Sample records for nadh dehydrogenase activity

  1. An activity transition from NADH dehydrogenase to NADH oxidase during protein denaturation.

    PubMed

    Huston, Scott; Collins, John; Sun, Fangfang; Zhang, Ting; Vaden, Timothy D; Zhang, Y-H Percival; Fu, Jinglin

    2018-05-01

    A decrease in the specific activity of an enzyme is commonly observed when the enzyme is inappropriately handled or is stored over an extended period. Here, we reported a functional transition of an FMN-bound diaphorase (FMN-DI) that happened during the long-term storage process. It was found that FMN-DI did not simply lose its β-nicotinamide adenine diphosphate (NADH) dehydrogenase activity after a long-time storage, but obtained a new enzyme activity of NADH oxidase. Further mechanistic studies suggested that the alteration of the binding strength of an FMN cofactor with a DI protein could be responsible for this functional switch of the enzyme. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

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

    NASA Astrophysics Data System (ADS)

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

    2017-03-01

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

  3. A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD(+) regeneration.

    PubMed

    Wang, Jizhong; Yang, Chengli; Chen, Xing; Bao, Bingxin; Zhang, Xuan; Li, Dali; Du, Xingfan; Shi, Ruofu; Yang, Junfang; Zhu, Ronghui

    2016-08-01

    To find an efficient and cheap system for NAD(+) regeneration A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K m value for NADH of 10 μM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD(+) regeneration was established. The system is attractive in that the O2 consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD(+) was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C CONCLUSION: The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD(+) regeneration.

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

    NASA Technical Reports Server (NTRS)

    Hochstein, Lawrence I.; Dalton, Bonnie P.

    1973-01-01

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

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

    USDA-ARS?s Scientific Manuscript database

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

  6. [Involvement of hydrogen peroxide in the regulation of coexpression of alternative oxidase and rotenone-insensitive NADH dehydrogenase in tomato leaves and calluses].

    PubMed

    Eprintsev, A T; Mal'tseva, E V; Shatskikh, A S; Popov, V N

    2011-01-01

    The involvement of active oxygen forms in the regulation of the expression of mitochondrial respiratory chain components, which are not related to energy storing, has been in vitro and in vivo studied in Lycopersicum esculentum L. The highest level of transcription of genes encoding alternative oxidase and NADH dehydrogenase has been observed in green tomato leaves. It has been shown that even low H2O2 concentrations activate both aoxlalpha and ndb1 genes, encoding alternative oxidase and external mitochondrial rotenone-insensitive NADH dehydrogenase, respectively. According to our results, in the case of an oxidative stress, alternative oxidase and NADH dehydrogenase are coexpressed in tomato plant tissues, and active oxygen forms serve as the secondary messengers of their coexpression.

  7. Effect of micromolar Ca2+ on NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex and possible role of Ca2+ in signal amplification.

    PubMed

    Lawlis, V B; Roche, T E

    1980-11-20

    NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex was compared at 10 microM free Ca2+ or in the absence of Ca2+ (i.e., less than 1.0 nM free Ca2+). In the presence of Ca2+, NADH inhibition was appreciably decreased for a wide range of NADH:NAD+ ratios. A half-maximal decrease in NADH inhibition occurred at slightly less than 1 microM free Ca/+ (as determined with EGTA-Ca buffers). Of necessity this was observed on top of an effect of Ca2+ on the S0.5 for alpha-ketoglutarate which was decreased by Ca2+ with a half-maximal effect at a similar concentration. The effect of Ca2+ on NADH inhibition was not observed in assays of the dihydrolipoyl dehydrogenase component (using dihydrolipoamide as a substrate) or in assays of bovine kidney pyruvate dehydrogenase complex. This indicates that the overall reaction catalyzed by the alpha-ketoglutarate dehydrogenase complex is required to elicit the effect of Ca2+ on NADH inhibition. At a fixed alpha-ketoglutarate concentration (50 microM), removal of Ca2+ reduced the activity of the alpha-ketoglutarate dehydrogenase complex by 8.5-fold (due to an increase in S0.5 for alpha-ketoglutarate) and, in the presence of different NADH:NAD+ ratios, decreased the activity of the complex by 50 to 100-fold. Effects of the phosphate potential (ATP/ADPxPi) or a combination of the phosphate potential and NADH:NAD+ ratio are also described. The possibility that the level of intramitochondrial free Ca/+ serves as a signal amplifier normally coupled to the energy state of mitochondria is discussed.

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

    USDA-ARS?s Scientific Manuscript database

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

  9. Expression of the yeast NADH dehydrogenase Ndi1 in Drosophila confers increased lifespan independently of dietary restriction

    PubMed Central

    Sanz, Alberto; Soikkeli, Mikko; Portero-Otín, Manuel; Wilson, Angela; Kemppainen, Esko; McIlroy, George; Ellilä, Simo; Kemppainen, Kia K.; Tuomela, Tea; Lakanmaa, Matti; Kiviranta, Essi; Stefanatos, Rhoda; Dufour, Eric; Hutz, Bettina; Naudí, Alba; Jové, Mariona; Zeb, Akbar; Vartiainen, Suvi; Matsuno-Yagi, Akemi; Yagi, Takao; Rustin, Pierre; Pamplona, Reinald; Jacobs, Howard T.

    2010-01-01

    Mutations in mitochondrial oxidative phosphorylation complex I are associated with multiple pathologies, and complex I has been proposed as a crucial regulator of animal longevity. In yeast, the single-subunit NADH dehydrogenase Ndi1 serves as a non-proton-translocating alternative enzyme that replaces complex I, bringing about the reoxidation of intramitochondrial NADH. We have created transgenic strains of Drosophila that express yeast NDI1 ubiquitously. Mitochondrial extracts from NDI1-expressing flies displayed a rotenone-insensitive NADH dehydrogenase activity, and functionality of the enzyme in vivo was confirmed by the rescue of lethality resulting from RNAi knockdown of complex I. NDI1 expression increased median, mean, and maximum lifespan independently of dietary restriction, and with no change in sirtuin activity. NDI1 expression mitigated the aging associated decline in respiratory capacity and the accompanying increase in mitochondrial reactive oxygen species production, and resulted in decreased accumulation of markers of oxidative damage in aged flies. Our results support a central role of mitochondrial oxidative phosphorylation complex I in influencing longevity via oxidative stress, independently of pathways connected to nutrition and growth signaling. PMID:20435911

  10. Characterization of the NADH:ubiquinone oxidoreductase (complex I) in the trypanosomatid Phytomonas serpens (Kinetoplastida).

    PubMed

    Cermáková, Petra; Verner, Zdenek; Man, Petr; Lukes, Julius; Horváth, Anton

    2007-06-01

    NADH dehydrogenase activity was characterized in the mitochondrial lysates of Phytomonas serpens, a trypanosomatid flagellate parasitizing plants. Two different high molecular weight NADH dehydrogenases were characterized by native PAGE and detected by direct in-gel activity staining. The association of NADH dehydrogenase activities with two distinct multisubunit complexes was revealed in the second dimension performed under denaturing conditions. One subunit present in both complexes cross-reacted with the antibody against the 39 kDa subunit of bovine complex I. Out of several subunits analyzed by MS, one contained a domain characteristic for the LYR family subunit of the NADH:ubiquinone oxidoreductases. Spectrophotometric measurement of the NADH:ubiquinone 10 and NADH:ferricyanide dehydrogenase activities revealed their different sensitivities to rotenone, piericidin, and diphenyl iodonium.

  11. Regulation of NADH/CoQ oxidoreductase: do phosphorylation events affect activity?

    PubMed

    Maj, Mary C; Raha, Sandeep; Myint, Tomoko; Robinson, Brian H

    2004-01-01

    We had previously suggested that phosphorylation of proteins by mitochondrial kinases regulate the activity of NADH/CoQ oxidoreductase. Initial data showed that pyruvate dehydrogenase kinase (PDK) and cAMP-dependent protein kinase A (PKA) phosphorylate mitochondrial membrane proteins. Upon phosphorylation with crude PDK, mitochondria appeared to be deficient in NADH/cytochrome c reductase activity associated with increased superoxide production. Conversely, phosphorylation by PKA resulted in increased NADH/cytochrome c reductase activity and decreased superoxide formation. Current data confirms PKA involvement in regulating Complex I activity through phosphorylation of an 18 kDa subunit. Beef heart NADH/ cytochrome c reductase activity increases to 150% of control upon incubation with PKA and ATP-gamma-S. We have cloned the four human isoforms of PDK and purified beef heart Complex I. Incubation of mitochondria with PDK isoforms and ATP did not alter Complex I activity or superoxide production. Radiolabeling of mitochondria and purified Complex I with PDK failed to reveal phosphorylated proteins.

  12. 2,4-Dichlorophenoxyacetic Acid Inhibits the Outer Membrane NADH Dehydrogenase of Plant Mitochondria 1

    PubMed Central

    Mannella, Carmen A.; Bonner, Walter D.

    1978-01-01

    The NADH dehydrogenase of potato (Solanum tuberosum) and mung bean (Phaseolus aureus) outer mitochondrial membranes is specifically inhibited by both 2,4-dichlorophenoxyacetic and 2,4,5-trichlorophenoxyacetic acids but not by the natural auxin indole-3-acetic acid. PMID:16660539

  13. NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production.

    PubMed

    Maia, Luisa; Duarte, Rui O; Ponces-Freire, Ana; Moura, José J G; Mira, Lurdes

    2007-08-01

    To characterise the NADH oxidase activity of both xanthine dehydrogenase (XD) and xanthine oxidase (XO) forms of rat liver xanthine oxidoreductase (XOR) and to evaluate the potential role of this mammalian enzyme as an O2*- source, kinetics and electron paramagnetic resonance (EPR) spectroscopic studies were performed. A steady-state kinetics study of XD showed that it catalyses NADH oxidation, leading to the formation of one O2*- molecule and half a H(2)O(2) molecule per NADH molecule, at rates 3 times those observed for XO (29.2 +/- 1.6 and 9.38 +/- 0.31 min(-1), respectively). EPR spectra of NADH-reduced XD and XO were qualitatively similar, but they were quantitatively quite different. While NADH efficiently reduced XD, only a great excess of NADH reduced XO. In agreement with reductive titration data, the XD specificity constant for NADH (8.73 +/- 1.36 microM(-1) min(-1)) was found to be higher than that of the XO specificity constant (1.07 +/- 0.09 microM(-1) min(-1)). It was confirmed that, for the reducing substrate xanthine, rat liver XD is also a better O2*- source than XO. These data show that the dehydrogenase form of liver XOR is, thus, intrinsically more efficient at generating O2*- than the oxidase form, independently of the reducing substrate. Most importantly, for comparative purposes, human liver XO activity towards NADH oxidation was also studied, and the kinetics parameters obtained were found to be very similar to those of the XO form of rat liver XOR, foreseeing potential applications of rat liver XOR as a model of the human liver enzyme.

  14. URF6, Last Unidentified Reading Frame of Human mtDNA, Codes for an NADH Dehydrogenase Subunit

    NASA Astrophysics Data System (ADS)

    Chomyn, Anne; Cleeter, Michael W. J.; Ragan, C. Ian; Riley, Marcia; Doolittle, Russell F.; Attardi, Giuseppe

    1986-10-01

    The polypeptide encoded in URF6, the last unassigned reading frame of human mitochondrial DNA, has been identified with antibodies to peptides predicted from the DNA sequence. Antibodies prepared against highly purified respiratory chain NADH dehydrogenase from beef heart or against the cytoplasmically synthesized 49-kilodalton iron-sulfur subunit isolated from this enzyme complex, when added to a deoxycholate or a Triton X-100 mitochondrial lysate of HeLa cells, specifically precipitated the URF6 product together with the six other URF products previously identified as subunits of NADH dehydrogenase. These results strongly point to the URF6 product as being another subunit of this enzyme complex. Thus, almost 60% of the protein coding capacity of mammalian mitochondrial DNA is utilized for the assembly of the first enzyme complex of the respiratory chain. The absence of such information in yeast mitochondrial DNA dramatizes the variability in gene content of different mitochondrial genomes.

  15. Coupled ferredoxin and crotonyl coenzyme A (CoA) reduction with NADH catalyzed by the butyryl-CoA dehydrogenase/Etf complex from Clostridium kluyveri.

    PubMed

    Li, Fuli; Hinderberger, Julia; Seedorf, Henning; Zhang, Jin; Buckel, Wolfgang; Thauer, Rudolf K

    2008-02-01

    Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (acetyl-CoA)- and ferredoxin-dependent formation of H2 from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of acetyl-CoA with NADH via crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E0' = -410 mV) with NADH (E0' = -320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E0' = -10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH + 1 oxidized ferredoxin + 1 crotonyl-CoA = 2 NAD+ + 1 ferredoxin reduced by two electrons + 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper.

  16. The Kinetic Reaction Mechanism of the Vibrio cholerae Sodium-dependent NADH Dehydrogenase*♦

    PubMed Central

    Tuz, Karina; Mezic, Katherine G.; Xu, Tianhao; Barquera, Blanca; Juárez, Oscar

    2015-01-01

    The sodium-dependent NADH dehydrogenase (Na+-NQR) is the main ion transporter in Vibrio cholerae. Its activity is linked to the operation of the respiratory chain and is essential for the development of the pathogenic phenotype. Previous studies have described different aspects of the enzyme, including the electron transfer pathways, sodium pumping structures, cofactor and subunit composition, among others. However, the mechanism of the enzyme remains to be completely elucidated. In this work, we have studied the kinetic mechanism of Na+-NQR with the use of steady state kinetics and stopped flow analysis. Na+-NQR follows a hexa-uni ping-pong mechanism, in which NADH acts as the first substrate, reacts with the enzyme, and the oxidized NAD leaves the catalytic site. In this conformation, the enzyme is able to capture two sodium ions and transport them to the external side of the membrane. In the last step, ubiquinone is bound and reduced, and ubiquinol is released. Our data also demonstrate that the catalytic cycle involves two redox states, the three- and five-electron reduced forms. A model that gathers all available information is proposed to explain the kinetic mechanism of Na+-NQR. This model provides a background to understand the current structural and functional information. PMID:26004776

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

    PubMed Central

    Rutter, G A; Denton, R M

    1988-01-01

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

  18. Coupled Ferredoxin and Crotonyl Coenzyme A (CoA) Reduction with NADH Catalyzed by the Butyryl-CoA Dehydrogenase/Etf Complex from Clostridium kluyveri▿ †

    PubMed Central

    Li, Fuli; Hinderberger, Julia; Seedorf, Henning; Zhang, Jin; Buckel, Wolfgang; Thauer, Rudolf K.

    2008-01-01

    Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (acetyl-CoA)- and ferredoxin-dependent formation of H2 from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of acetyl-CoA with NADH via crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E0′ = −410 mV) with NADH (E0′ = −320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E0′ = −10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH + 1 oxidized ferredoxin + 1 crotonyl-CoA = 2 NAD+ + 1 ferredoxin reduced by two electrons + 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper. PMID:17993531

  19. Determination of the in vivo NAD:NADH ratio in Saccharomyces cerevisiae under anaerobic conditions, using alcohol dehydrogenase as sensor reaction.

    PubMed

    Bekers, K M; Heijnen, J J; van Gulik, W M

    2015-08-01

    With the current quantitative metabolomics techniques, only whole-cell concentrations of NAD and NADH can be quantified. These measurements cannot provide information on the in vivo redox state of the cells, which is determined by the ratio of the free forms only. In this work we quantified free NAD:NADH ratios in yeast under anaerobic conditions, using alcohol dehydrogenase (ADH) and the lumped reaction of glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase as sensor reactions. We showed that, with an alternative accurate acetaldehyde determination method, based on rapid sampling, instantaneous derivatization with 2,4 diaminophenol hydrazine (DNPH) and quantification with HPLC, the ADH-catalysed oxidation of ethanol to acetaldehyde can be applied as a relatively fast and simple sensor reaction to quantify the free NAD:NADH ratio under anaerobic conditions. We evaluated the applicability of ADH as a sensor reaction in the yeast Saccharomyces cerevisiae, grown in anaerobic glucose-limited chemostats under steady-state and dynamic conditions. The results found in this study showed that the cytosolic redox status (NAD:NADH ratio) of yeast is at least one order of magnitude lower, and is thus much more reduced, under anaerobic conditions compared to aerobic glucose-limited steady-state conditions. The more reduced state of the cytosol under anaerobic conditions has major implications for (central) metabolism. Accurate determination of the free NAD:NADH ratio is therefore of importance for the unravelling of in vivo enzyme kinetics and to judge accurately the thermodynamic reversibility of each redox reaction. Copyright © 2015 John Wiley & Sons, Ltd.

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

    USDA-ARS?s Scientific Manuscript database

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

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

    PubMed

    Hecht, K; Wrba, A; Jaenicke, R

    1989-07-15

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

  2. A probe for NADH and H2O2 amperometric detection at low applied potential for oxidase and dehydrogenase based biosensor applications.

    PubMed

    Ricci, Francesco; Amine, Aziz; Moscone, Danila; Palleschi, Giuseppe

    2007-01-15

    Modified screen-printed electrodes for amperometric detection of H(2)O(2) and nicotinamide adenine dinucleotide (NADH) at low applied potential are presented in this paper. The sensors are obtained by modifying the working electrode surface with Prussian Blue, a well known electrochemical mediator for H(2)O(2) reduction. The coupling of this sensor with phenazine methosulfate (PMS) in the working solution gives the possibility of measuring both NAD(P)H and H(2)O(2). PMS reacts with NADH producing PMSH, which in the presence of oxygen, gives an equimolar amount of H(2)O(2). This allows the measurement of both analytes with similar sensitivity (357 mA mol(-1)L cm(-2) for H(2)O(2) and 336 mA mol(-1)L cm(-2) for NADH) and LOD (5x10(-7)mol L(-1) for H(2)O(2) and NADH) and opens the possibility of a whole series of biosensor applications. In this paper, results obtained with a variety of dehydrogenase enzymes (alcohol, malic, lactate, glucose, glycerol and glutamate) for the detection of enzymatic substrates or enzymatic activity are presented demonstrating the suitability of the proposed method for future biosensor applications.

  3. Calcium signaling in brain mitochondria: interplay of malate aspartate NADH shuttle and calcium uniporter/mitochondrial dehydrogenase pathways.

    PubMed

    Contreras, Laura; Satrústegui, Jorgina

    2009-03-13

    Ca2+ signaling in mitochondria has been mainly attributed to Ca2+ entry to the matrix through the Ca2+ uniporter and activation of mitochondrial matrix dehydrogenases. However, mitochondria can also sense increases in cytosolic Ca2+ through a mechanism that involves the aspartate-glutamate carriers, extramitochondrial Ca2+ activation of the NADH malate-aspartate shuttle (MAS). Both pathways are linked through the shared substrate alpha-ketoglutarate (alphaKG). Here we have studied the interplay between the two pathways under conditions of Ca2+ activation. We show that alphaKG becomes limiting when Ca2+ enters in brain or heart mitochondria, but not liver mitochondria, resulting in a drop in alphaKG efflux through the oxoglutarate carrier and in a drop in MAS activity. Inhibition of alphaKG efflux and MAS activity by matrix Ca2+ in brain mitochondria was fully reversible upon Ca2+ efflux. Because of their differences in cytosolic calcium concentration requirements, the MAS and Ca2+ uniporter-mitochondrial dehydrogenase pathways are probably sequentially activated during a Ca2+ transient, and the inhibition of MAS at the center of the transient may provide an explanation for part of the increase in lactate observed in the stimulated brain in vivo.

