiphenyl, and 3,3′-dihydroxy-4,4′-chlorobiphenyl by biphenyl-induced Pandoraea pnomenusa B356 and by its biphenyl dioxygenase (BPDO). 4-Hydroxy-4′-chlorobiphenyl was hydroxylated principally through a 2,3-dioxygenation of the hydroxylated ring to generate 2,3-dihydro-2,3,4-trihydroxy-4′-chlorobiphenyl and 3,4-dihydroxy-4′-chlorobiphenyl after the removal of water. The former was further oxidized by the biphenyl dioxygenase to produce ultimately 3,4,5-trihydroxy-4′-chlorobiphenyl, a dead-end metabolite. 3-Hydroxy-4,4′-dichlorobiphenyl was oxygenated on both rings. Hydroxylation of the nonhydroxylated ring generated 2,3,3′-trihydroxy-4′-chlorobiphenyl with concomitant dechlorination, and 2,3,3′-trihydroxy-4′-chlorobiphenyl was ultimately metabolized to 2-hydroxy-4-chlorobenzoate, but hydroxylation of the hydroxylated ring generated dead-end metabolites. 3,3′-Dihydroxy-4,4′-dichlorobiphenyl was principally metabolized through a 2,3-dioxygenation to generate 2,3-dihydro-2,3,3′-trihydroxy-4,4′-dichlorobiphenyl, which was ultimately converted to 3-hydroxy-4-chlorobenzoate. Similar metabolites were produced when the biphenyl dioxygenase of Burkholderia xenovorans LB400 was used to catalyze the reactions, except that for the three substrates used, the BPDO of LB400 was less efficient than that of B356, and unlike that of B356, it was unable to further oxidize the initial reaction products. Together the data show that BPDO oxidation of doubly para-substituted hydroxychlorobiphenyls may generate nonnegligible amounts of dead-end metabolites. Therefore, biphenyl dioxygenase could produce metabolites other than those expected, corresponding to dihydrodihydroxy metabolites from initial doubly para-substituted substrates. This finding shows that a clear

  18. The second enzyme in pyrrolnitrin biosynthetic pathway is related to the heme-dependent dioxygenase superfamily†

    PubMed Central

    De Laurentis, Walter; Khim, Leang; Anderson, J.L. Ross; Adam, Ariane; Johnson, Kenneth A.; Phillips, Robert S.; Chapman, Stephen K.; van Pee, Karl-Heinz; Naismith, James H.

    2012-01-01

    Pyrrolnitrin is a commonly used and clinically effective treatment for fungal infections and provides the structural basis for the more widely used fludioxinil. The pyrrolnitrin biosynthetic pathway consists of four chemical steps, the second of which is the rearrangement of 7-chloro-tryptophan by the enzyme PrnB, a reaction that is so far unprecedented in biochemistry. When expressed in Pseudomonas fluorescens, PrnB is red in color due to the fact that it contains 1 mole of heme b per mole of protein. The crystal structure unexpectedly establishes PrnB as a member of the heme-dependent dioxygenase superfamily with significant structural but not sequence homology to the two-domain indoleamine 2,3-dioxygenase enzyme (IDO). The heme-binding domain is also structurally similar to that of tryptophan 2,3-dioxygenase (TDO). Here we report the binary complex structures of PrnB with D- and L-tryptophan and D- and L-chloro-tryptophan. The structures identify a common hydrophobic pocket for the indole ring but exhibit unusual heme ligation and substrate binding when compared with that observed in the TDO crystal structures. Our solution studies support the heme ligation observed in the crystal structures. Purification of the hexahistidine-tagged PrnB yields homogeneous protein that only displays in vitro activity with 7-chloro-L-tryptophan after reactivation with crude extract from the host strain, suggesting that an as yet unknown cofactor is required for activity. Mutation of the proximal heme ligand results, not surprisingly, in inactive enzyme. Redox titrations show that PrnB displays a significantly different reduction potential to that of IDO or TDO, indicating possible differences in the PrnB catalytic cycle. This is confirmed by the absence of tryptophan dioxygenase activity in PrnB, although a stable oxyferrous adduct (which is the first intermediate in the TDO/IDO catalytic cycle) can be generated. We propose that PrnB shares a key catalytic step with TDO and IDO

  19. Trichloroethylene removal and oxidation toxicity mediated by toluene dioxygenase of Pseudomonas putida

    SciTech Connect

    Heald, S.; Jenkins, R.O.

    1994-12-01

    Trichloroethylene (TCE) is a major ground water contaminant and potential health hazard in drinking water. This paper reports on the cometabolism of TCE by a wild-type strain of Pseudomonas putida containing an inducible toluene dioxygenase enzyme. The results show rapid TCE removal by the strain but severe oxidation toxicity and rapid cell death. This is also the first report of enhanced capacity of bacterial cells to remove TCE in the presence of dithiothreitol. Presented also is evidence for induction of toluene degradation by TCE. 17 refs., 2 figs., 2 tabs.

  20. Metabolism of Doubly para-Substituted Hydroxychlorobiphenyls by Bacterial Biphenyl Dioxygenases.

    PubMed

    Pham, Thi Thanh My; Sondossi, Mohammad; Sylvestre, Michel

    2015-07-01

    In this work, we examined the profile of metabolites produced from the doubly para-substituted biphenyl analogs 4,4'-dihydroxybiphenyl, 4-hydroxy-4'-chlorobiphenyl, 3-hydroxy-4,4'-dichlorobiphenyl, and 3,3'-dihydroxy-4,4'-chlorobiphenyl by biphenyl-induced Pandoraea pnomenusa B356 and by its biphenyl dioxygenase (BPDO). 4-Hydroxy-4'-chlorobiphenyl was hydroxylated principally through a 2,3-dioxygenation of the hydroxylated ring to generate 2,3-dihydro-2,3,4-trihydroxy-4'-chlorobiphenyl and 3,4-dihydroxy-4'-chlorobiphenyl after the removal of water. The former was further oxidized by the biphenyl dioxygenase to produce ultimately 3,4,5-trihydroxy-4'-chlorobiphenyl, a dead-end metabolite. 3-Hydroxy-4,4'-dichlorobiphenyl was oxygenated on both rings. Hydroxylation of the nonhydroxylated ring generated 2,3,3'-trihydroxy-4'-chlorobiphenyl with concomitant dechlorination, and 2,3,3'-trihydroxy-4'-chlorobiphenyl was ultimately metabolized to 2-hydroxy-4-chlorobenzoate, but hydroxylation of the hydroxylated ring generated dead-end metabolites. 3,3'-Dihydroxy-4,4'-dichlorobiphenyl was principally metabolized through a 2,3-dioxygenation to generate 2,3-dihydro-2,3,3'-trihydroxy-4,4'-dichlorobiphenyl, which was ultimately converted to 3-hydroxy-4-chlorobenzoate. Similar metabolites were produced when the biphenyl dioxygenase of Burkholderia xenovorans LB400 was used to catalyze the reactions, except that for the three substrates used, the BPDO of LB400 was less efficient than that of B356, and unlike that of B356, it was unable to further oxidize the initial reaction products. Together the data show that BPDO oxidation of doubly para-substituted hydroxychlorobiphenyls may generate nonnegligible amounts of dead-end metabolites. Therefore, biphenyl dioxygenase could produce metabolites other than those expected, corresponding to dihydrodihydroxy metabolites from initial doubly para-substituted substrates. This finding shows that a clear picture of the fate of polychlorinated

  1. Compound-Specific Isotope Analysis of Nitroaromatic Contaminant Transformations by Nitroarene Dioxygenases

    NASA Astrophysics Data System (ADS)

    Pati, Sarah G.; Kohler, Hans-Peter E.; Hofstetter, Thomas B.

    2014-05-01

    Dioxygenation is an important biochemical reaction that often initiates the mineralization of recalcitrant organic contaminants such as nitroaromatic explosives, chlorinated benzenes, and polycyclic aromatic hydrocarbons. However, to assess the extent of dioxygenation in contaminated environments is difficult because of competing transformation processes and further reactions of the dioxygenation products. Compound-specific isotope analysis (CSIA) offers a new approach to reliably quantify biodegradation initiated by dioxygenation based on changes in stable isotope ratios of the pollutant. For CSIA it is essential to know the kinetic isotope effects (KIEs) pertinent to the dioxygenation mechanism of organic contaminants. Unfortunately, the range of KIEs of such reactions is poorly constrained although many dioxygenase enzymes with a broad substrate specificity have been reported. Dioxygenase enzymes usually exhibit complex reaction kinetics involving multiple substrates and substrate-specific binding modes which makes the determination of KIEs challenging. The goal of this study was to explore the magnitude and variability of 13C-, 2H-, and 15N-KIEs for the dioxygenation of one contaminant class, that is nitroaromatic contaminants (NACs). To this end, we investigated the C, H, and N isotope fractionation during the dioxygenation of nitrobenzene (NB), 2-nitrotoluene (2-NT), and 3-nitrotoluene (3-NT) by pure cultures, E. coli clones, cell extracts, and purified enzymes. From isotope fractionations measured in the substrates and reaction products, we determined dioxygenation KIEs for different combinations of the three substrates with nitrobenzene dioxygenase (NBDO) and 2-nitrotoluene dioxygenase (2NTDO). The 13C-, 2H-, and 15N-KIEs for the dioxygenation of NB by NBDO were consistent for all experimental systems considered (i.e., Comamonas sp. Strain JS765, E. coli clones, cell extracts of E. coli clones, and purified NBDO). This observation suggests that the isotope

  2. Heterologous Expression and Characterization of Two 1-Hydroxy-2-Naphthoic Acid Dioxygenases from Arthrobacter phenanthrenivorans

    PubMed Central

    Kavakiotis, Konstantinos; Kallimanis, Aristeidis; Kyrpides, Nikos C.; Drainas, Constantin; Koukkou, Anna-Irini

    2012-01-01

    A protein fraction exhibiting 1-hydroxy-2-naphthoic acid (1-H2NA) dioxygenase activity was purified via ion exchange, hydrophobic interactions, and gel filtration chromatography from Arthrobacter phenanthrenivorans sp. nov. strain Sphe3 isolated from a Greek creosote-oil-polluted site. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and tandem MS (MS-MS) analysis revealed that the amino acid sequences of oligopeptides of the major 45-kDa protein species, as analyzed by SDS-PAGE and silver staining, comprising 29% of the whole sequence, exhibited strong homology with 1-H2NA dioxygenase of Nocardioides sp. strain KP7. A BLAST search of the recently sequenced Sphe3 genome revealed two putative open reading frames, named diox1 and diox2, showing 90% nucleotide identity to each other and 85% identity at the amino acid level with the Nocardia sp. homologue. diox1 was found on an indigenous Sphe3 plasmid, whereas diox2 was located on the chromosome. Both genes were induced by the presence of phenanthrene used as a sole carbon and energy source, and as expected, both were subject to carbon catabolite repression. The relative RNA transcription level of the chromosomal (diox2) gene was significantly higher than that of its plasmid (diox1) homologue. Both diox1 and diox2 putative genes were PCR amplified, cloned, and overexpressed in Escherichia coli. Recombinant E. coli cells expressed 1-H2NA dioxygenase activity. Recombinant enzymes exhibited Michaelis-Menten kinetics with an apparent Km of 35 μM for Diox1 and 29 μM for Diox2, whereas they showed similar kinetic turnover characteristics with Kcat/Km values of 11 × 106 M−1 s−1 and 12 × 106 M−1 s−1, respectively. Occurrence of two diox1 and diox2 homologues in the Sphe3 genome implies that a replicative transposition event has contributed to the evolution of 1-H2NA dioxygenase in A. phenanthrenivorans. PMID:22101055

  3. Pyruvate stabilizes electrocardiographic and hemodynamic function in pigs recovering from cardiac arrest.

    PubMed

    Cherry, Brandon H; Nguyen, Anh Q; Hollrah, Roger A; Williams, Arthur G; Hoxha, Besim; Olivencia-Yurvati, Albert H; Mallet, Robert T

    2015-12-01

    Cardiac electromechanical dysfunction may compromise recovery of patients who are initially resuscitated from cardiac arrest, and effective treatments remain elusive. Pyruvate, a natural intermediary metabolite, energy substrate, and antioxidant, has been found to protect the heart from ischemia-reperfusion injury. This study tested the hypothesis that pyruvate-enriched resuscitation restores hemodynamic, metabolic, and electrolyte homeostasis following cardiac arrest. Forty-two Yorkshire swine underwent pacing-induced ventricular fibrillation and, after 6 min pre-intervention arrest, 4 min precordial compressions followed by transthoracic countershocks. After defibrillation and recovery of spontaneous circulation, the pigs were monitored for another 4 h. Sodium pyruvate or NaCl were infused i.v. (0.1 mmol·kg(-1)·min(-1)) throughout precordial compressions and the first 60 min recovery. In 8 of the 24 NaCl-infused swine, the first countershock converted ventricular fibrillation to pulseless electrical activity unresponsive to subsequent countershocks, but only 1 of 18 pyruvate-treated swine developed pulseless electrical activity (relative risk 0.17; 95% confidence interval 0.13-0.22). Pyruvate treatment also lowered the dosage of vasoconstrictor phenylephrine required to maintain systemic arterial pressure at 15-60 min recovery, hastened clearance of excess glucose, elevated arterial bicarbonate, and raised arterial pH; these statistically significant effects persisted up to 3 h after sodium pyruvate infusion, while infusion-induced hypernatremia subsided. These results demonstrate that pyruvate-enriched resuscitation achieves electrocardiographic and hemodynamic stability in swine during the initial recovery from cardiac arrest. Such metabolically based treatment may offer an effective strategy to support cardiac electromechanical recovery immediately after cardiac arrest. PMID:26088865

  4. New Insights on the Mechanism of the K+-Independent Activity of Crenarchaeota Pyruvate Kinases

    PubMed Central

    De la Vega-Ruíz, Gustavo; Domínguez-Ramírez, Lenin; Riveros-Rosas, Héctor; Guerrero-Mendiola, Carlos; Torres-Larios, Alfredo; Hernández-Alcántara, Gloria; García-Trejo, José J.; Ramírez-Silva, Leticia

    2015-01-01

    Eukarya pyruvate kinases have glutamate at position 117 (numbered according to the rabbit muscle enzyme), whereas in Bacteria have either glutamate or lysine and in Archaea have other residues. Glutamate at this position makes pyruvate kinases K+-dependent, whereas lysine confers K+-independence because the positively charged residue substitutes for the monovalent cation charge. Interestingly, pyruvate kinases from two characterized Crenarchaeota exhibit K+-independent activity, despite having serine at the equivalent position. To better understand pyruvate kinase catalytic activity in the absence of K+ or an internal positive charge, the Thermofilum pendens pyruvate kinase (valine at the equivalent position) was characterized. The enzyme activity was K+-independent. The kinetic mechanism was random order with a rapid equilibrium, which is equal to the mechanism of the rabbit muscle enzyme in the presence of K+ or the mutant E117K in the absence of K+. Thus, the substrate binding order of the T. pendens enzyme was independent despite lacking an internal positive charge. Thermal stability studies of this enzyme showed two calorimetric transitions, one attributable to the A and C domains (Tm of 99.2°C), and the other (Tm of 105.2°C) associated with the B domain. In contrast, the rabbit muscle enzyme exhibits a single calorimetric transition (Tm of 65.2°C). The calorimetric and kinetic data indicate that the B domain of this hyperthermophilic enzyme is more stable than the rest of the protein with a conformation that induces the catalytic readiness of the enzyme. B domain interactions of pyruvate kinases that have been determined in Pyrobaculum aerophilum and modeled in T. pendens were compared with those of the rabbit muscle enzyme. The results show that intra- and interdomain interactions of the Crenarchaeota enzymes may account for their higher B domain stability. Thus the structural arrangement of the T. pendens pyruvate kinase could allow charge

  5. L-Lactate transport into rat heart mitochondria and reconstruction of the L-lactate/pyruvate shuttle.

    PubMed Central

    Valenti, Daniela; de Bari, Lidia; Atlante, Anna; Passarella, Salvatore

    2002-01-01

    In vitro reconstruction of the L-lactate/pyruvate shuttle has been performed, which allows NADH oxidation outside rat heart mitochondria. Such a shuttle occurs due to the combined action of the novel mitochondrial L-lactate/pyruvate antiporter, which differs from the monocarboxylate carrier, and the mitochondrial L-lactate dehydrogenase. The rate of L-lactate/pyruvate antiport proved to regulate the shuttle in vitro. PMID:11988081

  6. L-Lactate transport into rat heart mitochondria and reconstruction of the L-lactate/pyruvate shuttle.

    PubMed

    Valenti, Daniela; de Bari, Lidia; Atlante, Anna; Passarella, Salvatore

    2002-05-15

    In vitro reconstruction of the L-lactate/pyruvate shuttle has been performed, which allows NADH oxidation outside rat heart mitochondria. Such a shuttle occurs due to the combined action of the novel mitochondrial L-lactate/pyruvate antiporter, which differs from the monocarboxylate carrier, and the mitochondrial L-lactate dehydrogenase. The rate of L-lactate/pyruvate antiport proved to regulate the shuttle in vitro. PMID:11988081

  7. The pyruvate dehydrogenase complex in cancer: An old metabolic gatekeeper regulated by new pathways and pharmacological agents.

    PubMed

    Saunier, Elise; Benelli, Chantal; Bortoli, Sylvie

    2016-02-15

    Cancer cells exhibit an altered metabolism which is characterized by a preference for aerobic glycolysis more than mitochondrial oxidation of pyruvate. This provides anabolic support and selective growth advantage for cancer cells. Recently, a new concept has arisen suggesting that these metabolic changes may be due, in part, to an attenuated mitochondrial function which results from the inhibition of the pyruvate dehydrogenase complex (PDC). This mitochondrial complex links glycolysis to the Krebs cycle and the current understanding of its regulation involves the cyclic phosphorylation and dephosphorylation by specific pyruvate dehydrogenase kinases (PDKs) and pyruvate dehydrogenase phosphatases (PDPs). PMID:25868605

  8. Isoform Switch of Pyruvate Kinase M1 Indeed Occurs but Not to Pyruvate Kinase M2 in Human Tumorigenesis

    PubMed Central

    Zhan, Cheng; Yan, Li; Wang, Lin; Ma, Jun; Jiang, Wei; Zhang, Yongxing; Shi, Yu; Wang, Qun

    2015-01-01

    Muscle type of pyruvate kinase (PKM) is one of the key mediators of the Warburg effect and tumor metabolism. Due to alternative splicing, there are at least 12 known isoforms of the PKM gene, of which PKM1 and PKM2 are two major isoforms with only a 23 amino acid sequenced difference but quite different characteristics and functions. It was previously thought the isoform switch from PKM1 to PKM2 resulted in high PKM2 expression in tumors, providing a great advantage to tumor cells. However, this traditional view was challenged by two recent studies; one study claimed that this isoform switch does not occur during the Warburg effect; the other study asserted that the isoform switch is tissue-specific. Here, we re-analyzed the RNA sequencing data of 25 types of human tumors from The Cancer Genome Atlas Data Portal, and confirmed that PKM2 was the major isoform in the tumors and was highly elevated in addition to the entire PKM gene. We further demonstrated that the expression level of PKM1 significantly declined even though there was substantially increased expression of the entire PKM gene. The proportion of PKM1 in total transcript variants also significantly declined in tumors but the proportion of PKM2 did not change accordingly. Therefore, we conclude that the isoform switch of PKM1 does indeed occur, but it switches to other isoforms rather than PKM2. Considering the change in the expression levels of PKM1, PKM2 and the entire PKM gene, we propose that the upregulation of PKM2 is primarily due to elevated transcriptional levels of the entire PKM gene, instead of the isoform switch. PMID:25738776

  9. Efficient production and secretion of pyruvate from Halomonas sp. KM-1 under aerobic conditions.

    PubMed

    Kawata, Yoshikazu; Nishimura, Taku; Matsushita, Isao; Tsubota, Jun

    2016-03-01

    The alkaliphilic, halophilic bacterium Halomonas sp. KM-1 can utilize both hexose and pentose sugars for the intracellular storage of bioplastic poly-(R)-3-hydroxybutyric acid (PHB) under aerobic conditions. In this study, we investigated the effects of the sodium nitrate concentration on PHB accumulation in the KM-1 strain. Unexpectedly, we observed the secretion of pyruvate, a central intermediate in carbon- and energy-metabolism processes in all organisms; therefore, pyruvate is widely used as a starting material in the industrial biosynthesis of pharmaceuticals and is employed for the production of crop-protection agents, polymers, cosmetics, and food additives. We then further analyzed pyruvate productivity following changes in culture temperature and the buffer concentration. In 48-h batch-cultivation experiments, we found that wild-type Halomonas sp. KM-1 secreted 63.3 g/L pyruvate at a rate of 1.32 g/(L·h), comparable to the results of former studies using mutant and recombinant microorganisms. Thus, these data provided important insights into the production of pyruvate using this novel strain. PMID:26989057

  10. Modeling of the pyruvate production with Escherichia coli in a fed-batch bioreactor.

    PubMed

    Zelić, B; Vasić-Racki, D; Wandrey, C; Takors, R

    2004-07-01

    A family of 10 competing, unstructured models has been developed to model cell growth, substrate consumption, and product formation of the pyruvate producing strain Escherichia coli YYC202 ldhA::Kan strain used in fed-batch processes. The strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate (using glucose as the carbon source) resulting in an acetate auxotrophy during growth in glucose minimal medium. Parameter estimation was carried out using data from fed-batch fermentation performed at constant glucose feed rates of q(VG)=10 mL h(-1). Acetate was fed according to the previously developed feeding strategy. While the model identification was realized by least-square fit, the model discrimination was based on the model selection criterion (MSC). The validation of model parameters was performed applying data from two different fed-batch experiments with glucose feed rate q(VG)=20 and 30 mL h(-1), respectively. Consequently, the most suitable model was identified that reflected the pyruvate and biomass curves adequately by considering a pyruvate inhibited growth (Jerusalimsky approach) and pyruvate inhibited product formation (described by modified Luedeking-Piret/Levenspiel term). PMID:15085423

  11. Phosphorylation Status of Pyruvate Dehydrogenase Distinguishes Metabolic Phenotypes of Cultured Rat Brain Astrocytes and Neurons

    PubMed Central

    HALIM, NADER D.; McFATE, THOMAS; MOHYELDIN, AHMED; OKAGAKI, PETER; KOROTCHKINA, LIOUBOV G; PATEL, MULCHAND S; JEOUNG, NAM HO; HARRIS, ROBERT A.; SCHELL, MICHAEL J.; VERMA, AJAY

    2010-01-01

    Glucose metabolism in nervous tissue has been proposed to occur in a compartmentalized manner with astrocytes contributing largely to glycolysis and neurons being the primary site of glucose oxidation. However, mammalian astrocytes and neurons both contain mitochondria and it remains unclear why in culture neurons oxidize glucose, lactate, and pyruvate to a much larger extent than astrocytes. The objective of this study was to determine whether pyruvate metabolism is differentially regulated in cultured neurons vs. astrocytes. Expression of all components of the pyruvate dehydrogenase complex (PDC), the rate-limiting step for pyruvate entry into the Krebs cycle, was determined in cultured astrocytes and neurons. In addition, regulation of PDC enzymatic activity in the two cell types via protein phosphorylation was examined. We show that all components of the PDC are expressed in both cell types in culture but that PDC activity is kept strongly inhibited in astrocytes through phosphorylation of the pyruvate dehydrogenase alpha subunit (PDHα). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorlyated PDHα. Dephosphorylation of astrocytic PDHα restores PDC activity and lowers lactate production. Our findings suggest that the glucose metabolism of astrocytes and neurons may be far more flexible than previously believed. PMID:20544852

  12. Propionate Increases Hepatic Pyruvate Cycling and Anaplerosis and Alters Mitochondrial Metabolism*

    PubMed Central

    Perry, Rachel J.; Borders, Candace B.; Cline, Gary W.; Zhang, Xian-Man; Alves, Tiago C.; Petersen, Kitt Falk; Rothman, Douglas L.; Kibbey, Richard G.; Shulman, Gerald I.

    2016-01-01

    In mammals, pyruvate kinase (PK) plays a key role in regulating the balance between glycolysis and gluconeogenesis; however, in vivo regulation of PK flux by gluconeogenic hormones and substrates is poorly understood. To this end, we developed a novel NMR-liquid chromatography/tandem-mass spectrometry (LC-MS/MS) method to directly assess pyruvate cycling relative to mitochondrial pyruvate metabolism (VPyr-Cyc/VMito) in vivo using [3-13C]lactate as a tracer. Using this approach, VPyr-Cyc/VMito was only 6% in overnight fasted rats. In contrast, when propionate was infused simultaneously at doses previously used as a tracer, it increased VPyr-Cyc/VMito by 20–30-fold, increased hepatic TCA metabolite concentrations 2–3-fold, and increased endogenous glucose production rates by 20–100%. The physiologic stimuli, glucagon and epinephrine, both increased hepatic glucose production, but only glucagon suppressed VPyr-Cyc/VMito. These data show that under fasting conditions, when hepatic gluconeogenesis is stimulated, pyruvate recycling is relatively low in liver compared with VMito flux and that liver metabolism, in particular pyruvate cycling, is sensitive to propionate making it an unsuitable tracer to assess hepatic glycolytic, gluconeogenic, and mitochondrial metabolism in vivo. PMID:27002151

  13. Targeting Tumor Metabolism for Cancer Treatment: Is Pyruvate Dehydrogenase Kinases (PDKs) a Viable Anticancer Target?

    PubMed Central

    Zhang, Wen; Zhang, Shao-Lin; Hu, Xiaohui; Tam, Kin Yip

    2015-01-01

    Cancer remains a lethal threat to global lives. Development of novel anticancer therapeutics is still a challenge to scientists in the field of biomedicine. In cancer cells, the metabolic features are significantly different from those of normal ones, which are hallmarks of several malignancies. Recent studies brought atypical cellular metabolism, such as aerobic glycolysis or the Warburg effect, into the scientific limelight. Targeting these altered metabolic pathways in cancer cells presents a promising therapeutic strategy. Pyruvate dehydrogenase kinases (PDKs), key enzymes in the pathway of glucose metabolism, could inactivate the pyruvate dehydrogenase complex (PDC) by phosphorylating it and preserving the substrates pyruvate, lactate and alanine for gluconeogenesis. Overexpression of PDKs could block the oxidative decarboxylation of pyruvate to satisfy high oxygen demand in cancer cells, while inhibition of PDKs could upregulate the activity of PDC and rectify the balance between the demand and supply of oxygen, which could lead to cancer cell death. Thus, inhibitors targeting PDKs represent a promising strategy for cancer treatment by acting on glycolytic tumors while showing minimal side effects on the oxidative healthy organs. This review considers the role of PDKs as regulator of PDC that catalyzes the oxidative decarboxylation of pyruvate in mitochondrion. It is concluded that PDKs are solid therapeutic targets. Inhibition of PDKs could be an attractive therapeutic approach for the development of anti-cancer drugs. PMID:26681918

  14. Mitochondrial Pyruvate Import Promotes Long-Term Survival of Antibody-Secreting Plasma Cells.

    PubMed

    Lam, Wing Y; Becker, Amy M; Kennerly, Krista M; Wong, Rachel; Curtis, Jonathan D; Llufrio, Elizabeth M; McCommis, Kyle S; Fahrmann, Johannes; Pizzato, Hannah A; Nunley, Ryan M; Lee, Jieun; Wolfgang, Michael J; Patti, Gary J; Finck, Brian N; Pearce, Erika L; Bhattacharya, Deepta

    2016-07-19

    Durable antibody production after vaccination or infection is mediated by long-lived plasma cells (LLPCs). Pathways that specifically allow LLPCs to persist remain unknown. Through bioenergetic profiling, we found that human and mouse LLPCs could robustly engage pyruvate-dependent respiration, whereas their short-lived counterparts could not. LLPCs took up more glucose than did short-lived plasma cells (SLPCs) in vivo, and this glucose was essential for the generation of pyruvate. Glucose was primarily used to glycosylate antibodies, but glycolysis could be promoted by stimuli such as low ATP levels and the resultant pyruvate used for respiration by LLPCs. Deletion of Mpc2, which encodes an essential component of the mitochondrial pyruvate carrier, led to a progressive loss of LLPCs and of vaccine-specific antibodies in vivo. Thus, glucose uptake and mitochondrial pyruvate import prevent bioenergetic crises and allow LLPCs to persist. Immunizations that maximize these plasma cell metabolic properties might thus provide enduring antibody-mediated immunity. PMID:27396958

  15. Central Role of Pyruvate Kinase in Carbon Co-catabolism of Mycobacterium tuberculosis.

    PubMed

    Noy, Tahel; Vergnolle, Olivia; Hartman, Travis E; Rhee, Kyu Y; Jacobs, William R; Berney, Michael; Blanchard, John S

    2016-03-25

    Mycobacterium tuberculosis (Mtb) displays a high degree of metabolic plasticity to adapt to challenging host environments. Genetic evidence suggests thatMtbrelies mainly on fatty acid catabolism in the host. However,Mtbalso maintains a functional glycolytic pathway and its role in the cellular metabolism ofMtbhas yet to be understood. Pyruvate kinase catalyzes the last and rate-limiting step in glycolysis and theMtbgenome harbors one putative pyruvate kinase (pykA, Rv1617). Here we show thatpykAencodes an active pyruvate kinase that is allosterically activated by glucose 6-phosphate (Glc-6-P) and adenosine monophosphate (AMP). Deletion ofpykApreventsMtbgrowth in the presence of fermentable carbon sources and has a cidal effect in the presence of glucose that correlates with elevated levels of the toxic catabolite methylglyoxal. Growth attenuation was also observed in media containing a combination of short chain fatty acids and glucose and surprisingly, in media containing odd and even chain fatty acids alone. Untargeted high sensitivity metabolomics revealed that inactivation of pyruvate kinase leads to accumulation of phosphoenolpyruvate (P-enolpyruvate), citrate, and aconitate, which was consistent with allosteric inhibition of isocitrate dehydrogenase by P-enolpyruvate. This metabolic block could be relieved by addition of the α-ketoglutarate precursor glutamate. Taken together, our study identifies an essential role of pyruvate kinase in preventing metabolic block during carbon co-catabolism inMtb. PMID:26858255

  16. Propionate Increases Hepatic Pyruvate Cycling and Anaplerosis and Alters Mitochondrial Metabolism.

    PubMed

    Perry, Rachel J; Borders, Candace B; Cline, Gary W; Zhang, Xian-Man; Alves, Tiago C; Petersen, Kitt Falk; Rothman, Douglas L; Kibbey, Richard G; Shulman, Gerald I

    2016-06-01

    In mammals, pyruvate kinase (PK) plays a key role in regulating the balance between glycolysis and gluconeogenesis; however, in vivo regulation of PK flux by gluconeogenic hormones and substrates is poorly understood. To this end, we developed a novel NMR-liquid chromatography/tandem-mass spectrometry (LC-MS/MS) method to directly assess pyruvate cycling relative to mitochondrial pyruvate metabolism (VPyr-Cyc/VMito) in vivo using [3-(13)C]lactate as a tracer. Using this approach, VPyr-Cyc/VMito was only 6% in overnight fasted rats. In contrast, when propionate was infused simultaneously at doses previously used as a tracer, it increased VPyr-Cyc/VMito by 20-30-fold, increased hepatic TCA metabolite concentrations 2-3-fold, and increased endogenous glucose production rates by 20-100%. The physiologic stimuli, glucagon and epinephrine, both increased hepatic glucose production, but only glucagon suppressed VPyr-Cyc/VMito These data show that under fasting conditions, when hepatic gluconeogenesis is stimulated, pyruvate recycling is relatively low in liver compared with VMito flux and that liver metabolism, in particular pyruvate cycling, is sensitive to propionate making it an unsuitable tracer to assess hepatic glycolytic, gluconeogenic, and mitochondrial metabolism in vivo. PMID:27002151

  17. Utilization of Lactate Isomers by Propionibacterium freudenreichii subsp. shermanii: Regulatory Role for Intracellular Pyruvate

    PubMed Central

    Crow, Vaughan L.

    1986-01-01

    Five strains of Propionibacterium freudenreichii subsp. shermanii utilized the l-(+) isomer of lactate at a faster rate than they did the d-(−) isomer when grown with a mixture of lactate isomers under a variety of conditions. ATCC 9614, grown anaerobically in defined medium containing 160 mM dl-lactate, utilized only 4 and 15% of the d-(−)-lactate by the time 50 and 90%, respectively, of the l-(+)-lactate was used. The intracellular pyruvate concentration was high (>100 mM) in the initial stages of lactate utilization, when either dl-lactate or the l-(+) isomer was the starting substrate. The concentration of this intermediate dropped during dl-lactate fermentation such that when only d-(−)-lactate remained, the concentration was <20 mM. When only the d-(−) isomer was initially present, a similar relatively low concentration of intracellular pyruvate was present, even at the start of lactate utilization. The NAD+-independent lactate dehydrogenase activities in extracts showed different kinetic properties with regard to pyruvate inhibition, depending upon the lactate isomer present. Pyruvate gave a competitive inhibitor pattern with l-(+)-lactate and a mixed-type inhibitor pattern with d-(−)-lactate. It is suggested that these properties of the lactate dehydrogenases and the intracellular pyruvate concentrations explain the preferential use of the l-(+) isomer. PMID:16347134

  18. Molecular Insights into Substrate Recognition and Catalysis by Tryptophan 2,3-dioxygenase

    SciTech Connect

    Forouhar,F.; Ross Anderson, J.; Mowat, C.; Vorobiev, S.; Hussain, A.; Abashidze, M.; Bruckmann, C.; Thackray, S.; Seetharaman, J.; et al.

    2007-01-01

    Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) constitute an important, yet relatively poorly understood, family of heme-containing enzymes. Here, we report extensive structural and biochemical studies of the Xanthomonas campestris TDO and a related protein SO4414 from Shewanella oneidensis, including the structure at 1.6-{angstrom} resolution of the catalytically active, ferrous form of TDO in a binary complex with the substrate l-Trp. The carboxylate and ammonium moieties of tryptophan are recognized by electrostatic and hydrogen-bonding interactions with the enzyme and a propionate group of the heme, thus defining the l-stereospecificity. A second, possibly allosteric, l-Trp-binding site is present at the tetramer interface. The sixth coordination site of the heme-iron is vacant, providing a dioxygen-binding site that would also involve interactions with the ammonium moiety of l-Trp and the amide nitrogen of a glycine residue. The indole ring is positioned correctly for oxygenation at the C2 and C3 atoms. The active site is fully formed only in the binary complex, and biochemical experiments confirm this induced-fit behavior of the enzyme. The active site is completely devoid of water during catalysis, which is supported by our electrochemical studies showing significant stabilization of the enzyme upon substrate binding.