  4. Achromobacter denitrificans Strain YD35 Pyruvate Dehydrogenase Controls NADH Production To Allow Tolerance to Extremely High Nitrite Levels

    PubMed Central

    Doi, Yuki; Shimizu, Motoyuki; Fujita, Tomoya; Nakamura, Akira; Takizawa, Noboru

    2014-01-01

    We identified the extremely nitrite-tolerant bacterium Achromobacter denitrificans YD35 that can grow in complex medium containing 100 mM nitrite (NO2−) under aerobic conditions. Nitrite induced global proteomic changes and upregulated tricarboxylate (TCA) cycle enzymes as well as antioxidant proteins in YD35. Transposon mutagenesis generated NO2−-hypersensitive mutants of YD35 that had mutations at genes for aconitate hydratase and α-ketoglutarate dehydrogenase in the TCA cycle and a pyruvate dehydrogenase (Pdh) E1 component, indicating the importance of TCA cycle metabolism to NO2− tolerance. A mutant in which the pdh gene cluster was disrupted (Δpdh mutant) could not grow in the presence of 100 mM NO2−. Nitrite decreased the cellular NADH/NAD+ ratio and the cellular ATP level. These defects were more severe in the Δpdh mutant, indicating that Pdh contributes to upregulating cellular NADH and ATP and NO2−-tolerant growth. Exogenous acetate, which generates acetyl coenzyme A and then is metabolized by the TCA cycle, compensated for these defects caused by disruption of the pdh gene cluster and those caused by NO2−. These findings demonstrate a link between NO2− tolerance and pyruvate/acetate metabolism through the TCA cycle. The TCA cycle mechanism in YD35 enhances NADH production, and we consider that this contributes to a novel NO2−-tolerating mechanism in this strain. PMID:24413603

  5. Visible light-driven NADH regeneration sensitized by proflavine for biocatalysis.

    PubMed

    Nam, Dong Heon; Park, Chan Beum

    2012-06-18

    Harvest time: Proflavine drives the reduction of NAD(+) in the presence of a Rh-based electron mediator. Photoregenerated NADH was enzymatically active for oxidation by NADH-dependent L-glutamate dehydrogenase for the synthesis of L-glutamate. This work suggests that proflavine has the potential to become an efficient light-harvesting component in biocatalytic photosynthesis driven by solar energy. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Molecular, biochemical, and functional characterization of a Nudix hydrolase protein that stimulates the activity of a nicotinoprotein alcohol dehydrogenase.

    PubMed

    Kloosterman, Harm; Vrijbloed, Jan W; Dijkhuizen, Lubbert

    2002-09-20

    The cytoplasmic coenzyme NAD(+)-dependent alcohol (methanol) dehydrogenase (MDH) employed by Bacillus methanolicus during growth on C(1)-C(4) primary alcohols is a decameric protein with 1 Zn(2+)-ion and 1-2 Mg(2+)-ions plus a tightly bound NAD(H) cofactor per subunit (a nicotinoprotein). Mg(2+)-ions are essential for binding of NAD(H) cofactor in MDH protein expressed in Escherichia coli. The low coenzyme NAD(+)-dependent activity of MDH with C(1)-C(4) primary alcohols is strongly stimulated by a second B. methanolicus protein (ACT), provided that MDH contains NAD(H) cofactor and Mg(2+)-ions are present in the assay mixture. Characterization of the act gene revealed the presence of the highly conserved amino acid sequence motif typical of Nudix hydrolase proteins in the deduced ACT amino acid sequence. The act gene was successfully expressed in E. coli allowing purification and characterization of active ACT protein. MDH activation by ACT involved hydrolytic removal of the nicotinamide mononucleotide NMN(H) moiety of the NAD(H) cofactor of MDH, changing its Ping-Pong type of reaction mechanism into a ternary complex reaction mechanism. Increased cellular NADH/NAD(+) ratios may reduce the ACT-mediated activation of MDH, thus preventing accumulation of toxic aldehydes. This represents a novel mechanism for alcohol dehydrogenase activity regulation.

  7. Identification of a subunit of NADH-dehydrogenase as a p49/STRAP-binding protein.

    PubMed

    Zhang, Xiaomin; Azhar, Gohar; Helms, Scott; Zhong, Ying; Wei, Jeanne Y

    2008-01-29

    The p49/STRAP (or SRFBP1) protein was recently identified in our laboratory as a cofactor of serum response factor that contributes to the regulation of SRF target genes in the heart. In the present study, we report that NDUFAB1, a nuclear encoded subunit of NADH dehydrogenase, represented the majority of the cDNA clones that interacted with p49/STRAP in multiple screenings using the yeast two-hybrid system. The p49/STRAP and NDUFAB1 proteins interacted and co-localized with each other in the cell. The p49/STRAP protein contains four classic nuclear localization sequence motifs, and it was observed to be present predominantly in the nucleus. Overexpression of p49/STRAP altered the intracellular level of NAD, and reduced the NAD/NADH ratio. Overexpression of p49/STRAP also induced the deacetylation of serum response factor. These data suggest that p49/STRAP plays a role in the regulation of intracellular processes such as cardiac cellular metabolism, gene expression, and possibly aging.

  8. Identification of a subunit of NADH-dehydrogenase as a p49/STRAP-binding protein

    PubMed Central

    Zhang, Xiaomin; Azhar, Gohar; Helms, Scott; Zhong, Ying; Wei, Jeanne Y

    2008-01-01

    Background The p49/STRAP (or SRFBP1) protein was recently identified in our laboratory as a cofactor of serum response factor that contributes to the regulation of SRF target genes in the heart. Results In the present study, we report that NDUFAB1, a nuclear encoded subunit of NADH dehydrogenase, represented the majority of the cDNA clones that interacted with p49/STRAP in multiple screenings using the yeast two-hybrid system. The p49/STRAP and NDUFAB1 proteins interacted and co-localized with each other in the cell. The p49/STRAP protein contains four classic nuclear localization sequence motifs, and it was observed to be present predominantly in the nucleus. Overexpression of p49/STRAP altered the intracellular level of NAD, and reduced the NAD/NADH ratio. Overexpression of p49/STRAP also induced the deacetylation of serum response factor. Conclusion These data suggest that p49/STRAP plays a role in the regulation of intracellular processes such as cardiac cellular metabolism, gene expression, and possibly aging. PMID:18230186

  9. The plastid ndh genes code for an NADH-specific dehydrogenase: Isolation of a complex I analogue from pea thylakoid membranes

    PubMed Central

    Sazanov, Leonid A.; Burrows, Paul A.; Nixon, Peter J.

    1998-01-01

    The plastid genomes of several plants contain ndh genes—homologues of genes encoding subunits of the proton-pumping NADH:ubiquinone oxidoreductase, or complex I, involved in respiration in mitochondria and eubacteria. From sequence similarities with these genes, the ndh gene products have been suggested to form a large protein complex (Ndh complex); however, the structure and function of this complex remains to be established. Herein we report the isolation of the Ndh complex from the chloroplasts of the higher plant Pisum sativum. The purification procedure involved selective solubilization of the thylakoid membrane with dodecyl maltoside, followed by two anion-exchange chromatography steps and one size-exclusion chromatography step. The isolated Ndh complex has an apparent total molecular mass of approximately 550 kDa and according to SDS/PAGE consists of at least 16 subunits including NdhA, NdhI, NdhJ, NdhK, and NdhH, which were identified by N-terminal sequencing and immunoblotting. The Ndh complex showed an NADH- and deamino-NADH-specific dehydrogenase activity, characteristic of complex I, when either ferricyanide or the quinones menadione and duroquinone were used as electron acceptors. This study describes the isolation of the chloroplast analogue of the respiratory complex I and provides direct evidence for the function of the plastid Ndh complex as an NADH:plastoquinone oxidoreductase. Our results are compatible with a dual role for the Ndh complex in the chlororespiratory and cyclic photophosphorylation pathways. PMID:9448329

  10. Identification of the Catalytic Ubiquinone-binding Site of Vibrio cholerae Sodium-dependent NADH Dehydrogenase

    PubMed Central

    Tuz, Karina; Li, Chen; Fang, Xuan; Raba, Daniel A.; Liang, Pingdong; Minh, David D. L.; Juárez, Oscar

    2017-01-01

    The sodium-dependent NADH dehydrogenase (Na+-NQR) is a key component of the respiratory chain of diverse prokaryotic species, including pathogenic bacteria. Na+-NQR uses the energy released by electron transfer between NADH and ubiquinone (UQ) to pump sodium, producing a gradient that sustains many essential homeostatic processes as well as virulence factor secretion and the elimination of drugs. The location of the UQ binding site has been controversial, with two main hypotheses that suggest that this site could be located in the cytosolic subunit A or in the membrane-bound subunit B. In this work, we performed alanine scanning mutagenesis of aromatic residues located in transmembrane helices II, IV, and V of subunit B, near glycine residues 140 and 141. These two critical glycine residues form part of the structures that regulate the site's accessibility. Our results indicate that the elimination of phenylalanine residue 211 or 213 abolishes the UQ-dependent activity, produces a leak of electrons to oxygen, and completely blocks the binding of UQ and the inhibitor HQNO. Molecular docking calculations predict that UQ interacts with phenylalanine 211 and pinpoints the location of the binding site in the interface of subunits B and D. The mutagenesis and structural analysis allow us to propose a novel UQ-binding motif, which is completely different compared with the sites of other respiratory photosynthetic complexes. These results are essential to understanding the electron transfer pathways and mechanism of Na+-NQR catalysis. PMID:28053088

  11. Purification and Characterization of Two Distinct NAD(P)H Dehydrogenases from Onion (Allium cepa L.) Root Plasma Membrane.

    PubMed Central

    Serrano, A.; Cordoba, F.; Gonzalez-Reyes, J. A.; Navas, P.; Villalba, J. M.

    1994-01-01

    Highly purified plasma membrane fractions were obtained from onion (Allium cepa L.) roots and used as a source for purification of redox proteins. Plasma membranes solubilized with Triton X-100 contained two distinct polypeptides showing NAD(P)H-dependent dehydrogenase activities. Dehydrogenase I was purified by gel filtration in Sephacryl S-300 HR, ion-exchange chromatography in DEAE-Sepharose CL-6B, and dye-ligand affinity chromatography in Blue-Sepharose CL-6B after biospecific elution with NADH. Dehydrogenase I consisted of a single polypeptide of about 27 kD and an isoelectric point of about 6. Dehydrogenase II was purified from the DEAE-unbound fraction by chromatography in Blue-Sepharose CL-6B and affinity elution with NADH. Dehydrogenase II consisted of a single polypeptide of about 31 kD and an isoelectric point of about 8. Purified dehydrogenase I oxidized both NADPH and NADH, although higher rates of electron transfer were obtained with NADPH. Maximal activity was achieved with NADPH as donor and juglone or coenzyme Q as acceptor. Dehydrogenase II was specific for NADH and exhibited maximal activity with ferricyanide. Optimal pH for both dehydrogenases was about 6. Dehydrogenase I was moderately inhibited by dicumarol, thenoyltrifluoroacetone, and the thiol reagent N-ethyl-maleimide. A strong inhibition of dehydrogenase II was obtained with dicumarol, thenoyltrifluoroacetone, and the thiol reagent p-hydroxymercuribenzoate. PMID:12232306

  12. Crystallographic analysis and structure-guided engineering of NADPH-dependent Ralstonia sp. alcohol dehydrogenase toward NADH cosubstrate specificity.

    PubMed

    Lerchner, Alexandra; Jarasch, Alexander; Meining, Winfried; Schiefner, André; Skerra, Arne

    2013-11-01

    The NADP⁺-dependent alcohol dehydrogenase from Ralstonia sp. (RasADH) belongs to the protein superfamily of short-chain dehydrogenases/reductases (SDRs). As an enzyme that accepts different types of substrates--including bulky-bulky as well as small-bulky secondary alcohols or ketones--with high stereoselectivity, it offers potential as a biocatalyst for industrial biotechnology. To understand substrate and cosubstrate specificities of RasADH we determined the crystal structure of the apo-enzyme as well as its NADP⁺-bound state with resolutions down to 2.8 Å. RasADH displays a homotetrameric quaternary structure that can be described as a dimer of homodimers while in each subunit a seven-stranded parallel β-sheet, flanked by three α-helices on each side, forms a Rossmann fold-type dinucleotide binding domain. Docking of the well-known substrate (S)-1-phenylethanol clearly revealed the structural determinants of stereospecificity. To favor practical RasADH application in the context of established cofactor recycling systems, for example, those involving an NADH-dependent amino acid dehydrogenase, we attempted to rationally change its cosubstrate specificity from NADP⁺ to NAD⁺ utilizing the structural information that NADP⁺ specificity is largely governed by the residues Asn15, Gly37, Arg38, and Arg39. Furthermore, an extensive sequence alignment with homologous dehydrogenases that have different cosubstrate specificities revealed a modified general SDR motif ASNG (instead of NNAG) at positions 86-89 of RasADH. Consequently, we constructed mutant enzymes with one (G37D), four (N15G/G37D/R38V/R39S), and six (N15G/G37D/R38V/R39S/A86N/S88A) amino acid exchanges. RasADH (N15G/G37D/R38V/R39S) was better able to accept NAD⁺ while showing much reduced catalytic efficiency with NADP⁺, leading to a change in NADH/NADPH specificity by a factor of ∼3.6 million. © 2013 Wiley Periodicals, Inc.

  13. Redox Specificity of 2-Hydroxyacid-Coupled NAD+/NADH Dehydrogenases: A Study Exploiting “Reactive” Arginine as a Reporter of Protein Electrostatics

    PubMed Central

    Durani, Susheel

    2013-01-01

    With “reactive” arginine as a kinetic reporter, 2-hydroxyacid dehydrogenases are assessed in basis of their specialization as NAD+-reducing or NADH-oxidizing enzymes. Specifically, M4 and H4 lactate dehydrogenases (LDHs) and cytoplasmic and mitochondrial malate dehydrogenases (MDHs) are compared to assess if their coenzyme specificity may involve electrostatics of cationic or neutral nicotinamide structure as the basis. The enzymes from diverse eukaryote and prokaryote sources thus are assessed in “reactivity” of functionally-critical arginine as a function of salt concentration and pH. Electrostatic calculations were performed on “reactive” arginines and found good correspondence with experiment. The reductive and oxidative LDHs and MDHs are assessed in their count over ionizable residues and in placement details of the residues in their structures as proteins. The variants found to be high or low in ΔpKa of “reactive” arginine are found to be also strong or weak cations that preferentially oxidize NADH (neutral nicotinamide structure) or reduce NAD+ (cationic nicotinamide structure). The ionized groups of protein structure may thus be important to redox specificity of the enzyme on basis of electrostatic preference for the oxidized (cationic nicotinamide) or reduced (neutral nicotinamide) coenzyme. Detailed comparisons of isozymes establish that the residues contributing in their redox specificity are scrambled in structure of the reductive enzyme. PMID:24391777

  14. Bioinspired Design of Alcohol Dehydrogenase@nano TiO₂ Microreactors for Sustainable Cycling of NAD⁺/NADH Coenzyme.

    PubMed

    Lin, Sen; Sun, Shiyong; Wang, Ke; Shen, Kexuan; Ma, Biaobiao; Ren, Yuquan; Fan, Xiaoyu

    2018-02-24

    The bioinspired design and construction of enzyme@capsule microreactors with specific cell-like functionality has generated tremendous interest in recent years. Inspired by their fascinating complexity, scientists have endeavored to understand the essential aspects of a natural cell and create biomimicking microreactors so as to immobilize enzymes within the hierarchical structure of a microcapsule. In this study, simultaneous encapsulation of alcohol dehydrogenase (ADH) was achieved during the preparation of microcapsules by the Pickering emulsion method using amphiphilic modified TiO₂ nanoparticles (NPs) as building blocks for assembling the photocatalytic microcapsule membrane. The ADH@TiO₂ NP microreactors exhibited dual catalytic functions, i.e., spatially confined enzymatic catalysis and the membrane-associated photocatalytic oxidation under visible light. The sustainable cycling of nicotinamide adenine dinucleotide (NAD) coenzyme between NADH and NAD⁺ was realized by enzymatic regeneration of NADH from NAD⁺ reduction, and was provided in a form that enabled further photocatalytic oxidation to NAD⁺ under visible light. This bioinspired ADH@TiO₂ NP microreactor allowed the linking of a semiconductor mineral-based inorganic photosystem to enzymatic reactions. This is a first step toward the realization of sustainable biological cycling of NAD⁺/NADH coenzyme in synthetic functional microsystems operating under visible light irradiation.

  15. Macromolecular crowding effect upon in vitro enzyme kinetics: mixed activation-diffusion control of the oxidation of NADH by pyruvate catalyzed by lactate dehydrogenase.

    PubMed

    Balcells, Cristina; Pastor, Isabel; Vilaseca, Eudald; Madurga, Sergio; Cascante, Marta; Mas, Francesc

    2014-04-17

    Enzyme kinetics studies have been usually designed as dilute solution experiments, which differ substantially from in vivo conditions. However, cell cytosol is crowded with a high concentration of molecules having different shapes and sizes. The consequences of such crowding in enzymatic reactions remain unclear. The aim of the present study is to understand the effect of macromolecular crowding produced by dextran of different sizes and at diverse concentrations in the well-known reaction of oxidation of NADH by pyruvate catalyzed by L-lactate dehydrogenase (LDH). Our results indicate that the reaction rate is determined by both the occupied volume and the relative size of dextran obstacles with respect to the enzyme present in the reaction. Moreover, we analyzed the influence of macromolecular crowding on the Michaelis-Menten constants, vmax and Km. The obtained results show that only high concentrations and large sizes of dextran reduce both constants suggesting a mixed activation-diffusion control of this enzymatic reaction due to the dextran crowding action. From our knowledge, this is the first experimental study that depicts mixed activation-diffusion control in an enzymatic reaction due to the effect of crowding.

  16. Malate dehydrogenase of the cytosol. A kinetic investigation of the reaction mechanism and a comparison with lactate dehydrogenase.

    PubMed Central

    Lodola, A; Shore, J D; Parker, D M; Holbrook, J

    1978-01-01

    1. The mechanisms of the reduction of oxaloacetate and of 3-fluoro-oxaloacetate by NADH catalysed by cytoplasmic pig heart malate dehydrogenase (MDH) were investigated. 2. One mol of dimeric enzyme produces 1.7+/-0.4 mol of enzyme-bound NADH when mixed with saturating NAD+ and L-malate at a rate much higher than the subsequent turnover at pH 7.5. 3. Transient measurements of protein and nucleotide fluorescence show that the steady-state complex in the forward direction is MDH-NADH and in the reverse direction MDH-NADH-oxaloacetate. 4. The rate of dissociation of MDH-NADH was measured and is the same as Vmax. in the forward direction at pH 7.5. Both NADH-binding sites are kinetically equivalent. The rate of dissociation varies with pH, as does the equilibrium binding constant for NADH. 5. 3-Fluoro-oxaloacetate is composed of three forms (F1, F2 and S) of which F1 and F2 are immediately substrates for the enzyme. The third form, S, is not a substrate, but when the F forms are used up form S slowly and non-enzymically equilibrates to yield the active substrate forms. S is 2,2-dihydroxy-3-fluorosuccinate. 6. The steady-state compound during the reduction of form F1 is an enzyme form that does not contain NADH, probably MDH-NAD+-fluoromalate. The steady-state compound for form F2 is an enzyme form containing NADH, probably MDH-NADH-fluoro-oxaloacetate. 7. The rate-limiting reaction in the reduction of form F2 shows a deuterium isotope rate ratio of 4 when NADH is replaced by its deuterium analogue, and the rate-limiting reaction is concluded to be hydride transfer. 8. A novel titration was used to show that dimeric cytoplasmic malate dehydrogenase contains two sites that can rapidly reduce the F1 form of 3-fluoro-oxaloacetate. The enzyme shows 'all-of-the-sites' behaviour. 9. Partial mechanisms are proposed to explain the enzyme-catalysed transformations of the natural and the fluoro substrates. These mechanisms are similar to the mechanism of pig heart lactate

  17. Identification of a magnesium-dependent NAD(P)(H)-binding domain in the nicotinoprotein methanol dehydrogenase from Bacillus methanolicus.

    PubMed

    Hektor, Harm J; Kloosterman, Harm; Dijkhuizen, Lubbert

    2002-12-06

    The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn(2+) ion, one or two Mg(2+) ions, and a tightly bound cofactor NAD(H) per subunit. The Mg(2+) ions are essential for binding of cofactor NAD(H) in MDH. A B. methanolicus activator protein strongly stimulates the relatively low coenzyme NAD(+)-dependent MDH activity, involving hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) (Kloosterman, H., Vrijbloed, J. W., and Dijkhuizen, L. (2002) J. Biol. Chem. 277, 34785-34792). Members of family III of NAD(P)-dependent alcohol dehydrogenases contain three unique, conserved sequence motifs (domains A, B, and C). Domain C is thought to be involved in metal binding, whereas the functions of domains A and B are still unknown. This paper provides evidence that domain A constitutes (part of) a new magnesium-dependent NAD(P)(H)-binding domain. Site-directed mutants D100N and K103R lacked (most of the) bound cofactor NAD(H) and had lost all coenzyme NAD(+)-dependent MDH activity. Also mutants G95A and S97G were both impaired in cofactor NAD(H) binding but retained coenzyme NAD(+)-dependent MDH activity. Mutant G95A displayed a rather low MDH activity, whereas mutant S97G was insensitive to activator protein but displayed "fully activated" MDH reaction rates. The various roles of these amino acid residues in coenzyme and/or cofactor NAD(H) binding in MDH are discussed.

  18. Dissection of the Caffeate Respiratory Chain in the Acetogen Acetobacterium woodii: Identification of an Rnf-Type NADH Dehydrogenase as a Potential Coupling Site▿

    PubMed Central

    Imkamp, Frank; Biegel, Eva; Jayamani, Elamparithi; Buckel, Wolfgang; Müller, Volker

    2007-01-01

    The anaerobic acetogenic bacterium Acetobacterium woodii couples caffeate reduction with electrons derived from hydrogen to the synthesis of ATP by a chemiosmotic mechanism with sodium ions as coupling ions, a process referred to as caffeate respiration. We addressed the nature of the hitherto unknown enzymatic activities involved in this process and their cellular localization. Cell extract of A. woodii catalyzes H2-dependent caffeate reduction. This reaction is strictly ATP dependent but can be activated also by acetyl coenzyme A (CoA), indicating that there is formation of caffeyl-CoA prior to reduction. Two-dimensional gel electrophoresis revealed proteins present only in caffeate-grown cells. Two proteins were identified by electrospray ionization-mass spectrometry/mass spectrometry, and the encoding genes were cloned. These proteins are very similar to subunits α (EtfA) and β (EtfB) of electron transfer flavoproteins present in various anaerobic bacteria. Western blot analysis demonstrated that they are induced by caffeate and localized in the cytoplasm. Etf proteins are known electron carriers that shuttle electrons from NADH to different acceptors. Indeed, NADH was used as an electron donor for cytosolic caffeate reduction. Since the hydrogenase was soluble and used ferredoxin as an electron acceptor, the missing link was a ferredoxin:NAD+ oxidoreductase. This activity could be determined and, interestingly, was membrane bound. A search for genes that could encode this activity revealed DNA fragments encoding subunits C and D of a membrane-bound Rnf-type NADH dehydrogenase that is a potential Na+ pump. These data suggest the following electron transport chain: H2 → ferredoxin → NAD+ → Etf → caffeyl-CoA reductase. They also imply that the sodium motive step in the chain is the ferredoxin-dependent NAD+ reduction catalyzed by Rnf. PMID:17873051

  19. Cofactor-Dependent Aldose Dehydrogenase of Rhodopseudomonas spheroides

    PubMed Central

    Niederpruem, Donald J.; Doudoroff, Michael

    1965-01-01

    Niederpruem, Donald J. (University of California, Berkeley), and Michael Doudoroff. Cofactor-dependent aldose dehydrogenase of Rhodopseudomonas spheroides. J. Bacteriol. 89:697–705. 1965.—Particulate enzyme preparations of cell extracts of Rhodopseudomonas spheroides possess constitutive dehydrogenase and oxidase activities for aldose sugars, reduced nicotinamide adenine dinucleotide (NADH2), and succinate. The dehydrogenation of aldoses requires an unidentified cofactor which is not required for the oxidation of succinate nor of NADH2. The cofactor is present in the particulate fraction of aerobic cells, but is unavailable to the enzyme system. It can be liberated by boiling or by treatment with salts at high concentration. The cofactor also appears in the soluble fraction of aerobic cells, but only after exponential growth has ceased. Extracts of cells grown anaerobically in the light possess the apoenzyme, but not the cofactor, for aldose oxidation. Cofactor activity was found in extracts of Bacterium anitratum (= Moraxella sp.) but not in Escherichia coli, Pseudomonas fluorescens, yeast, or mouse liver. In 0.075 m tris(hydroxymethyl)aminomethane-phosphoric acid buffer (pH 7.3), the oxidation of NADH2 was stimulated and succinoxidase was inhibited by high salt concentrations. PMID:14273648

  20. Lambda Red-mediated mutagenesis and efficient large scale affinity purification of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I).

    PubMed

    Pohl, Thomas; Uhlmann, Mareike; Kaufenstein, Miriam; Friedrich, Thorsten

    2007-09-18

    The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. The Escherichia coli complex I consists of 13 different subunits named NuoA-N (from NADH:ubiquinone oxidoreductase), that are coded by the genes of the nuo-operon. Genetic manipulation of the operon is difficult due to its enormous size. The enzymatic activity of variants is obscured by an alternative NADH dehydrogenase, and purification of the variants is hampered by their instability. To overcome these problems the entire E. coli nuo-operon was cloned and placed under control of the l-arabinose inducible promoter ParaBAD. The exposed N-terminus of subunit NuoF was chosen for engineering the complex with a hexahistidine-tag by lambda-Red-mediated recombineering. Overproduction of the complex from this construct in a strain which is devoid of any membrane-bound NADH dehydrogenase led to the assembly of a catalytically active complex causing the entire NADH oxidase activity of the cytoplasmic membranes. After solubilization with dodecyl maltoside the engineered complex binds to a Ni2+-iminodiacetic acid matrix allowing the purification of approximately 11 mg of complex I from 25 g of cells. The preparation is pure and monodisperse and comprises all known subunits and cofactors. It contains more lipids than earlier preparations due to the gentle and fast purification procedure. After reconstitution in proteoliposomes it couples the electron transfer with proton translocation in an inhibitor sensitive manner, thus meeting all prerequisites for structural and functional studies.

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

    PubMed

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

    2015-03-01

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

  2. Differential Role of Glutamate Dehydrogenase in Nitrogen Metabolism of Maize Tissues 1

    PubMed Central

    Loyola-Vargas, Victor Manuel; de Jimenez, Estela Sanchez

    1984-01-01

    Both calli and plantlets of maize (Zea mays L. var Tuxpeño 1) were exposed to specific nitrogen sources, and the aminative (NADH) and deaminative (NAD+) glutamate dehydrogenase activities were measured at various periods of time in homogenates of calli, roots, and leaves. A differential effect of the nitrogen sources on the tissues tested was observed. In callus tissue, glutamate, ammonium, and urea inhibited glutamate dehydrogenase (GDH) activity. The amination and deamination reactions also showed different ratios of activity under different nitrogen sources. In roots, ammonium and glutamine produced an increase in GDH-NADH activity whereas the same metabolites were inhibitory of this activity in leaves. These data suggest the presence of isoenzymes or conformers of GDH, specific for each tissue, whose activities vary depending on the nutritional requirements of the tissue and the state of differentiation. PMID:16663876

  3. Mitochondrial NADH Fluorescence is Enhanced by Complex I Binding

    PubMed Central

    Blinova, Ksenia; Levine, Rodney L.; Boja, Emily S.; Griffiths, Gary L.; Shi, Zhen-Dan; Ruddy, Brian; Balaban, Robert S.

    2012-01-01

    Mitochondrial NADH fluorescence has been a useful tool in evaluating mitochondrial energetics both in vitro and in vivo. Mitochondrial NADH fluorescence is enhanced several fold in the matrix through extended fluorescence lifetimes (EFL). However, the actual binding sites responsible for NADH EFL are unknown. We tested the hypothesis that NADH binding to Complex I is a significant source of mitochondrial NADH fluorescence enhancement. To test this hypothesis, the effect of Complex I binding on NADH fluorescence efficiency was evaluated in purified protein, and in native gels of the entire porcine heart mitochondria proteome. To avoid the oxidation of NADH in these preparations, we conducted the binding experiments under anoxic conditions in a specially designed apparatus. Purified intact Complex I enhanced NADH fluorescence in native gels approximately 10 fold. However, no enhancement was detected in denatured individual Complex I subunit proteins. In the Clear and Ghost native gels of the entire mitochondrial proteome, NADH fluorescence enhancement was localized to regions where NADH oxidation occurred in the presence of oxygen. Inhibitor and mass spectroscopy studies revealed that the fluorescence enhancement was specific to Complex I proteins. No fluorescence enhancement was detected for MDH or other dehydrogenases in this assay system, at physiological mole fractions of the matrix proteins. These data suggest that NADH associated with Complex I significantly contributes to the overall mitochondrial NADH fluorescence signal and provides an explanation for the well established close correlation of mitochondrial NADH fluorescence and the metabolic state. PMID:18702505

  4. Paper-Based Device for Rapid Visualization of NADH Based on Dissolution of Gold Nanoparticles.

    PubMed

    Liang, Pingping; Yu, Haixiang; Guntupalli, Bhargav; Xiao, Yi

    2015-07-15

    We describe a paper-based device that enables rapid and sensitive room-temperature detection of dihydronicotinamide adenine dinucleotide (NADH) via a colorimetric readout and demonstrate its value for monitoring NAD+-driven enzymatic reactions. Our system is based on NADH-mediated inhibition of gold nanoparticle (AuNPs) dissolution in a Au3+-cetyltrimethylammonium bromide (CTAB) solution. We fabricated a device consisting of a mixed cellulose ester paper featuring a wax-encircled, AuNP-coated film atop a cotton absorbent layer sandwiched between two plastic cover layers. In the absence of NADH, the Au3+-CTAB complex dissolves the AuNP layer completely, generating a white color in the test zone. In the presence of NADH, Au3+ is rapidly reduced to Au+, greatly decreasing the dissolution of AuNPs and yielding a red color that becomes stronger at increasing concentrations of NADH. This device exploits capillary force-assisted vertical diffusion, allowing us to apply a 25 μL sample to a surface-confined test zone to achieve a detection limit of 12.5 μM NADH. We used the enzyme glucose dehydrogenase as a model to demonstrate that our paper-based device can monitor NAD+-driven biochemical processes with and without selective dehydrogenase inhibitors by naked-eye observation within 4 min at room temperature in a small sample volume. We believe that our paper-based device could offer a valuable and low-cost analytical tool for monitoring NAD+-associated enzymatic reactions and screening for dehydrogenase inhibitors in a variety of testing contexts.

  5. L-Malate dehydrogenase activity in the reductive arm of the incomplete citric acid cycle of Nitrosomonas europaea.

    PubMed

    Deutch, Charles E

    2013-11-01

    The autotrophic nitrifying bacterium Nitrosomonas europaea does not synthesize 2-oxoglutarate (α-ketoglutarate) dehydrogenase under aerobic conditions and so has an incomplete citric acid cycle. L-malate (S-malate) dehydrogenase (MDH) from N. europaea was predicted to show similarity to the NADP(+)-dependent enzymes from chloroplasts and was separated from the NAD(+)-dependent proteins from most other bacteria or mitochondria. MDH activity in a soluble fraction from N. europaea ATCC 19718 was measured spectrophotometrically and exhibited simple Michaelis-Menten kinetics. In the reductive direction, activity with NADH increased from pH 6.0 to 8.5 but activity with NADPH was consistently lower and decreased with pH. At pH 7.0, the K m for oxaloacetate was 20 μM; the K m for NADH was 22 μM but that for NADPH was at least 10 times higher. In the oxidative direction, activity with NAD(+) increased with pH but there was very little activity with NADP(+). At pH 7.0, the K m for L-malate was 5 mM and the K m for NAD(+) was 24 μM. The reductive activity was quite insensitive to inhibition by L-malate but the oxidative activity was very sensitive to oxaloacetate. MDH activity was not strongly activated or inhibited by glycolytic or citric acid cycle metabolites, adenine nucleotides, NaCl concentrations, or most metal ions, but increased with temperature up to about 55 °C. The reductive activity was consistently 10-20 times higher than the oxidative activity. These results indicate that the L-malate dehydrogenase in N. europaea is similar to other NAD(+)-dependent MDHs (EC 1.1.1.37) but physiologically adapted for its role in a reductive biosynthetic sequence.

  6. Effects of Al(III) and Nano-Al13 Species on Malate Dehydrogenase Activity

    PubMed Central

    Yang, Xiaodi; Cai, Ling; Peng, Yu; Li, Huihui; Chen, Rong Fu; Shen, Ren Fang

    2011-01-01

    The effects of different aluminum species on malate dehydrogenase (MDH) activity were investigated by monitoring amperometric i-t curves for the oxidation of NADH at low overpotential using a functionalized multi-wall nanotube (MWNT) modified glass carbon electrode (GCE). The results showed that Al(III) and Al13 can activate the enzymatic activity of MDH, and the activation reaches maximum levels as the Al(III) and Al13 concentration increase. Our study also found that the effects of Al(III) and Al13 on the activity of MDH depended on the pH value and aluminum speciation. Electrochemical and circular dichroism spectra methods were applied to study the effects of nano-sized aluminum compounds on biomolecules. PMID:22163924

  7. Effects of Al(III) and nano-Al13 species on malate dehydrogenase activity.

    PubMed

    Yang, Xiaodi; Cai, Ling; Peng, Yu; Li, Huihui; Chen, Rong Fu; Shen, Ren Fang

    2011-01-01

    The effects of different aluminum species on malate dehydrogenase (MDH) activity were investigated by monitoring amperometric i-t curves for the oxidation of NADH at low overpotential using a functionalized multi-wall nanotube (MWNT) modified glass carbon electrode (GCE). The results showed that Al(III) and Al(13) can activate the enzymatic activity of MDH, and the activation reaches maximum levels as the Al(III) and Al(13) concentration increase. Our study also found that the effects of Al(III) and Al(13) on the activity of MDH depended on the pH value and aluminum speciation. Electrochemical and circular dichroism spectra methods were applied to study the effects of nano-sized aluminum compounds on biomolecules.

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

    PubMed

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

    2015-12-01

    Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  9. Interaction of cytoplasmic dehydrogenases: quantitation of pathways of ethanol metabolism.

    PubMed

    Vind, C; Grunnet, N

    1983-01-01

    The interaction between xylitol, alcohol and lactate dehydrogenase has been studied in hepatocytes from rats by applying specifically tritiated substrates. A simple model, describing the metabolic fate of tritium from [2-3H] xylitol and (1R) [1-3H]ethanol is presented. The model allows calculation of the specific radioactivity of free, cytosolic NADH, based on transfer of tritium to lactate, glucose and water. From the initial labelling rate of lactate and the specific radioactivity of cytosolic NADH, we have determined the reversible flow through the lactate dehydrogenase catalyzed reaction to 1-5 mumol/min . g wet wt. The results suggest that xylitol, alcohol and lactate dehydrogenase share the same pool of NAD(H) in the cytoplasma. This finding allows estimation of the ethanol oxidation rate by the non-alcohol dehydrogenase pathways from the relative yield of tritium in water and glucose. The calculations are based on a comparison of the fate of the 1-pro-R hydrogen of ethanol and the hydrogen bound to carbon 2 of xylitol or carbon 2 of lactate under identical conditions.

  10. Insight into the stereospecificity of short-chain thermus thermophilus alcohol dehydrogenase showing pro-S hydride transfer and prelog enantioselectivity.

    PubMed

    Pennacchio, Angela; Giordano, Assunta; Esposito, Luciana; Langella, Emma; Rossi, Mosè; Raia, Carlo A

    2010-04-01

    The stereochemistry of the hydride transfer in reactions catalyzed by NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus HB27 was determined by means of (1)H-NMR spectroscopy. The enzyme transfers the pro-S hydrogen of [4R-(2)H]NADH and exhibits Prelog specificity. Enzyme-substrate docking calculations provided structural details about the enantioselectivity of this thermophilic enzyme. These results give additional insights into the diverse active site architectures of the largely versatile short-chain dehydrogenase superfamily enzymes. A feasible protocol for the synthesis of [4R-(2)H]NADH with high yield was also set up by enzymatic oxidation of 2-propanol-d(8) catalyzed by Bacillus stearothermophilus alcohol dehydrogenase.

  11. The alternative NADH dehydrogenase is present in mitochondria of some animal taxa.

    PubMed

    Matus-Ortega, Macario Genaro; Salmerón-Santiago, Karina Gabriela; Flores-Herrera, Oscar; Guerra-Sánchez, Guadalupe; Martínez, Federico; Rendón, Juan Luis; Pardo, Juan Pablo

    2011-09-01

    The distribution of the alternative NADH dehydrogenase (NDH-2) in the living world was explored. The enzyme, although present in representatives of all living kingdoms, does not have a universal distribution. With the exception of ε-proteobacteria, the enzyme was found in all eubacterial groups. In contrast with the known presence of the NDH-2 in Archaea, the alternative oxidase (AOX) is absent in this group. With regard to the Eukarya domain, the NDH-2 was found in representatives of Protista, Fungi, Plantae, and Animalia. In the latter, however, the presence of the enzyme was restricted to some primitive Metazoa (Placozoa and Cnidaria), and two members of the Deuterostomate lineage of the Bilateria (Echinodermata and Urochordata). No evidence for the presence of the NDH-2 was found in any representative of the Protostomate branch of the Bilateria, contrasting with the existence of the AOX in this same group. It is worth mentioning that those animal species containing the NDH-2 also have an AOX. The actual distribution of the NDH-2 in the various living kingdoms is discussed within the framework of the endosymbiotic theory; in addition, a hypothesis is proposed to explain the disappearance of the alternative NDH-2 and AOX from the majority of the animals. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Extremely high intracellular concentration of glucose-6-phosphate and NAD(H) in Deinococcus radiodurans.

    PubMed

    Yamashiro, Takumi; Murata, Kousaku; Kawai, Shigeyuki

    2017-03-01

    Deinococcus radiodurans is highly resistant to ionizing radiation and UV radiation, and oxidative stress caused by such radiations. NADP(H) seems to be important for this resistance (Slade and Radman, Microbiol Mol Biol Rev 75:133-191; Slade, Radman, Microbiol Mol Biol Rev 75:133-191, 2011), but the mechanism underlying the generation of NADP(H) or NAD(H) in D. radiodurans has not fully been addressed. Intracellular concentrations of NAD + , NADH, NADP + , and NADPH in D. radiodurans are also not determined yet. We found that cell extracts of D. radiodurans catalyzed reduction of NAD(P) + in vitro, indicating that D. radiodurans cells contain both enzymes and a high concentration of substrates for this activity. The enzyme and the substrate were attributed to glucose-6-phosphate dehydrogenase and glucose-6-phosphate of which intracellular concentration was extremely high. Unexpectedly, the intracellular concentration of NAD(H) was also much greater than that of NADP(H), suggesting some significant roles of NADH. These unusual features of this bacterium would shed light on a new aspect of physiology of this bacterium.

  13. Live cell imaging of cytosolic NADH/NAD+ ratio in hepatocytes and liver slices.

    PubMed

    Masia, Ricard; McCarty, William J; Lahmann, Carolina; Luther, Jay; Chung, Raymond T; Yarmush, Martin L; Yellen, Gary

    2018-01-01

    Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD + ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD + ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD + ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD + ratio, and this increase is mitigated by the presence of NAD + -generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD + ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD + ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real

  14. Type 2 NADH Dehydrogenases in the Cyanobacterium Synechocystis sp. Strain PCC 6803 Are Involved in Regulation Rather Than Respiration

    PubMed Central

    Howitt, Crispin A.; Udall, Pacer K.; Vermaas, Wim F. J.