  19. Crystal Structure of the Non-heme Iron Dioxygenase PtlH in Pentalenolactone Biosynthesis

    PubMed Central

    You, Zheng; Omura, Satoshi; Ikeda, Haruo; Cane, David E.; Jogl, Gerwald

    2010-01-01

    The non-heme iron dioxygenase PtlH from the soil organism Streptomyces avermitilis is a member of the iron(II)/α-ketoglutarate–dependent dioxygenase superfamily and catalyzes an essential reaction in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone. To investigate the structural basis for substrate recognition and catalysis, we have determined the X-ray crystal structure of PtlH in several complexes with the cofactors iron, α-ketoglutarate, and the non-reactive enantiomer of the substrate, ent-1-deoxypentalenic acid, in four different crystal forms to up to 1.31 Å resolution. The overall structure of PtlH forms a double-stranded barrel helix fold and the cofactor-binding site for iron and α-keto-glutarate is similar to other double-stranded barrel helix fold enzymes. Additional secondary structure elements that contribute to the substrate-binding site in PtlH are not conserved in other double-stranded barrel helix fold enzymes. Binding of the substrate enantiomer induces a reorganization of the monoclinic crystal lattice leading to a disorder-order transition of a C-terminal α–helix. The newly formed helix blocks the major access to the active site and effectively traps the bound substrate. Kinetic analysis of wild type and site-directed mutant proteins confirms a critical function of two arginine residues in substrate binding, while simulated docking of the enzymatic reaction product reveals the likely orientation of bound substrate. PMID:17942405

  20. Oxidation of alkyl nitronates catalyzed by 2-nitropropane dioxygenase from Hansenula mrakii.

    PubMed

    Mijatovic, Slavica; Gadda, Giovanni

    2008-05-01

    2-Nitropropane dioxygenase from Hansenula mrakii was expressed in Escherichia coli cells and purified in active and stable form using 60% saturation of ammonium sulfate and a single chromatographic step onto a DEAE column. MALDI-TOF mass spectrometric and spectrophotometric analyses of the flavin extracted by heat or acid denaturation of the enzyme indicated that FMN, and not FAD as erroneously reported previously, is present in a 1:1 stoichiometry with the protein. Inductively coupled plasma mass spectrometric analysis of the enzyme established that H. mrakii 2-nitropropane dioxygenase contains negligible amounts of iron, manganese, zinc, and copper ions, which are not catalytically relevant. Anaerobic substrate reduction and kinetic data using a Clark oxygen electrode to measure rates of oxygen consumption indicated that the enzyme is active on a broad range of alkyl nitronates, with a marked preference for unbranched substrates over propyl-2-nitronate. Interestingly, the enzyme reacts poorly, if at all, with nitroalkanes, as suggested by lack of both anaerobic reduction of the enzyme-bound flavin and consumption of oxygen with nitroethane, nitrobutane, and 2-nitropropane. Finally, both the tight binding of sulfite (K(d)=90 microM, at pH 8 and 15 degrees C) to the enzyme and the formation of the anionic flavosemiquinone upon anaerobic incubation with alkyl nitronates are consistent with the presence of a positively charged group in proximity of the N1-C2=O atoms of the FMN cofactor. PMID:18329375

  1. The expression and prognostic relevance of indoleamine 2,3-dioxygenase in tongue squamous cell carcinoma.

    PubMed

    Seppälä, Miia; Halme, Elina; Tiilikainen, Lauri; Luukkainen, Annika; Laranne, Jussi; Rautiainen, Markus; Huhtala, Heini; Paavonen, Timo; Toppila-Salmi, Sanna

    2016-07-01

    Conclusion IDO might be useful for predicting progression of primary tumor stage T2 and T3 in tongue squamous cell carcinoma (TSCC), but does not seem like a specific biomarker for diagnosing TSCC and predicting patient survival. Objectives Indoleamine 2,3-dioxygenase (IDO) is expressed in many cells and it catabolises the essential amino acid tryptophan to kynurenine. IDO acts as an immune modulator through suppression of T-cell immunity and other pathways. In cancer cells, IDO has been proposed to promote tumor progression by enabling malignant cells to escape from the immune system. The aim of this study was to evaluate the association and prognostic relevance of IDO expression in TSCC. Method One hundred and eight retrospective tongue and lymph node specimens were stained immunohistochemically with monoclonal antibody anti-indoleamine 2,3-dioxygenase. The relative abundance of IDO positive epithelial cells, IDO staining intensity, and inflammation were assessed semi-quantitatively with light microscopy. Results IDO was expressed stronger in tongue hyperplasia than in TSCC. However, IDO expression associated with poor survival in the sub-groups with primary tumor stage T2-T4 and in the sub-group with strong inflammation in tumors' invasive front. PMID:26982018

  2. Nitrobenzofurazan derivatives of N'-hydroxyamidines as potent inhibitors of indoleamine-2,3-dioxygenase 1.

    PubMed

    Paul, Saurav; Roy, Ashalata; Deka, Suman Jyoti; Panda, Subhankar; Trivedi, Vishal; Manna, Debasis

    2016-10-01

    Tryptophan metabolism through the kynurenine pathway is considered as a crucial mechanism in immune tolerance. Indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in tryptophan catabolism in the immune system and it is also considered as an important therapeutic target for the treatment of cancer and other diseases that are linked with kynurenine pathway. In this study, a series of nitrobenzofurazan derivatives of N'-hydroxybenzimidamides (1) and N'-hydroxy-2-phenylacetimidamides (2) were synthesized and their inhibitory activities against human IDO1 enzyme were tested using in-vitro and cellular enzyme activity assay. The optimization leads to the identification of potent compounds, 1d, 2i and 2k (IC50 = 39-80 nM), which are either competitive or uncompetitive inhibitors of IDO1 enzyme. These compounds also showed IDO1 inhibition potencies in the nanomolar range (IC50 = 50-71 nM) in MDA-MB-231 cells with no/negligible amount of cytotoxicity. The stronger selectivity of the potent compounds for IDO1 enzyme over tryptophan 2,3-dioxygenase (TDO) enzyme (312-1593-fold) also makes them very attractive for further immunotherapeutic applications. PMID:27267006

  3. The metabolic and developmental roles of carotenoid cleavage dioxygenase4 from potato.

    PubMed

    Campbell, Raymond; Ducreux, Laurence J M; Morris, Wayne L; Morris, Jenny A; Suttle, Jeffrey C; Ramsay, Gavin; Bryan, Glenn J; Hedley, Pete E; Taylor, Mark A

    2010-10-01

    The factors that regulate storage organ carotenoid content remain to be fully elucidated, despite the nutritional and economic importance of this class of compound. Recent findings suggest that carotenoid pool size is determined, at least in part, by the activity of carotenoid cleavage dioxygenases. The aim of this study was to investigate whether Carotenoid Cleavage Dioxygenase4 (CCD4) activity affects potato (Solanum tuberosum) tuber carotenoid content. Microarray analysis revealed elevated expression of the potato CCD4 gene in mature tubers from white-fleshed cultivars compared with higher carotenoid yellow-fleshed tubers. The expression level of the potato CCD4 gene was down-regulated using an RNA interference (RNAi) approach in stable transgenic lines. Down-regulation in tubers resulted in an increased carotenoid content, 2- to 5-fold higher than in control plants. The increase in carotenoid content was mainly due to elevated violaxanthin content, implying that this carotenoid may act as the in vivo substrate. Although transcript level was also reduced in plant organs other than tubers, such as leaves, stems, and roots , there was no change in carotenoid content in these organs. However, carotenoid levels were elevated in flower petals from RNAi lines. As well as changes in tuber carotenoid content, tubers from RNAi lines exhibited phenotypes such as heat sprouting, formation of chain tubers, and an elongated shape. These results suggest that the product of the CCD4 reaction may be an important factor in tuber heat responses. PMID:20688977

  4. Crystallization and preliminary X-ray crystallographic analysis of hydroquinone dioxygenase from Sphingomonas sp. TTNP3

    PubMed Central

    Da Vela, Stefano; Ferraroni, Marta; Kolvenbach, Boris A.; Keller, Eva; Corvini, Philippe F. X.; Scozzafava, Andrea; Briganti, Fabrizio

    2012-01-01

    Hydroquinone dioxygenase (HQDO), a novel FeII ring-fission dioxygenase from Sphingomonas sp. strain TTNP3 which oxidizes a wide range of hydroquinones to the corresponding 4-hydroxymuconic semialdehydes, has been crystallized. The enzyme is an α2β2 heterotetramer constituted of two subunits of 19 and 38 kDa. Diffraction-quality crystals of HQDO were obtained using the sitting-drop vapour-diffusion method at 277 K from a solution consisting of 16% PEG 4000, 0.3 M MgCl2, 0.1 M Tris pH 8.5. The crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 88.4, b = 125.4, c = 90.8 Å, β = 105.3°. The asymmetric unit contained two heterotetramers, i.e. four copies of each of the two different subunits related by noncrystallographic 222 symmetry. A complete data set extending to a maximum resolution of 2.5 Å was collected at 100 K using a wavelength of 0.980 Å. PMID:22691794

  5. Bacterial dioxygenase- and monooxygenase-catalysed sulfoxidation of benzo[b]thiophenes.

    PubMed

    Boyd, Derek R; Sharma, Narain D; McMurray, Brian; Haughey, Simon A; Allen, Christopher C R; Hamilton, John T G; McRoberts, W Colin; O'Ferrall, Rory A More; Nikodinovic-Runic, Jasmina; Coulombel, Lydie A; O'Connor, Kevin E

    2012-01-28

    Asymmetric heteroatom oxidation of benzo[b]thiophenes to yield the corresponding sulfoxides was catalysed by toluene dioxygenase (TDO), naphthalene dioxygenase (NDO) and styrene monooxygenase (SMO) enzymes present in P. putida mutant and E. coli recombinant whole cells. TDO-catalysed oxidation yielded the relatively unstable benzo[b]thiophene sulfoxide; its dimerization, followed by dehydrogenation, resulted in the isolation of stable tetracyclic sulfoxides as minor products with cis-dihydrodiols being the dominant metabolites. SMO mainly catalysed the formation of enantioenriched benzo[b]thiophene sulfoxide and 2-methyl benzo[b]thiophene sulfoxides which racemized at ambient temperature. The barriers to pyramidal sulfur inversion of 2- and 3-methyl benzo[b]thiophene sulfoxide metabolites, obtained using TDO and NDO as biocatalysts, were found to be ca.: 25-27 kcal mol(-1). The absolute configurations of the benzo[b]thiophene sulfoxides were determined by ECD spectroscopy, X-ray crystallography and stereochemical correlation. A site-directed mutant E. coli strain containing an engineered form of NDO, was found to change the regioselectivity toward preferential oxidation of the thiophene ring rather than the benzene ring. PMID:22134441

  6. Pivotal role of anthranilate dioxygenase genes in the adaptation of Burkholderia multivorans ATCC 17616 in soil.

    PubMed

    Nishiyama, Eri; Ohtsubo, Yoshiyuki; Yamamoto, Yasuhiro; Nagata, Yuji; Tsuda, Masataka

    2012-05-01

    In our recent screen for soil-induced genes, the expression of andA operon (andAcAdAbAa) for anthranilate catabolism in Burkholderia multivorans ATCC 17616 was found to increase dramatically in a soil sample (Nishiyama et al., Environ Microbiol 12: 2539, 2010). The operon was preceded by andR encoding a putative transcriptional regulator for the andA operon. In this study, the andA promoter was induced by tryptophan and anthranilate in an andR-dependent manner. The andA promoter in a deletion mutant lacking tryptophan dioxygenase (one of enzymes for the catabolism of tryptophan to anthranilate) did not respond to tryptophan, indicating that not tryptophan but anthranilate is the effector of AndR. Although both anthranilate and tryptophan were under the detection levels in the soil sample, andA promoter showed higher activity in the soil sample than in a laboratory medium. Such induction required andR and was moderately dependent on the ferric uptake regulator (Fur). The proliferation ability of andAc mutant in the sterile soil was low compared with the co-incubated wild-type cells. These findings suggested that in the soil environment, anthranilate dioxygenase genes are induced by AndR and Fur, and play a pivotal role in the proliferation in the soil environment. PMID:22360670

  7. Distribution of naphthalene dioxygenase genes in crude oil-contaminated soils.

    PubMed

    Yang, Yuyin; Wang, Jie; Liao, Jingqiu; Xie, Shuguang; Huang, Yi

    2014-11-01

    Polycyclic aromatic hydrocarbons (PAHs) are one of the major pollutants in soils in oil exploring areas. Biodegradation is the major process for natural elimination of PAHs from contaminated soils. Functional genes can be used as biomarkers to assess the biodegradation potential of indigenous microbial populations. However, little is known about the distribution of PAH-degrading genes in the environment. The links between environmental parameters and the distribution of PAH metabolic genes remain essentially unclear. The present study investigated the abundance and diversity of naphthalene dioxygenase genes in the oil-contaminated soils in the Shengli Oil Field (China). Spatial variations in the density and diversity of naphthalene dioxygenase genes occurred in this area. Four different sequence genotypes were observed in the contaminated soils, with the predominance of novel PAH-degrading genes. Pearson's correlation analysis illustrated that gene abundance had positive correlations with the levels of total organic carbon and aromatic hydrocarbons, while gene diversity showed a negative correlation with the level of polar aromatics. This work could provide some new insights toward the distribution of PAH metabolic genes and PAH biodegradation potential in oil-contaminated ecosystems. PMID:25008984

  8. The role of placental indoleamine 2,3-dioxygenase in human pregnancy

    PubMed Central

    2013-01-01

    Munn et al. made a scientific observation of major biological importance. For the first time they showed that in the mammal the fetus does survive an immune attack mounted by the mother, and that the mechanism responsible for the survival depends on the fetus and placenta 'actively' defending itself from attack by maternal T cells by means of an enzyme indoleamine 2,3-dioxygenase (EC 1.13.11.42) dependent localised depletion of L-tryptophan. These findings raise critical questions for disease and its prevention during human pregnancy. Specifically, the role of this mechanism (discovered in mouse) in the human, and the extent to which defective activation of this process is responsible for major clinical diseases are unknown. Therefore some key facts about this enzyme expressed in the human placenta have been studied in order to test whether Munn et al.'s findings in mouse are met for human pregnancy. This short review attempts to describe our experimental work on human placental indoleamine 2,3-dioxygenase. PMID:24328005

  9. Leucoplast Pyruvate Kinase from Developing Castor Oil Seeds 1

    PubMed Central

    Plaxton, William C.

    1991-01-01

    Leucoplast pyruvate kinase (PKp; EC 2.7.1.40) from endosperm of developing castor oil seeds (Ricinus communis L. cv Baker 296) appears to be highly susceptible to limited degradation by a cysteine endopeptidase during the purification of the enzyme or incubation of clarified homogenates at 4°C. Purified castor seed PKp was previously reported to consist of immunologically related 57.5 and 44 kilodalton subunits (Plaxton WC, Dennis DT, Knowles VL [1990] Plant Physiol 94: 1528-1534). By contrast, immunoreactive polypeptides of about 63.5 and 54 kilodaltons were observed when a western blot of an extract prepared under denaturing conditions was probed with affinity purified rabbit anti-(castor seed PKp) immunoglobulin G. Proteolytic activity against PKp was estimated by the disappearance of the 63.5 and 54 kilodalton subunits and the concomitant appearance of lower molecular mass immunoreactive degradation products during the incubation of clarified homogenates at 4°C. The presence of 2 millimolar dithiothreitol accelerated the degradation of PKp. The conservation of the 63.5 and 54 kilodalton subunits was observed after extraction of the enzyme in the presence of 1 millimolar p-hydroxymecuribenzoate, or 1 millimolar Nα-p-tosyl-l-lysine chloromethyl ketone, or 10 millimolar iodoacetate. These results reveal that a cysteine endopeptidase was responsible for the in vitro proteolysis of PKp. This endopeptidase is present throughout all stages of endosperm development. Its PKp-degrading activity, however, appears to be most pronounced in preparations from older endosperm. When lysates of purified leucoplasts were incubated at 4°C for up to 21 hours, no degradation of PKp was observed; this indicated an extra-leucoplastic localization for the cysteine endopeptidase. Although the in vivo subunit structure of PKp remains uniform throughout all stages of endosperm development, the large decrease in PK activity that accompanies castor seed maturation coincides with a

  10. Kinetic studies on the regulation of rabbit liver pyruvate kinase

    PubMed Central

    Irving, M. G.; Williams, J. F.

    1973-01-01

    Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K+ and optimum activity was recorded with 30mm-K+, 4mm-MgADP−, with a MgADP−/ADP2− ratio of 50:1, but inhibition occurred with K+ concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg2+ was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (nH=2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent Km for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis–Menten response was obtained when phosphoenolpyruvate was the variable substrate (Km=0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg2+. PMID:4722439

  11. Modeling non-linear kinetics of hyperpolarized [1-(13)C] pyruvate in the crystalloid-perfused rat heart.

    PubMed

    Mariotti, E; Orton, M R; Eerbeek, O; Ashruf, J F; Zuurbier, C J; Southworth, R; Eykyn, T R

    2016-04-01

    Hyperpolarized (13)C MR measurements have the potential to display non-linear kinetics. We have developed an approach to describe possible non-first-order kinetics of hyperpolarized [1-(13)C] pyruvate employing a system of differential equations that agrees with the principle of conservation of mass of the hyperpolarized signal. Simultaneous fitting to a second-order model for conversion of [1-(13)C] pyruvate to bicarbonate, lactate and alanine was well described in the isolated rat heart perfused with Krebs buffer containing glucose as sole energy substrate, or glucose supplemented with pyruvate. Second-order modeling yielded significantly improved fits of pyruvate-bicarbonate kinetics compared with the more traditionally used first-order model and suggested time-dependent decreases in pyruvate-bicarbonate flux. Second-order modeling gave time-dependent changes in forward and reverse reaction kinetics of pyruvate-lactate exchange and pyruvate-alanine exchange in both groups of hearts during the infusion of pyruvate; however, the fits were not significantly improved with respect to a traditional first-order model. The mechanism giving rise to second-order pyruvate dehydrogenase (PDH) kinetics was explored experimentally using surface fluorescence measurements of nicotinamide adenine dinucleotide reduced form (NADH) performed under the same conditions, demonstrating a significant increase of NADH during pyruvate infusion. This suggests a simultaneous depletion of available mitochondrial NAD(+) (the cofactor for PDH), consistent with the non-linear nature of the kinetics. NADH levels returned to baseline following cessation of the pyruvate infusion, suggesting this to be a transient effect. PMID:26777799

  12. Thiamine Pyrophosphokinase Deficiency in Encephalopathic Children with Defects in the Pyruvate Oxidation Pathway

    PubMed Central

    Mayr, Johannes A.; Freisinger, Peter; Schlachter, Kurt; Rolinski, Boris; Zimmermann, Franz A.; Scheffner, Thomas; Haack, Tobias B.; Koch, Johannes; Ahting, Uwe; Prokisch, Holger; Sperl, Wolfgang

    2011-01-01

    Thiamine pyrophosphate (TPP) is an essential cofactor of the cytosolic transketolase and of three mitochondrial enzymes involved in the oxidative decarboxylation of either pyruvate, α-ketoglutarate or branched chain amino acids. Thiamine is taken up by specific transporters into the cell and converted to the active TPP by thiamine pyrophosphokinase (TPK) in the cytosol from where it can be transported into mitochondria. Here, we report five individuals from three families presenting with variable degrees of ataxia, psychomotor retardation, progressive dystonia, and lactic acidosis. Investigation of the mitochondrial energy metabolism showed reduced oxidation of pyruvate but normal pyruvate dehydrogenase complex activity in the presence of excess TPP. A reduced concentration of TPP was found in the muscle and blood. Mutation analysis of TPK1 uncovered three missense, one splice-site, and one frameshift mutation resulting in decreased TPK protein levels. PMID:22152682

  13. Inactivation of pyruvate dehydrogenase kinase 2 by mitochondrial reactive oxygen species.

    PubMed

    Hurd, Thomas R; Collins, Yvonne; Abakumova, Irina; Chouchani, Edward T; Baranowski, Bartlomiej; Fearnley, Ian M; Prime, Tracy A; Murphy, Michael P; James, Andrew M

    2012-10-12

    Reactive oxygen species are byproducts of mitochondrial respiration and thus potential regulators of mitochondrial function. Pyruvate dehydrogenase kinase 2 (PDHK2) inhibits the pyruvate dehydrogenase complex, thereby regulating entry of carbohydrates into the tricarboxylic acid (TCA) cycle. Here we show that PDHK2 activity is inhibited by low levels of hydrogen peroxide (H(2)O(2)) generated by the respiratory chain. This occurs via reversible oxidation of cysteine residues 45 and 392 on PDHK2 and results in increased pyruvate dehydrogenase complex activity. H(2)O(2) derives from superoxide (O(2)(.)), and we show that conditions that inhibit PDHK2 also inactivate the TCA cycle enzyme, aconitase. These findings suggest that under conditions of high mitochondrial O(2)(.) production, such as may occur under nutrient excess and low ATP demand, the increase in O(2)() and H(2)O(2) may provide feedback signals to modulate mitochondrial metabolism. PMID:22910903

  14. Inactivation of Pyruvate Dehydrogenase Kinase 2 by Mitochondrial Reactive Oxygen Species*

    PubMed Central

    Hurd, Thomas R.; Collins, Yvonne; Abakumova, Irina; Chouchani, Edward T.; Baranowski, Bartlomiej; Fearnley, Ian M.; Prime, Tracy A.; Murphy, Michael P.; James, Andrew M.

    2012-01-01

    Reactive oxygen species are byproducts of mitochondrial respiration and thus potential regulators of mitochondrial function. Pyruvate dehydrogenase kinase 2 (PDHK2) inhibits the pyruvate dehydrogenase complex, thereby regulating entry of carbohydrates into the tricarboxylic acid (TCA) cycle. Here we show that PDHK2 activity is inhibited by low levels of hydrogen peroxide (H2O2) generated by the respiratory chain. This occurs via reversible oxidation of cysteine residues 45 and 392 on PDHK2 and results in increased pyruvate dehydrogenase complex activity. H2O2 derives from superoxide (O2˙̄), and we show that conditions that inhibit PDHK2 also inactivate the TCA cycle enzyme, aconitase. These findings suggest that under conditions of high mitochondrial O2˙̄ production, such as may occur under nutrient excess and low ATP demand, the increase in O2˙̄ and H2O2 may provide feedback signals to modulate mitochondrial metabolism. PMID:22910903

  15. Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism

    PubMed Central

    Hitosugi, Taro; Fan, Jun; Chung, Tae-Wook; Lythgoe, Katherine; Wang, Xu; Xie, Jianxin; Ge, Qingyuan; Gu, Ting-Lei; Polakiewicz, Roberto D.; Roesel, Johannes L.; Chen, Zhuo (Georgia); Boggon, Titus J.; Lonial, Sagar; Fu, Haian; Khuri, Fadlo R.; Kang, Sumin; Chen, Jing

    2011-01-01

    SUMMARY Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate, and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth. PMID:22195962

  16. YjgF is required for isoleucine biosynthesis when Salmonella enterica is grown on pyruvate medium.

    PubMed

    Christopherson, Melissa R; Schmitz, G E; Downs, Diana M

    2008-04-01

    The YjgF/YER057c/UK114 family of proteins is conserved across the three domains of life, yet no biochemical function has been clearly defined for any member of this family. In Salmonella enterica, a deletion of yjgF results in a requirement for isoleucine when the mutant strain is grown in glucose-serine or pyruvate medium. Feedback inhibition of IlvA is required for the curative effect of isoleucine on glucose-serine medium. On pyruvate medium, yjgF mutants are unable to synthesize enough isoleucine for growth. From this study, we conclude that the isoleucine requirement of a yjgF mutant on pyruvate is a consequence of the decreased transaminase B (IlvE) activity that has previously been characterized in these mutants. PMID:18296521

  17. Respiratory Response of Acer pseudoplatanus Cells to Pyruvate and 2,4-Dinitrophenol 1

    PubMed Central

    Givan, Curtis V.; Torrey, John G.

    1968-01-01

    The endogenous respiration rate of unstarved cultured cells of Acer pseudo-platanus L. is markedly stimulated by 2,4-dinitrophenol. Pyruvate is also stimulatory but to a lesser degree than dinitrophenol. Exogenously supplied sugars cause no short-term stimulation. Pyruvate does not enhance the elevated rate of O2 uptake in the presence of dinitrophenol but does cause additional CO2 evolution. The endogenous concentration of pyruvate is elevated in the presence of dinitrophenol. These observations suggest that the rate of O2 uptake by the unstarved intact cells is limited by the rate of glycolysis and that rate of glycolysis is regulated by the intracellular concentration of adenine nucleotides or inorganic phosphate. Dinitrophenol stimulation of endogenous respiration is due in part to an indirect acceleration of glycolysis but also to a more direct facilitation of oxidation in the presence of excess mitochondrial substrate. PMID:16656818

  18. Pyruvate carboxylation enables growth of SDH-deficient cells by supporting aspartate biosynthesis.

    PubMed

    Cardaci, Simone; Zheng, Liang; MacKay, Gillian; van den Broek, Niels J F; MacKenzie, Elaine D; Nixon, Colin; Stevenson, David; Tumanov, Sergey; Bulusu, Vinay; Kamphorst, Jurre J; Vazquez, Alexei; Fleming, Stewart; Schiavi, Francesca; Kalna, Gabriela; Blyth, Karen; Strathdee, Douglas; Gottlieb, Eyal

    2015-10-01

    Succinate dehydrogenase (SDH) is a heterotetrameric nuclear-encoded complex responsible for the oxidation of succinate to fumarate in the tricarboxylic acid cycle. Loss-of-function mutations in any of the SDH genes are associated with cancer formation. However, the impact of SDH loss on cell metabolism and the mechanisms enabling growth of SDH-defective cells are largely unknown. Here, we generated Sdhb-ablated kidney mouse cells and used comparative metabolomics and stable-isotope-labelling approaches to identify nutritional requirements and metabolic adaptations to SDH loss. We found that lack of SDH activity commits cells to consume extracellular pyruvate, which sustains Warburg-like bioenergetic features. We further demonstrated that pyruvate carboxylation diverts glucose-derived carbons into aspartate biosynthesis, thus sustaining cell growth. By identifying pyruvate carboxylase as essential for the proliferation and tumorigenic capacity of SDH-deficient cells, this study revealed a metabolic vulnerability for potential future treatment of SDH-associated malignancies. PMID:26302408

  19. Metabolism of 1-aminoethylphosphinate generates acetylphosphinate, a potent inhibitor of pyruvate dehydrogenase.

    PubMed Central

    Laber, B; Amrhein, N

    1987-01-01

    The alanine analogue 1-aminoethylphosphinate [H3C-CH(NH2)-PO2H2] effectively inhibited anthocyanin synthesis in buckwheat hypocotyls and caused an increase in the concentrations of alanine and alanine-derived metabolites. Aminotransferase inhibitors partially alleviated the effects of the analogue. 1-Aminoethylphosphinate did not affect the growth of Klebsiella pneumoniae under anaerobic conditions, but under aerobic conditions it inhibited growth and caused the massive excretion of pyruvate. The analogue inhibited the pyruvate dehydrogenase complex in vitro in the presence of an aminotransferase activity. The transamination product of 1-aminoethylphosphinate, acetylphosphinate (H3C-CO-PO2H2), was found to inhibit the pyruvate dehydrogenase complex in a time-dependent reaction that followed first-order and saturation kinetics and required the presence of thiamin pyrophosphate. PMID:3325039

  20. Metabolism of 1-aminoethylphosphinate generates acetylphosphinate, a potent inhibitor of pyruvate dehydrogenase.

    PubMed

    Laber, B; Amrhein, N

    1987-12-01

    The alanine analogue 1-aminoethylphosphinate [H3C-CH(NH2)-PO2H2] effectively inhibited anthocyanin synthesis in buckwheat hypocotyls and caused an increase in the concentrations of alanine and alanine-derived metabolites. Aminotransferase inhibitors partially alleviated the effects of the analogue. 1-Aminoethylphosphinate did not affect the growth of Klebsiella pneumoniae under anaerobic conditions, but under aerobic conditions it inhibited growth and caused the massive excretion of pyruvate. The analogue inhibited the pyruvate dehydrogenase complex in vitro in the presence of an aminotransferase activity. The transamination product of 1-aminoethylphosphinate, acetylphosphinate (H3C-CO-PO2H2), was found to inhibit the pyruvate dehydrogenase complex in a time-dependent reaction that followed first-order and saturation kinetics and required the presence of thiamin pyrophosphate. PMID:3325039

  1. Abundance, Dynamics, and Biogeographic Distribution of Seven Polycyclic Aromatic Hydrocarbon Dioxygenase Gene Variants in Coastal Sediments of Patagonia

    PubMed Central

    Marcos, Magalí S.; Lozada, Mariana; Di Marzio, Walter D.

    2012-01-01

    Novel polycyclic aromatic hydrocarbon dioxygenase gene variants were present in abundances similar to or higher than those of phnA1 from Cycloclasticus spp. at a chronically polluted subantarctic coastal marine environment in Patagonia. These novel gene variants were detected over a 6-year time span and were also present in sediments from temperate Patagonian sites. PMID:22226948

  2. Isolation and characterization of a novel strain of Stenotrophomonas maltophilia possessing various dioxygenases for monocyclic hydrocarbon degradation

    PubMed Central

    Urszula, Guzik; Izabela, Greń; Danuta, Wojcieszyńska; Sylwia, Łabużek

    2009-01-01

    A Gram-negative bacterium, designated as strain KB2, was isolated from activated sludge and was found to utilize different aromatic substrates as sole carbon and energy source. On the basis of morphological and physiochemical characteristics and 16S rRNA gene sequence analysis, the isolated strain KB2 was identified as Stenotrophomonas maltophilia. Strain KB2 is from among different Stenotrophomonas maltophilia strains the first one described as exhibiting the activities of three types of dioxygenases depending on the structure of the inducer. The cells grown on benzoate and catechol showed mainly catechol 1,2-dioxygenase activity. The activity of 2,3-dioxygenase was detected after phenol induction. Protocatechuate 3,4-dioxygenase was found in crude cell extracts of this strain after incubation with 4-hydroxybenzoic acid, protocatechuic acid and vanillic acid. Because of broad spectrum of dioxygenases’ types that Stenotrophomonas maltophilia KB2 can exhibit, this strain appears to be very powerful and useful tool in the biotreatment of wastewaters and in soil decontamination. PMID:24031359

  3. Relative red blood cell enzyme levels as a clue to the diagnosis of pyruvate kinase deficiency.

    PubMed

    Titapiwatanakun, Ruetima; Hoyer, James D; Crain, Karen; Arndt, Carola A S

    2008-12-01

    Evaluation of two patients with transfusion dependent anemia revealed RBC pyruvate kinase to be 33% and 41% of the mean normal value, with normal or high values of other RBC enzymes. Parental PK activities were just below normal in three of four of the parents. Subsequent DNA analysis revealed both patients to be compound heterozygotes for PKLR gene mutations, two of which are previously undescribed. Borderline low pyruvate kinase activities with increased in other RBC enzyme activities should prompt consideration of measurement of parental enzyme activities, and confirmation by DNA analysis if available. PMID:18726918

  4. Pyruvate and Metabolic Flexibility: Illuminating a Path Toward Selective Cancer Therapies.

    PubMed

    Olson, Kristofor A; Schell, John C; Rutter, Jared

    2016-03-01

    Dysregulated metabolism is an emerging hallmark of cancer, and there is abundant interest in developing therapies to selectively target these aberrant metabolic phenotypes. Sitting at the decision-point between mitochondrial carbohydrate oxidation and aerobic glycolysis (i.e., the 'Warburg effect'), the synthesis and consumption of pyruvate is tightly controlled and is often differentially regulated in cancer cells. This review examines recent efforts toward understanding and targeting mitochondrial pyruvate metabolism, and addresses some of the successes, pitfalls, and significant challenges of metabolic therapy to date. PMID:26873641

  5. High-field dissolution dynamic nuclear polarization of [1-(13)C]pyruvic acid.

    PubMed

    Yoshihara, Hikari A I; Can, Emine; Karlsson, Magnus; Lerche, Mathilde H; Schwitter, Juerg; Comment, Arnaud

    2016-05-14

    [1-(13)C]pyruvate is the most widely used hyperpolarized metabolic magnetic resonance imaging agent. Using a custom-built 7.0 T polarizer operating at 1.0 K and trityl radical-doped [1-(13)C]pyruvic acid, unextrapolated solution-state (13)C polarization greater than 60% was measured after dissolution and rapid transfer to a spectrometer magnet, demonstrating the signal enhancement attainable using optimized hardware. Slower rates of polarization under these conditions can be largely overcome with higher radical concentrations. PMID:27093499

  6. Chronic Pyruvate Supplementation Increases Exploratory Activity and Brain Energy Reserves in Young and Middle-Aged Mice

    PubMed Central

    Koivisto, Hennariikka; Leinonen, Henri; Puurula, Mari; Hafez, Hani Sayed; Barrera, Glenda Alquicer; Stridh, Malin H.; Waagepetersen, Helle S.; Tiainen, Mika; Soininen, Pasi; Zilberter, Yuri; Tanila, Heikki

    2016-01-01

    Numerous studies have reported neuroprotective effects of pyruvate when given in systemic injections. Impaired glucose uptake and metabolism are found in Alzheimer’s disease (AD) and in AD mouse models. We tested whether dietary pyruvate supplementation is able to provide added energy supply to brain and thereby attenuate aging- or AD-related cognitive impairment. Mice received ~800 mg/kg/day Na-pyruvate in their chow for 2–6 months. In middle-aged wild-type mice and in 6.5-month-old APP/PS1 mice, pyruvate facilitated spatial learning and increased exploration of a novel odor. However, in passive avoidance task for fear memory, the treatment group was clearly impaired. Independent of age, long-term pyruvate increased explorative behavior, which likely explains the paradoxical impairment in passive avoidance. We also assessed pyruvate effects on body weight, muscle force, and endurance, and found no effects. Metabolic postmortem assays revealed increased energy compounds in nuclear magnetic resonance spectroscopy as well as increased brain glycogen storages in the pyruvate group. Pyruvate supplementation may counteract aging-related behavioral impairment, but its beneficial effect seems related to increased explorative activity rather than direct memory enhancement. PMID:27014054

  7. Acetylphosphinate is the most potent mechanism-based substrate-like inhibitor of both the human and E. coli pyruvate dehydrogenase components of the pyruvate dehydrogenase complex

    PubMed Central

    Nemeria, Natalia S.; Korotchkina, Lioubov G.; Chakraborty, Sumit; Patel, Mulchand S.; Jordan, Frank

    2006-01-01

    Two analogues of pyruvate, acetylphosphinate and acetylmethylphosphinate were tested as inhibitors of the E1 (pyruvate dehydrogenase) component of the human and Escherichia coli pyruvate dehydrogenase complexes. This is the first instance of such studies on the human enzyme. The acetylphosphinate is a stronger inhibitor of both enzymes (Ki< 1 μM) than acetylmethylphosphinate. Both inhibitors are found to be reversible tight-binding inhibitors. With both inhibitors and with both enzymes, the inhibition apparently takes place by formation of a C2α-phosphinolactylthiamin diphosphate derivative, a covalent adduct of the inhibitor and the coenzyme, mimicking the behavior of substrate and forming a stable analogue of the C2α-lactylthiamin diphosphate. Formation of the intermediate analogue in each case is confirmed by the appearance of a positive circular dichroism signal in the 305-306 nm range, attributed to the 1',4'-iminopyrimidine tautomeric form of the coenzyme. It is further shown that the αHis63 residue of the human E1 has a role in the formation of C2α-lactylthiamin diphosphate since the αHis63Ala variant is only modestly inhibited by either inhibitor, nor did either compound generate the circular dichroism bands assigned to different tautomeric forms of the 4'-aminopyrimidine ring of the coenzyme seen with the wild type enzyme. Interestingly, opposite enantiomers of the carboligase side product acetoin are produced by the human and bacterial enzymes. PMID:17070897

  8. Acetylphosphinate is the most potent mechanism-based substrate-like inhibitor of both the human and Escherichia coli pyruvate dehydrogenase components of the pyruvate dehydrogenase complex.

    PubMed

    Nemeria, Natalia S; Korotchkina, Lioubov G; Chakraborty, Sumit; Patel, Mulchand S; Jordan, Frank

    2006-12-01

    Two analogues of pyruvate, acetylphosphinate and acetylmethylphosphinate were tested as inhibitors of the E1 (pyruvate dehydrogenase) component of the human and Escherichia coli pyruvate dehydrogenase complexes. This is the first instance of such studies on the human enzyme. The acetylphosphinate is a stronger inhibitor of both enzymes (Ki < 1 microM) than acetylmethylphosphinate. Both inhibitors are found to be reversible tight-binding inhibitors. With both inhibitors and with both enzymes, the inhibition apparently takes place by formation of a C2alpha-phosphinolactylthiamin diphosphate derivative, a covalent adduct of the inhibitor and the coenzyme, mimicking the behavior of substrate and forming a stable analogue of the C2alpha-lactylthiamin diphosphate. Formation of the intermediate analogue in each case is confirmed by the appearance of a positive circular dichroism band in the 305-306 nm range, attributed to the 1',4'-iminopyrimidine tautomeric form of the coenzyme. It is further shown that the alphaHis63 residue of the human E1 has a role in the formation of C2alpha-lactylthiamin diphosphate since the alphaHis63Ala variant is only modestly inhibited by either inhibitor, nor did either compound generate the circular dichroism bands assigned to different tautomeric forms of the 4'-aminopyrimidine ring of the coenzyme seen with the wild-type enzyme. Interestingly, opposite enantiomers of the carboligase side product acetoin are produced by the human and bacterial enzymes. PMID:17070897

  9. Involvement of a flavosemiquinone in the enzymatic oxidation of nitroalkanes catalyzed by 2-nitropropane dioxygenase.

    PubMed

    Francis, Kevin; Russell, Bethany; Gadda, Giovanni

    2005-02-18

    2-Nitropropane dioxygenase (EC 1.13.11.32) catalyzes the oxidation of nitroalkanes into their corresponding carbonyl compounds and nitrite. In this study, the ncd-2 gene encoding for the enzyme in Neurospora crassa was cloned, expressed in Escherichia coli, and the resulting enzyme was purified. Size exclusion chromatography, heat denaturation, and mass spectroscopic analyses showed that 2-nitropropane dioxygenase is a homodimer of 80 kDa, containing a mole of non-covalently bound FMN per mole of subunit, and is devoid of iron. With neutral nitroalkanes and anionic nitronates other than propyl-1- and propyl-2-nitronate, for which a non-enzymatic free radical reaction involving superoxide was established using superoxide dismutase, substrate oxidation occurs within the enzyme active site. The enzyme was more specific for nitronates than nitroalkanes, as suggested by the second order rate constant k(cat)/K(m) determined with 2-nitropropane and primary nitroalkanes with alkyl chain lengths between 2 and 6 carbons. The steady state kinetic mechanism with 2-nitropropane, nitroethane, nitrobutane, and nitrohexane, in either the neutral or anionic form, was determined to be sequential, consistent with oxygen reacting with a reduced form of enzyme before release of the carbonyl product. Enzyme-monitored turnover with ethyl nitronate as substrate indicated that the catalytically relevant reduced form of enzyme is an anionic flavin semiquinone, whose formation requires the substrate, but not molecular oxygen, as suggested by anaerobic substrate reduction with nitroethane or ethyl nitronate. Substrate deuterium kinetic isotope effects with 1,2-[(2)H(4)]nitroethane and 1,1,2-[(2)H(3) ethyl nitronate at pH 8 yielded normal and inverse effects on the k(cat)/K(m) value, respectively, and were negligible on the k(cat) value. The k(cat)/K(m) and k(cat) pH profiles with anionic nitronates showed the requirement of an acid, whereas those for neutral nitroalkanes were consistent with

  10. Breeding of a cyclic imide-assimilating bacterium, Pseudomonas putida s52, for high efficiency production of pyruvate.

    PubMed

    Hibi, Makoto; Horinouchi, Nobuyuki; Tu, Weihao; Soong, Chee-Leong; Ito, Masashi; Segawa, Toshinori; Mu, Xiaoqing; Hagishita, Tairo; Yokozeki, Kenzo; Shimizu, Sakayu; Ogawa, Jun

    2013-01-01

    A succinimide-assimilating bacterium, Pseudomonas putida s52, was found to be a potent producer of pyruvate from fumarate. Using washed cells from P. putida s52 as catalyst, 400 mM pyruvate was produced from 500 mM fumarate in a 36-h reaction. Bromopyruvate, a malic enzyme inhibitor, was used for the selection of mutants with higher pyruvate productivity. A bromopyruvate-resistant mutant, P. putida 15160, was found to be an effective catalyst for pyruvate production. Moreover, under batch bioreactor conditions, 767 mM of pyruvate was successfully produced from 1,000 mM fumarate in a 72-h reaction with washed cells from P. putida 15160 as catalyst. PMID:23924711

  11. Validation of the In Vivo Assessment of Pyruvate Dehydrogenase Activity Using Hyperpolarized 13C-Magnetic Resonance Spectroscopy

    PubMed Central

    Dodd, Michael S.; Heather, Lisa C.; Carter, Emma E.; Cochlin, Lowri E.; Nagel, Simon; Sibson, Nicola R.; Radda, George K.; Clarke, Kieran; Tyler, Damian J.

    2015-01-01

    Aim Many diseases of the heart are characterised by changes in substrate utilisation, which is in part regulated by the activity of the enzyme pyruvate dehydrogenase (PDH). Consequently, there is much interest in the in vivo evaluation of PDH activity in a range of physiological and pathological states to obtain information regarding the metabolic mechanisms of cardiac diseases. Hyperpolarized [1-13C]pyruvate, detected using MRS, is a novel technique for evaluating PDH flux non-invasively. PDH flux has been assumed to directly reflect in vivo PDH activity, although to date this assumption remains unproven. Methods Control animals and animals undergoing interventions known to modulate PDH activity, namely high fat feeding and dichloroacetate infusion, were used to investigate the relationship between in vivo hyperpolarized MRS measurements of PDH flux and ex vivo measurements of PDH enzyme activity (PDHa). Further, the plasma concentrations of pyruvate and other important metabolites were evaluated following pyruvate infusion to assess the metabolic consequences of the pyruvate infusion during hyperpolarized MRS experiments. Results Hyperpolarized MRS measurements of PDH flux significantly correlated with ex vivo measurements of PDHa, confirming that PDH activity directly influences the in vivo flux of hyperpolarized pyruvate through cardiac PDH. The maximum plasma concentration of pyruvate reached during hyperpolarized MRS experiments was ~250 μM, equivalent to physiological pyruvate concentrations reached during exercise or with dietary interventions. Concentrations of other metabolites, including lactate, glucose and β-hydroxybutyrate (BHB), did not vary during the 60 s following pyruvate infusion. Hence, during the 60 s data acquisition period, metabolism was minimally affected by pyruvate infusion. PMID:20799252

  12. Indoleamine 2,3-dioxygenase depletes tryptophan, activates general control non-derepressible 2 kinase and down-regulates key enzymes involved in fatty acid synthesis in primary human CD4+ T cells.

    PubMed

    Eleftheriadis, Theodoros; Pissas, Georgios; Antoniadi, Georgia; Liakopoulos, Vassilios; Stefanidis, Ioannis

    2015-10-01

    Indoleamine 2,3-dioxygenase (IDO) is expressed in antigen-presenting cells and exerts immunosuppressive effects on CD4(+) T cells. One mechanism is through the inhibition of aerobic glycolysis. Another prerequisite for T-cell proliferation and differentiation into effector cells is increased fatty acid (FA) synthesis. The effect of IDO on enzymes involved in FA synthesis was evaluated in primary human cells both in mixed lymphocyte reactions in the presence or not of the IDO inhibitor 1-dl-methyl-tryptophan, and in stimulated CD4(+) T cells in the presence or not of the general control non-derepressible 2 (GCN2) kinase activator tryptophanol (TRP). IDO or TRP inhibited cell proliferation. By assessing the level of GCN2 kinase or mammalian target of rapamycin complex 1 substrates along with a kynurenine free system we showed that IDO exerts its effect mainly through activation of GCN2 kinase. IDO or TRP down-regulated ATP-citrate lyase and acetyl coenzyme A carboxylase 1, key enzymes involved in FA synthesis. Also, IDO or TRP altered the expression of enzymes that control the availability of carbon atoms for FA synthesis, such as lactate dehydrogenase-A, pyruvate dehydrogenase, glutaminase 1 and glutaminase 2, in a way that inhibits FA synthesis. In conclusion, IDO through GCN2 kinase activation inhibits CD4(+) T-cell proliferation and down-regulates key enzymes that directly or indirectly promote FA synthesis, a prerequisite for CD4(+) T-cell proliferation and differentiation into effector cell lineages. PMID:26147366

  13. 2-Diazo-1-(4-hydroxyphenyl)ethanone: A Versatile Photochemical and Synthetic Reagenta

    PubMed Central

    Senadheera, Sanjeewa N.; Evans, Anthony S.; Toscano, John P.; Givens, Richard S.

    2014-01-01

    α-Diazo arylketones are well-known substrates for Wolff rearrangement to phenylacetic acids through a ketene intermediate by either thermal or photochemical activation. Likewise, α-substituted p-hydroxyphenacyl (pHP) esters are substrates for photo-Favorskii rerrangements to phenylacetic acids by a different pathway that purportedly involves a cyclopropanone intermediate. In this paper, we show that the photolysis of a series of α-diazo-p-hydroxyacetophenones and p-hydroxyphenacyl (pHP) α-esters both generate the identical rearranged phenylacetates as major products. Since α-diazo-p-hydroxyacetophenone (1a, pHP N2) contains all the necessary functionalities for either Wolff or Favorskii rearrangement, we were prompted to probe this intriguing mechanistic dichotomy under conditions favorable to the photo-Favorskii reangement, i.e., photolysis in hydroxylic media. An investigation of the mechanism for conversion of 1a to p-hydroxyphenyl acetic acid (4a) using time-resolved infrared (TRIR) spectroscopy clearly demonstrates the formation of a ketene intermediate that is subsequently trapped by solvent or nucleophiles. The photoreaction of 1a is quenched by oxygen and sensitized by triplet sensitizers and the quantum yields for 1a–c range from 0.19 to a robust 0.25. The lifetime of the triplet, determined by Stern-Volmer quenching, is 15 ns with a rate for appearance of 4a of k = 7,1 × 106 s−1 in aq. acetonitrile (1:1 v:v). These studies establish that the primary rearrangement pathway for 1a involves ketene formation in accordance with the photo-Wolff rearrangement. Furthermore we have also demonstrated the synthetic utility of 1a as an esterification and etherification reagent with a variety of substituted α-diazo-p-hydroxyacetophenones, using them as synthons for efficiently coupling it to acids and phenols to produce pHP protect substrates. PMID:24305682

  14. 2-Oxoglutarate-dependent dioxygenases in the biosynthesis of simple coumarins

    PubMed Central

    Shimizu, Bun-Ichi

    2014-01-01

    Coumarins are natural plant products that have been the subject of extensive phytochemical and pharmacological research studies in the past few decades. The core structure of coumarins is derived from the respective cinnamates via ortho-hydroxylation of the aromatic ring, trans/cis isomerization, and lactonization. Various substitution patterns of coumarins have been reported, whereas the biosynthesis of coumarins remains elusive. Ortho-hydroxylation is a key step in simple coumarin biosynthesis as a branch point from the lignin biosynthetic pathway. 2-Oxoglutarate-dependent dioxygenases (2OGDs) from plants convert cinnamate derivatives into simple coumarins through the process of ortho-hydroxylation. This review describes the 2OGDs involved in coumarin biosynthesis and their substrate specificities. PMID:25404933

  15. Functional diversity of 2-oxoglutarate/Fe(II)-dependent dioxygenases in plant metabolism

    PubMed Central

    Farrow, Scott C.; Facchini, Peter J.

    2014-01-01

    Oxidative enzymes catalyze many different reactions in plant metabolism. Among this suite of enzymes are the 2-oxoglutarate/Fe(II)-dependent dioxygenases (2-ODDs). Cytochromes P450 (CYPs) as often considered the most versatile oxidative enzymes in nature, but the diversity and complexity of reactions catalyzed by 2-ODDs is superior to the CYPs. The list of oxidative reactions catalyzed by 2-ODDs includes hydroxylations, demethylations, desaturations, ring closure, ring cleavage, epimerization, rearrangement, halogenation, and demethylenation. Furthermore, recent work, including the discovery of 2-ODDs involved in epigenetic regulation, and others catalyzing several characteristic steps in specialized metabolic pathways, support the argument that 2-ODDs are among the most versatile and important oxidizing biological catalysts. In this review, we survey and summarize the pertinent literature with a focus on several key reactions catalyzed by 2-ODDs, and discuss the significance and impact of these enzymes in plant metabolism. PMID:25346740

  16. 1,2,3-Triazoles as inhibitors of indoleamine 2,3-dioxygenase 2 (IDO2).

    PubMed

    Röhrig, Ute F; Majjigapu, Somi Reddy; Caldelari, Daniela; Dilek, Nahzli; Reichenbach, Patrick; Ascencao, Kelly; Irving, Melita; Coukos, George; Vogel, Pierre; Zoete, Vincent; Michielin, Olivier

    2016-09-01

    Indoleamine 2,3-dioxygenase 2 (IDO2) is a potential therapeutic target for the treatment of diseases that involve immune escape such as cancer. In contrast to IDO1, only a very limited number of inhibitors have been described for IDO2 due to inherent difficulties in expressing and purifying a functionally active, soluble form of the enzyme. Starting from our previously discovered highly efficient 4-aryl-1,2,3-triazole IDO1 inhibitor scaffold, we used computational structure-based methods to design inhibitors of IDO2 which we then tested in cellular assays. Our approach yielded low molecular weight inhibitors of IDO2, the most active displaying an IC50 value of 51μM for mIDO2, and twofold selectivity over hIDO1. These compounds could be useful as molecular probes to investigate the biological role of IDO2, and could inspire the design of new IDO2 inhibitors. PMID:27469130

  17. Enhanced degradation of polychlorinated biphenyls by directed evolution of biphenyl dioxygenase

    SciTech Connect

    Kumamaru, Tetsuya; Suenaga, Hikaru; Mitsuoka, Mariko; Watanabe, Takahito; Furukawa, Kensuke

    1998-07-01

    Biphenyl dioxygenases (BP Dox) from different organisms, which are involved in the initial oxygenation and subsequent degradation of polychlorinated biphenyls (PCB), are similar in structure but have different functions. The large subunit of BP Dox, encoded by the bphA1 gene, is crucial for substrate selectivity. Using the process of DNA shuffling, the authors randomly recombined the bphA1 genes of Pseudomonas pseudoalcaligenes KF707 and Burkholderia cepacia LB400 and selected for genes that expressed proteins with altered function. Upon expression in Escherichia coli, some of these evolved genes exhibited enhanced degradation capacity, not only for PCB and related biphenyl compounds, but for single aromatic hydrocarbons such as benzene and toluene, which are poor substrates for the original BP Dox.

  18. Homogentisate 1,2 Dioxygenase is Expressed in Human Osteoarticular Cells: Implications in Alkaptonuria

    PubMed Central

    Laschi, Marcella; Tinti, Laura; Braconi, Daniela; Millucci, Lia; Ghezzi, Lorenzo; Amato, Loredana; Selvi, Enrico; Spreafico, Adriano; Bernardini, Giulia; Santucci, Annalisa

    2012-01-01

    Alkaptonuria (AKU) results from defective homogentisate1,2-dioxygenase (HGD), causing degenerative arthropathy. The deposition of ochronotic pigment in joints is so far attributed to homogentisic acid produced by the liver, circulating in the blood and accumulating locally. Human normal and AKU osteoarticular cells were tested for HGD gene expression by RT-PCR, mono- and 2D-Western blotting. HGD gene expression was revealed in chondrocytes, synoviocytes, osteoblasts. Furthermore, HGD expression was confirmed by Western blotting, that also revealed the presence of five enzymatic molecular species. Our findings indicate that AKU osteoarticular cells produce the ochronotic pigment in loco and this may strongly contribute to induction of ochronotic arthropathy. J. Cell. Physiol. 227: 3254–3257, 2012. © 2011 Wiley Periodicals, Inc. PMID:22105303

  19. Homogentisate 1,2 dioxygenase is expressed in brain: implications in alkaptonuria.

    PubMed

    Bernardini, Giulia; Laschi, Marcella; Geminiani, Michela; Braconi, Daniela; Vannuccini, Elisa; Lupetti, Pietro; Manetti, Fabrizio; Millucci, Lia; Santucci, Annalisa

    2015-09-01

    Alkaptonuria is an ultra-rare autosomal recessive disease developed from the lack of homogentisate 1,2-dioxygenase (HGD) activity, causing an accumulation in connective tissues of homogentisic acid (HGA) and its oxidized derivatives in polymerized form. The deposition of ochronotic pigment has been so far attributed to homogentisic acid produced by the liver, circulating in the blood, and accumulating locally. In the present paper, we report the expression of HGD in the brain. Mouse and human brain tissues were positively tested for HGD gene expression by western blotting. Furthermore, HGD expression was confirmed in human neuronal cells that also revealed the presence of six HGD molecular species. Moreover, once cultured in HGA excess, human neuronal cells produced ochronotic pigment and amyloid. Our findings indicate that alkaptonuric brain cells produce the ochronotic pigment in loco and this may contribute to induction of neurological complications. PMID:25762405

  20. Homogentisate 1,2 dioxygenase is expressed in human osteoarticular cells: implications in alkaptonuria.

    PubMed

    Laschi, Marcella; Tinti, Laura; Braconi, Daniela; Millucci, Lia; Ghezzi, Lorenzo; Amato, Loredana; Selvi, Enrico; Spreafico, Adriano; Bernardini, Giulia; Santucci, Annalisa

    2012-09-01

    Alkaptonuria (AKU) results from defective homogentisate1,2-dioxygenase (HGD), causing degenerative arthropathy. The deposition of ochronotic pigment in joints is so far attributed to homogentisic acid produced by the liver, circulating in the blood and accumulating locally. Human normal and AKU osteoarticular cells were tested for HGD gene expression by RT-PCR, mono- and 2D-Western blotting. HGD gene expression was revealed in chondrocytes, synoviocytes, osteoblasts. Furthermore, HGD expression was confirmed by Western blotting, that also revealed the presence of five enzymatic molecular species. Our findings indicate that AKU osteoarticular cells produce the ochronotic pigment in loco and this may strongly contribute to induction of ochronotic arthropathy. PMID:22105303

  1. Ferrous iron and α-ketoglutarate-dependent dioxygenases in the biosynthesis of microbial natural products.

    PubMed

    Wu, Long-Fei; Meng, Song; Tang, Gong-Li

    2016-05-01

    Apart from its vital role as the terminal electron acceptor in oxidative phosphorylation in nature, dioxygen also serves as a universal agent which diversifies natural products by oxidative transformations. Ferrous iron and α-ketoglutarate (αKG)-dependent dioxygenases (αKGDs) are versatile enzymes that use dioxygen as an oxidant to catalyse various reactions via CH bond activation, including hydroxylation, dealkylation, desaturation, epoxidation, epimerisation, halogenation, cyclisation, peroxide formation, and ring expansion/contraction reactions. This review updates the reported αKGDs that catalyse reactions related to microbial natural product biosynthesis in the past 10 years. We hope that the versatility of αKGDs shown here can serve as an inspiration for future engineering and catalyst design, which could provide alternative methods to meet the on-going demand for fine chemicals and pharmaceutics. PMID:26845569

  2. Dendritic cells, indoleamine 2,3 dioxygenase and acquired immune privilege

    PubMed Central

    Huang, Lei; Baban, Babak; Johnson, Burles A; Mellor, Andrew L.

    2013-01-01

    Dendritic cells (DCs) are specialized to stimulate T cell immunity. Paradoxically some DCs suppress T cell responses, and activate regulatory T cells. In this review we focus on a potent counter-regulatory pathway mediated by plasmacytoid DCs (pDCs) expressing the immunosuppressive enzyme indoleamine 2,3 dioxygenase (IDO). IDO-expressing pDCs inhibit effector T cell responses, activate regulatory T cells, and attenuate pro-inflammatory responses in settings of chronic inflammation that manifest in clinical syndromes such as infectious, allergic and autoimmune diseases, cancer, and transplantation. Thus IDO-expressing pDCs create immune privilege, and provide novel opportunities to improve immunotherapy in multiple disease syndromes. PMID:20367139

  3. Crystallization and Initial X-Ray Diffraction Analysis of Human Pyruvate Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Ciszak, Ewa; Korotchkina, Lioubov G.; Hong, Young-Soo; Joachimiak, Andrzj; Patel, Mulchand S.; Rose, M. Franklin

    2000-01-01

    Human pyruvate dehydrogenase (E1) is a component enzyme of the pyruvate dehydrogenase complex. The enzyme catalyzes the decarboxylation of pyruvate followed by a reductive acetylation of lipoyl groups of the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex. El is an alpha(sub 2)Beta(sub 2) tetrameric assembly of an approximate molecular mass of 154 kDa. The crystals of this recombinant enzyme have been grown from polyethylene glycol 3350 using vapor diffusion method at 295K. The crystals are characterized as orthorhombic, space group P2(sub 1)2(sub 1)2(sub 1), with cell parameters of a = 64.2, b = 126.9 and c = 190.2 A. Crystals diffracted to a minimum d-spacing of 2.5 A. The asymmetric unit contains one alpha(sub 2)Beta(sub 2) tetrameric El assembly, and self-rotation function analysis showed a pseudo-twofold symmetry relating the two monomers.

  4. Pyruvate dose response studies targeting the vital signs following hemorrhagic shock

    PubMed Central

    Sharma, Pushpa; Vyacheslav, Makler; Carissa, Chalut; Vanessa, Rodriguez; Bodo, Mike

    2015-01-01

    Objectives: To determine the optimal effective dose of sodium pyruvate in maintaining the vital signs following hemorrhagic shock (HS) in rats. Materials and Methods: Anesthetized, male Sprague-Dawley rats underwent computer-controlled HS for 30 minute followed by fluid resuscitation with either hypertonic saline, or sodium pyruvate solutions of 0.5 M, 1.0 M, 2.0 M, and 4.0 M at a rate of 5ml/kg/h (60 minute) and subsequent blood infusion (60 minute). The results were compared with sham and non- resuscitated groups. The animals were continuously monitored for mean arterial pressure, systolic and diastolic pressure, heart rate, pulse pressure, temperature, shock index and Kerdo index (KI). Results: The Sham group remained stable throughout the experiment. Non-resuscitated HS animals did not survive for the entire experiment due to non-viable vital signs and poor shock and KI. All fluids were effective in normalizing the vital signs when shed blood was used adjunctively. Sodium pyruvate 2.0 M was most effective, and 4.0 M solution was least effective in improving the vital signs after HS. Conclusions: Future studies should be directed to use 2.0 M sodium pyruvate adjuvant for resuscitation on multiorgan failure and survival rate in HS. PMID:26229300

  5. [The important role of vitamins in the over-production of pyruvic acid].

    PubMed

    Li, Y; Chen, J; Lun, S; Rui, X

    2000-10-01

    The effect of nicotinic acid, thiamine, pyridoxine, biotin and riboflavin on the production of pyruvic acid by Torulopsis glabrata WSH-IP303 with glucose as carbon source and NH4Cl as sole nitrogen source was investigated. By using orthogonal experiment method, thiamine was confirmed to be the most important factor affecting the production of pyruvic acid. Based on a certain concentration range of thiamine (0.01-0.015 mg/L), glucose consumption rate can be enhanced by increasing the concentration of nicotinic acid. When the concentration of nicotinic acid, thiamine, pyridoxine, biotin and riboflavin were 8, 0.015, 0.4, 0.04 and 0.1 mg/L, respectively, the concentration and yield to glucose of pyruvic acid reached 52.4 g/L and 0.525 g/g at 48 h in flask culture, respectively. Batch culture was conducted in a 2.5 L fermentor with initial glucose concentration of 120 g/L. By adopting the optimal concentration combination of vitamins, the concentration and yield to glucose of pyruvic acid reached 69.4 g/L and 0.593 g/g at 57.5 h, which were increased by 32.4% and 13% than the best results in flask culture, respectively. PMID:12548766

  6. NH4(+) triggers the release of astrocytic lactate via mitochondrial pyruvate shunting.

    PubMed

    Lerchundi, Rodrigo; Fernández-Moncada, Ignacio; Contreras-Baeza, Yasna; Sotelo-Hitschfeld, Tamara; Mächler, Philipp; Wyss, Matthias T; Stobart, Jillian; Baeza-Lehnert, Felipe; Alegría, Karin; Weber, Bruno; Barros, L Felipe

    2015-09-01

    Neural activity is accompanied by a transient mismatch between local glucose and oxygen metabolism, a phenomenon of physiological and pathophysiological importance termed aerobic glycolysis. Previous studies have proposed glutamate and K(+) as the neuronal signals that trigger aerobic glycolysis in astrocytes. Here we used a panel of genetically encoded FRET sensors in vitro and in vivo to investigate the participation of NH4(+), a by-product of catabolism that is also released by active neurons. Astrocytes in mixed cortical cultures responded to physiological levels of NH4(+) with an acute rise in cytosolic lactate followed by lactate release into the extracellular space, as detected by a lactate-sniffer. An acute increase in astrocytic lactate was also observed in acute hippocampal slices exposed to NH4(+) and in the somatosensory cortex of anesthetized mice in response to i.v. NH4(+). Unexpectedly, NH4(+) had no effect on astrocytic glucose consumption. Parallel measurements showed simultaneous cytosolic pyruvate accumulation and NADH depletion, suggesting the involvement of mitochondria. An inhibitor-stop technique confirmed a strong inhibition of mitochondrial pyruvate uptake that can be explained by mitochondrial matrix acidification. These results show that physiological NH4(+) diverts the flux of pyruvate from mitochondria to lactate production and release. Considering that NH4(+) is produced stoichiometrically with glutamate during excitatory neurotransmission, we propose that NH4(+) behaves as an intercellular signal and that pyruvate shunting contributes to aerobic lactate production by astrocytes. PMID:26286989

  7. The mitochondrial pyruvate carrier in health and disease: To carry or not to carry?

    PubMed

    Bender, Tom; Martinou, Jean-Claude

    2016-10-01

    Mitochondria play a key role in energy metabolism, hosting the machinery for oxidative phosphorylation, the most efficient cellular pathway for generating ATP. A major checkpoint in this process is the transport of pyruvate produced by cytosolic glycolysis into the mitochondrial matrix, which is accomplished by the recently identified mitochondrial pyruvate carrier (MPC). As the gatekeeper for pyruvate entry into mitochondria, the MPC is thought to be of fundamental importance in establishing the metabolic programming of a cell. This is especially relevant in the context of the aerobic glycolysis, also known as the Warburg effect, which is a hallmark in many types of cancer, and MPC loss of function promotes cancer growth. Moreover, mitochondrial pyruvate uptake is needed for efficient hepatic gluconeogenesis and the regulation of blood glucose levels. In this review we discuss recent advances in our knowledge of the MPC, and we argue that it may offer a promising target in diseases like cancer and type 2 diabetes. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou. PMID:26826034

  8. Sunlight-initiated Chemistry of Aqueous Pyruvic Acid: Building Complexity in the Origin of Life

    NASA Astrophysics Data System (ADS)

    Griffith, Elizabeth C.; Shoemaker, Richard K.; Vaida, Veronica

    2013-10-01

    Coupling chemical reactions to an energy source is a necessary step in the origin of life. Here, we utilize UV photons provided by a simulated sun to activate aqueous pyruvic acid and subsequently prompt chemical reactions mimicking some of the functions of modern metabolism. Pyruvic acid is interesting in a prebiotic context due to its prevalence in modern metabolism and its abiotic availability on early Earth. Here, pyruvic acid (CH3COCOOH, a C3 molecule) photochemically reacts to produce more complex molecules containing four or more carbon atoms. Acetoin (CH3CHOHCOCH3), a C4 molecule and a modern bacterial metabolite, is produced in this chemistry as well as lactic acid (CH3CHOHCOOH), a molecule which, when coupled with other abiotic chemical reaction pathways, can provide a regeneration pathway for pyruvic acid. This chemistry is discussed in the context of plausible environments on early Earth such as near the ocean surface and atmospheric aerosol particles. These environments allow for combination and exchange of reactants and products of other reaction environments (such as shallow hydrothermal vents). The result could be a contribution to the steady increase in chemical complexity requisite in the origin of life.

  9. SIRT3 DEACETYLATES AND INCREASES PYRUVATE DEHYDROGENASE ACTIVITY IN CANCER CELLS

    PubMed Central

    Wagner, Brett A.; Song, Ha Yong; Zhu, Yueming; Vassilopoulos, Athanassios; Jung, Barbara; Buettner, Garry R.; Gius, David

    2015-01-01

    Pyruvate dehydrogenase E1 alpha (PDHE1α or PDHA1) is the first component enzyme of the pyruvate dehydrogenase (PDH) complex (PDC) that transforms pyruvate, via pyruvate decarboxylation, into acetyl-CoA that is subsequently used by both the citric acid cycle and oxidative phosphorylation to generate ATP. As such, PDH links glycolysis and oxidative phosphorylation in normal as well as cancer cells. Herein we report that SIRT3 interacts with PDHA1 and directs its enzymatic activity via changes in protein acetylation. SIRT3 deacetylates PDHA1 lysine 321 (K321) and a PDHA1 mutant, mimicking a deacetylated lysine (PDHA1K321R) increases in PDH activity, as compared to the K321 acetylation mimic (PDHA1K321Q) or wild-type PDHA1. Finally, PDHA1K321Q exhibited a more transformed in vitro cellular phenotype as compared to PDHA1K321R. These results suggest that the acetylation of PDHA1 provides another layer of enzymatic regulation, in addition to phosphorylation, involving a reversible acetyl-lysine suggesting that the acetylome, as well as the kinome, links glycolysis to respiration. PMID:25152236

  10. Oxidation of an Adjacent Methionine Residue Inhibits Regulatory Seryl-phosphorylation of Pyruvate Dehydrogenase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A Met residue is located adjacent to phosphorylation site 1 in the sequences of mitochondrial pyruvate dehydrogenase E1alpha subunits. When synthetic peptides including site 1 were treated with Hydrogen peroxide, the Met residue was oxidized to methionine sulfoxide (MetSO), and the peptides were no...

  11. A Substrate-induced Biotin Binding Pocket in the Carboxyltransferase Domain of Pyruvate Carboxylase*

    PubMed Central

    Lietzan, Adam D.; St. Maurice, Martin

    2013-01-01

    Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the carboxyltransferase domain of PC from Rhizobium etli. Structures of the carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp590 and Tyr628 and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes. PMID:23698000

  12. EXPRESSION AND ASSEMBLY OF ARABIDOPSIS THALIANA PYRUVATE DEHYDROGENASE IN INSECT CELL CYTOPLASM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A vector was constructed for expression of Arabidopsis thaliana mitochondrial pyruvate dehydrogenase (E1) in the cytoplasm of Trichoplusia ni cells. The construct, pDDR101, comprises the mature-E1alpha coding sequence under control of the polh promoter, plus the mature-E1beta coding sequence under ...

  13. Embryonic Lethality of Mitochondrial Pyruvate Carrier 1 Deficient Mouse Can Be Rescued by a Ketogenic Diet.

    PubMed

    Vanderperre, Benoît; Herzig, Sébastien; Krznar, Petra; Hörl, Manuel; Ammar, Zeinab; Montessuit, Sylvie; Pierredon, Sandra; Zamboni, Nicola; Martinou, Jean-Claude

    2016-05-01

    Mitochondrial import of pyruvate by the mitochondrial pyruvate carrier (MPC) is a central step which links cytosolic and mitochondrial intermediary metabolism. To investigate the role of the MPC in mammalian physiology and development, we generated a mouse strain with complete loss of MPC1 expression. This resulted in embryonic lethality at around E13.5. Mouse embryonic fibroblasts (MEFs) derived from mutant mice displayed defective pyruvate-driven respiration as well as perturbed metabolic profiles, and both defects could be restored by reexpression of MPC1. Labeling experiments using 13C-labeled glucose and glutamine demonstrated that MPC deficiency causes increased glutaminolysis and reduced contribution of glucose-derived pyruvate to the TCA cycle. Morphological defects were observed in mutant embryonic brains, together with major alterations of their metabolome including lactic acidosis, diminished TCA cycle intermediates, energy deficit and a perturbed balance of neurotransmitters. Strikingly, these changes were reversed when the pregnant dams were fed a ketogenic diet, which provides acetyl-CoA directly to the TCA cycle and bypasses the need for a functional MPC. This allowed the normal gestation and development of MPC deficient pups, even though they all died within a few minutes post-delivery. This study establishes the MPC as a key player in regulating the metabolic state necessary for embryonic development, neurotransmitter balance and post-natal survival. PMID:27176894

  14. Embryonic Lethality of Mitochondrial Pyruvate Carrier 1 Deficient Mouse Can Be Rescued by a Ketogenic Diet

    PubMed Central

    Krznar, Petra; Hörl, Manuel; Ammar, Zeinab; Montessuit, Sylvie; Pierredon, Sandra; Zamboni, Nicola; Martinou, Jean-Claude

    2016-01-01

    Mitochondrial import of pyruvate by the mitochondrial pyruvate carrier (MPC) is a central step which links cytosolic and mitochondrial intermediary metabolism. To investigate the role of the MPC in mammalian physiology and development, we generated a mouse strain with complete loss of MPC1 expression. This resulted in embryonic lethality at around E13.5. Mouse embryonic fibroblasts (MEFs) derived from mutant mice displayed defective pyruvate-driven respiration as well as perturbed metabolic profiles, and both defects could be restored by reexpression of MPC1. Labeling experiments using 13C-labeled glucose and glutamine demonstrated that MPC deficiency causes increased glutaminolysis and reduced contribution of glucose-derived pyruvate to the TCA cycle. Morphological defects were observed in mutant embryonic brains, together with major alterations of their metabolome including lactic acidosis, diminished TCA cycle intermediates, energy deficit and a perturbed balance of neurotransmitters. Strikingly, these changes were reversed when the pregnant dams were fed a ketogenic diet, which provides acetyl-CoA directly to the TCA cycle and bypasses the need for a functional MPC. This allowed the normal gestation and development of MPC deficient pups, even though they all died within a few minutes post-delivery. This study establishes the MPC as a key player in regulating the metabolic state necessary for embryonic development, neurotransmitter balance and post-natal survival. PMID:27176894

  15. Pyruvate blocks blood-brain barrier disruption, lymphocyte infiltration and immune response in excitotoxic brain injury

    PubMed Central

    Ryu, Jae K; McLarnon, James G

    2016-01-01

    The effects of pyruvate, the end metabolite of glycolysis, on blood-brain barrier (BBB) impairment and immune reactivity were examined in the quinolinic acid (QA)-injected rat striatum. Extensive disruption of BBB was observed at 7 d post QA-injection as demonstrated by increased immunohistochemical staining using antibody against immunoglobulin G (IgG). Animals receiving pyruvate administration (500 mg/kg) with QA-injection exhibited reduced lgG immunoreactivity (by 45%) relative to QA alone. QA intrastriatal injection also resulted in marked increases in the number of infiltrating T-lymphocytes (by 70-fold) and expression of major histocompatibility complex (MHC-class II) (by 45-fold) relative to unlesioned control. Treatment with pyruvate significantly reduced infiltration of T-cells (by 68%) and MHC class II expression (by 48%) induced by QA. These results indicate that QA injection into rat striatum leads to impairment in BBB function with pyruvate administration reducing immune response and BBB leakiness in excitotoxic injury. PMID:27073744

  16. NH4+ triggers the release of astrocytic lactate via mitochondrial pyruvate shunting

    PubMed Central

    Lerchundi, Rodrigo; Fernández-Moncada, Ignacio; Contreras-Baeza, Yasna; Sotelo-Hitschfeld, Tamara; Mächler, Philipp; Wyss, Matthias T.; Stobart, Jillian; Baeza-Lehnert, Felipe; Alegría, Karin; Weber, Bruno; Barros, L. Felipe

    2015-01-01

    Neural activity is accompanied by a transient mismatch between local glucose and oxygen metabolism, a phenomenon of physiological and pathophysiological importance termed aerobic glycolysis. Previous studies have proposed glutamate and K+ as the neuronal signals that trigger aerobic glycolysis in astrocytes. Here we used a panel of genetically encoded FRET sensors in vitro and in vivo to investigate the participation of NH4+, a by-product of catabolism that is also released by active neurons. Astrocytes in mixed cortical cultures responded to physiological levels of NH4+ with an acute rise in cytosolic lactate followed by lactate release into the extracellular space, as detected by a lactate-sniffer. An acute increase in astrocytic lactate was also observed in acute hippocampal slices exposed to NH4+ and in the somatosensory cortex of anesthetized mice in response to i.v. NH4+. Unexpectedly, NH4+ had no effect on astrocytic glucose consumption. Parallel measurements showed simultaneous cytosolic pyruvate accumulation and NADH depletion, suggesting the involvement of mitochondria. An inhibitor-stop technique confirmed a strong inhibition of mitochondrial pyruvate uptake that can be explained by mitochondrial matrix acidification. These results show that physiological NH4+ diverts the flux of pyruvate from mitochondria to lactate production and release. Considering that NH4+ is produced stoichiometrically with glutamate during excitatory neurotransmission, we propose that NH4+ behaves as an intercellular signal and that pyruvate shunting contributes to aerobic lactate production by astrocytes. PMID:26286989

  17. In vivo 13 carbon metabolic imaging at 3T with hyperpolarized 13C-1-pyruvate.

    PubMed

    Kohler, S J; Yen, Y; Wolber, J; Chen, A P; Albers, M J; Bok, R; Zhang, V; Tropp, J; Nelson, S; Vigneron, D B; Kurhanewicz, J; Hurd, R E

    2007-07-01

    We present for the first time dynamic spectra and spectroscopic images acquired in normal rats at 3T following the injection of (13)C-1-pyruvate that was hyperpolarized by the dynamic nuclear polarization (DNP) method. Spectroscopic sampling was optimized for signal-to-noise ratio (SNR) and for spectral resolution of (13)C-1-pyruvate and its metabolic products (13)C-1-alanine, (13)C-1-lactate, and (13)C-bicarbonate. Dynamic spectra in rats were collected with a temporal resolution of 3 s from a 90-mm axial slab using a dual (1)H-(13)C quadrature birdcage coil to observe the combined effects of metabolism, flow, and T(1) relaxation. In separate experiments, spectroscopic imaging data were obtained during a 17-s acquisition of a 20-mm axial slice centered on the rat kidney region to provide information on the spatial distribution of the metabolites. Conversion of pyruvate to lactate, alanine, and bicarbonate occurred within a minute of injection. Alanine was observed primarily in skeletal muscle and liver, while pyruvate, lactate, and bicarbonate concentrations were relatively high in the vasculature and kidneys. In contrast to earlier work at 1.5 T, bicarbonate was routinely observed in skeletal muscle as well as the kidney and vasculature. PMID:17659629

  18. Catalytic and regulatory properties of muscle pyruvate kinase from Cancer magister.

    PubMed

    Guderley, H; Hochachka, P W

    1980-06-01

    Despite the marked changes that crustacean muscle undergoes during the molt cycle, pyruvate kinase is present as the same form throughout the molt cycle. This pyruvate kinase was subject to feed-forward activation by fructose-1, 6 bisphosphate (FBP) as well as feed-back inhibition by MgATP. The enzyme showed a high affinity for phosphoenolpyruvate (Km = 0.1 mM) but showed no cooperativity in substrate binding. The addition of 0.05 mM FBP reduced the PEP Km to 0.05 mM. MgATP inhibition showed a Ki of 1.8 mM versus PEP. The inhibition due to MgATP could be reversed by FBP. Various other compounds inhibited the enzyme, including citrate, alpha-ketoglutarate, tryptophan, and malate, although at rather high levels. Measurements of the reversal of this pyruvate kinase, taken together with the low levels of phosphoenolpyruvate carboxykinase and pyruvate carboxylase, predict only minimal levels of gluconeogenic flux in crustacean muscle. PMID:7462968

  19. Formation of indigo and related compounds from indolecarboxylic acids by aromatic acid-degrading bacteria: chromogenic reactions for cloning genes encoding dioxygenases that act on aromatic acids.

    PubMed Central

    Eaton, R W; Chapman, P J

    1995-01-01

    The p-cumate-degrading strain Pseudomonas putida F1 and the m- and p-toluate-degrading strain P. putida mt-2 transform indole-2-carboxylate and indole-3-carboxylate to colored products identified here as indigo, indirubin, and isatin. A mechanism by which these products could be formed spontaneously following dioxygenase-catalyzed dihydroxylation of the indolecarboxylates is proposed. Indolecarboxylates were employed as chromogenic substrates for identifying recombinant bacteria carrying genes encoding p-cumate dioxygenase and toluate dioxygenase. Dioxygenase gene-carrying bacteria could be readily distinguished as dark green-blue colonies among other colorless recombinant Escherichia coli colonies on selective agar plates containing either indole-2-carboxylate or indole-3-carboxylate. PMID:7592495

  20. Nitrosyl hydride (HNO) replaces dioxygen in nitroxygenase activity of manganese quercetin dioxygenase

    PubMed Central

    Kumar, Murugaeson R.; Zapata, Adrian; Ramirez, Alejandro J.; Bowen, Sara K.; Francisco, Wilson A.; Farmer, Patrick J.