    1999-01-01

    Analysis of the genome of Synechocystis sp. strain PCC 6803 reveals three open reading frames (slr0851, slr1743, and sll1484) that may code for type 2 NAD(P)H dehydrogenases (NDH-2). The sequence similarity between the translated open reading frames and NDH-2s from other organisms is low, generally not exceeding 30% identity. However, NAD(P)H and flavin adenine dinucleotide binding motifs are conserved in all three putative NDH-2s in Synechocystis sp. strain PCC 6803. The three open reading frames were cloned, and deletion constructs were made for each. An expression construct containing one of the three open reading frames, slr1743, was able to functionally complement an Escherichia coli mutant lacking both NDH-1s and NDH-2s. Therefore, slr0851, slr1743, and sll1484 have been designated ndbA, ndbB, and ndbC, respectively. Strains that lacked one or more of the ndb genes were created in wild-type and photosystem (PS) I-less backgrounds. Deletion of ndb genes led to small changes in photoautotrophic growth rates and respiratory activities. Electron transfer rates into the plastoquinone pool in thylakoids in darkness were consistent with the presence of a small amount of NDH-2 activity in thylakoids. No difference was observed between wild-type and the Ndb-less strains in the banding patterns seen on native gels when stained for either NADH or NADPH dehydrogenase activity, indicating that the Ndb proteins do not accumulate to high levels. A striking phenotype of the PS I-less background strains lacking one or more of the NDH-2s is that they were able to grow at high light intensities that were lethal to the control strain but they retained normal PS II activity. We suggest that the Ndb proteins in Synechocystis sp. strain PCC 6803 are redox sensors and that they play a regulatory role responding to the redox state of the plastoquinone pool. PMID:10383967

  15. Alcohol Dehydrogenase and Ethanol in the Stems of Trees 1

    PubMed Central

    Kimmerer, Thomas W.; Stringer, Mary A.

    1988-01-01

    Anaerobic fermentation in plants is usually thought to be a transient phenomenon, brought about by environmental limitations to oxygen availability, or by structural constraints to oxygen transport. The vascular cambium of trees is separated from the air by the outer bark and secondary phloem, and we hypothesized that the cambium may experience sufficient hypoxia to induce anaerobic fermentation. We found high alcohol dehydrogenase activity in the cambium of several tree species. Mean activity of alcohol dehydrogenase in Populus deltoides was 165 micromoles NADH oxidized per minute per gram fresh weight in May. Pyruvate decarboxylase activity was also present in the cambium of P. deltoides, with mean activity of 26 micromoles NADH oxidized per minute per gram fresh weight in May. Lactate dehydrogenase activity was not present in any tree species we examined. Contrary to our expectation, alcohol dehydrogenase activity was inversely related to bark thickness in Acer saccharum and unrelated to bark thickness in two Populus species. Bark thickness may be less important in limiting oxygen availability to the cambium than is oxygen consumption by rapidly respiring phloem and cambium in actively growing trees. Ethanol was present in the vascular cambium of all species examined, with mean concentrations of 35 to 143 nanomoles per gram fresh weight, depending on species. Ethanol was also present in xylem sap and may have been released from the cambium into the transpiration stream. The presence in the cambium of the enzymes necessary for fermentation as well as the products of fermentation is evidence that respiration in the vascular cambium of trees may be oxygen-limited, but other biosynthetic origins of ethanol have not been ruled out. PMID:16666209

  16. Short-term regulation of the alpha-ketoglutarate dehydrogenase complex by energy-linked and some other effectors.

    PubMed

    Strumilo, S

    2005-07-01

    The question of regulation of alpha-ketoglutarate dehydrogenase complex (KGDHC) has been considered in the biochemical literature very rarely. Moreover, such information is not usually accurate, especially in biochemical textbooks. From the mini-review of research works published during the last 25 years, the following basic view is clear: a) animal KGDHC is very sensitive to ADP, P(i), and Ca2+; b) these positive effectors increase manifold the affinity of KGDHC to alpha-ketoglutarate; c) KGDHC is inhibited by ATP, NADH, and succinyl-CoA; d) the ATP effect is realized in several ways, probably mainly via opposition versus ADP activation; e) NADH, besides inhibiting dihydrolipoamide dehydrogenase component competitively versus NAD+, decreases the affinity of alpha-ketoglutarate dehydrogenase to substrate and inactivates it; f) thioredoxin protects KGDHC from self-inactivation during catalysis; g) bacterial and plant KGDHC is activated by AMP instead of ADP. These main effects form the basis of short-term regulation of KGDHC.

  17. Evaluation of functioning of mitochondrial electron transport chain with NADH and FAD autofluorescence

    PubMed

    Danylovych, H V

    2016-01-01

    We prove the feasibility of evaluation of mitochondrial electron transport chain function in isolated mitochondria of smooth muscle cells of rats from uterus using fluorescence of NADH and FAD coenzymes. We found the inversely directed changes in FAD and NADH fluorescence intensity under normal functioning of mitochondrial electron transport chain. The targeted effect of inhibitors of complex I, III and IV changed fluorescence of adenine nucleotides. Rotenone (5 μM) induced rapid increase in NADH fluorescence due to inhibition of complex I, without changing in dynamics of FAD fluorescence increase. Antimycin A, a complex III inhibitor, in concentration of 1 μg/ml caused sharp increase in NADH fluorescence and moderate increase in FAD fluorescence in comparison to control. NaN3 (5 mM), a complex IV inhibitor, and CCCP (10 μM), a protonophore, caused decrease in NADH and FAD fluorescence. Moreover, all the inhibitors caused mitochondria swelling. NO donors, e.g. 0.1 mM sodium nitroprusside and sodium nitrite similarly to the effects of sodium azide. Energy-dependent Ca2+ accumulation in mitochondrial matrix (in presence of oxidation substrates and Mg-ATP2- complex) is associated with pronounced drop in NADH and FAD fluorescence followed by increased fluorescence of adenine nucleotides, which may be primarily due to Ca2+- dependent activation of dehydrogenases of citric acid cycle. Therefore, the fluorescent signal of FAD and NADH indicates changes in oxidation state of these nucleotides in isolated mitochondria, which may be used to assay the potential of effectors of electron transport chain.

  18. Structural characterization of the thermostable Bradyrhizobium japonicumD-sorbitol dehydrogenase.

    PubMed

    Fredslund, Folmer; Otten, Harm; Gemperlein, Sabrina; Poulsen, Jens Christian N; Carius, Yvonne; Kohring, Gert Wieland; Lo Leggio, Leila

    2016-11-01

    Bradyrhizobium japonicum sorbitol dehydrogenase is NADH-dependent and is active at elevated temperatures. The best substrate is D-glucitol (a synonym for D-sorbitol), although L-glucitol is also accepted, giving it particular potential in industrial applications. Crystallization led to a hexagonal crystal form, with crystals diffracting to 2.9 Å resolution. In attempts to phase the data, a molecular-replacement solution based upon PDB entry 4nbu (33% identical in sequence to the target) was found. The solution contained one molecule in the asymmetric unit, but a tetramer similar to that found in other short-chain dehydrogenases, including the search model, could be reconstructed by applying crystallographic symmetry operations. The active site contains electron density consistent with D-glucitol and phosphate, but there was not clear evidence for the binding of NADH. In a search for the features that determine the thermostability of the enzyme, the T m for the orthologue from Rhodobacter sphaeroides, for which the structure was already known, was also determined, and this enzyme proved to be considerably less thermostable. A continuous β-sheet is formed between two monomers in the tetramer of the B. japonicum enzyme, a feature not generally shared by short-chain dehydrogenases, and which may contribute to thermostability, as may an increased Pro/Gly ratio.

  19. Enhancement of succinate yield by manipulating NADH/NAD+ ratio and ATP generation.

    PubMed

    Li, Jiaojiao; Li, Yikui; Cui, Zhiyong; Liang, Quanfeng; Qi, Qingsheng

    2017-04-01

    We previously engineered Escherichia coli YL104 to efficiently produce succinate from glucose. In this study, we investigated the relationships between the NADH/NAD + ratio, ATP level, and overall yield of succinate production by using glucose as the carbon source in YL104. First, the use of sole NADH dehydrogenases increased the overall yield of succinate by 7% and substantially decreased the NADH/NAD + ratio. Second, the soluble fumarate reductase from Saccharomyces cerevisiae was overexpressed to manipulate the anaerobic NADH/NAD + ratio and ATP level. Third, another strategy for reducing the ATP level was applied by introducing ATP futile cycling for improving succinate production. Finally, a combination of these methods exerted a synergistic effect on improving the overall yield of succinate, which was 39% higher than that of the previously engineered strain YL104. The study results indicated that regulation of the NADH/NAD + ratio and ATP level is an efficient strategy for succinate production.

  20. Malate-aspartate shuttle and exogenous NADH/cytochrome c electron transport pathway as two independent cytosolic reducing equivalent transfer systems.

    PubMed

    Abbrescia, Daniela Isabel; La Piana, Gianluigi; Lofrumento, Nicola Elio

    2012-02-15

    In mammalian cells aerobic oxidation of glucose requires reducing equivalents produced in glycolytic phase to be channelled into the phosphorylating respiratory chain for the reduction of molecular oxygen. Data never presented before show that the oxidation rate of exogenous NADH supported by the malate-aspartate shuttle system (reconstituted in vitro with isolated liver mitochondria) is comparable to the rate obtained on activation of the cytosolic NADH/cytochrome c electron transport pathway. The activities of these two reducing equivalent transport systems are independent of each other and additive. NADH oxidation induced by the malate-aspartate shuttle is inhibited by aminooxyacetate and by rotenone and/or antimycin A, two inhibitors of the respiratory chain, while the NADH/cytochrome c system remains insensitive to all of them. The two systems may simultaneously or mutually operate in the transfer of reducing equivalents from the cytosol to inside the mitochondria. In previous reports we suggested that the NADH/cytochrome c system is expected to be functioning in apoptotic cells characterized by the presence of cytochrome c in the cytosol. As additional new finding the activity of reconstituted shuttle system is linked to the amount of α-ketoglutarate generated inside the mitochondria by glutamate dehydrogenase rather than by aspartate aminotransferase. Copyright © 2011 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2010-03-01

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

  2. The unique kinetic behavior of the very large NAD-dependent glutamate dehydrogenase from Janthinobacterium lividum.

    PubMed

    Kawakami, Ryushi; Oyama, Masaki; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2010-01-01

    The kinetics of a very large NAD-dependent glutamate dehydrogenase from Janthinobacterium lividum showed positive cooperativity toward alpha-ketoglutarate and NADH, and the Michaelis-Menten type toward ammonium chloride in the absence of the catalytic activator, L-aspartate. An increase in the maximum activity accompanied the decrease in the S(0.5) values for alpha-ketoglutarate and NADH with the addition of L-aspartate, and the kinetic response for alpha-ketoglutarate changed completely to a typical Michaelis-Menten type in the presence of 10 mM L-aspartate.

  3. Inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex by reduced nicotinamide adenine dinucleotide in the presence or absence of calcium ion and effect of adenosine 5'-diphosphate on reduced nicotinamide adenine dinucleotide inhibition.

    PubMed

    Lawlis, V B; Roche, T E

    1981-04-28

    Micromolar Ca2+ markedly reduces NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex [Lawlis, V. B., & Roche, T. E. (1980) Mol. Cell. Biochem. 32, 147-152]. Product inhibition patterns from initial velocity studies conducted at less than 10(-9) M or at 1.5 X 10(-5) M Ca2+ with NAD+, CoA, or alpha-ketoglutarate as the variable substrate showed that NADH was a noncompetitive inhibitor with respect to each of these substrates, except at high NAD+ concentrations, where reciprocal plots were nonlinear and the inhibition pattern for NADH vs. NAD+ changed from a noncompetitive to a competitive pattern. From slope and intercept replots, 2-fold to 12-fold higher inhibition constants were estimated for inhibition by NADH vs. the various substrates in the presence of 1.5 X 10(-5) M Ca2+ than for inhibition at less than 10(-9) M Ca2+. These inhibition patterns and the lack of an effect of Ca2+ on the inhibition of the dihydrolipoyl dehydrogenase component suggested that Ca2+-modulated NADH inhibition occurs at an allosteric site with competitive binding at the site by high levels of NAD+. Decarboxylation of alpha-keto[1-14C]glutarate by the resolved alpha-ketoglutarate dehydrogenase component was investigated in the presence of 5.0 mM glyoxylate which served as an efficient acceptor. NADH (0.2 mM) or 1.0 mM ATP inhibited the partial reaction whereas 15 muM Ca2+, 1.0 mM ADP, or 10 mM NAD+ stimulated the partial reaction and reduced NADH inhibition of this reaction. Thus these effectors alter the activity of the alpha-ketoglutarate dehydrogenase complex by binding at allosteric sites on the alpha-ketoglutarate dehydrogenase component. Inhibition by NADH over a wide range of NADH/NAD+ ratios was measured under conditions in which the level of alpha-ketoglutarate was adjusted to give matching control activities at less than 10(-9) M Ca2+ or 1.5 X 10(-5) M Ca2+ in either the presence or the absence of 1.6 mM ADP. These studies establish that both Ca2+ and ADP

  4. A fiber-optic sorbitol biosensor based on NADH fluorescence detection toward rapid diagnosis of diabetic complications.

    PubMed

    Gessei, Tomoko; Arakawa, Takahiro; Kudo, Hiroyuki; Mitsubayashi, Kohji

    2015-09-21

    Accumulation of sorbitol in the tissue is known to cause microvascular diabetic complications. In this paper, a fiber-optic biosensor for sorbitol which is used as a biomarker of diabetic complications was developed and tested. The biosensor used a sorbitol dehydrogenase from microorganisms of the genus Flavimonas with high substrate specificity and detected the fluorescence of reduced nicotinamide adenine dinucleotide (NADH) by the enzymatic reaction. An ultraviolet light emitting diode (UV-LED) was used as the excitation light source of NADH. The fluorescence of NADH was detected using a spectrometer or a photomultiplier tube (PMT). The UV-LED and the photodetector were coupled using a Y-shaped optical fiber. In the experiment, an optical fiber probe with a sorbitol dehydrogenase immobilized membrane was placed in a cuvette filled with a phosphate buffer containing the oxidized form of nicotinamide adenine dinucleotide (NAD(+)). The changes in NADH fluorescence intensity were measured after adding a standard sorbitol solution. According to the experimental assessment, the calibration range of the sorbitol biosensor systems using a spectrometer and a PMT was 5.0-1000 μmol L(-1) and 1.0-1000 μmol L(-1), respectively. The sorbitol biosensor system using the sorbitol dehydrogenase from microorganisms of the genus Flavimonas has high selectivity and sensitivity compared with that from sheep liver. The sorbitol biosensor allows for point-of-care testing applications or daily health care tests for diabetes patients.

  5. The Regulation of Pyruvate Dehydrogenase Activity in Pea Leaf Mitochondria (The Effect of Respiration and Oxidative Phosphorylation).

    PubMed

    Moore, A. L.; Gemel, J.; Randall, D. D.

    1993-12-01

    The regulation of the pea (Pisum sativum) leaf mitochondrial pyruvate dehydrogenase complex by respiratory rate and oxidative phosphorylation has been investigated by measuring the respiratory activity, the redox poise of the quinone pool (Q-pool), and mitochondrial pyruvate dehydrogenase (mtPDC) activity under various metabolic conditions. It was found that, under state 4 conditions, mtPDC activity was unaffected by either the addition of succinate, 2-oxoglutarate, or glycine or the overall respiratory rate and redox poise of the Q-pool but was partially inhibited by NADH due to product inhibition. In the presence of ADP significant inactivation of PDC, which was sensitive to oligomycin, was observed with all substrates, apart from pyruvate, suggesting that inactivation was due to ATP formation. Inactivation of PDC by ADP addition was observed even in the presence of carboxyatractyloside, an inhibitor of the ATP/ADP translocator, suggesting that other mechanisms to facilitate the entry of adenylates, in addition to the adenylate carrier, must exist in plant mitochondria.

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

    PubMed Central

    Fahimi, H. Dariush; Karnovsky, Morris J.

    1966-01-01

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

  7. Single-walled carbon nanotubes covalently functionalized with polytyrosine: A new material for the development of NADH-based biosensors.

    PubMed

    Eguílaz, Marcos; Gutierrez, Fabiana; González-Domínguez, Jose Miguel; Martínez, María T; Rivas, Gustavo

    2016-12-15

    We report for the first time the use of single-walled carbon nanotubes (SWCNT) covalently functionalized with polytyrosine (Polytyr) (SWCNT-Polytyr) as a new electrode material for the development of nicotinamide adenine dinucleotide (NADH)-based biosensors. The oxidation of glassy carbon electrodes (GCE) modified with SWCNT-Polytyr at potentials high enough to oxidize the tyrosine residues have allowed the electrooxidation of NADH at low potentials due to the catalytic activity of the quinones generated from the primary oxidation of tyrosine without any additional redox mediator. The amperometric detection of NADH at 0.200V showed a sensitivity of (217±3)µAmM(-1)cm(-2) and a detection limit of 7.9nM. The excellent electrocatalytic activity of SWCNT-Polytyr towards NADH oxidation has also made possible the development of a sensitive ethanol biosensor through the immobilization of alcohol dehydrogenase (ADH) via Nafion entrapment, with excellent analytical characteristics (sensitivity of (5.8±0.1)µAmM(-1)cm(-2), detection limit of 0.67µM) and very successful application for the quantification of ethanol in different commercial beverages. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Increased work in cardiac trabeculae causes decreased mitochondrial NADH fluorescence followed by slow recovery.

    PubMed Central

    Brandes, R; Bers, D M

    1996-01-01

    The oxidative phosphorylation rate in isolated mitochondria is stimulated by increased [ADP], resulting in decreased [NADH]. In intact hearts, however, increased mechanical work has generally not been shown to cause an increase in [ADP]. Therefore, increased [NADH] has been suggested as an alternative for stimulating the phosphorylation rate. Such a rise in [NADH] could result from stimulation of various substrate dehydrogenases by increased intracellular [Ca2+] (e.g., during increased pacing frequency). We have monitored mitochondrial [NADH] in isolated rat ventricular trabeculae, using a novel fluorescence spectroscopy method where a native fluorescence signal was used to correct for motion artifacts. Work was controlled by increased pacing frequency and assessed using time-averaged force. At low-pacing rates (approximately 0.1 Hz), [NADH] immediately decreased during contraction and then slowly recovered (approximately 5 s) before the next contraction. At higher rates, [NADH] initially decreased by an amount related to pacing rate (i.e., work). However, during prolonged stimulation, [NADH] slowly (approximately 60 s) recovered to a new steady-state level below the initial level. We conclude that 1) during increased work, oxidative phosphorylation is not initially stimulated by increased mitochondrial [NADH]; and 2) increased pacing frequency slowly causes stimulation of NADH production. Images FIGURE 2 FIGURE 4 PMID:8842239

  9. Properties of a Purified Halophilic Malic Dehydrogenase

    PubMed Central

    Holmes, P. K.; Halvorson, H. Orin

    1965-01-01

    Holmes, P. K. (University of Illinois, Urbana), and H. Orin Halvorson. Properties of a purified halophilic malic dehydrogenase. J. Bacteriol. 90:316–326. 1965.—The malic dehydrogenase (MDH) from Halobacterium salinarium required high concentrations of monovalent ions for stability and activity. Studies of inactivation rates at different salt concentrations suggested that approximately 25% NaCl (w/v) is required to stabilize MDH. From 50 to 100% reactivation, depending on the salt concentration present during inactivation, could occur in 2.5 to 5 m NaCl or KCl. The optimal salt concentration for activity of MDH was a function of the pH, and ranged from 1 to 3 m NaCl or KCl. The effect of salt concentration on the pH-activity curves occurred chiefly below pH 7.0. Inactivation of MDH with heat or thiol reagents showed that the enzyme was more labile in the state induced by absence of salt. The activation of MDH by salts was attributed to a decreased rate of dissociation of MDH and reduced nicotinamide adenine dinucleotide (NADH2). The inactivation of the enzyme in the absence of salt could be largely prevented by the presence of NADH2. The S20.w of MDH decreased threefold at low salt concentrations. The enzyme was assumed to be in its native compact configuration only in the presence of a high concentration of salt. PMID:14329442

  10. The 2-Oxoacid Dehydrogenase Complexes in Mitochondria Can Produce Superoxide/Hydrogen Peroxide at Much Higher Rates Than Complex I*

    PubMed Central

    Quinlan, Casey L.; Goncalves, Renata L. S.; Hey-Mogensen, Martin; Yadava, Nagendra; Bunik, Victoria I.; Brand, Martin D.