    2011-01-01

    Quercetin dioxygenase (QDO) catalyzes the oxidation of the flavonol quercetin with dioxygen, cleaving the central heterocyclic ring and releasing CO. The QDO from Bacillus subtilis is unusual in that it has been shown to be active with several divalent metal cofactors such as Fe, Mn, and Co. Previous comparison of the catalytic activities suggest that Mn(II) is the preferred cofactor for this enzyme. We herein report the unprecedented substitution of nitrosyl hydride (HNO) for dioxygen in the activity of Mn-QDO, resulting in the incorporation of both N and O atoms into the product. Turnover is demonstrated by consumption of quercetin and other related substrates under anaerobic conditions in the presence of HNO-releasing compounds and the enzyme. As with dioxygenase activity, a nonenzymatic base-catalyzed reaction of quercetin with HNO is observed above pH 7, but no enhancement of this basal reactivity is found upon addition of divalent metal salts. Unique and regioselective N-containing products (14N/15N) have been characterized by MS analysis for both the enzymatic and nonenzymatic reactions. Of the several metallo-QDO enzymes examined for nitroxygenase activity under anaerobic condition, only the Mn(II) is active; the Fe(II) and Co(II) substituted enzymes show little or no activity. This result represents an enzymatic catalysis which we denote nitroxygenase activity; the unique reactivity of the Mn-QDO suggests a metal-mediated electron transfer mechanism rather than metal activation of the substrate’s inherent base-catalyzed reactivity. PMID:22084064

  1. Metabolism of chlorobiphenyls by a variant biphenyl dioxygenase exhibiting enhanced activity toward dibenzofuran

    SciTech Connect

    Viger, Jean-Francois; Mohammadi, Mahmood; Barriault, Diane; Sylvestre, Michel

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Burkholderia xenovorans LB400 biphenyl dioxygenase (BphAE{sub LB400}) metabolizes PCBs. Black-Right-Pointing-Pointer Asn338Gln/Leu409Phe double mutation speeds up electron transfer of enzyme reaction. Black-Right-Pointing-Pointer We tested how the mutations affect the PCB-degrading abilities of BphAE{sub LB400} variants. Black-Right-Pointing-Pointer The same mutations also broaden the PCB substrate range of BphAE{sub LB400} variants. -- Abstract: The biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE{sub LB400}) catalyzes the dihydroxylation of biphenyl and of several polychlorinated biphenyls (PCBs) but it poorly oxidizes dibenzofuran. In this work we showed that BphAE{sub RR41}, a variant which was previously found to metabolize dibenzofuran more efficiently than its parent BphAE{sub LB400}, metabolized a broader range of PCBs than BphAE{sub LB400}. Hence, BphAE{sub RR41} was able to metabolize 2,6,2 Prime ,6 Prime -, 3,4,3 Prime ,5 Prime - and 2,4,3 Prime ,4 Prime -tetrachlorobiphenyl that BphAE{sub LB400} is unable to metabolize. BphAE{sub RR41} was obtained by changing Thr335Phe336Asn338Ile341Leu409 of BphAE{sub LB400} to Ala335Met336Gln338Val341Phe409. Site-directed mutagenesis was used to create combinations of each substitution, in order to assess their individual contributions. Data show that the same Asn338Glu/Leu409Phe substitution that enhanced the ability to metabolize dibenzofuran resulted in a broadening of the PCB substrates range of the enzyme. The role of these substitutions on regiospecificities toward selected PCBs is also discussed.

  2. Purification and characterization of the Comamonas testosteroni B-356 biphenyl dioxygenase components.

    PubMed Central

    Hurtubise, Y; Barriault, D; Powlowski, J; Sylvestre, M

    1995-01-01

    In this report, we describe some of the characteristics of the Comamonas testosteroni B-356 biphenyl (BPH)-chlorobiphenyl dioxygenase system, which includes the terminal oxygenase, an iron-sulfur protein (ISPBPH) made up of an alpha subunit (51 kDa) and a beta subunit (22 kDa) encoded by bphA and bphE, respectively; a ferredoxin (FERBPH; 12 kDa) encoded by bphF; and a ferredoxin reductase (REDBPH; 43 kDa) encoded by bphG. ISPBPH subunits were purified from B-356 cells grown on BPH. Since highly purified FERBPH and REDBPH were difficult to obtain from strain B-356, these two components were purified from recombinant Escherichia coli strains by using the His tag purification system. These His-tagged fusion proteins were shown to support BPH 2,3-dioxygenase activity in vitro when added to preparations of ISPBPH in the presence of NADH. FERBPH and REDBPH are thought to pass electrons from NADH to ISPBPH, which then activates molecular oxygen for insertion into the aromatic substrate. The reductase was found to contain approximately 1 mol of flavin adenine dinucleotide per mol of protein and was specific for NADH as an electron donor. The ferredoxin was found to contain a Rieske-type [2Fe-2S] center (epsilon 460, 7,455 M-1 cm-1) which was readily lost from the protein during purification and storage. In the presence of REDBPH and FERBPH, ISPBPH was able to convert BPH into both 2,3-dihydro-2,3-dihydroxybiphenyl and 3,4-dihydro-3,4-dihydroxybiphenyl. The significance of this observation is discussed. PMID:7592440

  3. Role of indoleamine 2,3-dioxygenase in health and disease.

    PubMed

    Yeung, Amanda W S; Terentis, Andrew C; King, Nicholas J C; Thomas, Shane R

    2015-10-01

    IDO1 (indoleamine 2,3-dioxygenase 1) is a member of a unique class of mammalian haem dioxygenases that catalyse the oxidative catabolism of the least-abundant essential amino acid, L-Trp (L-tryptophan), along the kynurenine pathway. Significant increases in knowledge have been recently gained with respect to understanding the fundamental biochemistry of IDO1 including its catalytic reaction mechanism, the scope of enzyme reactions it catalyses, the biochemical mechanisms controlling IDO1 expression and enzyme activity, and the discovery of enzyme inhibitors. Major advances in understanding the roles of IDO1 in physiology and disease have also been realised. IDO1 is recognised as a prominent immune regulatory enzyme capable of modulating immune cell activation status and phenotype via several molecular mechanisms including enzyme-dependent deprivation of L-Trp and its conversion into the aryl hydrocarbon receptor ligand kynurenine and other bioactive kynurenine pathway metabolites, or non-enzymatic cell signalling actions involving tyrosine phosphorylation of IDO1. Through these different modes of biochemical signalling, IDO1 regulates certain physiological functions (e.g. pregnancy) and modulates the pathogenesis and severity of diverse conditions including chronic inflammation, infectious disease, allergic and autoimmune disorders, transplantation, neuropathology and cancer. In the present review, we detail the current understanding of IDO1's catalytic actions and the biochemical mechanisms regulating IDO1 expression and activity. We also discuss the biological functions of IDO1 with a focus on the enzyme's immune-modulatory function, its medical implications in diverse pathological settings and its utility as a therapeutic target. PMID:26186743

  4. Characterization of a naphthalene dioxygenase endowed with an exceptionally broad substrate specificity toward polycyclic aromatic hydrocarbons.

    PubMed

    Jouanneau, Yves; Meyer, Christine; Jakoncic, Jean; Stojanoff, Vivian; Gaillard, Jacques

    2006-10-10

    In Sphingomonas CHY-1, a single ring-hydroxylating dioxygenase is responsible for the initial attack of a range of polycyclic aromatic hydrocarbons (PAHs) composed of up to five rings. The components of this enzyme were separately purified and characterized. The oxygenase component (ht-PhnI) was shown to contain one Rieske-type [2Fe-2S] cluster and one mononuclear Fe center per alpha subunit, based on EPR measurements and iron assay. Steady-state kinetic measurements revealed that the enzyme had a relatively low apparent Michaelis constant for naphthalene (K(m) = 0.92 +/- 0.15 microM) and an apparent specificity constant of 2.0 +/- 0.3 mM(-)(1) s(-)(1). Naphthalene was converted to the corresponding 1,2-dihydrodiol with stoichiometric oxidation of NADH. On the other hand, the oxidation of eight other PAHs occurred at slower rates and with coupling efficiencies that decreased with the enzyme reaction rate. Uncoupling was associated with hydrogen peroxide formation, which is potentially deleterious to cells and might inhibit PAH degradation. In single turnover reactions, ht-PhnI alone catalyzed PAH hydroxylation at a faster rate in the presence of organic solvent, suggesting that the transfer of substrate to the active site is a limiting factor. The four-ring PAHs chrysene and benz[a]anthracene were subjected to a double ring-dihydroxylation, giving rise to the formation of a significant proportion of bis-cis-dihydrodiols. In addition, the dihydroxylation of benz[a]anthracene yielded three dihydrodiols, the enzyme showing a preference for carbons in positions 1,2 and 10,11. This is the first characterization of a dioxygenase able to dihydroxylate PAHs made up of four and five rings. PMID:17014090

  5. Functional Metagenomics of a Biostimulated Petroleum-Contaminated Soil Reveals an Extraordinary Diversity of Extradiol Dioxygenases.

    PubMed

    Terrón-González, Laura; Martín-Cabello, Guadalupe; Ferrer, Manuel; Santero, Eduardo

    2016-04-01

    A metagenomic library of a petroleum-contaminated soil was constructed in a fosmid vector that allowed heterologous expression of metagenomic DNA. The library, consisting of 6.5 Gb of metagenomic DNA, was screened for extradiol dioxygenase (Edo) activity using catechol and 2,3-dihydroxybiphenyl as the substrates. Fifty-eight independent clones encoding extradiol dioxygenase activity were identified. Forty-one different Edo-encoding genes were identified. The population of Edo genes was not dominated by a particular gene or by highly similar genes; rather, the genes had an even distribution and high diversity. Phylogenetic analyses revealed that most of the genes could not be ascribed to previously defined subfamilies of Edos. Rather, the Edo genes led to the definition of 10 new subfamilies of type I Edos. Phylogenetic analysis of type II enzymes defined 7 families, 2 of which harbored the type II Edos that were found in this work. Particularly striking was the diversity found in family I.3 Edos; 15 out of the 17 sequences assigned to this family belonged to 7 newly defined subfamilies. A strong bias was found that depended on the substrate used for the screening: catechol mainly led to the detection of Edos belonging to the I.2 family, while 2,3-dihydroxybiphenyl led to the detection of most other Edos. Members of the I.2 family showed a clear substrate preference for monocyclic substrates, while those from the I.3 family showed a broader substrate range and high activity toward 2,3-dihydroxybiphenyl. This metagenomic analysis has substantially increased our knowledge of the existing biodiversity of Edos. PMID:26896130

  6. Characterization of a Naphthalene Dioxygenase Endowed with an Exceptionally Broad Substrate Specificity Toward Polycyclic Aromatic Hydrocarbons

    SciTech Connect

    Jouanneau,Y.; Meyer, C.; Jakoncic, J.; Stojanoff, V.; Gaillard, J.

    2006-01-01

    In Sphingomonas CHY-1, a single ring-hydroxylating dioxygenase is responsible for the initial attack of a range of polycyclic aromatic hydrocarbons (PAHs) composed of up to five rings. The components of this enzyme were separately purified and characterized. The oxygenase component (ht-PhnI) was shown to contain one Rieske-type [2Fe-2S] cluster and one mononuclear Fe center per {alpha} subunit, based on EPR measurements and iron assay. Steady-state kinetic measurements revealed that the enzyme had a relatively low apparent Michaelis constant for naphthalene (K{sub m} = 0.92 {+-} 0.15 {mu}M) and an apparent specificity constant of 2.0 {+-} 0.3 M{sup -1} s{sup -1}. Naphthalene was converted to the corresponding 1,2-dihydrodiol with stoichiometric oxidation of NADH. On the other hand, the oxidation of eight other PAHs occurred at slower rates and with coupling efficiencies that decreased with the enzyme reaction rate. Uncoupling was associated with hydrogen peroxide formation, which is potentially deleterious to cells and might inhibit PAH degradation. In single turnover reactions, ht-PhnI alone catalyzed PAH hydroxylation at a faster rate in the presence of organic solvent, suggesting that the transfer of substrate to the active site is a limiting factor. The four-ring PAHs chrysene and benz[a]anthracene were subjected to a double ring-dihydroxylation, giving rise to the formation of a significant proportion of bis-cis-dihydrodiols. In addition, the dihydroxylation of benz[a]anthracene yielded three dihydrodiols, the enzyme showing a preference for carbons in positions 1,2 and 10,11. This is the first characterization of a dioxygenase able to dihydroxylate PAHs made up of four and five rings.

  7. Rat liver cysteine dioxygenase (cysteine oxidase). Further purification, characterization, and analysis of the activation and inactivation.

    PubMed

    Yamaguchi, K; Hosokawa, Y; Kohashi, N; Kori, Y; Sakakibara, S; Ueda, I

    1978-02-01

    Rat liver cysteine dioxygenase has been purified to homogeneity. It is a single subunit protein having a molecular weight of 22,500 +/- 1,000, with a pI of 5.5. The enzyme purified was catalytically inactive and activated by anaerobic incubation with either L-cysteine or its analogues such as carboxymethyl-L-cysteine, carboxyethyl-L-cysteine, S-methyl-L-cysteine, D-cysteine, cysteamine, N-acetyl-L-cysteine, and DL-homocysteine. The enzyme thus activated with L-cysteine was rapidly inactivated under aerobic condition. This rapid inactivation was observed at 0 degrees C where no formation of either the reaction product cysteine sulfinate or the autoxidation product of cysteine, cystine, was detected. Further analysis shows that the inactivation of the activated enzyme was due to oxygen but unrelated to either the presence of substrate, enzyme turnover or accumulation of inhibitor produced during assay. A distinct rat liver cytoplasmic protein, called protein-A, could completely prevented the enzyme from the aerobic inactivation. The loss of activity during assay in the absence of protein-A was shown to be a first order decay process. From the plots of log(deltaproduct/min) versus time, the initial velocity (VO) and the velocity at 7 min (V7) were obtained. The apparent Km value for L-cysteine in the absence of protein-A was calculated from the initial velocity as 4.5 X 10(-4)M. Protein-A did not alter the apparent Km value for L-cysteine. The chelating agents such as o-phenanthroline, alpha,alpha'-dipyridyl, bathophenanthroline, 8-hydroxyquinoline, EGTA, and EDTA strongly inhibited the enzyme activity when these chelating agents were added before preactivation. The purified cystein dioxygenase contains 1 atom of iron per mol of enzyme protein. By the activation procedure, the enzyme became less susceptible to the heat denaturation, the inhibitory effects of chelating agents and the tryptic digestion. PMID:632231

  8. Spliceostatin hemiketal biosynthesis in Burkholderia spp. is catalyzed by an iron/α-ketoglutarate–dependent dioxygenase

    PubMed Central

    Eustáquio, Alessandra S.; Janso, Jeffrey E.; Ratnayake, Anokha S.; O’Donnell, Christopher J.; Koehn, Frank E.

    2014-01-01

    Spliceostatins are potent spliceosome inhibitors biosynthesized by a hybrid nonribosomal peptide synthetase−polyketide synthase (NRPS−PKS) system of the trans-acyl transferase (AT) type. Burkholderia sp. FERM BP-3421 produces hemiketal spliceostatins, such as FR901464, as well as analogs containing a terminal carboxylic acid. We provide genetic and biochemical evidence for hemiketal biosynthesis by oxidative decarboxylation rather than the previously hypothesized Baeyer–Villiger oxidative release postulated to be catalyzed by a flavin-dependent monooxygenase (FMO) activity internal to the last module of the PKS. Inactivation of Fe(II)/α-ketoglutarate–dependent dioxygenase gene fr9P led to loss of hemiketal congeners, whereas the mutant was still able to produce all major carboxylic acid-type compounds. FMO mutants, on the other hand, produced both hemiketal and carboxylic acid analogs containing an exocyclic methylene instead of an epoxide, indicating that the FMO is involved in epoxidation rather than Baeyer–Villiger oxidation. Moreover, recombinant Fr9P enzyme was shown to catalyze hydroxylation to form β-hydroxy acids, which upon decarboxylation led to hemiketal FR901464. Finally, a third oxygenase activity encoded in the biosynthetic gene cluster, the cytochrome P450 monooxygenase Fr9R, was assigned as a 4-hydroxylase based on gene inactivation results. Identification and deletion of the gene involved in hemiketal formation allowed us to generate a strain—the dioxygenase fr9P− mutant—that accumulates only the carboxylic acid-type spliceostatins, which are as potent as the hemiketal analogs, when derivatized to increase cell permeability, but are chemically more stable. PMID:25097259

  9. An Inner Membrane Dioxygenase that Generates the 2-Hydroxymyristate Moiety of Salmonella Lipid A

    PubMed Central

    Gibbons, Henry S.; Reynolds, C. Michael; Guan, Ziqiang; Raetz, Christian R.H.

    2009-01-01

    The lipid A residues of certain Gram-negative bacteria, including most strains of Salmonella and Pseudomonas, are esterified with one or two secondary S-2-hydroxyacyl chains. The S-2 hydroxylation process is O2-dependent in vivo, but the relevant enzymatic pathways have not been fully characterized because in vitro assays have not been developed. We previously reported that expression of the Salmonella lpxO gene confers upon Escherichia coli K-12 the ability to synthesize 2-hydroxymyristate modified lipid A (Gibbons, H. S., Lin, S., Cotter, R. J., and Raetz, C. R. H. J. Biol. Chem. 275, 32940–49, 2000). We now demonstrate that inactivation of lpxO, which encodes a putative Fe2+/O2/α-ketoglutarate-dependent dioxygenase, abolishes S-2-hydroxymyristate formation in S. typhimurium. Membranes of E. coli strains expressing lpxO are able to hydroxylate Kdo2-[4′-32P]-lipid A in vitro in the presence of Fe2+, O2, α-ketoglutarate, ascorbate and Triton X-100. The Fe2+ chelator 2,2′-bipyridyl inhibits the reaction. The product generated in vitro is a mono-hydroxylated Kdo2-lipid A derivative. The [4′-32P]-lipid A released by mild acid hydrolysis from the in vitro product migrates with authentic S-2-hydroxlyated lipid A isolated from 32P-labeled S. typhimurium cells. Electrospray ionization mass spectrometry and gas chromatography/mass spectrometry of the in vitro product are consistent with the 2-hydroxylation of the 3′-secondary myristoyl chain of Kdo2-lipid A. LpxO contains two predicted trans-membrane helices (one at each end of the protein), and its active site likely faces the cytoplasm. LpxO is an unusual example of an integral membrane protein that is a member of the Fe2+/O2/α-ketoglutarate-dependent dioxygenase family. PMID:18254598

  10. Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA.

    PubMed Central

    Garcia, I; Rodgers, M; Lenne, C; Rolland, A; Sailland, A; Matringe, M

    1997-01-01

    p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45-46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45-46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide. PMID:9271098

  11. Parallel induction of tetrahydrobiopterin biosynthesis and indoleamine 2,3-dioxygenase activity in human cells and cell lines by interferon-gamma.

    PubMed Central

    Werner, E R; Werner-Felmayer, G; Fuchs, D; Hausen, A; Reibnegger, G; Wachter, H

    1989-01-01

    In all of eight tested human cells and cell lines with inducible indoleamine 2,3-dioxygenase (EC 1.13.11.17) tetrahydrobiopterin biosynthesis was activated by interferon-gamma. This was demonstrated by GTP cyclohydrolase I (EC 3.5.4.16) activities and intracellular neopterin and biopterin concentrations. Pteridine synthesis was influenced by extracellular tryptophan. In T 24-cell extracts, submillimolar concentrations of tetrahydrobiopterin stimulated the indoleamine 2,3-dioxygenase reaction. PMID:2511835

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

    PubMed Central

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

    1993-01-01

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

  13. Role of pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during starvation

    PubMed Central

    Jeoung, Nam Ho; Wu, Pengfei; Joshi, Mandar A.; Jaskiewicz, Jerzy; Bock, Cheryl B.; Depaoli-Roach, Anna A.; Harris, Robert A.

    2006-01-01

    The PDC (pyruvate dehydrogenase complex) is strongly inhibited by phosphorylation during starvation to conserve substrates for gluconeogenesis. The role of PDHK4 (pyruvate dehydrogenase kinase isoenzyme 4) in regulation of PDC by this mechanism was investigated with PDHK4−/− mice (homozygous PDHK4 knockout mice). Starvation lowers blood glucose more in mice lacking PDHK4 than in wild-type mice. The activity state of PDC (percentage dephosphorylated and active) is greater in kidney, gastrocnemius muscle, diaphragm and heart but not in the liver of starved PDHK4−/− mice. Intermediates of the gluconeogenic pathway are lower in concentration in the liver of starved PDHK4−/− mice, consistent with a lower rate of gluconeogenesis due to a substrate supply limitation. The concentration of gluconeogenic substrates is lower in the blood of starved PDHK4−/− mice, consistent with reduced formation in peripheral tissues. Isolated diaphragms from starved PDHK4−/− mice accumulate less lactate and pyruvate because of a faster rate of pyruvate oxidation and a reduced rate of glycolysis. BCAAs (branched chain amino acids) are higher in the blood in starved PDHK4−/− mice, consistent with lower blood alanine levels and the importance of BCAAs as a source of amino groups for alanine formation. Non-esterified fatty acids are also elevated more in the blood of starved PDHK4−/− mice, consistent with lower rates of fatty acid oxidation due to increased rates of glucose and pyruvate oxidation due to greater PDC activity. Up-regulation of PDHK4 in tissues other than the liver is clearly important during starvation for regulation of PDC activity and glucose homoeostasis. PMID:16606348

  14. Pyruvate:Ferredoxin Oxidoreductase Is Coupled to Light-independent Hydrogen Production in Chlamydomonas reinhardtii*

    PubMed Central

    Noth, Jens; Krawietz, Danuta; Hemschemeier, Anja; Happe, Thomas

    2013-01-01

    In anaerobiosis, the green alga Chlamydomonas reinhardtii evolves molecular hydrogen (H2) as one of several fermentation products. H2 is generated mostly by the [Fe-Fe]-hydrogenase HYDA1, which uses plant type ferredoxin PETF/FDX1 (PETF) as an electron donor. Dark fermentation of the alga is mainly of the mixed acid type, because formate, ethanol, and acetate are generated by a pyruvate:formate lyase pathway similar to Escherichia coli. However, C. reinhardtii also possesses the pyruvate:ferredoxin oxidoreductase PFR1, which, like pyruvate:formate lyase and HYDA1, is localized in the chloroplast. PFR1 has long been suggested to be responsible for the low but significant H2 accumulation in the dark because the catalytic mechanism of pyruvate:ferredoxin oxidoreductase involves the reduction of ferredoxin. With the aim of proving the biochemical feasibility of the postulated reaction, we have heterologously expressed the PFR1 gene in E. coli. Purified recombinant PFR1 is able to transfer electrons from pyruvate to HYDA1, using the ferredoxins PETF and FDX2 as electron carriers. The high reactivity of PFR1 toward oxaloacetate indicates that in vivo, fermentation might also be coupled to an anaerobically active glyoxylate cycle. Our results suggest that C. reinhardtii employs a clostridial type H2 production pathway in the dark, especially because C. reinhardtii PFR1 was also able to allow H2 evolution in reaction mixtures containing Clostridium acetobutylicum 2[4Fe-4S]-ferredoxin and [Fe-Fe]-hydrogenase HYDA. PMID:23258532

  15. /sup 13/C NMR study of effects of fasting and diabetes on the metabolism of pyruvate in the tricarboxylic acid cycle and of the utilization of pyruvate and ethanol in lipogenesis in perfused rat liver

    SciTech Connect

    Cohen, S.M.

    1987-01-27

    /sup 13/C NMR has been used to study the competition of pyruvate dehydrogenase with pyruvate carboxylase for entry of pyruvate into the tricarboxylic acid (TCA) cycle in perfused liver from streptozotocin-diabetic and normal donor rats. The relative proportion of pyruvate entering the TCA cycle by these two routes was estimated from the /sup 13/C enrichments at the individual carbons of glutamate when (3-/sup 13/C)alanine was the only exogenous substrate present. In this way, the proportion of pyruvate entering by the pyruvate dehydrogenase route relative to the pyruvate carboxylase route was determined to be 1:1.2 +/- 0.1 in liver from fed controls, 1:7.7 +/- 2 in liver from 24-fasted controls, and 1:2.6 +/- 0.3 in diabetic liver. Pursuant to this observation that conversion of pyruvate to acetyl coenzyme A (acetyl-CoA) was greatest in perfused liver from fed controls, the incorporation of /sup 13/C label into fatty acids was monitored in this liver preparation. With the exception of the repeating methylene carbons, fatty acyl carbons labeled by (1-/sup 13/C)acetyl-CoA (from (2-/sup 13/C)pyruvate) gave rise to resonances distinguishable on the basis of chemical shift from those observed when label was introduced by (3-/sup 13/C)alanine plus (2-/sup 13/C)ethanol, which are converted to (2-/sup 13/C)acetyl-CoA. Thus, measurement of /sup 13/C enrichment at several specific sites in the fatty acyl chains in time-resolved spectra of perfused liver offers a novel way of monitoring the kinetics of the biosynthesis of fatty acids. In addition to obtaining the rate of lipogenesis, it was possible to distinguish the contributions of chain elongation from those of the de novo synthesis pathway and to estimate the average chain length of the /sup 13/C-labeled fatty acids produced.

  16. Altered redox status in Escherichia coli cells enhances pyruvate production in pH-adjusting culture with a fermenter.

    PubMed

    Ojima, Yoshihiro; Matsuo, Nahoko; Suparman, Asep; Suryadarma, Prayoga; Taya, Masahito

    2014-03-01

    Improvements in pyruvate production process were examined using Escherichia coli BW25113Dpta/ pHfdh strain carrying the formate dehydrogenase gene of Mycobacterium vaccae to change the redox status of the cells. Glucose and formate concentrations, and oxygenation levels determined previously in a shake-flask culture were applied for pyruvate production in a 1 l fermenter. However, pyruvate was not produced under the examined conditions. Detailed pH measurements during the fermenter culture using CaCO3 revealed that maintaining the pH value around 6.0 plays an important role in stabilizing the pyruvate accumulation. In the pH-adjusting culture around 6.0 with NaOH solution, the concentration and yield of pyruvate were 8.96 g l-1 and 0.48 g pyruvate g glucose-1, respectively, which were significantly higher than the values reported in the shake-flask culture (6.79 g l-1 and 0.32 g pyruvate g glucose-1). PMID:23797477

  17. Pyruvate and Lactate Metabolism by Shewanella oneidensis MR-1 under Fermentation, Oxygen Limitation, and Fumarate Respiration Conditions

    SciTech Connect

    Pinchuk, Grigoriy E.; Geydebrekht, Oleg V.; Hill, Eric A.; Reed, Jennifer L.; Konopka, Allan; Beliaev, Alex S.; Fredrickson, Jim K.

    2011-12-30

    Shewanella oneidensis MR-1 is a facultative anaerobe growing by coupling organic matter oxidation to reduction of wide range of electron acceptors. Here we quantitatively assessed lactate and pyruvate metabolism of these bacteria under three distinct conditions: electron acceptor limited growth on lactate with O2 and fumarate, and pyruvate fermentation, which does not sustain growth but allows cells to survive for prolonged period. Using physiological and genetic approaches combined with flux balance analysis, we showed that the proportion of ATP produced by substrate-level phosphorylation varied from 33% to 72.5% of all ATP needed for growth depending on the electron acceptor nature and availability. While being indispensible for growth, respiration of fumarate does not contribute much to ATP generation and likely serves to remove formate, a product of pyruvate formate-lyase-catalyzed pyruvate disproportionation. Under both tested respiratory conditions S. oneidensis MR-1 carried out incomplete substrate oxidation, and TCA cycle did not contribute significantly to substrate oxidation. Pyruvate dehydrogenase reaction was not involved in lactate metabolism under O2 limitation, however was important for anaerobic growth probably supplying reducing equivalents for biosynthesis. Unexpectedly, obtained results suggest that pyruvate fermentation by S. oneidensis MR-1 cells represents a combination between substrate-level phosphorylation and a respiratory process, where pyruvate serves as electron donor and electron acceptor. Pyruvate reduction to lactate at the expense of formate oxidation is catalyzed by recently described new type of oxidative NAD(P)H independent D-lactate dehydrogenase (Dld-II). Based on involved enzymes localization we hypothesize that pyruvate reduction coupled to formate oxidation may be accompanied by proton motive force generation.

  18. Pyruvate and Lactate Metabolism by Shewanella oneidensis MR-1 under Fermentation, Oxygen Limitation, and Fumarate Respiration Conditions

    SciTech Connect

    Pinchuk, Grigoriy E.; Geydebrekht, Oleg V.; Hill, Eric A.; Reed, Jennifer L.; Konopka, Allan; Beliaev, Alex S.; Fredrickson, Jim K.

    2011-12-01

    Shewanella oneidensis MR-1 is a facultative anaerobe that derives energy by coupling organic matter oxidation to the reduction of wide range of electron acceptors. Here, we quantitatively assessed lactate and pyruvate metabolism of MR-1 under three distinct conditions: electron acceptor limited growth on lactate with O2; lactate with fumarate; and pyruvate fermentation. The latter does not support growth but provides energy for cell survival. Using physiological and genetic approaches combined with flux balance analysis, we showed that the proportion of ATP produced by substrate-level phosphorylation varied from 33% to 72.5% of that needed for growth depending on the electron acceptor nature and availability. While being indispensible for growth, respiration of fumarate does not contribute significantly to ATP generation and likely serves to remove formate, a product of pyruvate formate-lyase-catalyzed pyruvate disproportionation. Under both tested respiratory conditions S. oneidensis MR-1 carried out incomplete substrate oxidation, whereby the TCA cycle did not contribute significantly. Pyruvate dehydrogenase was not involved in lactate metabolism under O2 limitation but was required for anaerobic growth likely by supplying reducing equivalents for biosynthesis. The results suggest that pyruvate fermentation by S. oneidensis MR-1 cells represents a combination of substrate-level phosphorylation and respiration, where pyruvate serves as electron donor and electron acceptor. Pyruvate reduction to lactate at the expense of formate oxidation is catalyzed by recently described new type of oxidative NAD(P)H independent D-lactate dehydrogenase (Dld-II). The results further indicate that pyruvate reduction coupled to formate oxidation may be accompanied by proton motive force generation.

  19. Pyruvate Oxidoreductases Involved in Glycolytic Anaerobic Metabolism of Polychaetes from the Continental Shelf off Central-South Chile

    NASA Astrophysics Data System (ADS)

    González, R. R.; Quiñones, R. A.

    2000-10-01

    The presence of low oxygen conditions in extensive areas of the continental shelf off central-south Chile has important effects on the biochemical adaptations of the organisms living in this ecosystem. Polychaetes assemblages cohabit on the shelf with an extensively distributed prokaryotic community made up of giant filamentous sulfur bacteria (mainly Thioploca sp.). The aim of this research was to characterize the pyruvate oxidoreductases enzymes involved in the biochemical adaptation of these benthic polychaetes. Nine polychaete species ( Paraprionospio pinnata, Nephtys ferruginea, Glycera americana, Haploscoloplos sp., Lumbrineris composita, Sigambra bassi, Aricidea pigmentata , Cossura chilensis, and Pectinaria chilensis) were assayed for lactic dehydrogenase (LDH), octopine dehydrogenase (OPDH), strombine dehydrogenase (STRDH) and alanopine dehydrogenase (ALPDH). Each species had a characteristic number of the pyruvate oxidoreductases assayed ranging from 4 in Paraprionospio pinnata to 1 in Pectinaria chilensis . The pyruvate saturation curves obtained for the enzymes from all species analysed, except L. composita, suggest that NADH can be oxidized at different rates depending on the amino acid used in the reaction with pyruvate. Our results indicate that organisms having more that one pyruvate oxidoreductase present a greater metabolic capacity to cope with functional and environmental hypoxia because these enzymes would better regulate the pyruvate consumption rate during the transition period. Thus, the dominance of Paraprionospio pinnata in the study area and its worldwide distribution is consistent with its higher number of pyruvate oxidoreductases with different pyruvate consumption rates involved in anaerobic metabolism. Finally, a positive allometric relationship was found between body size and the specific activity of ALPDH, STRDH, and maximum pyruvate oxidoreductase specific activity. This latter result suggests a positive scaling of the specific

  20. Crystallization and preliminary X-ray analysis of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase from Arthrobacter nitroguajacolicus Rü61a: a cofactor-devoid dioxygenase of the α/β-hydrolase-fold superfamily

    SciTech Connect

    Steiner, Roberto A.; Frerichs-Deeken, Ursula; Fetzner, Susanne

    2007-05-01

    Preliminary crystallographic data are reported for 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) from A. nitroguajacolicus Rü61a. 1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) is a cofactor-devoid dioxygenase that is involved in the anthranilate pathway of quinaldine degradation. HOD has been proposed to belong to the α/β-hydrolase-fold superfamily of enzymes. N-terminally His{sub 6}-tagged HOD has been crystallized by the hanging-drop vapour-diffusion method using sodium/potassium tartrate as a precipitant and CuCl{sub 2} as an additive. The structure was solved by the single anomalous dispersion (SAD) technique using data collected to 3.5 Å resolution at the Cu absorption peak wavelength. The crystals belong to the primitive tetragonal space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 153.788, c = 120.872 Å.

  1. Enzymatic degradation of aromatic hydrocarbon intermediates using a recombinant dioxygenase immobilized onto surfactant-activated carbon nanotube.

    PubMed

    Suma, Yanasinee; Lim, Heejun; Kwean, Oh Sung; Cho, Suyeon; Yang, Junwon; Kim, Yohan; Kang, Christina S; Kim, Han S

    2016-06-01

    This study examined the enzymatic decomposition of aromatic hydrocarbon intermediates (catechol, 4-chlorocatechol, and 3-methylcatechol) using a dioxygenase immobilized onto single-walled carbon nanotube (SWCNT). The surfaces of SWCNTs were activated with surfactants. The dioxygenase was obtained by recombinant technique: the corresponding gene was cloned from Arthrobacter chlorophenolicus A6, and the enzyme was overexpressed and purified subsequently. The enzyme immobilization yield was 62%, and the high level of enzyme activity was preserved (60-79%) after enzyme immobilization. Kinetic analyses showed that the substrate utilization rates and the catalytic efficiencies of the immobilized enzyme for all substrates (target aromatic hydrocarbon intermediates) tested were similar to those of the free enzyme, indicating that the loss of enzyme activity was minimal during enzyme immobilization. The immobilized enzyme was more stable than the free enzyme against abrupt changes in pH, temperature, and ionic strength. Moreover, it retained high enzyme activity even after repetitive use. PMID:26810145

  2. The Complete Reaction Mechanism of Indoleamine 2,3-Dioxygenase as Revealed by QM/MM Simulations

    PubMed Central

    Capece, Luciana; Lewis-Ballester, Ariel; Yeh, Syun-Ru; Estrin, Dario A.; Marti, Marcelo A.

    2012-01-01

    Indoleamine 2,3 dioxygenase (IDO) and tryptophan dioxygenase (TDO) are two heme-proteins that catalyze the oxidation reaction of tryptophan (Trp) to N-formylkynurenine (NFK). Human IDO (hIDO) has recently been recognized as a potent anti-cancer drug target, a fact that triggered intense research on the reaction and inhibition mechanisms of hIDO. Our recent studies revealed that the dioxygenase reaction catalyzed by hIDO and TDO is initiated by addition of the ferric iron-bound superoxide to the C2=C3 bond of Trp to form a ferryl and Trp-epoxide intermediate, via a 2-indolenylperoxo radical transition state. The data demonstrate that the two atoms of dioxygen are inserted into the substrate in a stepwise fashion, challenging the paradigm of heme-based dioxygenase chemistry. In the current study, we used QM/MM methods to decipher the mechanism by which the second ferryl oxygen is inserted into the Trp-epoxide to form the NFK product in hIDO. Our results show that the most energetically favored pathway involves proton transfer from Trp-NH3+ to the epoxide oxygen, triggering epoxide ring-opening and a concerted nucleophilic attack of the ferryl oxygen to the C2 of Trp that leads to a meta-stable reaction intermediate. This intermediate subsequently converts to NFK, following C2-C3 bond cleavage and the associated back proton transfer from the oxygen to the amino group of Trp. A comparative study with Xantomonas campestris TDO (xcTDO) indicates that the reaction follows a similar pathway, although subtle differences distinguishing the two enzyme reactions are evident. The results underscore the importance of the NH3+ group of Trp in the two-step ferryl-based mechanism of hIDO and xcTDO, by acting as an acid catalyst to facilitate the epoxide ring-opening reaction and ferryl oxygen addition to the indole ring. PMID:22196056

  3. Regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene by naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

    PubMed Central

    Resnick, S M; Gibson, D T

    1996-01-01

    The regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene was examined with mutant and recombinant strains expressing naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. The initial oxidation products were isolated and identified by gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry. Salicylate-induced cells of Pseudomonas sp. strain 9816/11 and isopropyl-beta-D-thiogalactopyranoside-induced cells of Escherichia coli JM109(DE3)(pDTG141) oxidized fluorene to (+)-(3S,4R)-cis-3,4-dihydroxy-3,4-dihydrofluorene (80 to 90% relative yield; > 95% enantiomeric excess [ee]) and 9-fluorenol (< 10% yield). The same cells oxidized dibenzofuran to (1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzofuran (60 to 70% yield; > 95% ee) and (3S,4R)-cis-3, 4-dihydroxy-3,4-dihydrodibenzofuran (30 to 40% yield; > 95% ee). Induced cells of both strains, as well as the purified dioxygenase, also oxidized dibenzothiophene to (+)-(1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzothiophene (84 to 87% yield; > 95% ee) and dibenzothiophene sulfoxide (< 15% yield). The major reaction catalyzed by naphthalene dioxygenase with each substrate was stereospecific dihydroxylation in which the cis-dihydrodiols were of identical regiochemistry and of R configuration at the benzylic center adjacent to the bridgehead carbon atom. The regiospecific oxidation of dibenzofuran differed from that of the other substrates in that a significant amount of the minor cis-3,4-dihydrodiol regioisomer was formed. The results indicate that although the absolute stereochemistry of the cis-diene diols was the same, the nature of the bridging atom or heteroatom influenced the regiospecificity of the reactions catalyzed by naphthalene dioxygenase. PMID:8899998

  4. Phthalate catabolic gene cluster is linked to the angular dioxygenase gene in Terrabacter sp. strain DBF63.

    PubMed

    Habe, H; Miyakoshi, M; Chung, J; Kasuga, K; Yoshida, T; Nojiri, H; Omori, T

    2003-03-01

    Phthalate is a metabolic intermediate of the pathway of fluorene (FN) degradation via angular dioxygenation. A gene cluster responsible for the conversion of phthalate to protocatechuate was cloned from the dibenzofuran (DF)- and FN-degrading bacterium Terrabacter sp. strain DBF63 and sequenced. The genes encoding seven catabolic enzymes, oxygenase large subunit of phthalate 3,4-dioxygenase (phtA1), oxygenase small subunit of phthalate 3,4-dioxygenase (phtA2), cis-3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase (phtB), [3Fe-4S] or [4Fe-4S] type of ferredoxin (phtA3), ferredoxin reductase (phtA4), 3,4-dihydroxyphthalate decarboxylase (phtC) and putative regulatory protein (phtR), were found in the upstream region of the angular dioxygenase gene (dbfA1A2), encoded in this order. Escherichia coli carrying phtA1A2BA3A4 genes converted phthalate to 3,4-dihydroxyphthalate, and the 3,4-dihydroxyphthalate decarboxylase activity by E. coli cells carrying phtC was finally detected with the introduction of a Shine-Dalgarno sequence in the upstream region of its initiation codon. Homology analysis on the upstream region of the pht gene cluster revealed that there was an insertion sequence (IS) (ISTesp2; ORF14 and its flanking region), part of which was almost 100% identical to the orf1 and its flanking region adjacent to the extradiol dioxygenase gene ( bphC1) involved in the DF degradation of Terrabacter sp. strain DPO360 [Schmid et al. (1997) J Bacteriol 179:53-62]. This suggests that ISTesp2 plays a role in the metabolism of aromatic compounds in Terrabacter sp. strains DBF63 and DPO360. PMID:12658514

  5. Catalytic mechanism of cofactor-free dioxygenases and how they circumvent spin-forbidden oxygenation of their substrates.

    PubMed

    Hernández-Ortega, Aitor; Quesne, Matthew G; Bui, Soi; Heyes, Derren J; Steiner, Roberto A; Scrutton, Nigel S; de Visser, Sam P

    2015-06-17

    Dioxygenases catalyze a diverse range of biological reactions by incorporating molecular oxygen into organic substrates. Typically, they use transition metals or organic cofactors for catalysis. Bacterial 1-H-3-hydroxy-4-oxoquinaldine-2,4-dioxygenase (HOD) catalyzes the spin-forbidden transfer of dioxygen to its N-heteroaromatic substrate in the absence of any cofactor. We combined kinetics, spectroscopic and computational approaches to establish a novel reaction mechanism. The present work gives insight into the rate limiting steps in the reaction mechanism, the effect of first-coordination sphere amino acids as well as electron-donating/electron-withdrawing substituents on the substrate. We highlight the role of active site residues Ser101/Trp160/His251 and their involvement in the reaction mechanism. The work shows, for the first time, that the reaction is initiated by triplet dioxygen and its binding to deprotonated substrate and only thereafter a spin state crossing to the singlet spin state occurs. As revealed by steady- and transient-state kinetics the oxygen-dependent steps are rate-limiting, whereas Trp160 and His251 are essential residues for catalysis and contribute to substrate positioning and activation, respectively. Computational modeling further confirms the experimental observations and rationalizes the electron transfer pathways, and the effect of substrate and substrate binding pocket residues. Finally, we make a direct comparison with iron-based dioxygenases and explain the mechanistic and electronic differences with cofactor-free dioxygenases. Our multidisciplinary study confirms that the oxygenation reaction can take place in absence of any cofactor by a unique mechanism in which the specially designed fit-for-purpose active-site architecture modulates substrate reactivity toward oxygen. PMID:25988744

  6. Reactivity of pyruvic acid and its derivatives towards reactive oxygen species.

    PubMed

    Kładna, Aleksandra; Marchlewicz, Mariola; Piechowska, Teresa; Kruk, Irena; Aboul-Enein, Hassan Y

    2015-11-01

    Pyruvic acid and its derivatives occurring in most biological systems are known to exhibit several pharmacological properties, such as anti-inflammatory, neuroprotective or anticancer, many of which are suggested to originate from their antioxidant and free radical scavenger activity. The therapeutic potential of these compounds is a matter of particular interest, due to their mechanisms of action, particularly their possible antioxidant behaviour. Here, we report the results of a study of the effect of pyruvic acid (PA), ethyl pyruvate (EP) and sodium pyruvate (SP) on reactions generating reactive oxygen species (ROS), such as superoxide anion radicals, hydroxyl radicals and singlet oxygen, and their total antioxidant capacity. Chemiluminescence (CL) and spectrophotometry techniques were employed. The pyruvate analogues studied were found to inhibit the CL signal arising from superoxide anion radicals in a dose-dependent manner with IC50 = 0.0197 ± 0.002 mM for EP and IC50 = 69.2 ± 5.2 mM for PA. These compounds exhibited a dose-dependent decrease in the CL signal of the luminol + H2O2 system over the range 0.5-10 mM with IC50 values of 1.71 ± 0.12 mM for PA, 3.85 ± 0.21 mM for EP and 22.91 ± 1.21 mM for SP. Furthermore, these compounds also inhibited hydroxyl radical-dependent deoxyribose degradation in a dose-dependent manner over the range 0.5-200 mM, with IC50 values of 33.2 ± 0.3 mM for SP, 116.1 ± 6.2 mM for EP and 168.2 ± 6.2 mM for PA. All the examined compounds also showed antioxidant capacity when estimated using the ferric-ferrozine assay. The results suggest that the antioxidant activities of pyruvate derivatives may reflect a direct effect on scavenging ROS and, in part, be responsible for their pharmacological actions. PMID:25754627

  7. How do components of real cloud water affect aqueous pyruvate oxidation?

    NASA Astrophysics Data System (ADS)

    Boris, Alexandra J.; Desyaterik, Yury; Collett, Jeffrey L.

    2014-06-01

    Chemical oxidation of dissolved volatile or semi-volatile organic compounds within fog and cloud droplets in the atmosphere could be a major pathway for secondary organic aerosol (SOA) formation. This proposed pathway consists of: (1) dissolution of organic chemicals from the gas phase into a droplet; (2) reaction with an aqueous phase oxidant to yield low volatility products; and (3) formation of particle phase organic matter as the droplet evaporates. The common approach to simulating aqueous SOA (aqSOA) reactions is photo-oxidation of laboratory standards in pure water. Reactions leading to aqSOA formation should be studied within real cloud and fog water to determine whether additional competing processes might alter apparent rates of reaction as indicated by rates of reactant loss or product formation. To evaluate and identify the origin of any cloud water matrix effects on one example of observed aqSOA production, pyruvate oxidation experiments simulating aqSOA formation were monitored within pure water, real cloud water samples, and an aqueous solution of inorganic salts. Two analysis methods were used: online electrospray ionization high-resolution time-of-flight mass spectrometry (ESI-HR-ToF-MS), and offline anion exchange chromatography (IC) with quantitative conductivity and qualitative ESI-HR-ToF-MS detection. The apparent rate of oxidation of pyruvate was slowed in cloud water matrices: overall measured degradation rates of pyruvate were lower than in pure water. This can be at least partially accounted for by the observed formation of pyruvate from reactions of other cloud water components. Organic constituents of cloud water also compete for oxidants and/or UV light, contributing to the observed slowed degradation rates of pyruvate. The oxidation of pyruvate was not significantly affected by the presence of inorganic anions (nitrate and sulfate) at cloud-relevant concentrations. Future bulk studies of aqSOA formation reactions using simplified

  8. Reprint of "How do components of real cloud water affect aqueous pyruvate oxidation?"

    NASA Astrophysics Data System (ADS)

    Boris, Alexandra J.; Desyaterik, Yury; Collett, Jeffrey L.

    2015-01-01

    Chemical oxidation of dissolved volatile or semi-volatile organic compounds within fog and cloud droplets in the atmosphere could be a major pathway for secondary organic aerosol (SOA) formation. This proposed pathway consists of: (1) dissolution of organic chemicals from the gas phase into a droplet; (2) reaction with an aqueous phase oxidant to yield low volatility products; and (3) formation of particle phase organic matter as the droplet evaporates. The common approach to simulating aqueous SOA (aqSOA) reactions is photo-oxidation of laboratory standards in pure water. Reactions leading to aqSOA formation should be studied within real cloud and fog water to determine whether additional competing processes might alter apparent rates of reaction as indicated by rates of reactant loss or product formation. To evaluate and identify the origin of any cloud water matrix effects on one example of observed aqSOA production, pyruvate oxidation experiments simulating aqSOA formation were monitored within pure water, real cloud water samples, and an aqueous solution of inorganic salts. Two analysis methods were used: online electrospray ionization high-resolution time-of-flight mass spectrometry (ESI-HR-ToF-MS), and offline anion exchange chromatography (IC) with quantitative conductivity and qualitative ESI-HR-ToF-MS detection. The apparent rate of oxidation of pyruvate was slowed in cloud water matrices: overall measured degradation rates of pyruvate were lower than in pure water. This can be at least partially accounted for by the observed formation of pyruvate from reactions of other cloud water components. Organic constituents of cloud water also compete for oxidants and/or UV light, contributing to the observed slowed degradation rates of pyruvate. The oxidation of pyruvate was not significantly affected by the presence of inorganic anions (nitrate and sulfate) at cloud-relevant concentrations. Future bulk studies of aqSOA formation reactions using simplified

  9. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs: Arg- and Gln-type Bacterial CDO Homologs

    DOE PAGESBeta

    Driggers, Camden M.; Hartman, Steven J.; Karplus, P. Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ~15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog ofmore » uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.« less

  10. Chemical intervention in bacterial lignin degradation pathways: Development of selective inhibitors for intradiol and extradiol catechol dioxygenases.

    PubMed

    Sainsbury, Paul D; Mineyeva, Yelena; Mycroft, Zoe; Bugg, Timothy D H

    2015-06-01

    Bacterial lignin degradation could be used to generate aromatic chemicals from the renewable resource lignin, provided that the breakdown pathways can be manipulated. In this study, selective inhibitors of enzymatic steps in bacterial degradation pathways were developed and tested for their effects upon lignin degradation. Screening of a collection of hydroxamic acid metallo-oxygenase inhibitors against two catechol dioxygenase enzymes, protocatechuate 3,4-dioxygenase (3,4-PCD) and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB), resulted in the identification of selective inhibitors D13 for 3,4-PCD (IC50 15μM) and D3 for MhpB (IC50 110μM). Application of D13 to Rhodococcus jostii RHA1 in minimal media containing ferulic acid led to the appearance of metabolic precursor protocatechuic acid at low concentration. Application of 1mM disulfiram, an inhibitor of mammalian aldehyde dehydrogenase, to R. jostii RHA1, gave rise to 4-carboxymuconolactone on the β-ketoadipate pathway, whereas in Pseudomonas fluorescens Pf-5 disulfiram treatment gave rise to a metabolite found to be glycine betaine aldehyde. PMID:25984987

  11. The Role of 4-Hydroxyphenylpyruvate Dioxygenase in Enhancement of Solid-Phase Electron Transfer by Shewanella oneidensis MR-1

    SciTech Connect

    Turick, Charles E.; Beliaev, Alex S.; Zakrajsek, Brian A.; Reardon, Catherine L.; Lowy, Daniel A.; Poppy, Tara E.; Maloney, Andrea; Ekechukwu, Amy A.

    2009-05-01

    ABSTRACT - While mechanistic details of dissimilatory metal reduction are far from being understood, it is postulated that the electron transfer to solid metal oxides is mediated by outer membrane associated c-type cytochromes and electron shuttling compounds. This study focuses on the production of homogensitate in Shewanella oneidensis MR-1, an intermediate of the tyrosine degradation pathway, which is a precursor of a redox cycling metabolite, pyomelanin. We determined that two enzymes involved in this pathway, 4-hydroxyphenylpyruvate dioxygenase (4HPPD) and homogentisate 1,2-dioxygenase are responsible for homogentisate production and oxidation, respectively. Inhibition of 4-HPPD activity with the specific inhibitor sulcotrione ([2-(2- chloro- 4- methane sulfonylbenzoyl)-1,3-cyclohexanedione), and deletion of melA, a gene encoding 4-HPPD, resulted in no pyomelanin production by S. oneidensis MR-1. Conversely, deletion of hmgA, which encodes the putative homogentisate 1,2-dioxygenase, resulted in pyomelanin overproduction. The efficiency and rates at which MR-1 reduces hydrous ferric oxide were directly linked to the ability of mutant strains to produce pyomelanin. Electrochemical studies with whole cells demonstrated that pyomelanin substantially increases the formal potential (E°') of S. oneidensis MR-1. Based on our findings, environmental production of pyomelanin likely contributes to an increased solid-phase metal reduction capacity in S. oneidensis MR-1.

  12. Indoleamine 2,3-dioxygenase 2 (IDO2) and the kynurenine pathway: characteristics and potential roles in health and disease.

    PubMed

    Fatokun, Amos A; Hunt, Nicholas H; Ball, Helen J

    2013-12-01

    The kynurenine pathway is the major route for the oxidative degradation of the amino acid tryptophan. Activity of the pathway is involved in several disease conditions, both in the periphery and the central nervous system, including cancer, inflammatory disorders, neurological conditions, psychiatric disorders and neurodegenerative diseases. Three enzymes are now known to catalyze the first and rate-limiting step in the catabolism of tryptophan along this pathway: tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO, subsequently named IDO1), both of which have been extensively studied, and a third enzyme, indoleamine 2,3-dioxygenase 2 (IDO2), a relative newcomer to the kynurenine pathway field. The adjuvant chemotherapeutic agent, 1-methyl-D-tryptophan, was intially suggested to target IDO2, implying involvement of IDO2 in tumorigenesis. Subsequently this compound has been suggested to have alternative actions and the physiological and pathophysiological roles of IDO2 are unclear. Targeted genetic interventions and selective inhibitors provide approaches for investigating the biology of IDO2. This review focuses on the current knowledge of IDO2 biology and discusses tools that will assist in further characterizing the enzymes of the kynurenine pathway. PMID:24105077

  13. Hydroquinone Dioxygenase from Pseudomonas fluorescens ACB: a Novel Member of the Family of Nonheme-Iron(II)-Dependent Dioxygenases▿

    PubMed Central

    Moonen, Mariëlle J. H.; Synowsky, Silvia A.; van den Berg, Willy A. M.; Westphal, Adrie H.; Heck, Albert J. R.; van den Heuvel, Robert H. H.; Fraaije, Marco W.; van Berkel, Willem J. H.

    2008-01-01

    Hydroquinone 1,2-dioxygenase (HQDO), an enzyme involved in the catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB, was purified to apparent homogeneity. Ligandation with 4-hydroxybenzoate prevented the enzyme from irreversible inactivation. HQDO was activated by iron(II) ions and catalyzed the ring fission of a wide range of hydroquinones to the corresponding 4-hydroxymuconic semialdehydes. HQDO was inactivated by 2,2′-dipyridyl, o-phenanthroline, and hydrogen peroxide and inhibited by phenolic compounds. The inhibition with 4-hydroxybenzoate (Ki = 14 μM) was competitive with hydroquinone. Online size-exclusion chromatography-mass spectrometry revealed that HQDO is an α2β2 heterotetramer of 112.4 kDa, which is composed of an α-subunit of 17.8 kDa and a β-subunit of 38.3 kDa. Each β-subunit binds one molecule of 4-hydroxybenzoate and one iron(II) ion. N-terminal sequencing and peptide mapping and sequencing based on matrix-assisted laser desorption ionization—two-stage time of flight analysis established that the HQDO subunits are encoded by neighboring open reading frames (hapC and hapD) of a gene cluster, implicated to be involved in 4-hydroxyacetophenone degradation. HQDO is a novel member of the family of nonheme-iron(II)-dependent dioxygenases. The enzyme shows insignificant sequence identity with known dioxygenases. PMID:18502867

  14. Crystallization and preliminary crystallographic analysis of the catechol 2,3-dioxygenase PheB from Bacillus stearothermophilus BR219

    SciTech Connect

    Sugimoto, Keisuke; Matsufuzi, Kazuki; Ohnuma, Hiroaki; Senda, Miki; Fukuda, Masao; Senda, Toshiya

    2006-02-01

    PheB, an extradiol-cleaving catecholic dioxygenase, was crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystal belongs to the orthorhombic system, space group P2{sub 1}2{sub 1}2{sub 1}, and diffracts to 2.3 Å resolution. Class II extradiol-cleaving catecholic dioxygenase, a key enzyme of aromatic compound degradation in bacteria, cleaves the aromatic ring of catechol by adding two O atoms. PheB is one of the class II extradiol-cleaving catecholic dioxygenases and shows a high substrate specificity for catechol derivatives, which have one aromatic ring. In order to reveal the mechanism of the substrate specificity of PheB, PheB has been crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The space group of the obtained crystal was P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 65.5, b = 119.2, c = 158.7 Å. The crystal diffracted to 2.3 Å resolution.

  15. THE ROLE OF 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE IN ENHANCEMENT OF SOLID-PHASE ELECTRON TRANSFER BY SHEWANELLA ONEIDENSIS MR-1

    SciTech Connect

    Turick, C; Amy Ekechukwu, A

    2007-06-01

    While mechanistic details of dissimilatory metal reduction are far from being understood, it is postulated that the electron transfer to solid metal oxides is mediated by outer membrane-associated c-type cytochromes and redox active electron shuttling compounds. This study focuses on the production of homogensitate in Shewanella oneidensis MR-1, an intermediate of tyrosine degradation pathway, which is a precursor of a redox cycling metabolite, pyomelanin. In this study, we determined that two enzymes involved in this pathway, 4-hydroxyphenylpyruvate dioxygenase (4HPPD) and homogentisate 1,2-dioxygenase are responsible for homogentisate production and oxidation, respectively. Inhibition of 4-HPPD activity with the specific inhibitor sulcotrione (2-(2-chloro-4-methane sulfonylbenzoyl)-1,3-cyclohexanedione), and deletion of melA, a gene encoding 4-HPPD, resulted in no pyomelanin production by S. oneidensis MR-1. Conversely, deletion of hmgA which encodes the putative homogentisate 1,2-dioxygenase, resulted in pyomelanin overproduction. The efficiency and rates, with which MR-1 reduces hydrous ferric oxide, were directly linked to the ability of mutant strains to produce pyomelanin. Electrochemical studies with whole cells demonstrated that pyomelanin substantially increases the formal potential (E{sup o}{prime}) of S. oneidensis MR-1. Based on this work, environmental production of pyomelanin likely contributes to an increased solid-phase metal reduction capacity in Shewanella oneidensis.

  16. An aryl dioxygenase shows remarkable double dioxygenation capacity for diverse bis-aryl compounds, provided they are carbocyclic.

    PubMed

    Overwin, Heike; González, Myriam; Méndez, Valentina; Seeger, Michael; Wray, Victor; Hofer, Bernd

    2016-09-01

    The bacterial dioxygenation of mono- or polycyclic aromatic compounds is an intensely studied field. However, only in a few cases has the repeated dioxygenation of a substrate possessing more than a single aromatic ring been described. We previously characterized the aryl-hydroxylating dioxygenase BphA-B4h, an artificial hybrid of the dioxygenases of the biphenyl degraders Burkholderia xenovorans LB400 and Pseudomonas sp. strain B4-Magdeburg, which contains the active site of the latter enzyme, as an exceptionally powerful biocatalyst. We now show that this dioxygenase possesses a remarkable capacity for the double dioxygenation of various bicyclic aromatic compounds, provided that they are carbocyclic. Two groups of biphenyl analogues were examined: series A compounds containing one heterocyclic aromatic ring and series B compounds containing two homocyclic aromatic rings. Whereas all of the seven partially heterocyclic biphenyl analogues were solely dioxygenated in the homocyclic ring, four of the six carbocyclic bis-aryls were converted into ortho,meta-hydroxylated bis-dihydrodiols. Potential reasons for failure of heterocyclic dioxygenations are discussed. The obtained bis-dihydrodiols may, as we also show here, be enzymatically re-aromatized to yield the corresponding tetraphenols. This opens a way to a range of new polyphenolic products, a class of compounds known to exert multiple biological activities. Several of the obtained compounds are novel molecules. PMID:27147529

  17. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs: Arg- and Gln-type Bacterial CDO Homologs

    SciTech Connect

    Driggers, Camden M.; Hartman, Steven J.; Karplus, P. Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ~15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.

  18. Challenges in diagnosing a metabolic disorder: error of pyruvate metabolism or drug induced?

    PubMed

    Mampilly, George Tomy; Mampilly, Tomy Kochuvareed; Christopher, Rita; Chandramohan, Neeradha; Janaki, Vijayalakshmy

    2014-06-01

    Certain drugs are known to cause metabolic changes resulting in altered metabolic profiles. We report here a case where a combination of antiepileptic drugs resulted in a profile that mimicked a metabolic disorder. A 16month-old female child on antiepileptic drugs (valproate and topiramate) was suspected to have the inherited metabolic disorder, dihydrolipoamide dehydrogenase deficiency, based on clinical symptoms and metabolic profile showing hyperalaninemia, elevated branched-chain amino acids, and lactate-pyruvate ratio. Suspecting that the observed metabolic changes could have also arised from medication, current medication was weaned off and replaced with levetiracetam, clonazepam, and levocarnitine (supportive therapy). Metabolic profiling conducted after 47 days showed normal alanine, branched-chain amino acids, ornithine, and lactate-pyruvate ratio, suggesting that the earlier abnormalities could have been medication induced. We stress that metabolic changes resulting from chronic medication should be considered while interpreting a positive result when investigating an inherited metabolic disorder. PMID:23439713

  19. Expression of Aeromonas caviae ST pyruvate dehydrogenase complex components mediate tellurite resistance in Escherichia coli

    SciTech Connect

    Castro, Miguel E.; Molina, Roberto C.; Diaz, Waldo A.; Pradenas, Gonzalo A.; Vasquez, Claudio C.

    2009-02-27

    Potassium tellurite (K{sub 2}TeO{sub 3}) is harmful to most organisms and specific mechanisms explaining its toxicity are not well known to date. We previously reported that the lpdA gene product of the tellurite-resistant environmental isolate Aeromonas caviae ST is involved in the reduction of tellurite to elemental tellurium. In this work, we show that expression of A. caviae ST aceE, aceF, and lpdA genes, encoding pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, respectively, results in tellurite resistance and decreased levels of tellurite-induced superoxide in Escherichia coli. In addition to oxidative damage resulting from tellurite exposure, a metabolic disorder would be simultaneously established in which the pyruvate dehydrogenase complex would represent an intracellular tellurite target. These results allow us to widen our vision regarding the molecular mechanisms involved in bacterial tellurite resistance by correlating tellurite toxicity and key enzymes of aerobic metabolism.

  20. Regulation of pyruvate dehydrogenase kinase expression by the farnesoid X receptor

    SciTech Connect

    Savkur, Rajesh S.; Bramlett, Kelli S.; Michael, Laura F.; Burris, Thomas P. . E-mail: burris_thomas_p@lilly.com

    2005-04-01

    The pyruvate dehydrogenase complex (PDC) functions as an important junction in intermediary metabolism by influencing the utilization of fat versus carbohydrate as a source of fuel. Activation of PDC is achieved by phosphatases, whereas, inactivation is catalyzed by pyruvate dehydrogenase kinases (PDKs). The expression of PDK4 is highly regulated by the glucocorticoid and peroxisome proliferator-activated receptors. We demonstrate that the farnesoid X receptor (FXR; NR1H4), which regulates a variety of genes involved in lipoprotein metabolism, also regulates the expression of PDK4. Treatment of rat hepatoma cells as well as human primary hepatocytes with FXR agonists stimulates the expression of PDK4 to levels comparable to those obtained with glucocorticoids. In addition, treatment of mice with an FXR agonist significantly increased hepatic PDK4 expression, while concomitantly decreasing plasma triglyceride levels. Thus, activation of FXR may suppress glycolysis and enhance oxidation of fatty acids via inactivation of the PDC by increasing PDK4 expression.

  1. Pro-haloacetate Nanoparticles for Efficient Cancer Therapy via Pyruvate Dehydrogenase Kinase Modulation

    NASA Astrophysics Data System (ADS)

    Misra, Santosh K.; Ye, Mao; Ostadhossein, Fatemeh; Pan, Dipanjan

    2016-06-01

    Anticancer agents based on haloacetic acids are developed for inhibition of pyruvate dehydrogenase kinase (PDK), an enzyme responsible for reversing the suppression of mitochondria-dependent apoptosis. Through molecular docking studies mono- and dihaloacetates are identified as potent PDK2 binders and matched their efficiency with dichloroacetic acid. In silico screening directed their conversion to phospholipid prodrugs, which were subsequently self-assembled to pro-haloacetate nanoparticles. Following a thorough physico-chemical characterization, the functional activity of these novel agents was established in wide ranges of human cancer cell lines in vitro and in vivo in rodents. Results indicated that the newly explored PDK modulators can act as efficient agent for cancer regression. A Pyruvate dehydrogenase (PDH) assay mechanistically confirmed that these agents trigger their activity through the mitochondria-dependent apoptosis.

  2. Pro-haloacetate Nanoparticles for Efficient Cancer Therapy via Pyruvate Dehydrogenase Kinase Modulation

    PubMed Central

    Misra, Santosh K.; Ye, Mao; Ostadhossein, Fatemeh; Pan, Dipanjan

    2016-01-01

    Anticancer agents based on haloacetic acids are developed for inhibition of pyruvate dehydrogenase kinase (PDK), an enzyme responsible for reversing the suppression of mitochondria-dependent apoptosis. Through molecular docking studies mono- and dihaloacetates are identified as potent PDK2 binders and matched their efficiency with dichloroacetic acid. In silico screening directed their conversion to phospholipid prodrugs, which were subsequently self-assembled to pro-haloacetate nanoparticles. Following a thorough physico-chemical characterization, the functional activity of these novel agents was established in wide ranges of human cancer cell lines in vitro and in vivo in rodents. Results indicated that the newly explored PDK modulators can act as efficient agent for cancer regression. A Pyruvate dehydrogenase (PDH) assay mechanistically confirmed that these agents trigger their activity through the mitochondria-dependent apoptosis. PMID:27323896

  3. A role for the mitochondrial pyruvate carrier as a repressor of the Warburg Effect and colon cancer cell growth

    PubMed Central

    Schell, John C.; Olson, Kristofor A.; Jiang, Lei; Hawkins, Amy J.; Van Vranken, Jonathan G.; Xie, Jianxin; Egnatchik, Robert A.; Earl, Espen G.; Deberardinis, Ralph J.; Rutter, Jared

    2014-01-01

    Summary Cancer cells are typically subject to profound metabolic alterations, including the Warburg effect wherein cancer cells oxidize a decreased fraction of the pyruvate generated from glycolysis. We show herein that the mitochondrial pyruvate carrier (MPC), composed of the products of the MPC1 and MPC2 genes, modulates fractional pyruvate oxidation. MPC1 is deleted or underexpressed in multiple cancers and correlates with poor prognosis. Cancer cells re-expressing MPC1 and MPC2 display increased mitochondrial pyruvate oxidation, with no changes in cell growth in adherent culture. MPC re-expression exerted profound effects in anchorage-independent growth conditions, however, including impaired colony formation in soft agar, spheroid formation, and xenograft growth. We also observed a decrease in markers of stemness and traced the growth effects of MPC expression to the stem cell compartment. We propose that reduced MPC activity is an important aspect of cancer metabolism, perhaps through altering the maintenance and fate of stem cells. PMID:25458841

  4. Effects of fasting on serial measurements of hyperpolarized [1-(13) C]pyruvate metabolism in tumors.

    PubMed

    Serrao, Eva M; Rodrigues, Tiago B; Gallagher, Ferdia A; Kettunen, Mikko I; Kennedy, Brett W C; Vowler, Sarah L; Burling, Keith A; Brindle, Kevin M

    2016-08-01

    Imaging of the metabolism of hyperpolarized [1-(13) C]pyruvate has shown considerable promise in preclinical studies in oncology, particularly for the assessment of early treatment response. The repeatability of measurements of (13) C label exchange between pyruvate and lactate was determined in a murine lymphoma model in fasted and non-fasted animals. The fasted state showed lower intra-individual variability, although the [1-(13) C]lactate/[1-(13) C]pyruvate signal ratio was significantly greater in fasted than in non-fasted mice, which may be explained by the higher tumor lactate concentrations in fasted animals. These results indicate that the fasted state may be preferable for the measurement of (13) C label exchange between pyruvate and lactate, as it reduces the variability and therefore should make it easier to detect the effects of therapy. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd. PMID:27309986

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  6. Effects of fasting on serial measurements of hyperpolarized [1‐13C]pyruvate metabolism in tumors

    PubMed Central

    Serrao, Eva M.; Rodrigues, Tiago B.; Gallagher, Ferdia A.; Kettunen, Mikko I.; Kennedy, Brett W. C.; Vowler, Sarah L.; Burling, Keith A.

    2016-01-01

    Imaging of the metabolism of hyperpolarized [1‐13C]pyruvate has shown considerable promise in preclinical studies in oncology, particularly for the assessment of early treatment response. The repeatability of measurements of 13C label exchange between pyruvate and lactate was determined in a murine lymphoma model in fasted and non‐fasted animals. The fasted state showed lower intra‐individual variability, although the [1‐13C]lactate/[1‐13C]pyruvate signal ratio was significantly greater in fasted than in non‐fasted mice, which may be explained by the higher tumor lactate concentrations in fasted animals. These results indicate that the fasted state may be preferable for the measurement of 13C label exchange between pyruvate and lactate, as it reduces the variability and therefore should make it easier to detect the effects of therapy. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd. PMID:27309986

  7. A rate-determining proton relay in the pyruvate kinase reaction

    SciTech Connect

    Rose, I.A.; Kuo, D.J.; Warms, J.V.B. )

    1991-01-22

    This study ascribes the large steady-state D{sub 2}O isotope effect on k{sub cat} of pyruvate kinase (PEP + ADP {yields} pyruvate + ATP) to the reprotonation of the product form of the enzyme for use in forming pyruvate. Previous tritium trapping experiments with muscle pyruvate kinase showed that the proton used for ketonization of enolpyruvate is derived from an enzyme pool that contains three kinetically equivalent hydrogens that could be trapped in a nontritiated chase medium by high levels of ADP and PEP. The exchange of this pool with the medium was rapid in the free enzyme prior to addition of PEP, and apparently much less in the completed complex. The present study shows that the competition constant, K{sub 1/2}, is decreased by {approximately}5-fold in D{sub 2}O, the same effect seen on k{sub cat} under conditions where k{sub cat}/K{sub m}, measured in the steady state, is not changed. The common effect of D{sub 2}O on k{sub cat} in the steady state and k{sub off}{sup T} in pulse/chase suggests that the forward reaction rate is determined by hydrogen transfer to the enzyme. Further evidence indicates that the kinetically important proton in question is the proton used for ketonization of enolpyruvate, the substrate proton. With Co, roughly three T of the pool can be trapped. With Mg, only one T can be trapped from the same pulse solution at the highest concentrations of PEP. Apparently two positions, possibly from a bound water molecule, are rapidly exchanged with water of the chase. The inference that lysine NH{sub 3{sup +}} is the proton donor group on the basis of the three T trapped is open to question by evidence that one or two of these come from relay positions.

  8. Malic Acid Production by Saccharomyces cerevisiae: Engineering of Pyruvate Carboxylation, Oxaloacetate Reduction, and Malate Export▿ †

    PubMed Central

    Zelle, Rintze M.; de Hulster, Erik; van Winden, Wouter A.; de Waard, Pieter; Dijkema, Cor; Winkler, Aaron A.; Geertman, Jan-Maarten A.; van Dijken, Johannes P.; Pronk, Jack T.; van Maris, Antonius J. A.

    2008-01-01

    Malic acid is a potential biomass-derivable “building block” for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO2-fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose)−1. A previously engineered glucose-tolerant, C2-independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2, (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene SpMAE1. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter−1 at a malate yield of 0.42 mol (mol glucose)−1. Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on 13C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved. PMID:18344340

  9. Effect of pyruvate dehydrogenase complex deficiency on L-lysine production with Corynebacterium glutamicum.

    PubMed

    Blombach, Bastian; Schreiner, Mark E; Moch, Matthias; Oldiges, Marco; Eikmanns, Bernhard J

    2007-09-01

    Intracellular precursor supply is a critical factor for amino acid productivity of Corynebacterium glutamicum. To test for the effect of improved pyruvate availability on L-lysine production, we deleted the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex (PDHC) in the L-lysine-producer C. glutamicum DM1729 and characterised the resulting strain DM1729-BB1 for growth and L-lysine production. Compared to the host strain, C. glutamicum DM1729-BB1 showed no PDHC activity, was acetate auxotrophic and, after complete consumption of the available carbon sources glucose and acetate, showed a more than 50% lower substrate-specific biomass yield (0.14 vs 0.33 mol C/mol C), an about fourfold higher biomass-specific L-lysine yield (5.27 vs 1.23 mmol/g cell dry weight) and a more than 40% higher substrate-specific L-lysine yield (0.13 vs 0.09 mol C/mol C). Overexpression of the pyruvate carboxylase or diaminopimelate dehydrogenase genes in C. glutamicum DM1729-BB1 resulted in a further increase in the biomass-specific L-lysine yield by 6 and 56%, respectively. In addition to L-lysine, significant amounts of pyruvate, L-alanine and L-valine were produced by C. glutamicum DM1729-BB1 and its derivatives, suggesting a surplus of precursor availability and a further potential to improve L-lysine production by engineering the L-lysine biosynthetic pathway. PMID:17333167

  10. Pyruvate treatment attenuates cerebral metabolic depression and neuronal loss after experimental traumatic brain injury.

    PubMed

    Moro, Nobuhiro; Ghavim, Sima S; Harris, Neil G; Hovda, David A; Sutton, Richard L

    2016-07-01

    Experimental traumatic brain injury (TBI) is known to produce an acute increase in cerebral glucose utilization, followed rapidly by a generalized cerebral metabolic depression. The current studies determined effects of single or multiple treatments with sodium pyruvate (SP; 1000mg/kg, i.p.) or ethyl pyruvate (EP; 40mg/kg, i.p.) on cerebral glucose metabolism and neuronal injury in rats with unilateral controlled cortical impact (CCI) injury. In Experiment 1 a single treatment was given immediately after CCI. SP significantly improved glucose metabolism in 3 of 13 brain regions while EP improved metabolism in 7 regions compared to saline-treated controls at 24h post-injury. Both SP and EP produced equivalent and significant reductions in dead/dying neurons in cortex and hippocampus at 24h post-CCI. In Experiment 2 SP or EP were administered immediately (time 0) and at 1, 3 and 6h post-CCI. Multiple SP treatments also significantly attenuated TBI-induced reductions in cerebral glucose metabolism (in 4 brain regions) 24h post-CCI, as did multiple injections of EP (in 4 regions). The four pyruvate treatments produced significant neuroprotection in cortex and hippocampus 1day after CCI, similar to that found with a single SP or EP treatment. Thus, early administration of pyruvate compounds enhanced cerebral glucose metabolism and neuronal survival, with 40mg/kg of EP being as effective as 1000mg/kg of SP, and multiple treatments within 6h of injury did not improve upon outcomes seen following a single treatment. PMID:27059390

  11. Chlamydomonas reinhardtii chloroplasts contain a homodimeric pyruvate:ferredoxin oxidoreductase that functions with FDX1.

    PubMed

    van Lis, Robert; Baffert, Carole; Couté, Yohann; Nitschke, Wolfgang; Atteia, Ariane

    2013-01-01

    Eukaryotic algae have long been known to live in anoxic environments, but interest in their anaerobic energy metabolism has only recently gained momentum, largely due to their utility in biofuel production. Chlamydomonas reinhardtii figures remarkably in this respect, because it efficiently produces hydrogen and its genome harbors many genes for anaerobic metabolic routes. Central to anaerobic energy metabolism in many unicellular eukaryotes (protists) is pyruvate:ferredoxin oxidoreductase (PFO), which decarboxylates pyruvate and forms acetyl-coenzyme A with concomitant reduction of low-potential ferredoxins or flavodoxins. Here, we report the biochemical properties of the homodimeric PFO of C. reinhardtii expressed in Escherichia coli. Electron paramagnetic resonance spectroscopy of the recombinant enzyme (Cr-rPFO) showed three distinct [4Fe-4S] iron-sulfur clusters and a thiamine pyrophosphate radical upon reduction by pyruvate. Purified Cr-rPFO exhibits a specific decarboxylase activity of 12 µmol pyruvate min⁻¹ mg⁻¹ protein using benzyl viologen as electron acceptor. Despite the fact that the enzyme is very oxygen sensitive, it localizes to the chloroplast. Among the six known chloroplast ferredoxins (FDX1-FDX6) in C. reinhardtii, FDX1 and FDX2 were the most efficient electron acceptors from Cr-rPFO, with comparable apparent K(m) values of approximately 4 µm. As revealed by immunoblotting, anaerobic conditions that lead to the induction of CrPFO did not increase levels of either FDX1 or FDX2. FDX1, being by far the most abundant ferredoxin, is thus likely the partner of PFO in C. reinhardtii. This finding postulates a direct link between CrPFO and hydrogenase and provides new opportunities to better study and engineer hydrogen production in this protist. PMID:23154536

  12. Regulation of pyc1 encoding pyruvate carboxylase isozyme I by nitrogen sources in Saccharomyces cerevisiae.

    PubMed

    Huet, C; Menendez, J; Gancedo, C; François, J M

    2000-12-01

    In Saccharomyces cerevisiae, the existence of PYC1 and PYC2 encoding cytosolic pyruvate carboxylase isoform I and II is rather puzzling, owing to the lack of potent differential gene regulation by the carbon sources. We report several findings indicating that these two genes are differentially regulated by the nature of the nitrogen source. In wild-type cells, the activity of pyruvate carboxylase, which is the sum of pyruvate carboxylase isoform I and II, was two- to fivefold lower in carbon medium containing aspartate, asparagine, glutamate or glutamine instead of ammonium as the nitrogen source, whereas it was 1.5- to threefold higher when the ammonium source was substituted by arginine, methionine, threonine or leucine. These enzymatic changes were independent of the nature of the carbon source and closely correlated to the changes in beta-galactosidase from PYC1-lacZ gene fusion and in PYC1 transcripts. Transfer of exponentially growing cells of the pyc2 mutant from an aspartate or a glutamate medium to an ammonium medium caused a fivefold increase in PYC1 mRNA in less than 30 min, whereas in the inverse experiment, PYC1 transcripts returned within 30 min to the low levels found in aspartate/glutamate medium. By contrast, these conditions affected neither the pyruvate carboxylase activity encoded by PYC2 nor PYC2 mRNA. Considering that changes in PYC1 expression inversely correlated with changes in alpha-ketoglutarate concentration or in alpha-ketoglutarate/glutamate ratio following the nitrogen shift experiments, and taking into account the pivotal role of this metabolite in ammonium assimilation, it is suggested that changes in alpha-ketoglutarate or in the alpha-ketoglutarate/glutamate ratio might be implicated in triggering the nitrogen effects on PYC1 expression. The physiological significance of the differential sensitivity of PYC1 and PYC2 genes with respect to the nitrogen source in the growth medium is also discussed. PMID:11082192

  13. Measurement of the acute metabolic response to hypoxia in rat tumours in vivo using magnetic resonance spectroscopy and hyperpolarised pyruvate

    PubMed Central

    Bluff, Joanne E.; Reynolds, Steven; Metcalf, Stephen; Alizadeh, Tooba; Kazan, Samira M.; Bucur, Adriana; Wholey, Emily G.; Bibby, Becky A.S.; Williams, Leigh; Paley, Martyn N.; Tozer, Gillian M.