    2014-01-01

    Several flavin-dependent enzymes of the mitochondrial matrix utilize NAD+ or NADH at about the same operating redox potential as the NADH/NAD+ pool and comprise the NADH/NAD+ isopotential enzyme group. Complex I (specifically the flavin, site IF) is often regarded as the major source of matrix superoxide/H2O2 production at this redox potential. However, the 2-oxoglutarate dehydrogenase (OGDH), branched-chain 2-oxoacid dehydrogenase (BCKDH), and pyruvate dehydrogenase (PDH) complexes are also capable of considerable superoxide/H2O2 production. To differentiate the superoxide/H2O2-producing capacities of these different mitochondrial sites in situ, we compared the observed rates of H2O2 production over a range of different NAD(P)H reduction levels in isolated skeletal muscle mitochondria under conditions that favored superoxide/H2O2 production from complex I, the OGDH complex, the BCKDH complex, or the PDH complex. The rates from all four complexes increased at higher NAD(P)H/NAD(P)+ ratios, although the 2-oxoacid dehydrogenase complexes produced superoxide/H2O2 at high rates only when oxidizing their specific 2-oxoacid substrates and not in the reverse reaction from NADH. At optimal conditions for each system, superoxide/H2O2 was produced by the OGDH complex at about twice the rate from the PDH complex, four times the rate from the BCKDH complex, and eight times the rate from site IF of complex I. Depending on the substrates present, the dominant sites of superoxide/H2O2 production at the level of NADH may be the OGDH and PDH complexes, but these activities may often be misattributed to complex I. PMID:24515115

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

    PubMed Central

    Burdette, D; Zeikus, J G

    1994-01-01

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

  12. [Membrane lipids and electron transfer. Effects of four detergents on NADH-ferricyanide reductase and NADH-cytochrome c reductase activities of potato tuber microsomes].

    PubMed

    Jolliot, A; Mazliak, P

    1977-10-17

    The NADH-ferricyanure reductase activity of Potato microsomes is stimulated by non ionic detergents (Triton X100 and Tween80) and is partially inhibited by ionic detergents (sodium-cholate and deoxycholate). All these four detergents progressively decreased the NADH-cytochrome c reductase in the following order: sodium deoxycholate greater than Triton X100 greater than sodium cholate greater than Tween80.

  13. Haloacetic Acid Water Disinfection Byproducts Affect Pyruvate Dehydrogenase Activity and Disrupt Cellular Metabolism.

    PubMed

    Dad, Azra; Jeong, Clara H; Wagner, Elizabeth D; Plewa, Michael J

    2018-02-06

    The disinfection of drinking water has been a major public health achievement. However, haloacetic acids (HAAs), generated as byproducts of water disinfection, are cytotoxic, genotoxic, mutagenic, carcinogenic, and teratogenic. Previous studies of monoHAA-induced genotoxicity and cell stress demonstrated that the toxicity was due to inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), leading to disruption of cellular metabolism and energy homeostasis. DiHAAs and triHAAs are also produced during water disinfection, and whether they share mechanisms of action with monoHAAs is unknown. In this study, we evaluated the effects of mono-, di-, and tri-HAAs on cellular GAPDH enzyme kinetics, cellular ATP levels, and pyruvate dehydrogenase complex (PDC) activity. Here, treatments conducted in Chinese hamster ovary (CHO) cells revealed differences among mono-, di-, and triHAAs in their molecular targets. The monoHAAs, iodoacetic acid and bromoacetic acid, were the strongest inhibitors of GAPDH and greatly reduced cellular ATP levels. Chloroacetic acid, diHAAs, and triHAAs were weaker inhibitors of GAPDH and some increased the levels of cellular ATP. HAAs also affected PDC activity, with most HAAs activating PDC. The primary finding of this work is that mono- versus multi-HAAs address different molecular targets, and the results are generally consistent with a model in which monoHAAs activate the PDC through GAPDH inhibition-mediated disruption in cellular metabolites, including altering ATP-to-ADP and NADH-to-NAD ratios. The monoHAA-mediated reduction in cellular metabolites results in accelerated PDC activity by way of metabolite-ratio-dependent PDC regulation. DiHAAs and triHAAs are weaker inhibitors of GAPDH, but many also increase cellular ATP levels, and we suggest that they increase PDC activity by inhibiting pyruvate dehydrogenase kinase.

  14. Auxin-activated NADH oxidase activity of soybean plasma membranes is distinct from the constitutive plasma membrane NADH oxidase and exhibits prion-like properties

    NASA Technical Reports Server (NTRS)

    Morre, D. James; Morre, Dorothy M.; Ternes, Philipp

    2003-01-01

    The hormone-stimulated and growth-related cell surface hydroquinone (NADH) oxidase activity of etiolated hypocotyls of soybeans oscillates with a period of about 24 min or 60 times per 24-h day. Plasma membranes of soybean hypocotyls contain two such NADH oxidase activities that have been resolved by purification on concanavalin A columns. One in the apparent molecular weight range of 14-17 kDa is stimulated by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The other is larger and unaffected by 2,4-D. The 2,4-D-stimulated activity absolutely requires 2,4-D for activity and exhibits a period length of about 24 min. Also exhibiting 24-min oscillations is the rate of cell enlargement induced by the addition of 2,4-D or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D or IAA addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges at the expense of the constitutive activity. A recruitment process analogous to that exhibited by prions is postulated to explain this behavior.

  15. Effect of CO2 on NADH production of denitrifying microbes via inhibiting carbon source transport and its metabolism.

    PubMed

    Wan, Rui; Chen, Yinguang; Zheng, Xiong; Su, Yinglong; Huang, Haining

    2018-06-15

    The potential effect of CO 2 on environmental microbes has drawn much attention recently. As an important section of the nitrogen cycle, biological denitrification requires electron donor to reduce nitrogen oxide. Nicotinamide adenine dinucleotide (NADH), which is formed during carbon source metabolism, is a widely reported electron donor for denitrification. Here we studied the effect of CO 2 on NADH production and carbon source utilization in the denitrifying microbe Paracoccus denitrificans. We observed that NADH level was decreased by 45.5% with the increase of CO 2 concentration from 0 to 30,000ppm, which was attributed to the significantly decreased utilization of carbon source (i.e., acetate). Further study showed that CO 2 inhibited carbon source utilization because of multiple negative influences: (1) suppressing the growth and viability of denitrifier cells, (2) weakening the driving force for carbon source transport by decreasing bacterial membrane potential, and (3) downregulating the expression of genes encoding key enzymes involved in intracellular carbon metabolism, such as citrate synthase, aconitate hydratase, isocitrate dehydrogenase, succinate dehydrogenase, and fumarate reductase. This study suggests that the inhibitory effect of CO 2 on NADH production in denitrifiers might deteriorate the denitrification performance in an elevated CO 2 climate scenario. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Impairment of NADH dehydrogenase and regulation of anaerobic metabolism by the small RNA RyhB and NadE for improved biohydrogen production in Enterobacter aerogenes.

    PubMed

    Wu, Yan; Hao, Yaqiao; Wei, Xuan; Shen, Qi; Ding, Xuanwei; Wang, Liyan; Zhao, Hongxin; Lu, Yuan

    2017-01-01

    Enterobacter aerogenes is a facultative anaerobe and is one of the most widely studied bacterial strains because of its ability to use a variety of substrates, to produce hydrogen at a high rate, and its high growth rate during dark fermentation. However, the rate of hydrogen production has not been optimized. In this present study, three strategies to improve hydrogen production in E. aerogenes , namely the disruption of nuoCDE , overexpression of the small RNA RyhB and of NadE to regulate global anaerobic metabolism, and the redistribution of metabolic flux. The goal of this study was to clarify the effect of nuoCDE , RyhB, and NadE on hydrogen production and how the perturbation of NADH influences the yield of hydrogen gas from E. aerogenes . NADH dehydrogenase activity was impaired by knocking out nuoCD or nuoCDE in E. aerogenes IAM1183 using the CRISPR-Cas9 system to explore the consequent effect on hydrogen production. The hydrogen yields from IAM1183-CD( ∆nuoC / ∆nuoD ) and IAM1183-CDE ( ∆nuoC / ∆nuoD / ∆nuoE ) increased, respectively, by 24.5 and 45.6% in batch culture (100 mL serum bottles). The hydrogen produced via the NADH pathway increased significantly in IAM1183-CDE, suggesting that nuoE plays an important role in regulating NADH concentration in E. aerogenes . Batch-cultivating experiments showed that by the overexpression of NadE (N), the hydrogen yields of IAM1183/N, IAM1183-CD/N, and IAM1183-CDE/N increased 1.06-, 1.35-, and 1.55-folds, respectively, compared with IAM1183. Particularly worth mentioning is that the strain IAM118-CDE/N reached 2.28 mol in H 2 yield, per mole of glucose consumed. IAN1183/R, IAM1183-CD/R, and IAM1183-CDE/R showed increasing H 2 yields in batch culture. Metabolic flux analysis indicated that increased expression of RyhB led to a significant shift in metabolic patterns. We further investigated IAM1183-CDE/N, which had the best hydrogen-producing traits, as a potential candidate for industry applications

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

    PubMed

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

    2013-05-01

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

  18. Metabolic engineering of an ATP-neutral Embden-Meyerhof-Parnas pathway in Corynebacterium glutamicum: growth restoration by an adaptive point mutation in NADH dehydrogenase.

    PubMed

    Komati Reddy, Gajendar; Lindner, Steffen N; Wendisch, Volker F

    2015-03-01

    Corynebacterium glutamicum uses the Embden-Meyerhof-Parnas pathway of glycolysis and gains 2 mol of ATP per mol of glucose by substrate-level phosphorylation (SLP). To engineer glycolysis without net ATP formation by SLP, endogenous phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was replaced by nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) from Clostridium acetobutylicum, which irreversibly converts glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) without generating ATP. As shown recently (S. Takeno, R. Murata, R. Kobayashi, S. Mitsuhashi, and M. Ikeda, Appl Environ Microbiol 76:7154-7160, 2010, http://dx.doi.org/10.1128/AEM.01464-10), this ATP-neutral, NADPH-generating glycolytic pathway did not allow for the growth of Corynebacterium glutamicum with glucose as the sole carbon source unless hitherto unknown suppressor mutations occurred; however, these mutations were not disclosed. In the present study, a suppressor mutation was identified, and it was shown that heterologous expression of udhA encoding soluble transhydrogenase from Escherichia coli partly restored growth, suggesting that growth was inhibited by NADPH accumulation. Moreover, genome sequence analysis of second-site suppressor mutants that were able to grow faster with glucose revealed a single point mutation in the gene of non-proton-pumping NADH:ubiquinone oxidoreductase (NDH-II) leading to the amino acid change D213G, which was shared by these suppressor mutants. Since related NDH-II enzymes accepting NADPH as the substrate possess asparagine or glutamine residues at this position, D213G, D213N, and D213Q variants of C. glutamicum NDH-II were constructed and were shown to oxidize NADPH in addition to NADH. Taking these findings together, ATP-neutral glycolysis by the replacement of endogenous NAD-dependent GAPDH with NADP-dependent GapN became possible via oxidation of NADPH formed in this pathway by mutant NADPH

  19. Increased Production of Hydrogen Peroxide by Lactobacillus delbrueckii subsp. bulgaricus upon Aeration: Involvement of an NADH Oxidase in Oxidative Stress

    PubMed Central

    Marty-Teysset, C.; de la Torre, F.; Garel, J.-R.

    2000-01-01

    The growth of Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii subsp. bulgaricus) on lactose was altered upon aerating the cultures by agitation. Aeration caused the bacteria to enter early into stationary phase, thus reducing markedly the biomass production but without modifying the maximum growth rate. The early entry into stationary phase of aerated cultures was probably related to the accumulation of hydrogen peroxide in the medium. Indeed, the concentration of hydrogen peroxide in aerated cultures was two to three times higher than in unaerated ones. Also, a similar shift from exponential to stationary phase could be induced in unaerated cultures by adding increasing concentrations of hydrogen peroxide. A significant fraction of the hydrogen peroxide produced by L. delbrueckii subsp. bulgaricus originated from the reduction of molecular oxygen by NADH catalyzed by an NADH:H2O2 oxidase. The specific activity of this NADH oxidase was the same in aerated and unaerated cultures, suggesting that the amount of this enzyme was not directly regulated by oxygen. Aeration did not change the homolactic character of lactose fermentation by L. delbrueckii subsp. bulgaricus and most of the NADH was reoxidized by lactate dehydrogenase with pyruvate. This indicated that NADH oxidase had no (or a very small) energetic role and could be involved in eliminating oxygen. PMID:10618234

  20. Biochemical characterization of ethanol-dependent reduction of furfural by alcohol dehydrogenases.

    PubMed

    Li, Qunrui; Metthew Lam, L K; Xun, Luying

    2011-11-01

    Lignocellulosic biomass is usually converted to hydrolysates, which consist of sugars and sugar derivatives, such as furfural. Before yeast ferments sugars to ethanol, it reduces toxic furfural to non-inhibitory furfuryl alcohol in a prolonged lag phase. Bioreduction of furfural may shorten the lag phase. Cupriavidus necator JMP134 rapidly reduces furfural with a Zn-dependent alcohol dehydrogenase (FurX) at the expense of ethanol (Li et al. 2011). The mechanism of the ethanol-dependent reduction of furfural by FurX and three homologous alcohol dehydrogenases was investigated. The reduction consisted of two individual reactions: ethanol-dependent reduction of NAD(+) to NADH and then NADH-dependent reduction of furfural to furfuryl alcohol. The kinetic parameters of the coupled reaction and the individual reactions were determined for the four enzymes. The data indicated that limited NADH was released in the coupled reaction. The enzymes had high affinities for NADH (e.g., K ( d ) of 0.043 μM for the FurX-NADH complex) and relatively low affinities for NAD(+) (e.g., K ( d ) of 87 μM for FurX-NAD(+)). The kinetic data suggest that the four enzymes are efficient "furfural reductases" with either ethanol or NADH as the reducing power. The standard free energy change (ΔG°') for ethanol-dependent reduction of furfural was determined to be -1.1 kJ mol(-1). The physiological benefit for ethanol-dependent reduction of furfural is likely to replace toxic and recalcitrant furfural with less toxic and more biodegradable acetaldehyde.

  1. Characterization of human DHRS6, an orphan short chain dehydrogenase/reductase enzyme: a novel, cytosolic type 2 R-beta-hydroxybutyrate dehydrogenase.

    PubMed

    Guo, Kunde; Lukacik, Petra; Papagrigoriou, Evangelos; Meier, Marc; Lee, Wen Hwa; Adamski, Jerzy; Oppermann, Udo

    2006-04-14

    Human DHRS6 is a previously uncharacterized member of the short chain dehydrogenases/reductase family and displays significant homologies to bacterial hydroxybutyrate dehydrogenases. Substrate screening reveals sole NAD(+)-dependent conversion of (R)-hydroxybutyrate to acetoacetate with K(m) values of about 10 mm, consistent with plasma levels of circulating ketone bodies in situations of starvation or ketoacidosis. The structure of human DHRS6 was determined at a resolution of 1.8 A in complex with NAD(H) and reveals a tetrameric organization with a short chain dehydrogenases/reductase-typical folding pattern. A highly conserved triad of Arg residues ("triple R" motif consisting of Arg(144), Arg(188), and Arg(205)) was found to bind a sulfate molecule at the active site. Docking analysis of R-beta-hydroxybutyrate into the active site reveals an experimentally consistent model of substrate carboxylate binding and catalytically competent orientation. GFP reporter gene analysis reveals a cytosolic localization upon transfection into mammalian cells. These data establish DHRS6 as a novel, cytosolic type 2 (R)-hydroxybutyrate dehydrogenase, distinct from its well characterized mitochondrial type 1 counterpart. The properties determined for DHRS6 suggest a possible physiological role in cytosolic ketone body utilization, either as a secondary system for energy supply in starvation or to generate precursors for lipid and sterol synthesis.

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

    PubMed

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

    2012-05-10

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

  3. In vitro assessment of anticholinesterase and NADH oxidase inhibitory activities of an edible fern, Diplazium esculentum.

    PubMed

    Roy, Subhrajyoti; Dutta, Somit; Chaudhuri, Tapas Kumar

    2015-07-01

    Diplazium esculentum is the most commonly consumed edible fern throughout Asia and Oceania. Several studies have been performed so far to determine different functional properties of this plant, but there have been no reports on the anticholinesterase and nicotinamide adenine dinucleotide (NADH) oxidase inhibitory activities of this plant. Therefore, the present study was conducted to determine the anticholinesterase and NADH oxidase inhibitory activities of 70% methanolic extract of D. esculentum. The D. esculentum extract was investigated for its acetylcholinesterase and NADH oxidase inhibitory activities as well as its free radical scavenging and total antioxidant activities in the linoleic acid system. The free radical scavenging activity of the extract was determined by the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) method. The total antioxidant activity of the extract was evaluated by ferric thiocyanate (FTC) and thiobarbituric acid (TBA) methods. The D. esculentum extract inhibited acetylcholinesterase and NADH oxidase in a dose-dependent manner, with IC50 values of 272.97±19.38 and 265.81±21.20 μg/mL, respectively. The extract also showed a potent DPPH radical scavenging activity with an IC50 value of 402.88±12.70 μg/mL. Moreover, the extract showed 27.41% and 33.22% of total antioxidant activities determined by FTC and TBA methods, respectively. Results indicated that 70% methanolic extract of D. esculentum effectively inhibited the enzymes acetylcholinesterase and NADH oxidase and acted as a potent antioxidant and free radical scavenger. These in vitro assays indicate that this plant extract is a significant source of natural antioxidants, which may be helpful in preventing the progression of various neurodegenerative disorders associated with oxidative stress.

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

    PubMed

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

    2015-03-01

    Modified electrodes using immobilized alcohol dehydrogenase enzymes for the efficient electroreduction of butyraldehyde to butanol are presented as an important step for the utilization of CO 2 -reduction products. Alcohol dehydrogenase was immobilized, embedded in an alginate-silicate hybrid gel, on a carbon felt (CF) electrode. The application of this enzyme to the reduction of an aldehyde to an alcohol with the aid of the coenzyme nicotinamide adenine dinucleotide (NADH), in analogy to the final step in the natural reduction cascade of CO 2 to alcohol, has been already reported. However, the use of such enzymatic reductions is limited because of the necessity of providing expensive NADH as a sacrificial electron and proton donor. Immobilization of such dehydrogenase enzymes on electrodes and direct pumping of electrons into the biocatalysts offers an easy and efficient way for the biochemical recycling of CO 2 to valuable chemicals or alternative synthetic fuels. We report the direct electrochemical addressing of immobilized alcohol dehydrogenase for the reduction of butyraldehyde to butanol without consumption of NADH. The selective reduction of butyraldehyde to butanol occurs at room temperature, ambient pressure and neutral pH. Production of butanol was detected by using liquid-injection gas chromatography and was estimated to occur with Faradaic efficiencies of around 40 %.

  5. Lack of complex I activity in human cells carrying a mutation in MtDNA-encoded ND4 subunit is corrected by the Saccharomyces cerevisiae NADH-quinone oxidoreductase (NDI1) gene.

    PubMed

    Bai, Y; Hájek, P; Chomyn, A; Chan, E; Seo, B B; Matsuno-Yagi, A; Yagi, T; Attardi, G

    2001-10-19

    The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA (mtDNA)-encoded subunit ND4. In particular, the NDI1 gene was introduced into the nuclear genome of the human 143B.TK(-) cell line derivative C4T, which carries a homoplasmic frameshift mutation in the ND4 gene. Two transformants with a low or high level of expression of the exogenous gene were chosen for a detailed analysis. In these cells the corresponding protein is localized in mitochondria, its NADH-binding site faces the matrix compartment as in yeast mitochondria, and in perfect correlation with its abundance restores partially or fully NADH-dependent respiration that is rotenone-insensitive, flavone-sensitive, and antimycin A-sensitive. Thus the yeast enzyme has become coupled to the downstream portion of the human respiratory chain. Furthermore, the P:O ratio with malate/glutamate-dependent respiration in the transformants is approximately two-thirds of that of the wild-type 143B.TK(-) cells, as expected from the lack of proton pumping activity in the yeast enzyme. Finally, whereas the original mutant cell line C4T fails to grow in medium containing galactose instead of glucose, the high NDI1-expressing transformant has a fully restored capacity to grow in galactose medium. The present observations substantially expand the potential of the yeast NDI1 gene for the therapy of mitochondrial diseases involving complex I deficiency.