    2015-01-01

    Purpose To estimate the rate constant for pyruvate to lactate conversion in tumours in response to a hypoxic challenge, using hyperpolarised 13C1-pyruvate and magnetic resonance spectroscopy. Methods and materials Hypoxic inspired gas was used to manipulate rat P22 fibrosarcoma oxygen tension (pO2), confirmed by luminescence decay of oxygen-sensitive probes. Hyperpolarised 13C1-pyruvate was injected into the femoral vein of anaesthetised rats and slice-localised 13C magnetic resonance (MR) spectra acquired. Spectral integral versus time curves for pyruvate and lactate were fitted to a precursor-product model to estimate the rate constant for tumour conversion of pyruvate to lactate (kpl). Mean arterial blood pressure (MABP) and oxygen tension (ArtpO2) were monitored. Pyruvate and lactate concentrations were measured in freeze-clamped tumours. Results MABP, ArtpO2 and tumour pO2 decreased significantly during hypoxia. kpl increased significantly (p < 0.01) from 0.029 ± 0.002 s−1 to 0.049 ± 0.006 s−1 (mean ± SEM) when animals breathing air were switched to hypoxic conditions, whereas pyruvate and lactate concentrations were minimally affected by hypoxia. Both ArtpO2 and MABP influenced the estimate of kpl, with a strong negative correlation between kpl and the product of ArtpO2 and MABP under hypoxia. Conclusion The rate constant for pyruvate to lactate conversion, kpl, responds significantly to a rapid reduction in tumour oxygenation. PMID:25824978

  14. Single sodium pyruvate ingestion modifies blood acid-base status and post-exercise lactate concentration in humans.

    PubMed

    Olek, Robert A; Kujach, Sylwester; Wnuk, Damian; Laskowski, Radoslaw

    2014-05-01

    This study examined the effect of a single sodium pyruvate ingestion on a blood acid-base status and exercise metabolism markers. Nine active, but non-specifically trained, male subjects participated in the double-blind, placebo-controlled, crossover study. One hour prior to the exercise, subjects ingested either 0.1 g·kg(-1) of body mass of a sodium pyruvate or placebo. The capillary blood samples were obtained at rest, 60 min after ingestion, and then three and 15 min after completing the workout protocol to analyze acid-base status and lactate, pyruvate, alanine, glucose concentrations. The pulmonary gas exchange, minute ventilation and the heart rate were measured during the exercise at a constant power output, corresponding to ~90% VO2max. The blood pH, bicarbonate and the base excess were significantly higher after sodium pyruvate ingestion than in the placebo trial. The blood lactate concentration was not different after the ingestion, but the post-exercise was significantly higher in the pyruvate trial (12.9 ± 0.9 mM) than in the placebo trial (10.6 ± 0.3 mM, p < 0.05) and remained elevated (nonsignificant) after 15 min of recovery. The blood pyruvate, alanine and glucose concentrations, as well as the overall pulmonary gas exchange during the exercise were not affected by the pyruvate ingestion. In conclusion, the sodium pyruvate ingestion one hour before workout modified the blood acid-base status and the lactate production during the exercise. PMID:24841105

  15. Cooperativity in highly aggregated enzyme systems. A slow transition model for the pyruvate dehydrogenase complex from Escherichia coli.

    PubMed

    Bisswanger, H

    1984-02-25

    Three models are compared describing cooperative phenomena in enzymatic reactions in order to explain sigmoidal saturation curves found with the pyruvate dehydrogenase complex from Escherichia coli: the concerted model, the sequential model, and the slow transition model. Both the concerted and the sequential model were considered especially with regard to the increasing number of identical interaction subunits (protomers) in order to get close to the situation found with the pyruvate dehydrogenase complex which consists of 24 protomers. Applying the sequential model to a great number of protomers results in a weak increase of the Hill coefficient, while, in addition to this effect, the concerted model drastically shifts the sigmoidal range of the saturation function to very low ligand concentrations. Such shift is seen with saturation curves of pyruvate and thiamine disphosphate with the pyruvate dehydrogenase complex and a good fit with theoretical curves derived from the concerted model is obtained. However, subcomplexes with a reduced number of protomers exhibited no change in saturation behavior, thus providing evidence against concerted conformational changes of all subunits of the enzyme complex. A scheme for the initial reaction of the pyruvate dehydrogenase complex based on slow transitions is presented and a rate equation has been derived. Ordered binding of thiamine diphosphate and pyruvate and a ligand-induced slow transition between a less active and a fully active enzyme form has been assumed. The curves simulated with this model are in agreement with all essential kinetic data, which are observed with the pyruvate dehydrogenase complex: the atypical shape of the saturation curves of pyruvate and thiamine diphosphate, the respective Hill coefficients and Michaelis constants, the hyperbolic binding behavior of thiamine diphosphate, and the inhibition pattern found for acetyl coenzyme A. PMID:6365912

  16. Single Sodium Pyruvate Ingestion Modifies Blood Acid-Base Status and Post-Exercise Lactate Concentration in Humans

    PubMed Central

    Olek, Robert A.; Kujach, Sylwester; Wnuk, Damian; Laskowski, Radoslaw

    2014-01-01

    This study examined the effect of a single sodium pyruvate ingestion on a blood acid-base status and exercise metabolism markers. Nine active, but non-specifically trained, male subjects participated in the double-blind, placebo-controlled, crossover study. One hour prior to the exercise, subjects ingested either 0.1 g·kg−1 of body mass of a sodium pyruvate or placebo. The capillary blood samples were obtained at rest, 60 min after ingestion, and then three and 15 min after completing the workout protocol to analyze acid-base status and lactate, pyruvate, alanine, glucose concentrations. The pulmonary gas exchange, minute ventilation and the heart rate were measured during the exercise at a constant power output, corresponding to ~90% O2max. The blood pH, bicarbonate and the base excess were significantly higher after sodium pyruvate ingestion than in the placebo trial. The blood lactate concentration was not different after the ingestion, but the post-exercise was significantly higher in the pyruvate trial (12.9 ± 0.9 mM) than in the placebo trial (10.6 ± 0.3 mM, p < 0.05) and remained elevated (nonsignificant) after 15 min of recovery. The blood pyruvate, alanine and glucose concentrations, as well as the overall pulmonary gas exchange during the exercise were not affected by the pyruvate ingestion. In conclusion, the sodium pyruvate ingestion one hour before workout modified the blood acid-base status and the lactate production during the exercise. PMID:24841105

  17. Acute porcine renal metabolic effect of endogastric soft drink administration assessed with hyperpolarized [1‐13c]pyruvate

    PubMed Central

    Hansen, Esben Søvsø Szocska; Kjærgaard, Uffe; Bertelsen, Lotte Bonde; Ringgaard, Steffen; Stødkilde‐Jørgensen, Hans

    2015-01-01

    Purpose Our aim was to determine the quantitative reproducibility of metabolic breakdown products in the kidney following intravenous injection of hyperpolarized [1‐13C]pyruvate and secondly to investigate the metabolic effect on the pyruvate metabolism of oral sucrose load using dissolution dynamic nuclear polarization. By this technique, metabolic alterations in several different metabolic related diseases and their metabolic treatment responses can be accessed. Methods In four healthy pigs the lactate‐to‐pyruvate, alanine‐to‐pyruvate and bicarbonate‐to‐pyruvate ratio was measured following administration of regular cola and consecutive injections of hyperpolarized [1‐13C]pyruvate four times within an hour. Results The overall lactate‐to‐pyruvate metabolic profile changed significantly over one hour following an acute sucrose load leading to a significant rise in blood glucose. Conclusion The reproducibility of hyperpolarized magnetic resonance spectroscopy in the healthy pig kidney demonstrated a repeatability of more than 94% for all metabolites and, furthermore, that the pyruvate to lactate conversion and the blood glucose level is elevated following endogastric sucrose administration. Magn Reson Med 74:558–563, 2015. © 2015 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. PMID:26014387

  18. Pyruvate dehydrogenase complex: metabolic link to ischemic brain injury and target of oxidative stress.

    PubMed

    Martin, Erica; Rosenthal, Robert E; Fiskum, Gary

    The mammalian pyruvate dehydrogenase complex (PDHC) is a mitochondrial matrix enzyme complex (greater than 7 million Daltons) that catalyzes the oxidative decarboxylation of pyruvate to form acetyl CoA, nicotinamide adenine dinucleotide (the reduced form, NADH), and CO(2). This reaction constitutes the bridge between anaerobic and aerobic cerebral energy metabolism. PDHC enzyme activity and immunoreactivity are lost in selectively vulnerable neurons after cerebral ischemia and reperfusion. Evidence from experiments carried out in vitro suggests that reperfusion-dependent loss of activity is caused by oxidative protein modifications. Impaired enzyme activity may explain the reduced cerebral glucose and oxygen consumption that occurs after cerebral ischemia. This hypothesis is supported by the hyperoxidation of mitochondrial electron transport chain components and NAD(H) that occurs during reperfusion, indicating that NADH production, rather than utilization, is rate limiting. Additional support comes from the findings that immediate postischemic administration of acetyl-L-carnitine both reduces brain lactate/pyruvate ratios and improves neurologic outcome after cardiac arrest in animals. As acetyl-L-carnitine is converted to acetyl CoA, the product of the PDHC reaction, it follows that impaired production of NADH is due to reduced activity of either PDHC or one or more steps in glycolysis. Impaired cerebral energy metabolism and PDHC activity are associated also with neurodegenerative disorders including Alzheimer's disease and Wernicke-Korsakoff syndrome, suggesting that this enzyme is an important link in the pathophysiology of both acute brain injury and chronic neurodegeneration. PMID:15562436

  19. Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

    SciTech Connect

    Anastasiou, Dimitrios; Yu, Yimin; Israelsen, William J.; Jiang, Jian-Kang; Boxer, Matthew B.; Hong, Bum Soo; Tempel, Wolfram; Dimov, Svetoslav; Shen, Min; Jha, Abhishek; Yang, Hua; Mattaini, Katherine R.; Metallo, Christian M.; Fiske, Brian P.; Courtney, Kevin D.; Malstrom, Scott; Khan, Tahsin M.; Kung, Charles; Skoumbourdis, Amanda P.; Veith, Henrike; Southall, Noel; Walsh, Martin J.; Brimacombe, Kyle R.; Leister, William; Lunt, Sophia Y.; Johnson, Zachary R.; Yen, Katharine E.; Kunii, Kaiko; Davidson, Shawn M.; Christofk, Heather R.; Austin, Christopher P.; Inglese, James; Harris, Marian H.; Asara, John M.; Stephanopoulos, Gregory; Salituro, Francesco G.; Jin, Shengfang; Dang, Lenny; Auld, Douglas S.; Park, Hee-Won; Cantley, Lewis C.; Thomas, Craig J.; Vander Heiden, Matthew G.

    2012-08-26

    Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. This data supports the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.

  20. Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

    PubMed Central

    Anastasiou, Dimitrios; Yu, Yimin; Israelsen, William J.; Jiang, Jian-kang; Boxer, Matthew B.; Hong, Bum Soo; Tempel, Wolfram; Dimov, Svetoslav; Shen, Min; Jha, Abhishek; Yang, Hua; Mattaini, Katherine R.; Metallo, Christian M.; Fiske, Brian P.; Courtney, Kevin D.; Malstrom, Scott; Khan, Tahsin M.; Kung, Charles; Skoumbourdis, Amanda P.; Veith, Henrike; Southall, Noel; Walsh, Martin J.; Brimacombe, Kyle R.; Leister, William; Lunt, Sophia Y.; Johnson, Zachary R.; Yen, Katharine E.; Kunii, Kaiko; Davidson, Shawn M.; Christofk, Heather R.; Austin, Christopher P.; Inglese, James; Harris, Marian H.; Asara, John M.; Stephanopoulos, Gregory; Salituro, Francesco G.; Jin, Shengfang; Dang, Lenny; Auld, Douglas S.; Park, Hee-Won; Cantley, Lewis C.; Thomas, Craig J.; Vander Heiden, Matthew G.

    2012-01-01

    Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. PKM2 interaction with phosphotyrosine-containing proteins inhibits enzyme activity and increases availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small molecule PKM2 activators inhibit growth of xenograft tumors. Structural studies reveal that small molecule activators bind PKM2 at the subunit interaction interface, a site distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small molecule activation of PKM2 can interfere with anabolic metabolism. PMID:22922757

  1. Lactate Contributes to Glyceroneogenesis and Glyconeogenesis in Skeletal Muscle by Reversal of Pyruvate Kinase.

    PubMed

    Jin, Eunsook S; Sherry, A Dean; Malloy, Craig R

    2015-12-18

    Phosphoenolpyruvate (PEP) generated from pyruvate is required for de novo synthesis of glycerol and glycogen in skeletal muscle. One possible pathway involves synthesis of PEP from the citric acid cycle intermediates via PEP carboxykinase, whereas another could involve reversal of pyruvate kinase (PK). Earlier studies have reported that reverse flux through PK can contribute carbon precursors for glycogen synthesis in muscle, but the physiological importance of this pathway remains uncertain especially in the setting of high plasma glucose. In addition, although PEP is a common intermediate for both glyconeogenesis and glyceroneogenesis, the importance of reverse PK in de novo glycerol synthesis has not been examined. Here we studied the contribution of reverse PK to synthesis of glycogen and the glycerol moiety of acylglycerols in skeletal muscle of animals with high plasma glucose. Rats received a single intraperitoneal bolus of glucose, glycerol, and lactate under a fed or fasted state. Only one of the three substrates was (13)C-labeled in each experiment. After 3 h of normal awake activity, the animals were sacrificed, and the contribution from each substrate to glycogen and the glycerol moiety of acylglycerols was evaluated. The fraction of (13)C labeling in glycogen and the glycerol moiety exceeded the possible contribution from either plasma glucose or muscle oxaloacetate. The reverse PK served as a common route for both glyconeogenesis and glyceroneogenesis in the skeletal muscle of rats with high plasma glucose. The activity of pyruvate carboxylase was low in muscle, and no PEP carboxykinase activity was detected. PMID:26491014

  2. Post-translational activation introduces a free radical into pyruvate formate-lyase.

    PubMed Central

    Knappe, J; Neugebauer, F A; Blaschkowski, H P; Gänzler, M

    1984-01-01

    Pyruvate formate-lyase (formate acetyltransferase; EC 2.3.1.54) of Escherichia coli cells is post-translationally interconverted between inactive and active forms. Conversion of the inactive to the active form is catalyzed by an Fe2+-dependent activating enzyme and requires adenosylmethionine and dihydroflavodoxin. This process is shown here to introduce a paramagnetic moiety into the structure of pyruvate formate-lyase. It displays an EPR signal at g = 2 with a doublet splitting of 1.5 mT and could comprise an organic free radical located on an amino acid residue of the polypeptide chain. Hypophosphite was discovered as a specific reagent that destroys both the enzyme radical and the enzyme activity; it becomes covalently bound to the protein. The enzymatic generation of the radical, which is linked to adenosylmethionine cleavage into 5'-deoxyadenosine and methionine, possibly occurs through an Fe-adenosyl complex. These results suggest a radical mechanism for the catalytic cycle of pyruvate formate-lyase. PMID:6369325

  3. Pyruvate carboxylation enables growth of SDH-deficient cells by supporting aspartate biosynthesis

    PubMed Central

    Cardaci, Simone; Zheng, Liang; MacKay, Gillian; van den Broek, Niels J.F.; MacKenzie, Elaine D.; Nixon, Colin; Stevenson, David; Tumanov, Sergey; Bulusu, Vinay; Kamphorst, Jurre J.; Vazquez, Alexei; Fleming, Stewart; Schiavi, Francesca; Kalna, Gabriela; Blyth, Karen; Strathdee, Douglas; Gottlieb, Eyal

    2015-01-01

    Succinate dehydrogenase (SDH) is a hetero-tetrameric nuclear-encoded complex responsible for the oxidation of succinate to fumarate in the tricarboxylic acid (TCA) cycle. Loss-of-function mutations in any of the SDH genes are associated with cancer formation. However, the impact of SDH loss on cell metabolism and the mechanisms enabling growth of SDH-defective cells are largely unknown. Here, we generated Sdhb-ablated kidney mouse cells and employed comparative metabolomics and stable isotope-labelling approaches to identify nutritional requirements and metabolic adaptations to SDH loss. We found that lack of SDH activity commits cells to consume extracellular pyruvate, which sustains Warburg-like bioenergetic features. We further demonstrated that pyruvate carboxylation diverts glucose-derived carbons into aspartate biosynthesis, thus sustaining cell growth. By identifying pyruvate carboxylase as an essential gene for the proliferation and tumorigenic capacity of SDH-deficient cells, this study revealed a metabolic vulnerability for potential future treatment of SDH-associated malignancies. PMID:26302408

  4. Pyruvate Kinase M2 Regulates Gene Transcription by Acting as A Protein Kinase

    PubMed Central

    Gao, Xueliang; Wang, Haizhen; Jenny, J. Yang; Liu, Xiaowei; Liu, Zhi-Ren

    2012-01-01

    Summary Pyruvate kinase isoform M2 (PKM2) is a glycolysis enzyme catalyzing conversion of phosphoenolpyruvate (PEP) to pyruvate with transferring a phosphate from PEP to ADP. We report here that PKM2 localizes to the cell nucleus. The levels of nuclear PKM2 correlate with cell proliferation. PKM2 activates transcription of MEK5 by phosphorylating stat3 at Y705. In vitro phosphorylation assays show that PKM2 is a protein kinase using PEP as phosphate donor. ADP competes with the protein substrate binding, indicating that the substrate may bind to the ADP site of PKM2. Our experiments suggest that PKM2 dimer is an active protein kinase, while the tetramer is an active pyruvate kinase. Expression a PKM2 mutant that exists as a dimer promotes cell proliferation, indicating that protein kinase activity of PKM2 plays a role in promoting cell proliferation. Our study reveals an important link between metabolism alteration and gene expression during tumor transformation and progression. PMID:22306293

  5. Regulation of yeast pyruvate kinase by ultrasensitive allostery independent of phosphorylation

    PubMed Central

    Xu, Yi-Fan; Zhao, Xin; Glass, David S.; Absalan, Farnaz; Perlman, David H.; Broach, James R.; Rabinowitz, Joshua D.

    2012-01-01

    Summary Allostery and covalent modification are major means of fast-acting metabolic regulation. Their relative roles in responding to environmental changes remain, however, unclear. Here we examine this issue, using as a case study the rapid decrease in pyruvate kinase flux in yeast upon glucose removal. The main pyruvate kinase isozyme (Cdc19) is phosphorylated in response to environmental cues. It also exhibits positively-cooperative (ultrasensitive) allosteric activation by fructose-1,6-bisphosphate (FBP). Glucose removal causes accumulation of Cdc19’s substrate, phosphoenolpyruvate. This response is retained in strains with altered protein-kinase-A or AMP-activated-protein-kinase activity or with CDC19 carrying mutated phosphorylation sites. In contrast, yeast engineered with a CDC19 point mutation that ablates FBP-based regulation fail to accumulate phosphoenolpyruvate. They also fail to grow on ethanol and slowly resume growth upon glucose upshift. Thus, while yeast pyruvate kinase is covalently modified in response to glucose availability, its activity is controlled almost exclusively by ultrasensitive allostery. PMID:22902555

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

    PubMed

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

    2014-12-01

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

  7. Pyruvate Dehydrogenase Complex: Metabolic Link to Ischemic Brain Injury and Target of Oxidative Stress

    PubMed Central

    Martin, Erica; Rosenthal, Robert E.; Fiskum, Gary

    2008-01-01

    The mammalian pyruvate dehydrogenase complex (PDHC) is a mitochondrial matrix enzyme complex (greater than 7 million Daltons) that catalyzes the oxidative decarboxylation of pyruvate to form acetyl CoA, nicotinamide adenine dinucleotide (the reduced form, NADH), and CO2. This reaction constitutes the bridge between anaerobic and aerobic cerebral energy metabolism. PDHC enzyme activity and immunoreactivity are lost in selectively vulnerable neurons after cerebral ischemia and reperfusion. Evidence from experiments carried out in vitro suggests that reperfusion-dependent loss of activity is caused by oxidative protein modifications. Impaired enzyme activity may explain the reduced cerebral glucose and oxygen consumption that occurs after cerebral ischemia. This hypothesis is supported by the hyperoxidation of mitochondrial electron transport chain components and NAD(H) that occurs during reperfusion, indicating that NADH production, rather than utilization, is rate limiting. Additional support comes from the findings that immediate postischemic administration of acetyl-l-carnitine both reduces brain lactate/pyruvate ratios and improves neurologic outcome after cardiac arrest in animals. As acetyl-l-carnitine is converted to acetyl CoA, the product of the PDHC reaction, it follows that impaired production of NADH is due to reduced activity of either PDHC or one or more steps in glycolysis. Impaired cerebral energy metabolism and PDHC activity are associated also with neurodegenerative disorders including Alzheimer's disease and Wernicke-Korsakoff syndrome, suggesting that this enzyme is an important link in the pathophysiology of both acute brain injury and chronic neurodegeneration. PMID:15562436

  8. Analysis of a pyruvic acid acetal-containing polysaccharide by the reductive-cleavage method.

    PubMed

    Zeller, S G; Gray, G R

    1991-04-24

    The applicability of the reductive-cleavage method to the analysis of polysaccharides bearing pyruvic acid acetals has been demonstrated. Direct reductive cleavage of fully methylated gum xanthan yielded the expected products, including 1,5-anhydro-4,6-O-[(S)-1-methoxycarbonylethylidene]-2,3-di-O-methy l-D- mannitol. The latter product was not observed when reductive cleavage was performed subsequent to reduction of ester groups in the fully methylated polysaccharide and mild hydrolysis to remove pyruvic acid acetal substituents. Instead, the latter experiment yielded 1,5-anhydro-2,3-di-O-methyl-D-mannitol, establishing the presence in the polysaccharide of terminal (nonreducing) D-mannopyranosyl groups bearing 4,6-O-(1-carboxyethylidene) substituents. The products of reductive cleavage were characterized, where appropriate, by comparison of the gas chromatographic retention times and chemical ionization- and electron ionization-mass spectra of their acetates to those of authentic standards. Alternatively, the products of reductive cleavage could be characterized without resort to comparison with authentic standards by analysis of the 1H-n.m.r. spectra of their benzoates, which were obtained in pure form by high-performance liquid chromatography. By either method of product characterization, this two-step procedure of analysis reveals the presence of pyruvic-acetal residues in polysaccharides and establishes both the identity of the sugar residue to which they are attached and their positions of attachment. PMID:1769016

  9. Nuclear pyruvate kinase M2 complex serves as a transcriptional coactivator of arylhydrocarbon receptor

    PubMed Central

    Matsuda, Shun; Adachi, Jun; Ihara, Masaru; Tanuma, Nobuhiro; Shima, Hiroshi; Kakizuka, Akira; Ikura, Masae; Ikura, Tsuyoshi; Matsuda, Tomonari

    2016-01-01

    Pyruvate kinase M2 (PKM2) and pyruvate dehydrogenase complex (PDC) regulate production of acetyl-CoA, which functions as an acetyl donor in diverse enzymatic reactions, including histone acetylation. However, the mechanism by which the acetyl-CoA required for histone acetylation is ensured in a gene context-dependent manner is not clear. Here we show that PKM2, the E2 subunit of PDC and histone acetyltransferase p300 constitute a complex on chromatin with arylhydrocarbon receptor (AhR), a transcription factor associated with xenobiotic metabolism. All of these factors are recruited to the enhancer of AhR-target genes, in an AhR-dependent manner. PKM2 contributes to enhancement of transcription of cytochrome P450 1A1 (CYP1A1), an AhR-target gene, acetylation at lysine 9 of histone H3 at the CYP1A1 enhancer. Site-directed mutagenesis of PKM2 indicates that this enhancement of histone acetylation requires the pyruvate kinase activity of the enzyme. Furthermore, we reveal that PDC activity is present in nuclei. Based on these findings, we propose a local acetyl-CoA production system in which PKM2 and PDC locally supply acetyl-CoA to p300 from abundant PEP for histone acetylation at the gene enhancer, and our data suggest that PKM2 sensitizes AhR-mediated detoxification in actively proliferating cells such as cancer and fetal cells. PMID:26405201

  10. Pyruvate carboxylase deficiency: clinical and biochemical response to anaplerotic diet therapy.

    PubMed

    Mochel, Fanny; DeLonlay, Pascale; Touati, Guy; Brunengraber, Henri; Kinman, Renee P; Rabier, Daniel; Roe, Charles R; Saudubray, Jean-Marie

    2005-04-01

    A six-day-old girl was referred for severe hepatic failure, dehydratation, axial hypotonia, and both lactic acidosis and ketoacidosis. Biotin-unresponsive pyruvate carboxylase deficiency type B was diagnosed. Triheptanoin, an odd-carbon triglyceride, was administrated as a source for acetyl-CoA and anaplerotic propionyl-CoA. Although this patient succumbed to a severe infection, during the six months interval of her anaplerotic and biochemical management, the following important observations were documented: (1) the immediate reversal (less than 48 h) of major hepatic failure with full correction of all biochemical abnormalities, (2) on citrate supplementation, the enhanced export from the liver of triheptanoin's metabolites, namely 5 carbon ketone bodies, increasing the availability of these anaplerotic substrates for peripheral organs, (3) the demonstration of the transport of C5 ketone bodies-representing alternative energetic fuel for the brain-across the blood-brain barrier, associated to increased levels of glutamine and free gamma-aminobutyric acid (f-GABA) in the cerebrospinal fluid. Considering that pyruvate carboxylase is a key enzyme for anaplerosis, besides the new perspectives brought by anaplerotic therapies in those rare pyruvate carboxylase deficiencies, this therapeutic trial also emphasizes the possible extended indications of triheptanoin in various diseases where the citric acid cycle is impaired. PMID:15781190

  11. The Importance of Polarity in the Evolution of the K+ Binding Site of Pyruvate Kinase

    PubMed Central

    Ramírez-Silva, Leticia; Guerrero-Mendiola, Carlos; Cabrera, Nallely

    2014-01-01

    In a previous phylogenetic study of the family of pyruvate kinase, we found one cluster with Glu117 and another with Lys117. Those sequences with Glu117 have Thr113 and are K+-dependent, whereas those with Lys117 have Leu113 and are K+-independent. The carbonyl oxygen of Thr113 is one of the residues that coordinate K+ in the active site. Even though the side chain of Thr113 does not participate in binding K+, the strict co-evolution between position 117 and 113 suggests that T113 may be the result of the evolutionary pressure to maintain the selectivity of pyruvate kinase activity for K+. Thus, we explored if the replacement of Thr113 by Leu alters the characteristics of the K+ binding site. We found that the polarity of the residue 113 is central in the partition of K+ into its site and that the substitution of Thr for Leu changes the ion selectivity for the monovalent cation with minor changes in the binding of the substrates. Therefore, Thr113 is instrumental in the selectivity of pyruvate kinase for K+. PMID:25474090

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

    PubMed Central

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

    2014-01-01

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

  13. Crystallographic study on the interaction of L-lactate oxidase with pyruvate at 1.9 A resolution

    SciTech Connect

    Li Shujie; Umena, Yasufumi; Yorita, Kazuko; Matsuoka, Takeshi; Kita, Akiko; Fukui, Kiyoshi; Morimoto, Yukio . E-mail: morimoto@rri.kyoto-u.ac.jp

    2007-07-13

    L-Lactate oxidase (LOX) from Aerococcus viridans catalyzes the oxidation of L-lactate to pyruvate by the molecular oxygen and belongs to a large family of 2-hydroxy acid-dependent flavoenzymes. To investigate the interaction of LOX with pyruvate in structural details and understand the chemical mechanism of flavin-dependent L-lactate dehydrogenation, the LOX-pyruvate complex was crystallized and the crystal structure of the complex has been solved at a resolution of 1.90 A. One pyruvate molecule bound to the active site and located near N5 position of FMN for subunits, A, B, and D in the asymmetric unit, were identified. The pyruvate molecule is stabilized by the interaction of its carboxylate group with the side-chain atoms of Tyr40, Arg181, His265, and Arg268, and of its keto-oxygen atom with the side-chain atoms of Tyr146, Tyr215, and His265. The {alpha}-carbon of pyruvate is found to be 3.13 A from the N5 atom of FMN at an angle of 105.4{sup o} from the flavin N5-N10 axis.

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

    PubMed Central

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

    1992-01-01

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

  15. An amino acid substitution in the pyruvate dehydrogenase E1{alpha} gene, affecting mitochondrial import of the precursor protein

    SciTech Connect

    Takakubo, F.; Thorburn, D.R.; Dahl, H.H.M.

    1995-10-01

    A mutation in the mitochondrial targeting sequence was characterized in a male patient with X chromosome-linked pyruvate dehydrogenase E1{alpha} deficiency. The mutation was a base substitution of G by C at nucleotide 134 in the mitochondrial targeting sequence of the PDHA1 gene, resulting in an arginine-to-proline substitution at codon 10 (R10P). Pyruvate dehydrogenase activity in cultured skin fibroblasts was 28% of the control value, and immunoblot analysis revealed a decreased level of pyruvate dehydrogenase E1{alpha}immunoreactivity. Chimeric constructs in which the normal and mutant pyruvate dehydrogenase E1{alpha} targeting sequences were attached to the mitochondrial matrix protein ornithine transcarbamylase were synthesized in a cell free translation system, and mitochondrial import of normal and mutant proteins was compared in vitro. The results show that ornithine transcarbamylase targeted by the mutant pyruvate dehydrogenase E1{alpha} sequence was translocated into the mitochondrial matrix at a reduced rate, suggesting that defective import is responsible for the reduced pyruvate dehydrogenase level in mitochondria. The mutation was also present in an affected brother and the mildly affected mother. The clinical presentations of this X chromosome-linked disorder in affected family members are discussed. To our knowledge, this is the first report of an amino acid substitution in a mitochondrial targeting sequence resulting in a human genetic disease. 58 refs., 5 figs., 1 tab.

  16. Computational Study of Catalytic Reaction of Quercetin 2,4-Dioxygenase.

    PubMed

    Saito, Toru; Kawakami, Takashi; Yamanaka, Shusuke; Okumura, Mitsutaka

    2015-06-11

    We present a quantum mechanics/molecular mechanics (QM/MM) and QM-only study on the oxidative ring-cleaving reaction of quercetin catalyzed by quercetin 2,4-dioxygenase (2,4-QD). 2,4-QD has a mononuclear type 2 copper center and incorporates two oxygen atoms at C2 and C4 positions of the substrate. It has not been clear whether dioxygen reacts with a copper ion or a substrate radical as the first step. We have found that dioxygen is more likely to bind to a Cu(2+) ion, involving the dissociation of the substrate from the copper ion. Then a Cu(2+)-alkylperoxo complex can be generated. Comparison of geometry and stability between QM-only and QM/MM results strongly indicates that steric effects of the protein environment contribute to maintain the orientation of the substrate dissociated from the copper center. The present QM/MM results also highlight that a prior rearrangement of the Cu(2+)-alkylperoxo complex and a subsequent hydrogen bond switching assisted by the movement of Glu73 can facilitate formation of an endoperoxide intermediate selectively. PMID:25990020

  17. Catalytic Activity of Human Indoleamine 2,3-Dioxygenase (hIDO1) at Low Oxygen

    PubMed Central

    Kolawole, Ayodele O.; Hixon, Brian P.; Dameron, Laura S.; Chrisman, Ian M.; Smirnov, Valeriy V.

    2015-01-01

    A cytokine-inducible extrahepatic human indoleamine 2,3-dioxygenase (hIDO1) catalyzes the first step of the kynurenine pathway. Immunosuppressive activity of hIDO1 in tumor cells weakens host T-cell immunity, contributing to the progression of cancer. Here we report on enzyme kinetics and catalytic mechanism of hIDO1, studied at varied levels of dioxygen (O2) and L-tryptophan (L-Trp). Using a cytochrome b5-based activating system, we measured the initial rates of O2 decay with a Clark-type oxygen electrode at physiologically-relevant levels of both substrates. Kinetics was also studied in the presence of two substrate analogs: 1-methyl-L-tryptophan and norharmane. Quantitative analysis supports a steady-state rather than a rapid equilibrium kinetic mechanism, where the rates of individual pathways, leading to a ternary complex, are significantly different, and the overall rate of catalysis depends on contributions of both routes. One path, where O2 binds to ferrous hIDO1 first, is faster than the second route, which starts with the binding of L-Trp. However, L-Trp complexation with free ferrous hIDO1 is more rapid than that of O2. As the level of L-Trp increases, the slower route becomes a significant contributor to the overall rate, resulting in observed substrate inhibition. PMID:25712221

  18. Genomewide Analysis of Carotenoid Cleavage Dioxygenases in Unicellular and Filamentous Cyanobacteria

    PubMed Central

    Cui, Hongli; Wang, Yinchu; Qin, Song

    2012-01-01

    Carotenoid cleavage dioxygenases (CCDs) are a group of enzymes that catalyze the oxidative cleavage steps from carotenoids to various carotenoid cleavage products. Some ccd genes have been identified and encoded enzymes functionally characterized in many higher plants, but little in cyanobacteria. We performed a comparative analysis of ccd sequences and explored their distribution, classification, phylogeny, evolution, and structure among 37 cyanobacteria. Totally 61 putative ccd sequences were identified, which are abundant in Acaryochloris marina MBIC 11017, filamentous N2-fixing cyanobacteria, and unicellular cyanobacterial Cyanothece. According to phylogenetic trees of 16S rDNA and CCD, nced and ccd8 genes occur later than the divergence of ccd7, apco, and ccd1. All CCD enzymes share conserved basic structure domains constituted by a single loop formed with seven β-strands and one helix. In this paper, a general framework of sequence-function-evolution connection for the ccd has been revealed, which may provide new insight for functional investigation. PMID:22474409

  19. Cysteine dioxygenase type 1 promotes adipogenesis via interaction with peroxisome proliferator-activated receptor gamma

    SciTech Connect

    Deng, Peng; Chen, Yi; Ji, Ning; Lin, Yunfeng; Yuan, Quan; Ye, Ling; Chen, Qianming

    2015-02-27

    Mammalian cysteine dioxygenase type 1 (CDO1) is an essential enzyme for taurine biosynthesis and the biodegradation of toxic cysteine. As previously suggested, Cdo1 may be a marker of liposarcoma progression and adipogenic differentiation, but the role of Cdo1 in adipogenesis has yet been reported. In this study, we found that the expression of Cdo1 is dramatically elevated during adipogenic differentiation of 3T3-L1 pre-adipocytes and mouse bone marrow-derived mesenchymal stem cells (mBMSCs). Conversely, knockdown of Cdo1 inhibited expression of adipogenic specific genes and lipid droplet formation in 3T3-L1 cells and mBMSCs. Mechanistically, we found Cdo1 interacted with Pparγ in response to adipogenic stimulus. Further, depletion of Cdo1 reduced the recruitment of Pparγ to the promoters of C/EBPα and Fabp4. Collectively, our finding indicates that Cdo1 may be a co-activator of Pparγ in adipogenesis, and may contribute to the development of disease associated with excessive adipose tissue. - Highlights: • Cdo1expression is highly up-regulated during adipogenic differentiation of 3T3-L1 and mBMSCs. • Depletion of Cdo1 inhibited expression of adipogenic specific genes and lipid droplet formation. • Cdo1interacts with Pparγ during adipogenesis. • Knockdown of Cdo1 inhibited Pparγ binding to the promoters of C/EBPα and Fabp4.

  20. Chemical rescue of the distal histidine mutants of tryptophan 2,3-dioxygenase.

    PubMed

    Geng, Jiafeng; Dornevil, Kednerlin; Liu, Aimin

    2012-07-25

    Tryptophan 2,3-dioxygenase (TDO) is a heme-dependent enzyme that catalyzes the oxidative degradation of L-tryptophan (L-Trp) to N-formylkynurenine (NFK). A highly conserved histidine residue in the distal heme pocket has attracted great attention in the mechanistic studies of TDO. However, a consensus has not been reached regarding whether and how this distal histidine plays a catalytic role after substrate binding. In this study, three mutant proteins, H72S, H72N, and Q73F were generated to investigate the function of the distal histidine residue in Cupriavidus metallidurans TDO (cmTDO). Spectroscopic characterizations, enzymatic kinetic analysis, and chemical rescue assays were employed to study the biochemical properties of the wild-type enzyme and the mutant proteins. Rapid kinetic methods were utilized to explore the molecular basis for the observed stimulation of catalytic activity by 2-methylimidazole in the His72 variants. The results indicate that the distal histidine plays multiple roles in cmTDO. First, His72 contributes to but is not essential for substrate binding. In addition, it shields the heme center from nonproductive binding of exogenous small ligand molecules (i.e., imidazole and its analogs) via steric hindrance. Most importantly, His72 participates in the subsequent chemical catalytic steps after substrate binding possibly by providing H-bonding interactions to the heme-bound oxygen. PMID:22742206

  1. Lignans from Carthamus tinctorius suppress tryptophan breakdown via indoleamine 2,3-dioxygenase.

    PubMed

    Kuehnl, Susanne; Schroecksnadel, Sebastian; Temml, Veronika; Gostner, Johanna M; Schennach, Harald; Schuster, Daniela; Schwaiger, Stefan; Rollinger, Judith M; Fuchs, Dietmar; Stuppner, Hermann

    2013-10-15

    Seed extracts of Carthamus tinctorius L. (Asteraceae), safflower, have been traditionally used to treat coronary disease, thrombotic disorders, and menstrual problems but also against cancer and depression. A possible effect of C. tinctorius compounds on tryptophan-degrading activity of enzyme indoleamine 2,3-dioxygenase (IDO) could explain many of its activities. To test for an effect of C. tinctorius extracts and isolated compounds on cytokine-induced IDO activity in immunocompetent cells in vitro methanol and ethylacetate seed extracts were prepared from cold pressed seed cakes of C. tinctorius and three lignan derivatives, trachelogenin, arctigenin and matairesinol were isolated. The influence on tryptophan breakdown was investigated in peripheral blood mononuclear cells (PBMCs). Effects were compared to neopterin production in the same cellular assay. Both seed extracts suppressed tryptophan breakdown in stimulated PBMC. The three structurally closely related isolates exerted differing suppressive activity on PBMC: arctigenin (IC50 26.5μM) and trachelogenin (IC50 of 57.4μM) showed higher activity than matairesinol (IC50 >200μM) to inhibit tryptophan breakdown. Effects on neopterin production were similar albeit generally less strong. Data show an immunosuppressive property of compounds which slows down IDO activity. The in vitro results support the view that some of the anti-inflammatory, anticancer and antidepressant properties of C. tinctorius lignans might relate to their suppressive influence on tryptophan breakdown. PMID:23867649

  2. One protein, two enzymes revisited: A structural entropy switch interconverts the two isoforms of acireductone dioxygenase

    PubMed Central

    Ju, Tingting; Goldsmith, Rachel Beaulieu; Chai, Sergio C.; Maroney, Michael J.; Pochapsky, Susan Sondej; Pochapsky, Thomas C.

    2006-01-01

    Summary Acireductone dioxygenase (ARD) catalyzes different reactions between O2 and 1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene (acireductone) depending upon the metal bound in the active site. Ni+2–ARD cleaves acireductone to formate, CO and methylthiopropionate. If Fe+2 is bound (ARD′), the same substrates yield methylthioketobutyrate and formate. The two forms differ in structure, and are chromatographically separable. Paramagnetism of Fe+2 renders the active site of ARD′ inaccessible to standard NMR methods. The structure of ARD′ has been determined using Fe+2 binding parameters determined by X-ray absorption spectroscopy and NMR restraints from H98S ARD, a metal-free diamagnetic protein that is isostructural with ARD′. ARD′ retains the β-sandwich fold of ARD, but a structural entropy switch increases order at one end of a two-helix system that bisects the β-sandwich and decreases order at the other upon interconversion of ARD and ARD′, causing loss of the C-terminal helix in ARD′ and rearrangements of residues involved in substrate orientation in the active site. PMID:16989860

  3. Thymic Indoleamine 2,3-Dioxygenase-Positive Eosinophils in Young Children

    PubMed Central

    Tulic, Meri K.; Sly, Peter D.; Andrews, David; Crook, Maxine; Davoine, Francis; Odemuyiwa, Solomon O.; Charles, Adrian; Hodder, Megan L.; Prescott, Susan L.; Holt, Patrick G.; Moqbel, Redwan

    2009-01-01

    Eosinophils expressing indoleamine 2, 3-dioxygenase (IDO) may contribute to T-helper cell (Th)2 predominance. To characterize human thymus IDO+ eosinophil ontogeny relative to Th2 regulatory gene expression, we processed surgically obtained thymi from 22 children (age: 7 days to 12 years) for immunohistochemistry and molecular analysis, and measured cytokine and kynurenine levels in tissue homogenates. Luna+ eosinophils (∼2% of total thymic cells) decreased in number with age (P = 0.02) and were IDO+. Thymic IDO immunoreactivity (P = 0.01) and kynurenine concentration (P = 0.01) decreased with age as well. In addition, constitutively-expressed interleukin (IL)-5 and IL-13 in thymus supernatants was highest in youngest children. Eosinophil numbers correlated positively with expression of the Th2 cytokines IL-5, IL-13 (r = 0.44, P = 0.002), and IL-4 (r = 0.46, P = 0.005), transcription factor signal transducer and activator of transcription-6 (r = 0.68, P = 0.001), and the chemokine receptor, CCR3 (r = 0.17, P = 0.04), but negatively with IL-17 mRNA (r = −0.57, P = 0.02) and toll-like receptor 4 expression (r = −0.74, P = 0.002). Taken together, these results suggest that functional thymic IDO+ eosinophils during human infant life may have an immunomodulatory role in Th2 immune responses. PMID:19815714

  4. Identification and expression pattern of a new carotenoid cleavage dioxygenase gene member from Bixa orellana

    PubMed Central

    Rodríguez-Ávila, N. L.; Narváez-Zapata, J. A.; Ramírez-Benítez, J. E.; Aguilar-Espinosa, M. L.; Rivera-Madrid, R.

    2011-01-01

    Carotenoid cleavage dioxygenases (CCDs) are a class of enzymes involved in the biosynthesis of a broad diversity of secondary metabolites known as apocarotenoids. In plants, CCDs are part of a genetic family with members which cleave specific double bonds of carotenoid molecules. CCDs are involved in the production of diverse and important metabolites such as vitamin A and abscisic acid (ABA). Bixa orellana L. is the main source of the natural pigment annatto or bixin, an apocarotenoid accumulated in large quantities in its seeds. Bixin biosynthesis has been studied and the involvement of a CCD has been confirmed in vitro. However, the CCD genes involved in the biosynthesis of the wide variety of apocarotenoids found in this plant have not been well documented. In this study, a new CCD1 gene member (BoCCD1) was identified and its expression was charaterized in different plant tissues of B. orellana plantlets and adult plants. The BoCCD1 sequence showed high homology with plant CCD1s involved mainly in the cleavage of carotenoids in several sites to generate multiple apocarotenoid products. Here, the expression profiles of the BoCCD1 gene were analysed and discussed in relation to total carotenoids and other important apocarotenoids such as bixin. PMID:21813796

  5. D2HGDH regulates alpha-ketoglutarate levels and dioxygenase function by modulating IDH2.

    PubMed

    Lin, An-Ping; Abbas, Saman; Kim, Sang-Woo; Ortega, Manoela; Bouamar, Hakim; Escobedo, Yissela; Varadarajan, Prakash; Qin, Yuejuan; Sudderth, Jessica; Schulz, Eduard; Deutsch, Alexander; Mohan, Sumitra; Ulz, Peter; Neumeister, Peter; Rakheja, Dinesh; Gao, Xiaoli; Hinck, Andrew; Weintraub, Susan T; DeBerardinis, Ralph J; Sill, Heinz; Dahia, Patricia L M; Aguiar, Ricardo C T

    2015-01-01

    Isocitrate dehydrogenases (IDH) convert isocitrate to alpha-ketoglutarate (α-KG). In cancer, mutant IDH1/2 reduces α-KG to D2-hydroxyglutarate (D2-HG) disrupting α-KG-dependent dioxygenases. However, the physiological relevance of controlling the interconversion of D2-HG into α-KG, mediated by D2-hydroxyglutarate dehydrogenase (D2HGDH), remains obscure. Here we show that wild-type D2HGDH elevates α-KG levels, influencing histone and DNA methylation, and HIF1α hydroxylation. Conversely, the D2HGDH mutants that we find in diffuse large B-cell lymphoma are enzymatically inert. D2-HG is a low-abundance metabolite, but we show that it can meaningfully elevate α-KG levels by positively modulating mitochondrial IDH activity and inducing IDH2 expression. Accordingly, genetic depletion of IDH2 abrogates D2HGDH effects, whereas ectopic IDH2 rescues D2HGDH-deficient cells. Our data link D2HGDH to cancer and describe an additional role for the enzyme: the regulation of IDH2 activity and α-KG-mediated epigenetic remodelling. These data further expose the intricacies of mitochondrial metabolism and inform on the pathogenesis of D2HGDH-deficient diseases. PMID:26178471

  6. The Role of Indoleamine 2, 3-Dioxygenase in Immune Suppression and Autoimmunity

    PubMed Central

    Mbongue, Jacques C.; Nicholas, Dequina A.; Torrez, Timothy W.; Kim, Nan-Sun; Firek, Anthony F.; Langridge, William H.R.

    2015-01-01

    Indoleamine 2, 3-dioxygenase (IDO) is the first and rate limiting catabolic enzyme in the degradation pathway of the essential amino acid tryptophan. By cleaving the aromatic indole ring of tryptophan, IDO initiates the production of a variety of tryptophan degradation products called “kynurenines” that are known to exert important immuno-regulatory functions. Because tryptophan must be supplied in the diet, regulation of tryptophan catabolism may exert profound effects by activating or inhibiting metabolism and immune responses. Important for survival, the regulation of IDO biosynthesis and its activity in cells of the immune system can critically alter their responses to immunological insults, such as infection, autoimmunity and cancer. In this review, we assess how IDO-mediated catabolism of tryptophan can modulate the immune system to arrest inflammation, suppress immunity to cancer and inhibit allergy, autoimmunity and the rejection of transplanted tissues. Finally, we examine how vaccines may enhance immune suppression of autoimmunity through the upregulation of IDO biosynthesis in human dendritic cells. PMID:26378585

  7. Broad 4-hydroxyphenylpyruvate dioxygenase inhibitor herbicide tolerance in soybean with an optimized enzyme and expression cassette.

    PubMed

    Siehl, Daniel L; Tao, Yumin; Albert, Henrik; Dong, Yuxia; Heckert, Matthew; Madrigal, Alfredo; Lincoln-Cabatu, Brishette; Lu, Jian; Fenwick, Tamara; Bermudez, Ericka; Sandoval, Marian; Horn, Caroline; Green, Jerry M; Hale, Theresa; Pagano, Peggy; Clark, Jenna; Udranszky, Ingrid A; Rizzo, Nancy; Bourett, Timothy; Howard, Richard J; Johnson, David H; Vogt, Mark; Akinsola, Goke; Castle, Linda A

    2014-11-01

    With an optimized expression cassette consisting of the soybean (Glycine max) native promoter modified for enhanced expression driving a chimeric gene coding for the soybean native amino-terminal 86 amino acids fused to an insensitive shuffled variant of maize (Zea mays) 4-hydroxyphenylpyruvate dioxygenase (HPPD), we achieved field tolerance in transgenic soybean plants to the HPPD-inhibiting herbicides mesotrione, isoxaflutole, and tembotrione. Directed evolution of maize HPPD was accomplished by progressively incorporating amino acids from naturally occurring diversity and novel substitutions identified by saturation mutagenesis, combined at random through shuffling. Localization of heterologously expressed HPPD mimicked that of the native enzyme, which was shown to be dually targeted to chloroplasts and the cytosol. Analysis of the native soybean HPPD gene revealed two transcription start sites, leading to transcripts encoding two HPPD polypeptides. The N-terminal region of the longer encoded peptide directs proteins to the chloroplast, while the short form remains in the cytosol. In contrast, maize HPPD was found almost exclusively in chloroplasts. Evolved HPPD enzymes showed insensitivity to five inhibitor herbicides. In 2013 field trials, transgenic soybean events made with optimized promoter and HPPD variant expression cassettes were tested with three herbicides and showed tolerance to four times the labeled rates of mesotrione and isoxaflutole and two times the labeled rates of tembotrione. PMID:25192697

  8. Substrate Recognition and Catalysis by the Cofactor-Independent Dioxygenase DpgC+

    SciTech Connect

    Fielding,E.; Widboom, P.; Bruner, S.

    2007-01-01

    The enzyme DpgC belongs to a small class of oxygenases not dependent on accessory cofactors for activity. DpgC is in the biosynthetic pathway for the nonproteinogenic amino acid 3, 5-dihydroxyphenylglycine in actinomycetes bacteria responsible for the production of the vancomycin/teicoplanin family of antibiotic natural products. The X-ray structure of DpgC confirmed the absence of cofactors and defined a novel hydrophobic dioxygen binding pocket adjacent to a bound substrate analogue. In this paper, the role specific amino acids play in substrate recognition and catalysis is examined through biochemical and structural characterization of site-specific enzyme mutations and alternate substrates. The results establish the importance of three amino acids, Arg254, Glu299, and Glu189, in the chemistry of DpgC. Arg254 and Glu189 join to form a specific contact with one of the phenolic hydroxyls of the substrate, and this interaction plays a key role in both substrate recognition and catalysis. The X-ray crystal structure of Arg254Lys was determined to address the role this residue plays in the chemistry. In addition, characterization of alternate substrate analogues demonstrates the presence and position of phenol groups are necessary for both enzyme recognition and downstream oxidation chemistry. Overall, this work defines the mechanism of substrate recognition and specificity by the cofactor-independent dioxygenase DpgC.

  9. The key role of water in the dioxygenase function of Escherichia coli flavohemoglobin.

    PubMed

    Ferreiro, Dardo N; Boechi, Leonardo; Estrin, Darío A; Martí, Marcelo A

    2013-02-01

    Flavohemoglobins (FHbs) are members of the globin superfamily, widely distributed among prokaryotes and eukaryotes that have been shown to carry out nitric oxide dioxygenase (NOD) activity. In prokaryotes, such as Escherichia coli, NOD activity is a defence mechanism against the NO release by the macrophages of the hosts' immune system during infection. Because of that, FHbs have been studied thoroughly and several drugs have been developed in an effort to fight infectious processes. Nevertheless, the protein's structural determinants involved in the NOD activity are still poorly understood. In this context, the aim of the present work is to unravel the molecular basis of FHbs structural dynamics-to-function relationship using state of the art computer simulation tools. In an effort to fulfill this goal, we studied three key processes that determine NOD activity, namely i) ligand migration into the active site ii) stabilization of the coordinated oxygen and iii) intra-protein electron transfer (ET). Our results allowed us to determine key factors related to all three processes like the presence of a long hydrophobic tunnel for ligand migration, the presence of a water mediated hydrogen bond to stabilize the coordinated oxygen and therefore achieve a high affinity, and the best possible ET paths between the FAD and the heme, where water molecules play an important role. Taken together the presented results close an important gap in our understanding of the wide and diverse globin structural-functional relationships. PMID:23220591

  10. Cysteine Dioxygenase 1 Is a Tumor Suppressor Gene Silenced by Promoter Methylation in Multiple Human Cancers

    PubMed Central

    Brait, Mariana; Ling, Shizhang; Nagpal, Jatin K.; Chang, Xiaofei; Park, Hannah Lui; Lee, Juna; Okamura, Jun; Yamashita, Keishi; Sidransky, David; Kim, Myoung Sook

    2012-01-01

    The human cysteine dioxygenase 1 (CDO1) gene is a non-heme structured, iron-containing metalloenzyme involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In our search for novel methylated gene promoters, we have analyzed differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2′-deoxycytidine. Among the genes identified, the CDO1 promoter was found to be differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues. In addition, a statistically significant difference in the frequency of CDO1 promoter methylation was observed between primary normal and tumor tissues derived from breast, esophagus, lung, bladder and stomach. Downregulation of CDO1 mRNA and protein levels were observed in cancer cell lines and tumors derived from these tissue types. Expression of CDO1 was tightly controlled by promoter methylation, suggesting that promoter methylation and silencing of CDO1 may be a common event in human carcinogenesis. Moreover, forced expression of full-length CDO1 in human cancer cells markedly decreased the tumor cell growth in an in vitro cell culture and/or an in vivo mouse model, whereas knockdown of CDO1 increased cell growth in culture. Our data implicate CDO1 as a novel tumor suppressor gene and a potentially valuable molecular marker for human cancer. PMID:23028699

  11. Toluene and ethylbenzene oxidation by purified naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

    PubMed Central

    Lee, K; Gibson, D T

    1996-01-01

    Purified naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 oxidized toluene to benzyl alcohol and benzaldehyde by reactions involving benzylic monooxygenation and dioxygen-dependent alcohol oxidation, respectively. Xylene and nitrotoluene isomers were also oxidized to substituted benzyl alcohol and benzaldehyde derivatives. NDO oxidized ethylbenzene sequentially through (S)-1-phenethyl alcohol (77% enantiomeric excess) and acetophenone to 2-hydroxyacetophenone. In addition, NDO also oxidized ethylbenzene through styrene to (R)-1-phenyl-1,2-ethanediol (74% enantiomeric excess) by reactions involving desaturation and dihydroxylation, respectively. Isotope experiments with 18O2, H2 18O, and D2O suggest that 1-phenethyl alcohol is oxidized to acetophenone by a minor reaction involving desaturation followed by tautomerization. The major reaction in the conversion of 1-phenethyl alcohol and benzyl alcohol to acetophenone and benzaldehyde, respectively, probably involves monohydroxylation to form a gem-diol intermediate which stereospecifically loses the incoming hydroxyl group to leave the carbonyl product. These results are compared with similar reactions catalyzed by cytochrome P-450. PMID:8795196

  12. Indoleamine 2,3 dioxygenase and regulation of T cell immunity

    SciTech Connect

    Mellor, Andrew . E-mail: amellor@mcg.edu

    2005-12-09

    Regulation of adaptive immune responses is critically important to allow the adaptive immune system to eradicate infections while causing minimal collateral damage to infected tissues, as well as preventing autoimmune disease mediated by self-reactive lymphocytes. Tumors and pathogens that cause persistent infections can subvert immunoregulatory processes to protect themselves from destruction by T cells, to the detriment of patients. A growing body of evidence supports the hypothesis that specialized subsets of dendritic cells expressing indoleamine 2,3 dioxygenase (IDO), which catalyzes oxidative catabolism of tryptophan, play critical roles in regulation of T cell-mediated immune responses. IDO-dependent T cell suppression by dendritic cells suggests that biochemical changes due to tryptophan catabolism have profound effects on T cell proliferation, differentiation, effector functions, and viability. This has critical implications for immunotherapeutic manipulations designed for patients with cancer and chronic infectious diseases. In this review, I focus on dendritic cells that can express IDO, and which acquire potent T cell regulatory functions as a consequence.

  13. Indoleamine 2,3 Dioxygenase as a Potential Therapeutic Target in Huntington’s Disease

    PubMed Central

    Mazarei, Gelareh; Leavitt, Blair R.

    2015-01-01

    Abstract Within the past decade, there has been increasing interest in the role of tryptophan (Trp) metabolites and the kynurenine pathway (KP) in diseases of the brain such as Huntington’s disease (HD). Evidence is accumulating to suggest that this pathway is imbalanced in neurologic disease states. The KP diverges into two branches that can lead to production of either neuroprotective or neurotoxic metabolites. In one branch, kynurenine (Kyn) produced as a result of tryptophan (Trp) catabolism is further metabolized to neurotoxic metabolites such as 3-hydroxykunurenine (3-HK) and quinolinic acid (QA). In the other branch, Kyn is converted to the neuroprotective metabolite kynurenic acid (KA). The enzyme Indoleamine 2,3 dioxygenase (IDO1) catalyzes the conversion of Trp into Kyn, the first and rate-limiting enzymatic step of the KP. This reaction takes place throughout the body in multiple cell types as a required step in the degradation of the essential amino acid Trp. Studies of IDO1 in brain have focused primarily on a potential role in depression, immune tolerance associated with brain tumours, and multiple sclerosis; however the role of this enzyme in neurodegenerative disease has garnered significant attention in recent years. This review will provide a summary of the current understanding of the role of IDO1 in Huntington’s disease and will assess this enzyme as a potential therapeutic target for HD. PMID:26397892

  14. Blockade of indoleamine 2,3-dioxygenase protects mice against lipopolysaccharide-induced endotoxin shock.

    PubMed

    Jung, In Duk; Lee, Min-Goo; Chang, Jeong Hyun; Lee, Jun Sik; Jeong, Young-Il; Lee, Chang-Min; Park, Won Sun; Han, Jin; Seo, Su-Kil; Lee, Sang Yong; Park, Yeong-Min

    2009-03-01

    Suppression of an excessive systemic inflammatory response is a promising and potent strategy for treating endotoxic sepsis. Indoleamine 2,3-dioxygenase (IDO), which is the rate-limiting enzyme for tryptophan catabolism, may play a critical role in various inflammatory disorders. In this study, we report a critical role for IDO in the dysregulated immune response associated with endotoxin shock. We found that IDO knockout (IDO(-/-)) mice and 1-methyl-D-tryptophan-treated, endotoxin-shocked mice had decreased levels of the cytokines, TNF-alpha, IL-6, and IL-12, and enhanced levels of IL-10. Blockade of IDO is thought to promote host survival in LPS-induced endotoxin shock, yet little is known about the molecular mechanisms that regulate IDO expression during endotoxin shock. In vitro and in vivo, IDO expression was increased by exogenous IL-12, but decreased by exogenous IL-10 in dendritic cells and splenic dendritic cells. Interestingly, whereas LPS-induced IL-12 levels in serum were higher than those of IL-10, the balance between serum IL-12 and IL-10 following challenge became reversed in IDO(-/-)- or 1-methyl-D-tryptophan-treated mice. Our findings demonstrate that the detrimental immune response to endotoxin shock may occur via IDO modulation. Restoring the IL-12 and IL-10 balance by blocking IDO represents a potential strategy for sepsis treatment. PMID:19234212

  15. Effects of indoleamine 2,3-dioxygenases in carbon tetrachloride-induced hepatitis model of rats.

    PubMed

    Li, Dan; Cai, Haidong; Hou, Min; Fu, Da; Ma, Yushui; Luo, Qiong; Yuan, Xueyu; Lv, Mingli; Zhang, Xiaoping; Cong, Xianling; Lv, Zhongwei

    2012-06-01

    Indoleamine 2,3-dioxygenase (IDO) converts tryptophan to l-kynurenine, and it is noted as a relevant molecule in promoting tolerance and suppressing adaptive immunity. In this study, to investigate the effects of IDO in carbon tetrachloride (CCl(4) )-induced hepatitis model, the levels of IDO enzymic activities in the mock group, the control group and the 1-methyl-D-tryptophan (1-MT)-treated group were confirmed by determination of l-kynurenine concentrations. Serum alanine aminotransferase levels in 1-MT-treated rats after CCl(4) injection significantly increased compared with those in mock and control groups. In CCl(4)-induced hepatitis models, tumour necrosis factor-α (TNF-α) is critical in the development of liver injury. The mRNA expression and secretion levels of TNF-α in the liver from 1-MT-treated rats were more enhanced compared with those in the mock and the control groups. Moreover, the levels of cytokine and chemokine from mock, control group and 1-MT-treated rats after treated with CCl(4) were analyzed by ELISA, and the level of interleukin-6 was found to increase in 1-MT-treated rats. It was concluded that the deficiency of IDO exacerbated liver injury in CCl(4)-induced hepatitis and its effect may be connected with TNF-α and interleukin-6. PMID:22249930

  16. Sphingobium Chlorophenolicum Dichlorohydroquinone Dioxygenase (PcpA) Is Alkaline Resistant and Thermally Stable

    PubMed Central

    Sun, Wanpeng; Sammynaiken, Ramaswami; Chen, Lifeng; Maley, Jason; Schatte, Gabriele; Zhou, Yijiang; Yang, Jian

    2011-01-01

    Dichlorohydroquinone dioxygenase (PcpA) is the ring-cleavage enzyme in the PCP biodegradation pathway in Sphingobium chlorophenolicum strain ATCC 39723. PcpA dehalogenates and oxidizes 2,6-dichlorohydroquinone to form 2-chloromaleylacetate, which is subsequently converted to succinyl coenzyme A and acetyl coenzyme A via 3-oxoadipate. Previous studies have shown that PcpA is highly substrate-specific and only uses 2,6-dichlorohydroquinone as its substrate. In the current study, we overexpressed and purified recombinant PcpA and showed that PcpA was highly alkaline resistant and thermally stable. PcpA exhibited two activity peaks at pH 7.0 and 10.0, respectively. The apparent kcat and Km were measured as 0.19 ± 0.01 s-1 and 0.24 ± 0.08 mM, respectively at pH 7.0, and 0.17 ± 0.01 s-1 and 0.77 ± 0.29 mM, respectively at pH 10.0. Electron paramagnetic resonance studies showed rapid oxidation of Fe(II) to Fe(III) in PcpA and the formation of a stable radical intermediate during the enzyme catalysis. The stable radical was predicted to be an epoxide type dichloro radical with the unpaired electron density localized on C3. PMID:22043174

  17. D2HGDH regulates alpha-ketoglutarate levels and dioxygenase function by modulating IDH2

    PubMed Central

    Lin, An-Ping; Abbas, Saman; Kim, Sang-Woo; Ortega, Manoela; Bouamar, Hakim; Escobedo, Yissela; Varadarajan, Prakash; Qin, Yuejuan; Sudderth, Jessica; Schulz, Eduard; Deutsch, Alexander; Mohan, Sumitra; Ulz, Peter; Neumeister, Peter; Rakheja, Dinesh; Gao, Xiaoli; Hinck, Andrew; Weintraub, Susan T.; DeBerardinis, Ralph J.; Sill, Heinz; Dahia, Patricia L. M.; Aguiar, Ricardo C. T.

    2015-01-01

    Isocitrate dehydrogenases (IDH) convert isocitrate to alpha-ketoglutarate (α-KG). In cancer, mutant IDH1/2 reduces α-KG to D2-hydroxyglutarate (D2-HG) disrupting α-KG-dependent dioxygenases. However, the physiological relevance of controlling the interconversion of D2-HG into α-KG, mediated by D2-hydroxyglutarate dehydrogenase (D2HGDH), remains obscure. Here we show that wild-type D2HGDH elevates α-KG levels, influencing histone and DNA methylation, and HIF1α hydroxylation. Conversely, the D2HGDH mutants that we find in diffuse large B-cell lymphoma are enzymatically inert. D2-HG is a low-abundance metabolite, but we show that it can meaningfully elevate α-KG levels by positively modulating mitochondrial IDH activity and inducing IDH2 expression. Accordingly, genetic depletion of IDH2 abrogates D2HGDH effects, whereas ectopic IDH2 rescues D2HGDH-deficient cells. Our data link D2HGDH to cancer and describe an additional role for the enzyme: the regulation of IDH2 activity and α-KG-mediated epigenetic remodelling. These data further expose the intricacies of mitochondrial metabolism and inform on the pathogenesis of D2HGDH-deficient diseases. PMID:26178471

  18. Degradation of trichloroethylene by toluene dioxygenase in whole-cell studies with Pseudomonas putida F1.

    PubMed Central

    Wackett, L P; Gibson, D T

    1988-01-01

    Toluene-induced cells of Pseudomonas putida F1 removed trichloroethylene from growth media at a significantly greater initial rate than the methanotroph Methylosinus trichosporium OB3b. With toluene-induced P. putida F1, the initial degradation rate varied linearly with trichloroethylene concentration over the range of 8 to 80 microM (1.05 to 10.5 ppm). At 80 microM (10.5 ppm) trichloroethylene and 30 degrees C, the initial rate was 1.8 nmol/min per mg of total cell protein, but the rate decreased rapidly with time. A series of mutant strains derived from P. putida F1 that are defective in the todC gene, which encodes the oxygenase component of toluene dioxygenase, failed to degrade trichloroethylene and to oxidize indole to indigo. A spontaneous revertant selected from a todC culture regained simultaneously the abilities to oxidize toluene, to form indigo, and to degrade trichloroethylene. The three isomeric dichloroethylenes were degraded by P. putida F1, but tetrachloroethylene, vinyl chloride, and ethylene were not removed from incubation mixtures. PMID:3415234

  19. Antiviral and immunoregulatory effects of indoleamine-2,3-dioxygenase in hepatitis C virus infection

    PubMed Central

    Lepiller, Quentin; Soulier, Eric; Li, Qisheng; Lambotin, Mélanie; Barths, Jochen; Fuchs, Dietmar; Stoll-Keller, Françoise; Liang, T. Jake; Barth, Heidi

    2015-01-01

    In patients with hepatitis C virus (HCV) infection, enhanced activity of indoleamine-2,3-dioxygenase 1 (IDO) has been reported. IDO - a tryptophan-catabolizing enzyme – has been considered as both an innate defence mechanism and an important regulator of the immune response. The molecular mechanism of IDO induction in HCV infection and its role in the antiviral immune response remain unknown. Using primary human hepatocytes, we show that HCV infection stimulates IDO expression. IDO gene induction was transient and coincided with the expression of type I and type III interferons (IFNs) and IFN-stimulated genes (ISGs) in HCV-infected hepatocytes. Overexpression of hepatic IDO prior to HCV infection markedly impaired HCV replication in hepatocytes, suggesting that IDO limits the spread of HCV within the liver. siRNA-mediated IDO knockdown revealed that IDO functions as an IFN-mediated anti-HCV effector. Hepatic IDO was most potently induced by IFN-γ and ongoing HCV replication could significantly upregulate IDO expression. IRF1 and STAT1 regulated hepatic IDO expression. Hepatic IDO expression also had a significant inhibitory effect on CD4+ T cell proliferation. Our data suggest that hepatic IDO plays a dual role during HCV infection by retarding viral replication and also regulating host immune responses. PMID:25792183

  20. Negative Impact of Hypoxia on Tryptophan 2,3-Dioxygenase Function

    PubMed Central

    Elbers, Frank; Woite, Claudia; Antoni, Valentina; Stein, Sara; Funakoshi, Hiroshi; Nakamura, Toshikazu; Schares, Gereon; Däubener, Walter

    2016-01-01

    Tryptophan is an essential amino acid for hosts and pathogens. The liver enzyme tryptophan 2,3-dioxygenase (TDO) provokes, by its ability to degrade tryptophan to N-formylkynurenine, the precursor of the immune-relevant kynurenines, direct and indirect antimicrobial and immunoregulatory states. Up to now these TDO-mediated broad-spectrum effector functions have never been observed under hypoxia in vitro, although physiologic oxygen concentrations in liver tissue are low, especially in case of infection. Here we analysed recombinant expressed human TDO and ex vivo murine TDO functions under different oxygen conditions and show that TDO-induced restrictions of clinically relevant pathogens (bacteria, parasites) and of T cell proliferation are abrogated under hypoxic conditions. We pinpointed the loss of TDO efficiency to the reduction of TDO activity, since cell survival and TDO protein levels were unaffected. In conclusion, the potent antimicrobial as well as immunoregulatory effects of TDO were substantially impaired under hypoxic conditions that pathophysiologically occur in vivo. This might be detrimental for the appropriate host immune response towards relevant pathogens. PMID:27563172

  1. Tryptophan-2,3-dioxygenase (TDO) inhibition ameliorates neurodegeneration by modulation of kynurenine pathway metabolites.

    PubMed

    Breda, Carlo; Sathyasaikumar, Korrapati V; Sograte Idrissi, Shama; Notarangelo, Francesca M; Estranero, Jasper G; Moore, Gareth G L; Green, Edward W; Kyriacou, Charalambos P; Schwarcz, Robert; Giorgini, Flaviano

    2016-05-10

    Metabolites of the kynurenine pathway (KP) of tryptophan (TRP) degradation have been closely linked to the pathogenesis of several neurodegenerative disorders. Recent work has highlighted the therapeutic potential of inhibiting two critical regulatory enzymes in this pathway-kynurenine-3-monooxygenase (KMO) and tryptophan-2,3-dioxygenase (TDO). Much evidence indicates that the efficacy of KMO inhibition arises from normalizing an imbalance between neurotoxic [3-hydroxykynurenine (3-HK); quinolinic acid (QUIN)] and neuroprotective [kynurenic acid (KYNA)] KP metabolites. However, it is not clear if TDO inhibition is protective via a similar mechanism or if this is instead due to increased levels of TRP-the substrate of TDO. Here, we find that increased levels of KYNA relative to 3-HK are likely central to the protection conferred by TDO inhibition in a fruit fly model of Huntington's disease and that TRP treatment strongly reduces neurodegeneration by shifting KP flux toward KYNA synthesis. In fly models of Alzheimer's and Parkinson's disease, we provide genetic evidence that inhibition of TDO or KMO improves locomotor performance and ameliorates shortened life span, as well as reducing neurodegeneration in Alzheimer's model flies. Critically, we find that treatment with a chemical TDO inhibitor is robustly protective in these models. Consequently, our work strongly supports targeting of the KP as a potential treatment strategy for several major neurodegenerative disorders and suggests that alterations in the levels of neuroactive KP metabolites could underlie several therapeutic benefits. PMID:27114543

  2. Insights into DNA hydroxymethylation in the honeybee from in-depth analyses of TET dioxygenase

    PubMed Central

    Wojciechowski, Marek; Rafalski, Dominik; Kucharski, Robert; Misztal, Katarzyna; Maleszka, Joanna; Bochtler, Matthias; Maleszka, Ryszard

    2014-01-01

    In mammals, a family of TET enzymes producing oxidized forms of 5-methylcytosine (5mC) plays an important role in modulating DNA demethylation dynamics. In contrast, nothing is known about the function of a single TET orthologue present in invertebrates. Here, we show that the honeybee TET (AmTET) catalytic domain has dioxygenase activity and converts 5mC to 5-hydroxymethylcytosine (5hmC) in a HEK293T cell assay. In vivo, the levels of 5hmC are condition-dependent and relatively low, but in testes and ovaries 5hmC is present at approximately 7–10% of the total level of 5mC, which is comparable to that reported for certain mammalian cells types. AmTET is alternatively spliced and highly expressed throughout development and in adult tissues with the highest expression found in adult brains. Our findings reveal an additional level of flexible genomic modifications in the honeybee that may be important for the selection of multiple pathways controlling contrasting phenotypic outcomes in this species. In a broader context, our study extends the current, mammalian-centred attention to TET-driven DNA hydroxymethylation to an easily manageable organism with attractive and unique biology. PMID:25100549

  3. Association of a functional Indoleamine 2,3-dioxygenase 2 genotype with specific immune responses

    PubMed Central

    Køllgaard, Tania; Klausen, Tobias Wirenfeldt; Idorn, Manja; Holmgaard, Rikke Bæk; Straten, Per thor; Andersen, Mads Hald

    2012-01-01

    Two frequent single-nucleotide-polymorphisms (SNPs) are present in the indoleamine 2,3-dioxygenase 2 (IDO2) gene that influence its enzymatic activity. Thus, one SNP (R248W) is associated with a reduction in IDO2 catalytic activity, whereas the other SNP (Y359stop) generates a premature stop codon abolishing activity completely. In the present study, we describe the presence of a specific cellular immune response in the periphery which correlated with the functional status of the IDO2 protein. Hence, the induction of IDO2-specific T cells in peripheral blood requires the presence of a functional IDO2 protein and, consequently, is restricted to individuals that are not homozygous for the stop codon. Furthermore, we detected stronger T-cell responses in donors with the homozygous Y wild type at position 359 when compared with the heterozygous genotype. Interestingly, we found a higher number of immune responses against IDO2 in patients homozygous for the 248W giving reduction in IDO2 activity compared with the 248R. Hence, spontaneous immune responses against IDO2 seem to be correlated with reduced enzymatic activity of IDO2. The patient IDO2 genotype may well influence the outcome of IDO2-based anti-cancer vaccination. PMID:22754762

  4. Functional expression of a valencene dioxygenase from Pleurotus sapidus in E. coli.

    PubMed

    Zelena, Kateryna; Krings, Ulrich; Berger, Ralf G

    2012-03-01

    Valencene dioxygenase (ValOx) from the edible basidiomycete Pleurotus sapidus converted the sesquiterpene (+)-valencene to the valuable grapefruit flavour (+)-nootkatone and to nootkatols through intermediate hydroperoxides. Expression of the enzyme was carried out in the cytosol and periplasm of Escherichia coli. The heterologous production led to high yields of inclusion bodies. The poor yield of soluble recombinant protein was improved by various strategies including cold shock expression, chaperone co-expression, and employment of mutant E. coli strains. Up to 60 mg of the biologically active, soluble ValOx was produced by cold shock under control of the cspA promoter at 8 °C in the BL21(DE3)Star strain and co-expression of the E. coli trigger factor. The recombinant enzyme, purified using the N-terminal His tag, showed the catalytic properties of the wild-type enzyme, as was confirmed by the LC-MS analysis of hydroperoxide intermediates and GC-MS analysis of the volatile products. PMID:22264428

  5. Suicide inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-halocatechols

    SciTech Connect

    Bartels, I.; Knackmuss, H.J.; Reineke, W.

    1984-03-01

    The inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-chloro- and 3-fluorocatechol and the iron-chelating agent Tiron (catechol-3,5-disulfonate) was studied. Whereas inactivation by Tiron is an oxygen-independent and mostly reversible process, inactivation by the 3-halocatechols was only observed in the presence of oxygen and was largely irreversible. The rate constants for inactivation (K/sub 2/) were 1.62 x 10/sup -3/ sec/sup -1/ for 3-chlorocatechol and 2.38 x 10/sup -3/ sec/sup -1/ for 3-fluorocatechol. The inhibitor constants (K/sub i/) were 23 ..mu..M for 3-chlorocatechol and 17 ..mu..M for 3-fluorocatechol. The kinetic data for 3-fluorocatechol could only be obtained in the presence of 2-mercaptoethanol. Besides inactivated enzyme, some 2-hydroxyhexa-2,4-dienoic acid as the actual suicide product of meta-cleavage. A side product of 3-fluorocatechol cleavage is a yellow compound with the spectral characteristics of a 2-hydroxy-6-oxohexa-2,4-dienoci acid indicating 1,6-cleavage. Rates of inactivation by 3-fluorocatechol were reduced in the presence of superoxide dismutase, catalase, formate, and mannitol, which implies that superoxide anion, hydrogen peroxide, and hydroxyl radical exhibit additional inactivation. 64 references.