  6. Characterization of the type 2 NADH:menaquinone oxidoreductases from Staphylococcus aureus and the bactericidal action of phenothiazines.

    PubMed

    Schurig-Briccio, Lici A; Yano, Takahiro; Rubin, Harvey; Gennis, Robert B

    2014-07-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is currently one of the principal multiple drug resistant bacterial pathogens causing serious infections, many of which are life-threatening. Consequently, new therapeutic targets are required to combat such infections. In the current work, we explore the type 2 Nicotinamide adenine dinucleotide reduced form (NADH) dehydrogenases (NDH-2s) as possible drug targets and look at the effects of phenothiazines, known to inhibit NDH-2 from Mycobacterium tuberculosis. NDH-2s are monotopic membrane proteins that catalyze the transfer of electrons from NADH via flavin adenine dinucleotide (FAD) to the quinone pool. They are required for maintaining the NADH/Nicotinamide adenine dinucleotide (NAD(+)) redox balance and contribute indirectly to the generation of proton motive force. NDH-2s are not present in mammals, but are the only form of respiratory NADH dehydrogenase in several pathogens, including S. aureus. In this work, the two putative ndh genes present in the S. aureus genome were identified, cloned and expressed, and the proteins were purified and characterized. Phenothiazines were shown to inhibit both of the S. aureus NDH-2s with half maximal inhibitory concentration (IC50) values as low as 8μM. However, evaluating the effects of phenothiazines on whole cells of S. aureus was complicated by the fact that they are also acting as uncouplers of oxidative phosphorylation. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Constitutive NADPH-dependent electron transferase activity of the Nox4 dehydrogenase domain.

    PubMed

    Nisimoto, Yukio; Jackson, Heather M; Ogawa, Hisamitsu; Kawahara, Tsukasa; Lambeth, J David

    2010-03-23

    NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47(phox) and p67(phox) and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a K(m) for NADPH of 55 +/- 10 microM. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of approximately 200 mol of H(2)O(2) min(-1) (mol of Nox4)(-1). A chimeric protein (Nox2/4) consisting of the Nox2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H(2)O(2) production in the absence of cytosolic regulatory subunits. In contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electron transferase activities. Nox1 DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction of cytochrome c (160 min(-1), largely superoxide dismutase-independent), ferricyanide (470 min(-1)), and other electron acceptors (artificial dyes and cytochrome b(5)). Rates were similar to those observed for H(2)O(2) production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and was seen with NADPH but not NADH. These results indicate that the Nox4 DH domain exists in an intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.

  8. A mutant of barley lacking NADH-hydroxypyruvate reductase

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

    Blackwell, R.; Lea, P.

    1989-04-01

    A mutant of barley, LaPr 88/29, deficient in peroxisomal NADH-hydroxypyruvate reductase (HPR) activity has been identified. Compared to the wild type the activities of NADH-HPR and NADPH-HPR were severely reduced but the mutant was still capable of fixing CO{sub 2} at rates equivalent to 75% of that of the wild type in air. Although lacking an enzyme in the main photorespiratory pathway, there appeared to be little disruption to photorespiratory metabolism as ammonia release, CO{sub 2} efflux and {sup 14}CO{sub 2} release from L-(U-{sup 14}C) serine were similar in both mutant and wild type. LaPr 88/29 has been used tomore » show that NADH-glyoxylate reductase (GR) and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-HPR activity is due to the NADH-HPR enzyme. Immunological studies, using antibodies raised against spinach HPR, have shown that the NADH-dependent enzyme protein is absent in LaPr 88/29 but there appears to be enhanced synthesis of the NADPH-dependent enzyme protein.« less

  9. Facilitation of NADH Electrooxidation at Treated Carbon Nanotubes

    PubMed Central

    Wooten, Marilyn; Gorski, Waldemar

    2010-01-01

    The relationship between the state of the surface of carbon nanotubes (CNT) and their electrochemical activity was investigated using the enzyme cofactor dihydronicotinamide adenine dinucleotide (NADH) as a redox probe. The boiling of CNT in water, while nondestructive, activated them toward the oxidation of NADH as indicated by a shift in the anodic peak potential of NADH (ENADH) from 0.4 to 0.0 V. The shift in ENADH was due to the redox mediation of NADH oxidation by traces of quinone species that were formed on the surface of treated CNT. The harsher treatment that comprised of microwaving of CNT in concentrated nitric acid had a similar effect on the ENADH and, additionally, it increased the anodic peak current of NADH. The latter correlated with the formation of defects on the surface of acid-microwaved CNT as indicated by their Raman spectra. The increase in current was discussed considering a role of surface mediators on the buckled graphene sheets of acid-microwaved CNT. The other carbon allotropes including the edge plane pyrolytic graphite, graphite powder, and glassy carbon did not display a comparable activation toward the oxidation of NADH. PMID:20088562

  10. Biochemical characterization of an L-tryptophan dehydrogenase from the photoautotrophic cyanobacterium Nostoc punctiforme.

    PubMed

    Ogura, Ryutaro; Wakamatsu, Taisuke; Mutaguchi, Yuta; Doi, Katsumi; Ohshima, Toshihisa

    2014-06-10

    An NAD(+)-dependent l-tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH) was cloned and overexpressed in Escherichia coli. The recombinant NpTrpDH with a C-terminal His6-tag was purified to homogeneity using a Ni-NTA agarose column, and was found to be a homodimer with a molecular mass of 76.1kDa. The enzyme required NAD(+) and NADH as cofactors for oxidative deamination and reductive amination, respectively, but not NADP(+) or NADPH. l-Trp was the preferred substrate for deamination, though l-Phe was deaminated at a much lower rate. The enzyme exclusively aminated 3-indolepyruvate; phenylpyruvate was inert. The pH optima for the deamination of l-Trp and amination of 3-indolpyruvate were 11.0 and 7.5, respectively. For deamination of l-Trp, maximum enzymatic activity was observed at 45°C. NpTrpDH retained more than 80% of its activity after incubation for 30min at pHs ranging from 5.0 to 11.5 or incubation for 10min at temperatures up to 40°C. Unlike l-Trp dehydrogenases from higher plants, NpTrpDH activity was not activated by metal ions. Typical Michaelis-Menten kinetics were observed for NAD(+) and l-Trp for oxidative deamination, but with reductive amination there was marked substrate inhibition by 3-indolepyruvate. NMR analysis of the hydrogen transfer from the C4 position of the nicotinamide moiety of NADH showed that NpTrpDH has a pro-S (B-type) stereospecificity similar to the Glu/Leu/Phe/Val dehydrogenase family. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Converting NADH to NAD+ by nicotinamide nucleotide transhydrogenase as a novel strategy against mitochondrial pathologies during aging.

    PubMed

    Olgun, Abdullah

    2009-08-01

    Mitochondrial DNA defects are involved supposedly via free radicals in many pathologies including aging and cancer. But, interestingly, free radical production was not found increased in prematurely aging mice having higher mutation rate in mtDNA. Therefore, some other mechanisms like the increase of mitochondrial NADH/NAD(+) and ubiquinol/ubiquinone ratios, can be in action in respiratory chain defects. NADH/NAD(+) ratio can be normalized by the activation or overexpression of nicotinamide nucleotide transhydrogenase (NNT), a mitochondrial enzyme catalyzing the following very important reaction: NADH + NADP(+ )<--> NADPH + NAD(+). The products NAD(+) and NADPH are required in many critical biological processes, e.g., NAD(+) is used by histone deacetylase Sir2 which regulates longevity in different species. NADPH is used in a number of biosynthesis reactions (e.g., reduced glutathione synthesis), and processes like apoptosis. Increased ubiquinol/ubiquinone ratio interferes the function of dihydroorotate dehydrogenase, the only mitochondrial enzyme involved in ubiquinone mediated de novo pyrimidine synthesis. Uridine and its prodrug triacetyluridine are used to compensate pyrimidine deficiency but their bioavailability is limited. Therefore, the normalization of the ubiquinol/ubiquinone ratio can be accomplished by allotopic expression of alternative oxidase, a mitochondrial ubiquinol oxidase which converts ubiquinol to ubiquinone.

  12. Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing Saccharomyces cerevisiae.

    PubMed

    Hou, Jin; Vemuri, Goutham N; Bao, Xiaoming; Olsson, Lisbeth

    2009-04-01

    During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation. However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO(2) to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall, although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated.

  13. Novel chiral tool, (R)-2-octanol dehydrogenase, from Pichia finlandica: purification, gene cloning, and application for optically active α-haloalcohols.

    PubMed

    Yamamoto, Hiroaki; Kudoh, Masatake

    2013-09-01

    A novel enantioselective alcohol dehydrogenase, (R)-2-octanol dehydrogenase (PfODH), was discovered among methylotrophic microorganisms. The enzyme was purified from Pichia finlandica and characterized. The molecular mass of the enzyme was estimated to be 83,000 and 30,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The enzyme was an NAD(+)-dependent secondary alcohol dehydrogenase and showed a strict enantioselectivity, very broad substrate specificity, and high tolerance to SH reagents. A gene-encoding PfODH was cloned and sequenced. The gene consisted of 765 nucleotides, coding polypeptides of 254 amino acids. The gene was singly expressed and coexpressed together with a formate dehydrogenase as an NADH regenerator in an Escherichia coli. Ethyl (S)-4-chloro-3-hydroxybutanoate and (S)-2-chloro-1-phenylethanol were synthesized using a whole-cell biocatalyst in more than 99 % optical purity.

  14. Preferential inhibition of the plasma membrane NADH oxidase (NOX) activity by diphenyleneiodonium chloride with NADPH as donor

    NASA Technical Reports Server (NTRS)

    Morre, D. James

    2002-01-01

    The cell-surface NADH oxidase (NOX) protein of plant and animal cells will utilize both NADH and NADPH as reduced electron donors for activity. The two activities are distinguished by a differential inhibition by the redox inhibitor diphenyleneiodonium chloride (DPI). Using both plasma membranes and cells, activity with NADPH as donor was markedly inhibited by DPI at submicromolar concentrations, whereas with NADH as donor, DPI was much less effective or had no effect on the activity. The possibility of the inhibition being the result of two different enzymes was eliminated by the use of a recombinant NOX protein. The findings support the concept that NOX proteins serve as terminal oxidases for plasma membrane electron transport involving cytosolic reduced pyridine nucleotides as the natural electron donors and with molecular oxygen as the electron acceptor.

  15. Coulometric determination of NAD+ and NADH in normal and cancer cells using LDH, RVC and a polymer mediator.

    PubMed

    Torabi, F; Ramanathan, K; Larsson, P O; Gorton, L; Svanberg, K; Okamoto, Y; Danielsson, B; Khayyami, M

    1999-11-15

    An electrochemical method for the measurement of NAD(+) and NADH in normal and cancer tissues using flow injection analysis (FIA) is reported. Reticulated vitreous carbon (RVC) electrodes with entrapped l-lactate dehydrogenase (LDH) and a new redox polymer containing covalently bound toluidine blue O (TBO) were employed for this purpose. Both NAD(+) and NADH were estimated coulometrically based on their reaction with LDH. The latter was immobilized on controlled pore glass (CPG) by cross-linking with glutaraldehyde and packed within the RVC. The concentrations of NAD(+) and NADH in the tissues, estimated using different electron mediators such as ferricyanide (FCN), meldola blue (MB) and TBO have also been compared. The effects of flow rate, pH, applied potential (versus Ag/AgCl reference) and adsorption of the mediators have also been investigated. Based on the measurements of NAD(+) and NADH in normal and cancer tissues it has been concluded that the NADH concentration is lower, while the NAD(+) concentration is higher in cancer tissues. Amongst the electron mediators TBO was found to be a more stable mediator for such measurements.

  16. Putting together a plasma membrane NADH oxidase: a tale of three laboratories.

    PubMed

    Löw, Hans; Crane, Frederick L; Morré, D James

    2012-11-01

    The observation that high cellular concentrations of NADH were associated with low adenylate cyclase activity led to a search for the mechanism of the effect. Since cyclase is in the plasma membrane, we considered the membrane might have a site for NADH action, and that NADH might be oxidized at that site. A test for NADH oxidase showed very low activity, which could be increased by adding growth factors. The plasma membrane oxidase was not inhibited by inhibitors of mitochondrial NADH oxidase such as cyanide, rotenone or antimycin. Stimulation of the plasma membrane oxidase by iso-proterenol or triiodothyronine was different from lack of stimulation in endoplasmic reticulum. After 25 years of research, three components of a trans membrane NADH oxidase have been discovered. Flavoprotein NADH coenzyme Q reductases (NADH cytochrome b reductase) on the inside, coenzyme Q in the middle, and a coenzyme Q oxidase on the outside as a terminal oxidase. The external oxidase segment is a copper protein with unique properties in timekeeping, protein disulfide isomerase and endogenous NADH oxidase activity, which affords a mechanism for control of cell growth by the overall NADH oxidase and the remarkable inhibition of oxidase activity and growth of cancer cells by a wide range of anti-tumor drugs. A second trans plasma membrane electron transport system has been found in voltage dependent anion channel (VDAC), which has NADH ferricyanide reductase activity. This activity must be considered in relation to ferricyanide stimulation of growth and increased VDAC antibodies in patients with autism. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Deep sequencing of the mitochondrial genome reveals common heteroplasmic sites in NADH dehydrogenase genes.

    PubMed

    Liu, Chunyu; Fetterman, Jessica L; Liu, Poching; Luo, Yan; Larson, Martin G; Vasan, Ramachandran S; Zhu, Jun; Levy, Daniel

    2018-03-01

    Increasing evidence implicates mitochondrial dysfunction in aging and age-related conditions. But little is known about the molecular basis for this connection. A possible cause may be mutations in the mitochondrial DNA (mtDNA), which are often heteroplasmic-the joint presence of different alleles at a single locus in the same individual. However, the involvement of mtDNA heteroplasmy in aging and age-related conditions has not been investigated thoroughly. We deep-sequenced the complete mtDNA genomes of 356 Framingham Heart Study participants (52% women, mean age 43, mean coverage 4570-fold), identified 2880 unique mutations and comprehensively annotated them by MITOMAP and PolyPhen-2. We discovered 11 heteroplasmic "hot" spots [NADH dehydrogenase (ND) subunit 1, 4, 5 and 6 genes, n = 7; cytochrome c oxidase I (COI), n = 2; 16S rRNA, n = 1; D-loop, n = 1] for which the alternative-to-reference allele ratios significantly increased with advancing age (Bonferroni correction p < 0.001). Four of these heteroplasmic mutations in ND and COI genes were predicted to be deleterious nonsynonymous mutations which may have direct impact on ATP production. We confirmed previous findings that healthy individuals carry many low-frequency heteroplasmy mutations with potentially deleterious effects. We hypothesize that the effect of a single deleterious heteroplasmy may be minimal due to a low mutant-to-wildtype allele ratio, whereas the aggregate effects of many deleterious mutations may cause changes in mitochondrial function and contribute to age-related diseases. The identification of age-related mtDNA mutations is an important step to understand the genetic architecture of age-related diseases and may uncover novel therapeutic targets for such diseases.

  18. NADH-ubiquinone oxidoreductase activity in the kinetoplasts of the plant trypanosomatid Phytomonas serpens.

    PubMed

    González-Halphen, Diego; Maslov, Dmitri A

    2004-03-01

    NADH-ubiquinone oxidoreductase activity is present in mitochondrial lysates of Phytomonas serpens. Rotenone at 2-10 microM inhibited the activity 50-75%, indicating that it belongs to respiratory complex I. The activity was also inhibited 50-60% in the presence of 10-30 nM atovaquone suggesting that inhibition of complex I represents a likely mechanism of the known antileishmanial activity of this drug. The complex was partially purified by chromatography on DEAE-Sepharose CL-6B and gel-filtration on Sepharose CL-2B. The NADH:ubiquinone oxidoreductase activity in this preparation was completely inactivated by 20 nM atovaquone. The partially purified complex was present in a low amount and its subunits could not be discerned by staining with Coomassie. However, one of its components, a homologue of the 39 kDa subunit of the bovine complex I, was identified immunochemically in the original lysate and in the partially purified material.

  19. Purification and characterization of an anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol.

    PubMed

    Meng, Fantao; Xu, Yan

    2010-04-01

    An anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol was purified by 26-fold to homogeneity. The enzyme had a homodimeric structure consisting of 49 kDa subunits, required NADPH, but not NADH, as a cofactor and was a Zn-independent short-chain dehydrogenase. Aliphatic methyl ketones (chain length > or =6 carbon atoms) and aromatic methyl ketones were the preferred substrates for the enzyme, the best being 2-octanone. Maximum enzyme activity with 2-octanone was at 45 degrees C and at pH 8.0.

  20. Metabolism of hydroxypyruvate in a mutant of barley lacking NADH-dependent hydroxypyruvate reductase, an important photorespiratory enzyme activity

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

    Murray, A.J.S.; Blackwell, R.D.; Lea, P.J.

    1989-09-01

    A mutant of barley (Hordeum vulgare L.), LaPr 88/29, deficient in NADH-dependent hydroxypyruvate reductase (HPR) activity has been isolated. The activities of both NADH (5%) and NADPH-dependent (19%) HPR were severely reduced in this mutant compared to the wild type. Although lacking an enzyme in the main carbon pathway of photorespiration, this mutant was capable of CO{sub 2} fixation rates equivalent to 75% of that of the wild type, in normal atmospheres and 50% O{sub 2}. There also appeared to be little disruption to the photorespiratory metabolism as ammonia release, CO{sub 2} efflux and {sup 14}CO{sub 2} release from L-(U-{supmore » 14}C)serine feeding were similar in both mutant and wild-type leaves. When leaves of LaPr 88/29 were fed either ({sup 14}C)serine or {sup 14}CO{sub 2}, the accumulation of radioactivity was in serine and not in hydroxypyruvate, although the mutant was still able to metabolize over 25% of the supplied ({sup 14}C)serine into sucrose. After 3 hours in air the soluble amino acid pool was almost totally dominated by serine and glycine. LaPr 88/29 has also been used to show that NADH-glyoxylate reductase and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-dependent HPR activity is due to the NADH-dependent enzyme. We also suggest that the alternative NADPH activity can metabolize a proportion, but not all, of the hydroxypyruvate produced during photorespiration and may thus form a useful backup to the NADH-dependent enzyme under conditions of maximal photorespiration.« less

  1. Antimalarial Activity of Potential Inhibitors of Plasmodium falciparum Lactate Dehydrogenase Enzyme Selected by Docking Studies

    PubMed Central

    Penna-Coutinho, Julia; Cortopassi, Wilian Augusto; Oliveira, Aline Alves; França, Tanos Celmar Costa; Krettli, Antoniana Ursine

    2011-01-01

    The Plasmodium falciparum lactate dehydrogenase enzyme (PfLDH) has been considered as a potential molecular target for antimalarials due to this parasite's dependence on glycolysis for energy production. Because the LDH enzymes found in P. vivax, P. malariae and P. ovale (pLDH) all exhibit ∼90% identity to PfLDH, it would be desirable to have new anti-pLDH drugs, particularly ones that are effective against P. falciparum, the most virulent species of human malaria. Our present work used docking studies to select potential inhibitors of pLDH, which were then tested for antimalarial activity against P. falciparum in vitro and P. berghei malaria in mice. A virtual screening in DrugBank for analogs of NADH (an essential cofactor to pLDH) and computational studies were undertaken, and the potential binding of the selected compounds to the PfLDH active site was analyzed using Molegro Virtual Docker software. Fifty compounds were selected based on their similarity to NADH. The compounds with the best binding energies (itraconazole, atorvastatin and posaconazole) were tested against P. falciparum chloroquine-resistant blood parasites. All three compounds proved to be active in two immunoenzymatic assays performed in parallel using monoclonals specific to PfLDH or a histidine rich protein (HRP2). The IC50 values for each drug in both tests were similar, were lowest for posaconazole (<5 µM) and were 40- and 100-fold less active than chloroquine. The compounds reduced P. berghei parasitemia in treated mice, in comparison to untreated controls; itraconazole was the least active compound. The results of these activity trials confirmed that molecular docking studies are an important strategy for discovering new antimalarial drugs. This approach is more practical and less expensive than discovering novel compounds that require studies on human toxicology, since these compounds are already commercially available and thus approved for human use. PMID:21779323

  2. Purification and Kinetics of Higher Plant NADH:Nitrate Reductase.

    PubMed

    Campbell, W H; Smarrelli, J

    1978-04-01

    Squash cotyledon (Cucurbita pepo L.) NADH:nitrate reductase (NR) was purified 150-fold with 50% recovery by a single step procedure based on the affinity of the NR for blue-Sepharose. Blue-Sepharose, which is prepared by direct coupling of Cibacron blue to Sepharose, appears to bind squash NR at the NADH site. The NR can be purified in 2 to 3 hours to a specific activity of 2 mumol of NADH oxidized/minute * milligram of protein. Corn (Zea mays L.) leaf NR was also purified to a specific activity of 6.9 mumol of NADH oxidized/minute * milligram of protein using a blue-Sepharose affinity step. The blue-Sepharose method offers the advantages of a rapid purification of plant NR to a high specific activity with reasonable recovery of total activity.The kinetic mechanism of higher plant NR was investigated using these highly purified squash and corn NR preparations. Based on initial velocity and product inhibition studies utilizing both enzymes, a two-site ping-pong mechanism is proposed for NR. This kinetic mechanism incorporates the concept of the reduced NR transferring electrons from the NADH site to a physically separated nitrate site.

  3. Overexpression of NADH-dependent fumarate reductase improves D-xylose fermentation in recombinant Saccharomyces cerevisiae.

    PubMed

    Salusjärvi, Laura; Kaunisto, Sanna; Holmström, Sami; Vehkomäki, Maija-Leena; Koivuranta, Kari; Pitkänen, Juha-Pekka; Ruohonen, Laura

    2013-12-01

    Deviation from optimal levels and ratios of redox cofactors NAD(H) and NADP(H) is common when microbes are metabolically engineered. The resulting redox imbalance often reduces the rate of substrate utilization as well as biomass and product formation. An example is the metabolism of D-xylose by recombinant Saccharomyces cerevisiae strains expressing xylose reductase and xylitol dehydrogenase encoding genes from Scheffersomyces stipitis. This pathway requires both NADPH and NAD(+). The effect of overexpressing the glycosomal NADH-dependent fumarate reductase (FRD) of Trypanosoma brucei in D-xylose-utilizing S. cerevisiae alone and together with an endogenous, cytosol directed NADH-kinase (POS5Δ17) was studied as one possible solution to overcome this imbalance. Expression of FRD and FRD + POS5Δ17 resulted in 60 and 23 % increase in ethanol yield, respectively, on D-xylose under anaerobic conditions. At the same time, xylitol yield decreased in the FRD strain suggesting an improvement in redox balance. We show that fumarate reductase of T. brucei can provide an important source of NAD(+) in yeast under anaerobic conditions, and can be useful for metabolic engineering strategies where the redox cofactors need to be balanced. The effects of FRD and NADH-kinase on aerobic and anaerobic D-xylose and D-glucose metabolism are discussed.

  4. Localization of Ubiquinone-8 in the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae*

    PubMed Central

    Casutt, Marco S.; Nedielkov, Ruslan; Wendelspiess, Severin; Vossler, Sara; Gerken, Uwe; Murai, Masatoshi; Miyoshi, Hideto; Möller, Heiko M.; Steuber, Julia

    2011-01-01

    Na+ is the second major coupling ion at membranes after protons, and many pathogenic bacteria use the sodium-motive force to their advantage. A prominent example is Vibrio cholerae, which relies on the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR) as the first complex in its respiratory chain. The Na+-NQR is a multisubunit, membrane-embedded NADH dehydrogenase that oxidizes NADH and reduces quinone to quinol. Existing models describing redox-driven Na+ translocation by the Na+-NQR are based on the assumption that the pump contains four flavins and one FeS cluster. Here we show that the large, peripheral NqrA subunit of the Na+-NQR binds one molecule of ubiquinone-8. Investigations of the dynamic interaction of NqrA with quinones by surface plasmon resonance and saturation transfer difference NMR reveal a high affinity, which is determined by the methoxy groups at the C-2 and C-3 positions of the quinone headgroup. Using photoactivatable quinone derivatives, it is demonstrated that ubiquinone-8 bound to NqrA occupies a functional site. A novel scheme of electron transfer in Na+-NQR is proposed that is initiated by NADH oxidation on subunit NqrF and leads to quinol formation on subunit NqrA. PMID:21885438

  5. Glyceraldehyde 3-phosphate dehydrogenase-telomere association correlates with redox status in Trypanosoma cruzi.

    PubMed

    Pariona-Llanos, Ricardo; Pavani, Raphael Souza; Reis, Marcelo; Noël, Vincent; Silber, Ariel Mariano; Armelin, Hugo Aguirre; Cano, Maria Isabel Nogueira; Elias, Maria Carolina

    2015-01-01

    Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a classical metabolic enzyme involved in energy production and plays a role in additional nuclear functions, including transcriptional control, recognition of misincorporated nucleotides in DNA and maintenance of telomere structure. Here, we show that the recombinant protein T. cruzi GAPDH (rTcGAPDH) binds single-stranded telomeric DNA. We demonstrate that the binding of GAPDH to telomeric DNA correlates with the balance between oxidized and reduced forms of nicotinamide adenine dinucleotides (NAD+/NADH). We observed that GAPDH-telomere association and NAD+/NADH balance changed throughout the T. cruzi life cycle. For example, in replicative epimastigote forms of T. cruzi, which show similar intracellular concentrations of NAD+ and NADH, GAPDH binds to telomeric DNA in vivo and this binding activity is inhibited by exogenous NAD+. In contrast, in the T. cruzi non-proliferative trypomastigote forms, which show higher NAD+ concentration, GAPDH was absent from telomeres. In addition, NAD+ abolishes physical interaction between recombinant GAPDH and synthetic telomere oligonucleotide in a cell free system, mimicking exogenous NAD+ that reduces GAPDH-telomere interaction in vivo. We propose that the balance in the NAD+/NADH ratio during T. cruzi life cycle homeostatically regulates GAPDH telomere association, suggesting that in trypanosomes redox status locally modulates GAPDH association with telomeric DNA.

  6. Glyceraldehyde 3-Phosphate Dehydrogenase-Telomere Association Correlates with Redox Status in Trypanosoma cruzi

    PubMed Central

    Pariona-Llanos, Ricardo; Pavani, Raphael Souza; Reis, Marcelo; Noël, Vincent; Silber, Ariel Mariano; Armelin, Hugo Aguirre; Cano, Maria Isabel Nogueira; Elias, Maria Carolina

    2015-01-01

    Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a classical metabolic enzyme involved in energy production and plays a role in additional nuclear functions, including transcriptional control, recognition of misincorporated nucleotides in DNA and maintenance of telomere structure. Here, we show that the recombinant protein T. cruzi GAPDH (rTcGAPDH) binds single-stranded telomeric DNA. We demonstrate that the binding of GAPDH to telomeric DNA correlates with the balance between oxidized and reduced forms of nicotinamide adenine dinucleotides (NAD+/NADH). We observed that GAPDH-telomere association and NAD+/NADH balance changed throughout the T. cruzi life cycle. For example, in replicative epimastigote forms of T. cruzi, which show similar intracellular concentrations of NAD+ and NADH, GAPDH binds to telomeric DNA in vivo and this binding activity is inhibited by exogenous NAD+. In contrast, in the T. cruzi non-proliferative trypomastigote forms, which show higher NAD+ concentration, GAPDH was absent from telomeres. In addition, NAD+ abolishes physical interaction between recombinant GAPDH and synthetic telomere oligonucleotide in a cell free system, mimicking exogenous NAD+ that reduces GAPDH-telomere interaction in vivo. We propose that the balance in the NAD+/NADH ratio during T. cruzi life cycle homeostatically regulates GAPDH telomere association, suggesting that in trypanosomes redox status locally modulates GAPDH association with telomeric DNA. PMID:25775131

  7. Reduced levels of NADH-dependent glutamate dehydrogenase decrease the glutamate content of ripe tomato fruit but have no effect on green fruit or leaves.

    PubMed

    Ferraro, Gisela; D'Angelo, Matilde; Sulpice, Ronan; Stitt, Mark; Valle, Estela M

    2015-06-01

    Glutamate (Glu) is a taste enhancer that contributes to the characteristic flavour of foods. In fruit of tomato (Solanum lycopersicum L.), the Glu content increases dramatically during the ripening process, becoming the most abundant free amino acid when the fruit become red. There is also a concomitant increase in NADH-dependent glutamate dehydrogenase (GDH) activity during the ripening transition. This enzyme is located in the mitochondria and catalyses the reversible amination of 2-oxoglutarate to Glu. To investigate the potential effect of GDH on Glu metabolism, the abundance of GDH was altered by artificial microRNA technology. Efficient silencing of all the endogenous SlGDH genes was achieved, leading to a dramatic decrease in total GDH activity. This decrease in GDH activity did not lead to any clear morphological or metabolic phenotype in leaves or green fruit. However, red fruit on the transgenic plants showed markedly reduced levels of Glu and a large increase in aspartate, glucose and fructose content in comparison to wild-type fruit. These results suggest that GDH is involved in the synthesis of Glu in tomato fruit during the ripening processes. This contrasts with the biological role ascribed to GDH in many other tissues and species. Overall, these findings suggest that GDH has a major effect on the control of metabolic composition during tomato fruit ripening, but not at other stages of development. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  8. Rational design of engineered microbial cell surface multi-enzyme co-display system for sustainable NADH regeneration from low-cost biomass.

    PubMed

    Han, Lei; Liang, Bo; Song, Jianxia

    2018-02-01

    As an important cofactor, NADH is essential for most redox reactions and biofuel cells. However, supply of exogenous NADH is challenged, due to the low production efficiency and high cost of NADH regeneration system, as well as low stability of NADH. Here, we constructed a novel cell surface multi-enzyme co-display system with ratio- and space-controllable manner as exogenous NADH regeneration system for the sustainable NADH production from low-cost biomass. Dockerin-fused glucoamylase (GA) and glucose dehydrogenase (GDH) were expressed and assembled on the engineered bacterial surfaces, which displayed protein scaffolds with various combinations of different cohesins. When the ratio of GA and GDH was 3:1, the NADH production rate of the whole-cell biocatalyst reached the highest level using starch as substrate, which was three times higher than that of mixture of free enzymes, indicating that the highly ordered spatial organization of enzymes would promote reactions, due to the ratio of enzymes and proximity effect. To confirm performance of the established NADH regeneration system, the highly efficient synthesis of L-lactic acid (L-LA) was conducted by the system and the yield of L-LA (16 g/L) was twice higher than that of the mixture of free enzymes. The multi-enzyme co-display system showed good stability in the cyclic utilization. In conclusion, the novel sustainable NADH system would provide a cost-effective strategy to regenerate cofactor from low-cost biomass.

  9. Aminobacter aminovorans NADH:flavin oxidoreductase His140: a highly conserved residue critical for NADH binding and utilization.

    PubMed

    Russell, Thomas R; Tu, Shiao-Chun

    2004-10-12

    Homodimeric FRD(Aa) Class I is an NADH:flavin oxidoreductase from Aminobacter aminovorans. It is unusual because it contains an FMN cofactor but utilizes a sequential-ordered kinetic mechanism. Because little is known about NADH-specific flavin reductases in general and FRD(Aa) in particular, this study aimed to further explore FRD(Aa) by identifying the functionalities of a key residue. A sequence alignment of FRD(Aa) with several known and hypothetical flavoproteins in the same subfamily reveals within the flavin reductase active-site domain a conserved GDH motif, which is believed to be responsible for the enzyme and NADH interaction. Mutation of the His140 in this GDH motif to alanine reduced FRD(Aa) activity to <3%. An ultrafiltration assay and fluorescence quenching demonstrated that H140A FRD(Aa) binds FMN in the same 1:1 stoichiometric ratio as the wild-type enzyme, but with slightly weakened affinity (K(d) = 0.9 microM). Anaerobic stopped-flow studies were carried out using both the native and mutated FRD(Aa). Similar to the native enzyme, H140A FRD(Aa) was also able to reduce the FMN cofactor by NADH although much less efficiently. Kinetic analysis of anaerobic reduction measurements indicated that the His140 residue of FRD(Aa) was essential to NADH binding, as well as important for the reduction of the FMN cofactor. For the native enzyme, the cofactor reduction was followed by at least one slower step in the catalytic pathway.

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

    PubMed

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

    2008-07-01

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

  11. Quinone Reduction by the Na+-Translocating NADH Dehydrogenase Promotes Extracellular Superoxide Production in Vibrio cholerae▿ †

    PubMed Central

    Lin, Po-Chi; Türk, Karin; Häse, Claudia C.; Fritz, Günter; Steuber, Julia

    2007-01-01

    The pathogenicity of Vibrio cholerae is influenced by sodium ions which are actively extruded from the cell by the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR). To study the function of the Na+-NQR in the respiratory chain of V. cholerae, we examined the formation of organic radicals and superoxide in a wild-type strain and a mutant strain lacking the Na+-NQR. Upon reduction with NADH, an organic radical was detected in native membranes by electron paramagnetic resonance spectroscopy which was assigned to ubisemiquinones generated by the Na+-NQR. The radical concentration increased from 0.2 mM at 0.08 mM Na+ to 0.4 mM at 14.7 mM Na+, indicating that the concentration of the coupling cation influences the redox state of the quinone pool in V. cholerae membranes. During respiration, V. cholerae cells produced extracellular superoxide with a specific activity of 10.2 nmol min−1 mg−1 in the wild type compared to 3.1 nmol min−1 mg−1 in the NQR deletion strain. Raising the Na+ concentration from 0.1 to 5 mM increased the rate of superoxide formation in the wild-type V. cholerae strain by at least 70%. Rates of respiratory H2O2 formation by wild-type V. cholerae cells (30.9 nmol min−1 mg−1) were threefold higher than rates observed with the mutant strain lacking the Na+-NQR (9.7 nmol min−1 mg−1). Our study shows that environmental Na+ could stimulate ubisemiquinone formation by the Na+-NQR and hereby enhance the production of reactive oxygen species formed during the autoxidation of reduced quinones. PMID:17322313

  12. Chaetomium thermophilum formate dehydrogenase has high activity in the reduction of hydrogen carbonate (HCO3 -) to formate.

    PubMed

    Aslan, Aşkın Sevinç; Valjakka, Jarkko; Ruupunen, Jouni; Yildirim, Deniz; Turner, Nicholas J; Turunen, Ossi; Binay, Barış

    2017-01-01

    While formate dehydrogenases (FDHs) have been used for cofactor recycling in chemoenzymatic synthesis, the ability of FDH to reduce CO 2 could also be utilized in the conversion of CO 2 to useful products via formate (HCOO - ). In this study, we investigated the reduction of CO 2 in the form of hydrogen carbonate (HCO 3 - ) to formate by FDHs from Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) in a NADH-dependent reaction. The catalytic performance with HCO 3 - as a substrate was evaluated by measuring the kinetic rates and conducting productivity assays. CtFDH showed a higher efficiency in converting HCO 3 - to formate than CmFDH, whereas CmFDH was better in the oxidation of formate. The pH optimum of the reduction was at pH 7-8. However, the high concentrations of HCO 3 - reduced the reaction rate. CtFDH was modeled in the presence of HCO 3 - showing that it fits to the active site. The active site setting for hydride transfer in CO 2 reduction was modeled. The hydride donated by NADH would form a favorable contact to the carbon atom of HCO 3 - , resulting in a surplus of electrons within the molecule. This would cause the complex formed by hydrogen carbonate and the hydride to break into formate and hydroxide ions. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Reverse electron transport effects on NADH formation and metmyoglobin reduction.

    PubMed

    Belskie, K M; Van Buiten, C B; Ramanathan, R; Mancini, R A

    2015-07-01

    The objective was to determine if NADH generated via reverse electron flow in beef mitochondria can be used for electron transport-mediated reduction and metmyoglobin reductase pathways. Beef mitochondria were isolated from bovine hearts (n=5) and reacted with combinations of succinate, NAD, and mitochondrial inhibitors to measure oxygen consumption and NADH formation. Mitochondria and metmyoglobin were reacted with succinate, NAD, and mitochondrial inhibitors to measure electron transport-mediated metmyoglobin reduction and metmyoglobin reductase activity. Addition of succinate and NAD increased oxygen consumption, NADH formation, electron transport-mediated metmyoglobin reduction, and reductase activity (p<0.05). Addition of antimycin A prevented electron flow beyond complex III, therefore, decreasing oxygen consumption and electron transport-mediated metmyoglobin reduction. Addition of rotenone prevented reverse electron flow, increased oxygen consumption, increased electron transport-mediated metmyoglobin reduction, and decreased NADH formation. Succinate and NAD can generate NADH in bovine tissue postmortem via reverse electron flow and this NADH can be used by both electron transport-mediated and metmyoglobin reductase pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Initial Evidence for Adaptive Selection on the NADH Subunit Two of Freshwater Dolphins by Analyses of Mitochondrial Genomes.

    PubMed

    Caballero, Susana; Duchêne, Sebastian; Garavito, Manuel F; Slikas, Beth; Baker, C Scott

    2015-01-01

    A small number of cetaceans have adapted to an entirely freshwater environment, having colonized rivers in Asia and South America from an ancestral origin in the marine environment. This includes the 'river dolphins', early divergence from the odontocete lineage, and two species of true dolphins (Family Delphinidae). Successful adaptation to the freshwater environment may have required increased demands in energy involved in processes such as the mitochondrial osmotic balance. For this reason, riverine odontocetes provide a compelling natural experiment in adaptation of mammals from marine to freshwater habitats. Here we present initial evidence of positive selection in the NADH dehydrogenase subunit 2 of riverine odontocetes by analyses of full mitochondrial genomes, using tests of selection and protein structure modeling. The codon model with highest statistical support corresponds to three discrete categories for amino acid sites, those under positive, neutral, and purifying selection. With this model we found positive selection at site 297 of the NADH dehydrogenase subunit 2 (dN/dS>1.0,) leading to a substitution of an Ala or Val from the ancestral state of Thr. A phylogenetic reconstruction of 27 cetacean mitogenomes showed that an Ala substitution has evolved at least four times in cetaceans, once or more in the three 'river dolphins' (Families Pontoporidae, Lipotidae and Inidae), once in the riverine Sotalia fluviatilis (but not in its marine sister taxa), once in the riverine Orcaella brevirostris from the Mekong River (but not in its marine sister taxa) and once in two other related marine dolphins. We located the position of this amino acid substitution in an alpha-helix channel in the trans-membrane domain in both the E. coli structure and Sotalia fluviatilis model. In E. coli this position is located in a helix implicated in a proton translocation channel of respiratory complex 1 and may have a similar role in the NADH dehydrogenases of cetaceans.