  6. Niacin metabolism and indoleamine 2,3-dioxygenase activation in malnourished patients with flaky paint dermatosis.

    PubMed

    Maltos, André Luiz; Portari, Guilherme Vannucchi; Moraes, Giselle Vanessa; Monteiro, Marina Casteli Rodrigues; Vannucchi, Helio; da Cunha, Daniel Ferreira

    2015-06-01

    Flaky paint dermatosis, characterized by extensive, often bilateral areas of flaking and pigmentation, mostly in sun unexposed areas is considered a feature of kwashiorkor in both children and adults, and must be differentiated from other dermatosis, including chapped and xerotica skin, and pellagra. In this case series we provide evidence that malnourished patients with flaky paint dermatosis and infection/inflammation shown laboratory data suggestive of indoleamine 2,3-dioxygenase (IDO) activation, besides decreased urinary excretion of N1-methylnicotinamide (N1 MN), a marker of pellagra. We study nine adult patients showing flaky paint dermatosis and clinical features of infection or inflammation, and increased serum C-reactive protein, characteristic of the presence of acute phase response syndrome. As a group, they had low or deficient urinary N1 MN excretion (0.52 ± 0.39 mg/g creatinine) compatible with pellagra. They also showed low serum tryptophan levels (<29 μmol/L) and a serum kynurenine/tryptophan ratio higher than 0.04, suggesting increased IDO expression and increase in the tryptophan oxidation. Findings suggest that some patients with flaky paint dermatosis showed laboratory data suggestive of IDO activation, besides decreased N1 MN urinary excretion. Taken together, the data support the idea that flaky paint dermatosis could be a skin manifestation of niacin deficiency. PMID:25933499

  7. The Human Enzyme That Converts Dietary Provitamin A Carotenoids to Vitamin A Is a Dioxygenase*

    PubMed Central

    dela Seña, Carlo; Riedl, Kenneth M.; Narayanasamy, Sureshbabu; Curley, Robert W.; Schwartz, Steven J.; Harrison, Earl H.

    2014-01-01

    β-Carotene 15–15′-oxygenase (BCO1) catalyzes the oxidative cleavage of dietary provitamin A carotenoids to retinal (vitamin A aldehyde). Aldehydes readily exchange their carbonyl oxygen with water, making oxygen labeling experiments challenging. BCO1 has been thought to be a monooxygenase, incorporating oxygen from O2 and H2O into its cleavage products. This was based on a study that used conditions that favored oxygen exchange with water. We incubated purified recombinant human BCO1 and β-carotene in either 16O2-H218O or 18O2-H216O medium for 15 min at 37 °C, and the relative amounts of 18O-retinal and 16O-retinal were measured by liquid chromatography-tandem mass spectrometry. At least 79% of the retinal produced by the reaction has the same oxygen isotope as the O2 gas used. Together with the data from 18O-retinal-H216O and 16O-retinal-H218O incubations to account for nonenzymatic oxygen exchange, our results show that BCO1 incorporates only oxygen from O2 into retinal. Thus, BCO1 is a dioxygenase. PMID:24668807

  8. Indoleamine-2,3-dioxygenase elevated in tumor-initiating cells is suppressed by mitocans.

    PubMed

    Stapelberg, Michael; Zobalova, Renata; Nguyen, Maria Nga; Walker, Tom; Stantic, Marina; Goodwin, Jacob; Pasdar, Elham Alizadeh; Thai, Thuan; Prokopova, Katerina; Yan, Bing; Hall, Susan; de Pennington, Nicholas; Thomas, Shane R; Grant, Gary; Stursa, Jan; Bajzikova, Martina; Meedeniya, Adrian C B; Truksa, Jaroslav; Ralph, Stephen J; Ansorge, Olaf; Dong, Lan-Feng; Neuzil, Jiri

    2014-02-01

    Tumor-initiating cells (TICs) often survive therapy and give rise to second-line tumors. We tested the plausibility of sphere cultures as models of TICs. Microarray data and microRNA data analysis confirmed the validity of spheres as models of TICs for breast and prostate cancer as well as mesothelioma cell lines. Microarray data analysis revealed the Trp pathway as the only pathway upregulated significantly in all types of studied TICs, with increased levels of indoleamine-2,3-dioxygenase-1 (IDO1), the rate-limiting enzyme of Trp metabolism along the kynurenine pathway. All types of TICs also expressed higher levels of the Trp uptake system consisting of CD98 and LAT1 with functional consequences. IDO1 expression was regulated via both transcriptional and posttranscriptional mechanisms, depending on the cancer type. Serial transplantation of TICs in mice resulted in gradually increased IDO1. Mitocans, represented by α-tocopheryl succinate and mitochondrially targeted vitamin E succinate (MitoVES), suppressed IDO1 in TICs. MitoVES suppressed IDO1 in TICs with functional mitochondrial complex II, involving transcriptional and posttranscriptional mechanisms. IDO1 increase and its suppression by VE analogues were replicated in TICs from primary human glioblastomas. Our work indicates that IDO1 is increased in TICs and that mitocans suppress the protein. PMID:24145120

  9. Emerging concepts on inhibitors of indoleamine 2,3-dioxygenase in rheumatic diseases.

    PubMed

    Filippini, P; Del Papa, N; Sambataro, D; Del Bufalo, A; Locatelli, F; Rutella, S

    2012-01-01

    The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) finely regulates both innate and adaptive immune responses through the degradation of the essential amino acid tryptophan into kynurenine and other downstream metabolites, which suppress effector T-cell function and promote the differentiation of regulatory T cells. A novel role for IDO1 as a signaling molecule and a modifier of innate inflammatory responses is now emerging. In particular, IDO1 can either support or antagonize inflammation in a context- and tissuedependent manner. Studies in experimental arthritis have unravelled a previously unappreciated role for IDO in controlling B-cell activation and autoantibody production. IDO dysregulation has been documented in patients with systemic lupus erythematosus, systemic sclerosis and Sjogren's syndrome, as well as in severe sepsis and chronic kidney disease. This article summarizes the contribution of IDO to the pathophysiology of inflammatory/autoimmune disorders, and discusses whether strategies to restore metabolic equilibrium in the kynurenine pathway might be pursued in diseases states such as rheumatoid arthritis and systemic sclerosis. PMID:22963664

  10. Salmonella overcomes tumor immune tolerance by inhibition of tumor indoleamine 2, 3-dioxygenase 1 expression.

    PubMed

    Kuan, Yu-Diao; Lee, Che-Hsin

    2016-01-01

    Over the past decades, Salmonella has been proven capable of inhibiting tumor growth. It can specifically target tumors and due to its facultative anaerobic property, can be more penetrative than other drug therapies. However, the molecular mechanism by which Salmonella inhibits tumor growth is still incompletely known. The antitumor therapeutic effect mediated by Salmonella is associated with an inflammatory immune response at the tumor site and a T cell-dependent immune response. Many tumors have been proven to have a high expression of indoleamine 2, 3-dioxygenase 1 (IDO), which is a rate-limiting enzyme that catalyzes tryptophan to kynurenine, thus causing immune tolerance within the tumor microenvironment. With decreased expression of IDO, increased immune response can be observed, which might be helpful when developing cancer immunotherapy. The expression of IDO was decreased after tumor cells were infected with Salmonella. In addition, Western blot analysis showed that the expression levels of phospho-protein kinase B (P-AKT), phospho-mammalian targets of rapamycin (P-mTOR), and phospho-p70 ribosomal s6 kinase (P-p70s6K) in tumor cells were decreased after Salmonella infection. In conclusion, our results indicate that Salmonella inhibits IDO expression and plays a crucial role in anti-tumor therapy, which might be a promising strategy combined with other cancer treatments. PMID:26517244

  11. Characterization of Bacillus thuringiensis l-Isoleucine Dioxygenase for Production of Useful Amino Acids▿†

    PubMed Central

    Hibi, Makoto; Kawashima, Takashi; Kodera, Tomohiro; Smirnov, Sergey V.; Sokolov, Pavel M.; Sugiyama, Masakazu; Shimizu, Sakayu; Yokozeki, Kenzo; Ogawa, Jun

    2011-01-01

    We determined the enzymatic characteristics of an industrially important biocatalyst, α-ketoglutarate-dependent l-isoleucine dioxygenase (IDO), which was found to be the enzyme responsible for the generation of (2S,3R,4S)-4-hydroxyisoleucine in Bacillus thuringiensis 2e2. Depending on the amino acid used as the substrate, IDO catalyzed three different types of oxidation reactions: hydroxylation, dehydrogenation, and sulfoxidation. IDO stereoselectively hydroxylated several hydrophobic aliphatic l-amino acids, as well as l-isoleucine, and produced (S)-3-hydroxy-l-allo-isoleucine, 4-hydroxy-l-leucine, (S)-4-hydroxy-l-norvaline, 4-hydroxy-l-norleucine, and 5-hydroxy-l-norleucine. The IDO reaction product of l-isoleucine, (2S,3R,4S)-4-hydroxyisoleucine, was again reacted with IDO and dehydrogenated into (2S,3R)-2-amino-3-methyl-4-ketopentanoate, which is also a metabolite found in B. thuringiensis 2e2. Interestingly, IDO catalyzed the sulfoxidation of some sulfur-containing l-amino acids and generated l-methionine sulfoxide and l-ethionine sulfoxide. Consequently, the effective production of various modified amino acids would be possible using IDO as the biocatalyst. PMID:21821743

  12. Characterization of Bacillus thuringiensis L-isoleucine dioxygenase for production of useful amino acids.

    PubMed

    Hibi, Makoto; Kawashima, Takashi; Kodera, Tomohiro; Smirnov, Sergey V; Sokolov, Pavel M; Sugiyama, Masakazu; Shimizu, Sakayu; Yokozeki, Kenzo; Ogawa, Jun

    2011-10-01

    We determined the enzymatic characteristics of an industrially important biocatalyst, α-ketoglutarate-dependent l-isoleucine dioxygenase (IDO), which was found to be the enzyme responsible for the generation of (2S,3R,4S)-4-hydroxyisoleucine in Bacillus thuringiensis 2e2. Depending on the amino acid used as the substrate, IDO catalyzed three different types of oxidation reactions: hydroxylation, dehydrogenation, and sulfoxidation. IDO stereoselectively hydroxylated several hydrophobic aliphatic l-amino acids, as well as l-isoleucine, and produced (S)-3-hydroxy-l-allo-isoleucine, 4-hydroxy-l-leucine, (S)-4-hydroxy-l-norvaline, 4-hydroxy-l-norleucine, and 5-hydroxy-l-norleucine. The IDO reaction product of l-isoleucine, (2S,3R,4S)-4-hydroxyisoleucine, was again reacted with IDO and dehydrogenated into (2S,3R)-2-amino-3-methyl-4-ketopentanoate, which is also a metabolite found in B. thuringiensis 2e2. Interestingly, IDO catalyzed the sulfoxidation of some sulfur-containing l-amino acids and generated l-methionine sulfoxide and l-ethionine sulfoxide. Consequently, the effective production of various modified amino acids would be possible using IDO as the biocatalyst. PMID:21821743

  13. The Role of Indoleamine 2, 3-Dioxygenase in Immune Suppression and Autoimmunity.

    PubMed

    Mbongue, Jacques C; Nicholas, Dequina A; Torrez, Timothy W; Kim, Nan-Sun; Firek, Anthony F; Langridge, William H R

    2015-01-01

    Indoleamine 2, 3-dioxygenase (IDO) is the first and rate limiting catabolic enzyme in the degradation pathway of the essential amino acid tryptophan. By cleaving the aromatic indole ring of tryptophan, IDO initiates the production of a variety of tryptophan degradation products called "kynurenines" that are known to exert important immuno-regulatory functions. Because tryptophan must be supplied in the diet, regulation of tryptophan catabolism may exert profound effects by activating or inhibiting metabolism and immune responses. Important for survival, the regulation of IDO biosynthesis and its activity in cells of the immune system can critically alter their responses to immunological insults, such as infection, autoimmunity and cancer. In this review, we assess how IDO-mediated catabolism of tryptophan can modulate the immune system to arrest inflammation, suppress immunity to cancer and inhibit allergy, autoimmunity and the rejection of transplanted tissues. Finally, we examine how vaccines may enhance immune suppression of autoimmunity through the upregulation of IDO biosynthesis in human dendritic cells. PMID:26378585

  14. Pyrazolone-quinazolone hybrids: a novel class of human 4-hydroxyphenylpyruvate dioxygenase inhibitors.

    PubMed

    Xu, Yu-Ling; Lin, Hong-Yan; Cao, Run-Jie; Ming, Ze-Zhong; Yang, Wen-Chao; Yang, Guang-Fu

    2014-10-01

    4-Hydroxyphenylpyruvate dioxygenase (HPPD), converting 4-hydroxyphenylpyruvate acid to homogentisate, is an important target for treating type I tyrosinemia and alkaptonuria due to its significant role in tyrosine catabolism. However, only one commercial drug, NTBC, also known as nitisinone, has been available for clinical use so far. Herein, we have elucidated the structure-based design of a series of pyrazolone-quinazolone hybrids that are novel potent human HPPD inhibitors through the successful integration of various techniques including computational simulations, organic synthesis, and biochemical characterization. Most of the new compounds displayed potent inhibitory activity against the recombinant human HPPD in nanomolar range. Compounds 3h and 3u were identified as the most potent candidates with Ki values of around 10 nM against human HPPD, about three-fold more potent than NTBC. Molecular modeling indicated that the interaction between the pyrazolone ring and ferrous ion, and the hydrophobic interaction of quinazolone with its surrounding residues, such as Phe347 and Phe364, contributed greatly to the high potency of these inhibitors. Therefore, compounds 3h and 3u could be potentially useful for the treatment of type I tyrosinemia and other diseases with defects in tyrosine degradation. PMID:25182962

  15. Indoleamine 2,3 Dioxygenase (IDO) Expression and Activity in Relapsing- Remitting Multiple Sclerosis

    PubMed Central

    Mancuso, Roberta; Hernis, Ambra; Agostini, Simone; Rovaris, Marco; Caputo, Domenico; Fuchs, Dietmar; Clerici, Mario

    2015-01-01

    Background Interferon gamma (IFN-γ) production induces the transcription of indoleamine 2,3 dioxygenase (IDO) resulting in the reduction of T-cell activation and proliferation through the depletion of tryptophan and the elicitation of Treg lymphocytes. IDO was shown to be involved in the pathogenesis of autoimmune diseases; we investigated whether changes in IDO gene expression and activity could be indicative of onset of relapse in multiple sclerosis (MS) patients. Methods IDO and interferon-γ (IFN-γ) gene expression, serum IDO activity (Kynurenine/Tryptophan ratio) and serum neopterin concentration – a protein released by macrophages upon IFN-γ stimulation – were measured in 51 individuals: 36 relapsing remitting (RR)-MS patients (21 in acute phase -AMS, 15 in stable phase -SMS) and 15 healthy controls (HC). PBMCs samples in AMS patients were collected before (BT-AMS) and during glucocorticoids-based therapy (DT-AMS). Results IDO expression was increased and IFN-γ was decreased (p<0.001) in BT-AMS compared to SMS patients. Glucocorticoids-induced disease remission resulted in a significant reduction of IDO and IFN-γ gene expression, IDO catalytic activity (p<0.001). Serum neopterin concentration followed the same trend as IDO expression and activity. Conclusions Measurement of IDO gene expression and activity in blood could be a useful marker to monitor the clinical course of RR-MS. Therapeutic interventions modulating IDO activity may be beneficial in MS. PMID:26110930

  16. The Crystal Structures of Zea mays and Arabidopsis 4-Hydroxyphenylpyruvate Dioxygenase

    PubMed Central

    Fritze, Iris M.; Linden, Lars; Freigang, Jörg; Auerbach, Günter; Huber, Robert; Steinbacher, Stefan

    2004-01-01

    The transformation of 4-hydroxyphenylpyruvate to homogentisate, catalyzed by 4-hydroxyphenylpyruvate dioxygenase (HPPD), plays an important role in degrading aromatic amino acids. As the reaction product homogentisate serves as aromatic precursor for prenylquinone synthesis in plants, the enzyme is an interesting target for herbicides. In this study we report the first x-ray structures of the plant HPPDs of Zea mays and Arabidopsis in their substrate-free form at 2.0 Å and 3.0 Å resolution, respectively. Previous biochemical characterizations have demonstrated that eukaryotic enzymes behave as homodimers in contrast to prokaryotic HPPDs, which are homotetramers. Plant and bacterial enzymes share the overall fold but use orthogonal surfaces for oligomerization. In addition, comparison of both structures provides direct evidence that the C-terminal helix gates substrate access to the active site around a nonheme ferrous iron center. In the Z. mays HPPD structure this helix packs into the active site, sequestering it completely from the solvent. In contrast, in the Arabidopsis structure this helix tilted by about 60° into the solvent and leaves the active site fully accessible. By elucidating the structure of plant HPPD enzymes we aim to provide a structural basis for the development of new herbicides. PMID:15084729

  17. Novel bacterial bioassay for a high-throughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors.

    PubMed

    Rocaboy-Faquet, Emilie; Noguer, Thierry; Romdhane, Sana; Bertrand, Cédric; Dayan, Franck Emmanuel; Barthelmebs, Lise

    2014-08-01

    Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is the molecular target of a range of synthetic β-triketone herbicides that are currently used commercially. Their mode of action is based on an irreversible inhibition of HPPD. Therefore, this inhibitory capacity was used to develop a whole-cell colorimetric bioassay with a recombinant Escherichia coli expressing a plant HPPD for the herbicide analysis of β-triketones. The principle of the bioassay is based on the ability of the recombinant E. coli clone to produce a soluble melanin-like pigment, from tyrosine catabolism through p-hydroxyphenylpyruvate and homogentisate. The addition of sulcotrione, a HPPD inhibitor, decreased the pigment production. With the aim to optimize the assay, the E. coli recombinant clone was immobilized in sol-gel or agarose matrix in a 96-well microplate format. The limit of detection for mesotrione, tembotrione, sulcotrione, and leptospermone was 0.069, 0.051, 0.038, and 20 μM, respectively, allowing to validate the whole-cell colorimetric bioassay as a simple and cost-effective alternative tool for laboratory use. The bioassay results from sulcotrione-spiked soil samples were confirmed with high-performance liquid chromatography. PMID:24816780

  18. Synthesis and Herbicidal Activity of Triketone-Quinoline Hybrids as Novel 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors.

    PubMed

    Wang, Da-Wei; Lin, Hong-Yan; Cao, Run-Jie; Chen, Tao; Wu, Feng-Xu; Hao, Ge-Fei; Chen, Qiong; Yang, Wen-Chao; Yang, Guang-Fu

    2015-06-17

    4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) is one of the most important targets for herbicide discovery. In the search for new HPPD inhibitors with novel scaffolds, triketone-quinoline hybrids were designed and subsequently optimized on the basis of the structure-activity relationship (SAR) studies. Most of the synthesized compounds displayed potent inhibition of Arabidopsis thaliana HPPD (AtHPPD), and some of them exhibited broad-spectrum and promising herbicidal activity at the rate of 150 g ai/ha by postemergence application. Most promisingly, compound III-l, 3-hydroxy-2-(2-methoxy-7-(methylthio)quinoline-3-carbonyl)cyclohex-2-enone (Ki = 0.009 μM, AtHPPD), had broader spectrum of weed control than mesotrione. Furthermore, compound III-l was much safer to maize at the rate of 150 g ai/ha than mesotrione, demonstrating its great potential as herbicide for weed control in maize fields. Therefore, triketone-quinoline hybrids may serve as new lead structures for novel herbicide discovery. PMID:26006257

  19. Lignans from Carthamus tinctorius suppress tryptophan breakdown via indoleamine 2,3-dioxygenase

    PubMed Central

    Kuehnl, Susanne; Schroecksnadel, Sebastian; Temml, Veronika; Gostner, Johanna M.; Schennach, Harald; Schuster, Daniela; Schwaiger, Stefan; Rollinger, Judith M.; Fuchs, Dietmar; Stuppner, Hermann

    2013-01-01

    Seed extracts of Carthamus tinctorius L. (Asteraceae), safflower, have been traditionally used to treat coronary disease, thrombotic disorders, and menstrual problems but also against cancer and depression. A possible effect of C. tinctorius compounds on tryptophan-degrading activity of enzyme indoleamine 2,3-dioxygenase (IDO) could explain many of its activities. To test for an effect of C. tinctorius extracts and isolated compounds on cytokine-induced IDO activity in immunocompetent cells in vitro methanol and ethylacetate seed extracts were prepared from cold pressed seed cakes of C. tinctorius and three lignan derivatives, trachelogenin, arctigenin and matairesinol were isolated. The influence on tryptophan breakdown was investigated in peripheral blood mononuclear cells (PBMCs). Effects were compared to neopterin production in the same cellular assay. Both seed extracts suppressed tryptophan breakdown in stimulated PBMC. The three structurally closely related isolates exerted differing suppressive activity on PBMC: arctigenin (IC50 26.5 μM) and trachelogenin (IC50 of 57.4 μM) showed higher activity than matairesinol (IC50 >200 μM) to inhibit tryptophan breakdown. Effects on neopterin production were similar albeit generally less strong. Data show an immunosuppressive property of compounds which slows down IDO activity. The in vitro results support the view that some of the anti-inflammatory, anti-cancer and antidepressant properties of C. tinctorius lignans might relate to their suppressive influence on tryptophan breakdown. PMID:23867649

  20. Tryptophan-2,3-dioxygenase (TDO) inhibition ameliorates neurodegeneration by modulation of kynurenine pathway metabolites

    PubMed Central

    Breda, Carlo; Sathyasaikumar, Korrapati V.; Sograte Idrissi, Shama; Notarangelo, Francesca M.; Estranero, Jasper G.; Moore, Gareth G. L.; Green, Edward W.; Kyriacou, Charalambos P.; Schwarcz, Robert; Giorgini, Flaviano

    2016-01-01

    Metabolites of the kynurenine pathway (KP) of tryptophan (TRP) degradation have been closely linked to the pathogenesis of several neurodegenerative disorders. Recent work has highlighted the therapeutic potential of inhibiting two critical regulatory enzymes in this pathway—kynurenine-3-monooxygenase (KMO) and tryptophan-2,3-dioxygenase (TDO). Much evidence indicates that the efficacy of KMO inhibition arises from normalizing an imbalance between neurotoxic [3-hydroxykynurenine (3-HK); quinolinic acid (QUIN)] and neuroprotective [kynurenic acid (KYNA)] KP metabolites. However, it is not clear if TDO inhibition is protective via a similar mechanism or if this is instead due to increased levels of TRP—the substrate of TDO. Here, we find that increased levels of KYNA relative to 3-HK are likely central to the protection conferred by TDO inhibition in a fruit fly model of Huntington’s disease and that TRP treatment strongly reduces neurodegeneration by shifting KP flux toward KYNA synthesis. In fly models of Alzheimer’s and Parkinson’s disease, we provide genetic evidence that inhibition of TDO or KMO improves locomotor performance and ameliorates shortened life span, as well as reducing neurodegeneration in Alzheimer's model flies. Critically, we find that treatment with a chemical TDO inhibitor is robustly protective in these models. Consequently, our work strongly supports targeting of the KP as a potential treatment strategy for several major neurodegenerative disorders and suggests that alterations in the levels of neuroactive KP metabolites could underlie several therapeutic benefits. PMID:27114543

  1. The use of dynamic nuclear polarization 13C-pyruvate MRS in cancer

    PubMed Central

    Gutte, Henrik; Hansen, Adam Espe; Johannesen, Helle Hjorth; Clemmensen, Andreas Ettrup; Ardenkjær-Larsen, Jan Henrik; Nielsen, Carsten Haagen; Kjær, Andreas

    2015-01-01

    In recent years there has been an immense development of new targeted anti-cancer drugs. For practicing precision medicine, a sensitive method imaging for non-invasive, assessment of early treatment response and for assisting in developing new drugs is warranted. Magnetic Resonance Spectroscopy (MRS) is a potent technique for non-invasive in vivo investigation of tissue chemistry and cellular metabolism. Hyperpolarization by Dynamic Nuclear Polarization (DNP) is capable of creating solutions of molecules with polarized nuclear spins in a range of biological molecules and has enabled the real-time investigation of in vivo metabolism. The development of this new method has been demonstrated to enhance the nuclear polarization more than 10,000-fold, thereby significantly increasing the sensitivity of the MRS with a spatial resolution to the millimeters and a temporal resolution at the subsecond range. Furthermore, the method enables measuring kinetics of conversion of substrates into cell metabolites and can be integrated with anatomical proton magnetic resonance imaging (MRI). Many nuclei and substrates have been hyperpolarized using the DNP method. Currently, the most widely used compound is 13C-pyruvate due to favoring technicalities. Intravenous injection of the hyperpolarized 13C-pyruvate results in appearance of 13C-lactate, 13C-alanine and 13C-bicarbonate resonance peaks depending on the tissue, disease and the metabolic state probed. In cancer, the lactate level is increased due to increased glycolysis. The use of DNP enhanced 13C-pyruvate has in preclinical studies shown to be a sensitive method for detecting cancer and for assessment of early treatment response in a variety of cancers. Recently, a first-in-man 31-patient study was conducted with the primary objective to assess the safety of hyperpolarized 13C-pyruvate in healthy subjects and prostate cancer patients. The study showed an elevated 13C-lactate/13C-pyruvate ratio in regions of biopsy

  2. Re-evaluation of dioxygenase gene phylogeny for the development and validation of a quantitative assay for environmental aromatic hydrocarbon degraders

    PubMed Central

    Meynet, Paola; Head, Ian M.; Werner, David; Davenport, Russell J.

    2015-01-01

    Rieske non-heme iron oxygenases enzymes have been widely studied, as they catalyse essential reactions initiating the bacterial degradation of organic compounds, for instance aromatic hydrocarbons. The genes encoding these enzymes offer a potential target for studying aromatic hydrocarbon-degrading organisms in the environment. However, previously reported primer sets that target dioxygenase gene sequences or the common conserved Rieske centre of aromatics dioxygenases have limited specificity and/or target non-dioxygenase genes. In this work, an extensive database of dioxygenase α-subunit gene sequences was constructed, and primer sets targeting the conserved Rieske centre were developed. The high specificity of the primers was confirmed by polymerase chain reaction analysis, agarose gel electrophoresis and sequencing. Quantitative polymerase chain reaction (qPCR) assays were also developed and optimized, following MIQE guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments). Comparison of the qPCR quantification of dioxygenases in spiked sediment samples and in pure cultures demonstrated an underestimation of the Ct value, and the requirement for a correction factor at gene abundances below 108 gene copies per g of sediment. Externally validated qPCR provides a valuable tool to monitor aromatic hydrocarbon degrader population abundances at contaminated sites. PMID:25944871

  3. Novel binding motif and new flexibility revealed by structural analyses of a pyruvate dehydrogenase-dihydrolipoyl acetyltransferase subcomplex from the Escherichia coli pyruvate dehydrogenase multienzyme complex.

    PubMed

    Arjunan, Palaniappa; Wang, Junjie; Nemeria, Natalia S; Reynolds, Shelley; Brown, Ian; Chandrasekhar, Krishnamoorthy; Calero, Guillermo; Jordan, Frank; Furey, William

    2014-10-24

    The Escherichia coli pyruvate dehydrogenase multienzyme complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subcomplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subcomplex, both of which probably have consequences in the overall multienzyme complex assembly. This represents the first structure of an E1p-E2p didomain subcomplex involving a homodimeric E1p, and the results may be applicable to a large range of complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subcomplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly. PMID:25210042

  4. Neuroprotective effect of ethyl pyruvate against Zn(2+) toxicity via NAD replenishment and direct Zn(2+) chelation.

    PubMed

    Kim, Seung-Woo; Lee, Hye-Kyung; Kim, Hyun-Ji; Yoon, Sung-Hwa; Lee, Ja-Kyeong

    2016-06-01

    Ethyl pyruvate (EP) is a simple aliphatic ester of pyruvic acid and has been shown to have robust protective effect in various pathological conditions. A variety of mechanisms have been reported to underlie the protective effects of EP, which include anti-inflammatory, anti-oxidative, and anti-apoptotic functions. Recently, we reported that EP suppressed high mobility group box 1 (HMGB1) release from primary microglial cells via direct Ca(2+) chelation. In the present study, we investigated whether and how EP chelates Zn(2+) in neurons when it is present at toxic levels. In cortical neurons treated with 40 μM of Zn(2+) for 24 h, both EP and pyruvate significantly suppressed neuronal cell death, although the potency of pyruvate was greater than that of EP, and that NAD replenishment contributed to the neuroprotective effects of both pyruvate and EP. However, when cortical neurons were exposed to acute treatment of Zn(2+) (400 μM, 15 min), EP, but not pyruvate, significantly suppressed neuronal death, despite the fact that NAD replenishment by EP was weaker than that by pyruvate. Spectrophotometric studies revealed that EP directly chelates Zn(2+), and this was confirmed in physiological contexts, such as, NMDA-treated primary cortical cultures and OGD-subjected hippocampal slice cultures, in which EP suppressed intracellular Zn(2+) elevation and neuronal cell death. In addition, EP markedly reduced the expressions of PARP-1 and of the NADPH oxidase subunit in Zn(2+)-treated primary cortical neurons, well known Zn(2+)-induced downstream processes. Together, these results show EP suppresses Zn(2+) induced neurotoxicity via dual functions, chelating Zn(2+) and promoting NAD replenishment. PMID:26850126

  5. Modeling non‐linear kinetics of hyperpolarized [1‐13C] pyruvate in the crystalloid‐perfused rat heart

    PubMed Central

    Mariotti, E.; Orton, M. R.; Eerbeek, O.; Ashruf, J. F.; Zuurbier, C. J.; Southworth, R.

    2016-01-01

    Hyperpolarized 13C MR measurements have the potential to display non‐linear kinetics. We have developed an approach to describe possible non‐first‐order kinetics of hyperpolarized [1‐13C] pyruvate employing a system of differential equations that agrees with the principle of conservation of mass of the hyperpolarized signal. Simultaneous fitting to a second‐order model for conversion of [1‐13C] pyruvate to bicarbonate, lactate and alanine was well described in the isolated rat heart perfused with Krebs buffer containing glucose as sole energy substrate, or glucose supplemented with pyruvate. Second‐order modeling yielded significantly improved fits of pyruvate–bicarbonate kinetics compared with the more traditionally used first‐order model and suggested time‐dependent decreases in pyruvate–bicarbonate flux. Second‐order modeling gave time‐dependent changes in forward and reverse reaction kinetics of pyruvate–lactate exchange and pyruvate–alanine exchange in both groups of hearts during the infusion of pyruvate; however, the fits were not significantly improved with respect to a traditional first‐order model. The mechanism giving rise to second‐order pyruvate dehydrogenase (PDH) kinetics was explored experimentally using surface fluorescence measurements of nicotinamide adenine dinucleotide reduced form (NADH) performed under the same conditions, demonstrating a significant increase of NADH during pyruvate infusion. This suggests a simultaneous depletion of available mitochondrial NAD+ (the cofactor for PDH), consistent with the non‐linear nature of the kinetics. NADH levels returned to baseline following cessation of the pyruvate infusion, suggesting this to be a transient effect. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd. PMID:26777799

  6. Crystallization and preliminary X-ray characterization of the 2,4′-dihydroxyaceto­phenone dioxygenase from Alcaligenes sp. 4HAP

    PubMed Central

    Beaven, G.; Bowyer, A.; Erskine, P.; Wood, S. P.; McCoy, A.; Coates, L.; Keegan, R.; Lebedev, A.; Hopper, D. J.; Kaderbhai, M. A.; Cooper, J. B.

    2014-01-01

    The enzyme 2,4′-dihydroxyacetophenone dioxygenase (or DAD) catalyses the conversion of 2,4′-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C—C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits each containing nonhaem iron and its sequence suggests that it belongs to the cupin family of dioxygenases. By the use of limited chymotrypsinolysis, the DAD enzyme from Alcaligenes sp. 4HAP has been crystallized in a form that diffracts synchrotron radiation to a resolution of 2.2 Å. PMID:24915102

  7. Crystallization and preliminary crystallographic analysis of the ferredoxin component of carbazole 1,9a-dioxygenase from Nocardioides aromaticivorans IC177

    SciTech Connect

    Inoue, Kengo; Ashikawa, Yuji; Usami, Yusuke; Noguchi, Haruko; Fujimoto, Zui; Yamane, Hisakazu; Nojiri, Hideaki

    2007-10-01

    The ferredoxin component of carbazole 1,9a-dioxygenase from N. aromaticivorans IC177 was crystallized and diffraction data were collected to 2.0 Å resolution. Carbazole 1,9a-dioxygenase (CARDO) catalyzes the dihydroxylation of carbazole by angular position (C9a) carbon bonding to the imino nitrogen and its adjacent C1 carbon. CARDO consists of a terminal oxygenase component and two electron-transfer components: ferredoxin and ferredoxin reductase. The ferredoxin component of carbazole 1,9a-dioxygenase from Nocardioides aromaticivorans IC177 was crystallized at 293 K using the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant. The crystals, which were improved by macroseeding, diffract to 2.0 Å resolution and belong to space group P4{sub 1}2{sub 1}2.

  8. Crystallization and preliminary X-ray diffraction studies of the terminal oxygenase component of carbazole 1,9a-dioxygenase from Nocardioides aromaticivorans IC177

    SciTech Connect

    Inoue, Kengo; Ashikawa, Yuji; Usami, Yusuke; Noguchi, Haruko; Fujimoto, Zui; Yamane, Hisakazu; Nojiri, Hideaki

    2006-12-01

    The terminal oxygenase component of carbazole 1,9a-dioxygenase from N. aromaticivorans IC177 was crystallized and diffraction data were collected to 2.30 Å resolution. Carbazole 1,9a-dioxygenase (CARDO) catalyzes the dihydroxylation of carbazole by angular-position (C9a) carbon bonding to the imino nitrogen and its adjacent C1 carbon. CARDO consists of a terminal oxygenase component and two electron-transfer components: ferredoxin and ferredoxin reductase. The terminal oxygenase component (43.9 kDa) of carbazole 1,9a-dioxygenase from Nocardioides aromaticivorans IC177 was crystallized at 293 K using the hanging-drop vapour-diffusion method with PEG 8000 as the precipitant. The crystals diffract to 2.3 Å resolution and belong to space group C2.

  9. Peptide mapping and amino acid sequencing of two catechol 1,2-dioxygenases (CD I1 and CD I2) from Acinetobacter lwoffii K24.

    PubMed

    Kim, S I; Ha, K S

    1997-10-31

    The partial amino acid sequences of two catechol 1,2-dioxygenases (CD I1 and CD I2) from Acinetobacter lwoffii K24 have been determined by analysis of peptides after cleavages with endopeptidase Lys-C, endopeptidase Glu-C, trypsin, and chemicals (cyanogen bromide and BNPS-skatole). They include 248 amino acid sequences (4 fragments) of CD I1 and 211 amino acid sequences (5 fragments) of CD I2. Two enzymes have more than 50% sequence homology with type I catechol 1,2-dioxygenases and less than 30% sequence homology with type II catechol 1,2-dioxygenases. Two enzymes have similar hydropathy profiles in the N-terminal region, suggesting that they have similar secondary structures. PMID:9387151

  10. Characterizing the Promiscuity of LigAB, a Lignin Catabolite Degrading Extradiol Dioxygenase from Sphingomonas paucimobilis SYK-6

    PubMed Central

    Barry, Kevin P.; Taylor, Erika A.

    2014-01-01

    LigAB from Sphingomonas paucimobilis SYK-6 is the only structurally characterized dioxygenase of the largely uncharacterized superfamily of Type II extradiol dioxygenases (EDO). This enzyme catalyzes the oxidative ring-opening of protocatechuate (3,4-dihydroxybenzoic acid or PCA) in a pathway allowing the degradation of lignin derived aromatic compounds (LDACs). LigAB has also been shown to utilize two other LDACs from the same metabolic pathway as substrates, gallate, and 3-O-methyl gallate; however, kcat/KM had not been reported for any of these compounds. In order to assess the catalytic efficiency and get insights into the observed promiscuity of this enzyme, steady-state kinetic analyses were performed for LigAB with these and a library of related compounds. The dioxygenation of PCA by LigAB was highly efficient, with a kcat of 51 s−1 and a kcat/KM of 4.26 × 106 M−1s−1. LigAB demonstrated the ability to use a variety of catecholic molecules as substrates beyond the previously identified gallate and 3-O-methyl gallate, including 3,4-dihydroxybenzamide, homoprotocatechuate, catechol, and 3,4-dihydroxybenzonitrile. Interestingly, 3,4-dihydroxybenzamide (DHBAm) behaves in a manner similar to that of the preferred benzoic acid substrates, with a kcat/Km value only ~4-fold lower than that for gallate and ~10-fold higher than that for 3-O-methyl gallate. All of these most active substrates demonstrate mechanistic inactivation of LigAB. Additionally, DHBAm exhibits potent product inhibition that leads to an inactive enzyme, being more highly deactivating at lower substrate concentration, a phenomena that, to our knowledge, has not been reported for another dioxygenase substrate/product pair. These results provide valuable catalytic insight into the reactions catalyzed by LigAB and make it the first Type II EDO that is fully characterized both structurally and kinetically. PMID:23977959

  11. The Reaumuria trigyna leucoanthocyanidin dioxygenase (RtLDOX) gene complements anthocyanidin synthesis and increases the salt tolerance potential of a transgenic Arabidopsis LDOX mutant.

    PubMed

    Zhang, Huirong; Du, Chao; Wang, Yan; Wang, Jia; Zheng, Linlin; Wang, Yingchun

    2016-09-01

    Reaumuria trigyna is a typical, native desert halophyte that grows under extreme conditions in Inner Mongolia. In a previous transcriptomic profiling analysis, flavonoid pathway-related genes in R. trigyna showed significant differences in transcript abundance under salt stress. Leucoanthocyanidin dioxygenase (LDOX, EC 1.14.11.19) is on