  15. Alpha-ketoglutarate dehydrogenase: a target and generator of oxidative stress

    PubMed Central

    Tretter, Laszlo; Adam-Vizi, Vera

    2005-01-01

    Alpha-ketoglutarate dehydrogenase (α-KGDH) is a highly regulated enzyme, which could determine the metabolic flux through the Krebs cycle. It catalyses the conversion of α-ketoglutarate to succinyl-CoA and produces NADH directly providing electrons for the respiratory chain. α-KGDH is sensitive to reactive oxygen species (ROS) and inhibition of this enzyme could be critical in the metabolic deficiency induced by oxidative stress. Aconitase in the Krebs cycle is more vulnerable than α-KGDH to ROS but as long as α-KGDH is functional NADH generation in the Krebs cycle is maintained. NADH supply to the respiratory chain is limited only when α-KGDH is also inhibited by ROS. In addition being a key target, α-KGDH is able to generate ROS during its catalytic function, which is regulated by the NADH/NAD+ ratio. The pathological relevance of these two features of α-KGDH is discussed in this review, particularly in relation to neurodegeneration, as an impaired function of this enzyme has been found to be characteristic for several neurodegenerative diseases. PMID:16321804

  16. Life without complex I: proteome analyses of an Arabidopsis mutant lacking the mitochondrial NADH dehydrogenase complex

    PubMed Central

    Fromm, Steffanie; Senkler, Jennifer; Eubel, Holger; Peterhänsel, Christoph; Braun, Hans-Peter

    2016-01-01

    The mitochondrial NADH dehydrogenase complex (complex I) is of particular importance for the respiratory chain in mitochondria. It is the major electron entry site for the mitochondrial electron transport chain (mETC) and therefore of great significance for mitochondrial ATP generation. We recently described an Arabidopsis thaliana double-mutant lacking the genes encoding the carbonic anhydrases CA1 and CA2, which both form part of a plant-specific ‘carbonic anhydrase domain’ of mitochondrial complex I. The mutant lacks complex I completely. Here we report extended analyses for systematically characterizing the proteome of the ca1ca2 mutant. Using various proteomic tools, we show that lack of complex I causes reorganization of the cellular respiration system. Reduced electron entry into the respiratory chain at the first segment of the mETC leads to induction of complexes II and IV as well as alternative oxidase. Increased electron entry at later segments of the mETC requires an increase in oxidation of organic substrates. This is reflected by higher abundance of proteins involved in glycolysis, the tricarboxylic acid cycle and branched-chain amino acid catabolism. Proteins involved in the light reaction of photosynthesis, the Calvin cycle, tetrapyrrole biosynthesis, and photorespiration are clearly reduced, contributing to the significant delay in growth and development of the double-mutant. Finally, enzymes involved in defense against reactive oxygen species and stress symptoms are much induced. These together with previously reported insights into the function of plant complex I, which were obtained by analysing other complex I mutants, are integrated in order to comprehensively describe ‘life without complex I’. PMID:27122571

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

    PubMed

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

    2015-04-01

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

  18. The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase*

    PubMed Central

    Glasser, Nathaniel R.; Wang, Benjamin X.; Hoy, Julie A.; Newman, Dianne K.

    2017-01-01

    Phenazines are a class of redox-active molecules produced by diverse bacteria and archaea. Many of the biological functions of phenazines, such as mediating signaling, iron acquisition, and redox homeostasis, derive from their redox activity. Although prior studies have focused on extracellular phenazine oxidation by oxygen and iron, here we report a search for reductants and catalysts of intracellular phenazine reduction in Pseudomonas aeruginosa. Enzymatic assays in cell-free lysate, together with crude fractionation and chemical inhibition, indicate that P. aeruginosa contains multiple enzymes that catalyze the reduction of the endogenous phenazines pyocyanin and phenazine-1-carboxylic acid in both cytosolic and membrane fractions. We used chemical inhibitors to target general enzyme classes and found that an inhibitor of flavoproteins and heme-containing proteins, diphenyleneiodonium, effectively inhibited phenazine reduction in vitro, suggesting that most phenazine reduction derives from these enzymes. Using natively purified proteins, we demonstrate that the pyruvate and α-ketoglutarate dehydrogenase complexes directly catalyze phenazine reduction with pyruvate or α-ketoglutarate as electron donors. Both complexes transfer electrons to phenazines through the common subunit dihydrolipoamide dehydrogenase, a flavoprotein encoded by the gene lpdG. Although we were unable to co-crystallize LpdG with an endogenous phenazine, we report its X-ray crystal structure in the apo-form (refined to 1.35 Å), bound to NAD+ (1.45 Å), and bound to NADH (1.79 Å). In contrast to the notion that phenazines support intracellular redox homeostasis by oxidizing NADH, our work suggests that phenazines may substitute for NAD+ in LpdG and other enzymes, achieving the same end by a different mechanism. PMID:28174304

  19. The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase.

    PubMed

    Glasser, Nathaniel R; Wang, Benjamin X; Hoy, Julie A; Newman, Dianne K

    2017-03-31

    Phenazines are a class of redox-active molecules produced by diverse bacteria and archaea. Many of the biological functions of phenazines, such as mediating signaling, iron acquisition, and redox homeostasis, derive from their redox activity. Although prior studies have focused on extracellular phenazine oxidation by oxygen and iron, here we report a search for reductants and catalysts of intracellular phenazine reduction in Pseudomonas aeruginosa Enzymatic assays in cell-free lysate, together with crude fractionation and chemical inhibition, indicate that P. aeruginosa contains multiple enzymes that catalyze the reduction of the endogenous phenazines pyocyanin and phenazine-1-carboxylic acid in both cytosolic and membrane fractions. We used chemical inhibitors to target general enzyme classes and found that an inhibitor of flavoproteins and heme-containing proteins, diphenyleneiodonium, effectively inhibited phenazine reduction in vitro , suggesting that most phenazine reduction derives from these enzymes. Using natively purified proteins, we demonstrate that the pyruvate and α-ketoglutarate dehydrogenase complexes directly catalyze phenazine reduction with pyruvate or α-ketoglutarate as electron donors. Both complexes transfer electrons to phenazines through the common subunit dihydrolipoamide dehydrogenase, a flavoprotein encoded by the gene lpdG Although we were unable to co-crystallize LpdG with an endogenous phenazine, we report its X-ray crystal structure in the apo-form (refined to 1.35 Å), bound to NAD + (1.45 Å), and bound to NADH (1.79 Å). In contrast to the notion that phenazines support intracellular redox homeostasis by oxidizing NADH, our work suggests that phenazines may substitute for NAD + in LpdG and other enzymes, achieving the same end by a different mechanism. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    PubMed

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

    2015-05-01

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

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

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

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

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

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

    DOE PAGES

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

    2015-04-25

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

  3. Plant mitochondrial pyruvate dehydrogenase complex: purification and identification of catalytic components in potato.

    PubMed Central

    Millar, A H; Knorpp, C; Leaver, C J; Hill, S A

    1998-01-01

    The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv. Romano) tuber mitochondria was purified 40-fold to a specific activity of 5.60 micromol/min per mg of protein. The activity of the complex depended on pyruvate, divalent cations, NAD+ and CoA and was competitively inhibited by both NADH and acetyl-CoA. SDS/PAGE revealed the complex consisted of seven polypeptide bands with apparent molecular masses of 78, 60, 58, 55, 43, 41 and 37 kDa. N-terminal sequencing revealed that the 78 kDa protein was dihydrolipoamide transacetylase (E2), the 58 kDa protein was dihydrolipoamide dehydrogenase (E3), the 43 and 41 kDa proteins were alpha subunits of pyruvate dehydrogenase, and the 37 kDa protein was the beta subunit of pyruvate dehydrogenase. N-terminal sequencing of the 55 kDa protein band yielded two protein sequences: one was another E3; the other was similar to the sequence of E2 from plant and yeast sources but was distinctly different from the sequence of the 78 kDa protein. Incubation of the mPDC with [2-14C]pyruvate resulted in the acetylation of both the 78 and 55 kDa proteins. PMID:9729464

  4. Improving ethanol yield in acetate-reducing Saccharomyces cerevisiae by cofactor engineering of 6-phosphogluconate dehydrogenase and deletion of ALD6.

    PubMed

    Papapetridis, Ioannis; van Dijk, Marlous; Dobbe, Arthur P A; Metz, Benjamin; Pronk, Jack T; van Maris, Antonius J A

    2016-04-26

    Acetic acid, an inhibitor of sugar fermentation by yeast, is invariably present in lignocellulosic hydrolysates which are used or considered as feedstocks for yeast-based bioethanol production. Saccharomyces cerevisiae strains have been constructed, in which anaerobic reduction of acetic acid to ethanol replaces glycerol formation as a mechanism for reoxidizing NADH formed in biosynthesis. An increase in the amount of acetate that can be reduced to ethanol should further decrease acetic acid concentrations and enable higher ethanol yields in industrial processes based on lignocellulosic feedstocks. The stoichiometric requirement of acetate reduction for NADH implies that increased generation of NADH in cytosolic biosynthetic reactions should enhance acetate consumption. Replacement of the native NADP(+)-dependent 6-phosphogluconate dehydrogenase in S. cerevisiae by a prokaryotic NAD(+)-dependent enzyme resulted in increased cytosolic NADH formation, as demonstrated by a ca. 15% increase in the glycerol yield on glucose in anaerobic cultures. Additional deletion of ALD6, which encodes an NADP(+)-dependent acetaldehyde dehydrogenase, led to a 39% increase in the glycerol yield compared to a non-engineered strain. Subsequent replacement of glycerol formation by an acetate reduction pathway resulted in a 44% increase of acetate consumption per amount of biomass formed, as compared to an engineered, acetate-reducing strain that expressed the native 6-phosphogluconate dehydrogenase and ALD6. Compared to a non-acetate reducing reference strain under the same conditions, this resulted in a ca. 13% increase in the ethanol yield on glucose. The combination of NAD(+)-dependent 6-phosphogluconate dehydrogenase expression and deletion of ALD6 resulted in a marked increase in the amount of acetate that was consumed in these proof-of-principle experiments, and this concept is ready for further testing in industrial strains as well as in hydrolysates. Altering the cofactor

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

    PubMed

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

    2016-11-25

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

  6. Purification and Partial Characterization of Two Soluble NAD(P)H Dehydrogenases from Arum maculatum Mitochondria 1

    PubMed Central

    Chauveau, Michèle; Lance, Claude

    1991-01-01

    Two enzyme systems carrying out the oxidation of NAD(P)H in the presence of various electron acceptors have been isolated and partially characterized from the supernatant of frozen-thawed mitochondria from Arum maculatum spadices. The two systems contain flavoproteins and differ by their ability to oxidize NADH or NADPH, optimum pH and pI values, sensitivity to Ca2+ and EGTA, denaturation by 4 molar urea, molecular mass, and number of subunits. These properties, together with methodological considerations, are compatible with the location of these enzyme activities on the outer surface of the inner mitochondrial membrane, and support the hypothesis of the existence of two separate dehydrogenases responsible for the mitochondrial oxidation of cytosolic NADH and NADPH. Images Figure 1 Figure 3 Figure 7 PMID:16668075

  7. The FlxABCD-HdrABC proteins correspond to a novel NADH dehydrogenase/heterodisulfide reductase widespread in anaerobic bacteria and involved in ethanol metabolism in Desulfovibrio vulgaris Hildenborough.

    PubMed

    Ramos, Ana Raquel; Grein, Fabian; Oliveira, Gonçalo P; Venceslau, Sofia S; Keller, Kimberly L; Wall, Judy D; Pereira, Inês A C

    2015-07-01

    Flavin-based electron bifurcation (FBEB) is an important mechanism for the energy metabolism of anaerobes. A new family of NADH dehydrogenases, the flavin oxidoreductase (FlxABCD, previously called FloxABCD), was proposed to perform FBEB in sulphate-reducing organisms coupled with heterodisulfide reductase (HdrABC). We found that the hdrABC-flxABCD gene cluster is widespread among anaerobic bacteria, pointing to a general and important role in their bioenergetics. In this work, we studied FlxABCD of Desulfovibrio vulgaris Hildenborough. The hdr-flx genes are part of the same transcriptional unit and are increased in transcription during growth in ethanol-sulfate, and to a less extent during pyruvate fermentation. Two mutant strains were generated: one where expression of the hdr-flx genes was interrupted and another lacking the flxA gene. Both strains were unable to grow with ethanol-sulfate, whereas growth was restored in a flxA-complemented strain. The mutant strains also produced very reduced amounts of ethanol compared with the wild type during pyruvate fermentation. Our results show that in D. vulgaris, the FlxABCD-HdrABC proteins are essential for NADH oxidation during growth on ethanol, probably involving a FBEB mechanism that leads to reduction of ferredoxin and the small protein DsrC, while in fermentation they operate in reverse, reducing NAD(+) for ethanol production. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  8. Tyrosine phosphorylation of dihydrolipoamide dehydrogenase as a potential cadmium target and its inhibitory role in regulating mouse sperm motility.

    PubMed

    Li, Xinhong; Wang, Lirui; Li, Yuhua; Fu, Jieli; Zhen, Linqing; Yang, Qiangzhen; Li, Sisi; Zhang, Yukun

    2016-05-16

    Cadmium (Cd) is reported to reduce sperm motility and functions. However, the molecular mechanisms of Cd-induced toxicity remain largely unknown, presenting a major knowledge gap in research on reproductive toxicology. In the present study, we identified a candidate protein, dihydrolipoamide dehydrogenase (DLD), which is a post-pyruvate metabolic enzyme, exhibiting tyrosine phosphorylation in mouse sperm exposed to Cd both in vivo and in vitro. Immunoprecipitation assay demonstrated DLD was phosphorylated in tyrosine residues without altered expression after Cd treatment, which further confirmed our identified result. However, the tyrosine phosphorylation of DLD did not participate in mouse sperm capacitation and Bovine Serum Albumin (BSA) effectively prevented the tyrosine phosphorylation of DLD. Moreover, Cd-induced tyrosine phosphorylation of DLD lowered its dehydrogenase activity and meanwhile, Nicotinamide Adenine Dinucleotide Hydrogen (NADH) content, Adenosine Triphosphate (ATP) production and sperm motility were all inhibited by Cd. Interestingly, when the tyrosine phosphorylation of DLD was blocked by BSA, the decrease of DLD activity, NADH and ATP content as well as sperm motility was also suppressed simultaneously. These results suggested that Cd-induced tyrosine phosphorylation of DLD inhibited its activity and thus suppressed the tricarboxylic acid (TCA) cycle, which resulted in the reduction of NADH and hence the ATP production generated through oxidative phosphorylation (OPHOXS). Taken together, our results revealed that Cd induced DLD tyrosine phosphorylation, in response to regulate TCA metabolic pathway, which reduced ATP levels and these negative effects led to decreased sperm motility. This study provided new understanding of the mechanisms contributing to the harmful effects of Cd on the motility and function of spermatozoa. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  9. Generation of reactive oxygen species in the reaction catalyzed by alpha-ketoglutarate dehydrogenase.

    PubMed

    Tretter, Laszlo; Adam-Vizi, Vera

    2004-09-08

    Alpha-ketoglutarate dehydrogenase (alpha-KGDH), a key enzyme in the Krebs' cycle, is a crucial early target of oxidative stress (Tretter and Adam-Vizi, 2000). The present study demonstrates that alpha-KGDH is able to generate H(2)O(2) and, thus, could also be a source of reactive oxygen species (ROS) in mitochondria. Isolated alpha-KGDH with coenzyme A (HS-CoA) and thiamine pyrophosphate started to produce H(2)O(2) after addition of alpha-ketoglutarate in the absence of nicotinamide adenine dinucleotide-oxidized (NAD(+)). NAD(+), which proved to be a powerful inhibitor of alpha-KGDH-mediated H(2)O(2) formation, switched the H(2)O(2) forming mode of the enzyme to the catalytic [nicotinamide adenine dinucleotide-reduced (NADH) forming] mode. In contrast, NADH stimulated H(2)O(2) formation by alpha-KGDH, and for this, neither alpha-ketoglutarate nor HS-CoA were required. When all of the substrates and cofactors of the enzyme were present, the NADH/NAD(+) ratio determined the rate of H(2)O(2) production. The higher the NADH/NAD(+) ratio the higher the rate of H(2)O(2) production. H(2)O(2) production as well as the catalytic function of the enzyme was activated by Ca(2+). In synaptosomes, using alpha-ketoglutarate as respiratory substrate, the rate of H(2)O(2) production increased by 2.5-fold, and aconitase activity decreased, indicating that alpha-KGDH can generate H(2)O(2) in in situ mitochondria. Given the NADH/NAD(+) ratio as a key regulator of H(2)O(2) production by alpha-KGDH, it is suggested that production of ROS could be significant not only in the respiratory chain but also in the Krebs' cycle when oxidation of NADH is impaired. Thus alpha-KGDH is not only a target of ROS but could significantly contribute to generation of oxidative stress in the mitochondria.

  10. Adrenal 11-beta hydroxysteroid dehydrogenase activity in response to stress.

    PubMed

    Zallocchi, Marisa; Matković, Laura; Damasco, María C

    2004-06-01

    This work studied the effect of stresses produced by simulated gavage or gavage with 200 mmol/L HCl two hours before adrenal extraction, on the activities of the 11beta-hydroxysteroid dehydrogenase 1 and 11beta-hydroxysteroid dehydrogenase 2 isoforms present in the rat adrenal gland. These activities were determined on immediately prepared adrenal microsomes following incubations with 3H-corticosterone and NAD+ or NADP+. 11-dehydrocorticosterone was measured as an end-product by TLC, and controls were adrenal microsomes from rats kept under basal (unstressed) conditions. 11beta-hydroxysteroid dehydrogenase 1 activity, but not 11beta-hydroxysteroid dehydrogenase 2 activity, was increased under both stress-conditions. Homeostatically, the stimulation of 11beta-hydroxysteroid dehydrogenase 1 activity would increase the supply of glucocorticoids. These, in turn, would activate the enzyme phenylethanolamine N-methyl transferase, thereby improving the synthesis of epinephrine as part of the stress-response.

  11. Engineering a synthetic anaerobic respiration for reduction of xylose to xylitol using NADH output of glucose catabolism by Escherichia coli AI21.

    PubMed

    Iverson, Andrew; Garza, Erin; Manow, Ryan; Wang, Jinhua; Gao, Yuanyuan; Grayburn, Scott; Zhou, Shengde

    2016-04-16

    Anaerobic rather than aerobic fermentation is preferred for conversion of biomass derived sugars to high value redox-neutral and reduced commodities. This will likely result in a higher yield of substrate to product conversion and decrease production cost since substrate often accounts for a significant portion of the overall cost. To this goal, metabolic pathway engineering has been used to optimize substrate carbon flow to target products. This approach works well for the production of redox neutral products such as lactic acid from redox neutral sugars using the reducing power NADH (nicotinamide adenine dinucleotide, reduced) generated from glycolysis (2 NADH per glucose equivalent). Nevertheless, greater than two NADH per glucose catabolized is needed for the production of reduced products (such as xylitol) from redox neutral sugars by anaerobic fermentation. The Escherichia coli strain AI05 (ΔfrdBC ΔldhA ΔackA Δ(focA-pflB) ΔadhE ΔptsG ΔpdhR::pflBp 6-(aceEF-lpd)), previously engineered for reduction of xylose to xylitol using reducing power (NADH equivalent) of glucose catabolism, was further engineered by 1) deleting xylAB operon (encoding for xylose isomerase and xylulokinase) to prevent xylose from entering the pentose phosphate pathway; 2) anaerobically expressing the sdhCDAB-sucABCD operon (encoding for succinate dehydrogenase, α-ketoglutarate dehydrogenase and succinyl-CoA synthetase) to enable an anaerobically functional tricarboxcylic acid cycle with a theoretical 10 NAD(P)H equivalent per glucose catabolized. These reducing equivalents can be oxidized by synthetic respiration via xylose reduction, producing xylitol. The resulting strain, AI21 (pAI02), achieved a 96 % xylose to xylitol conversion, with a yield of 6 xylitol per glucose catabolized (molar yield of xylitol per glucose consumed (YRPG) = 6). This represents a 33 % improvement in xylose to xylitol conversion, and a 63 % increase in xylitol yield per glucose catabolized over

  12. Enzymatic properties of the membrane-bound NADH oxidase system in the aerobic respiratory chain of Bacillus cereus.

    PubMed

    Kim, Man Suk; Kim, Young Jae

    2004-11-30

    Membranes prepared from Bacillus cereus KCTC 3674, grown aerobically on a complex medium, oxidized NADH exclusively, whereas deamino-NADH was little oxidized. The respiratory chain-linked NADH oxidase exhibited an apparent K(m) value of approximately 65 microM for NADH. The maximum activity of the NADH oxidase was obtained at about pH 8.5 in the presence of 0.1 M KCl (or NaCl). Respiratory chain inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) inhibited the activity of the NADH oxidase by about 90% at a concentration of 40 microM. Interestingly, rotenone and capsaicin inhibited the activity of the NADH oxidase by about 60% at a concentration of 40 microM and the activity was also highly sensitive to Ag(+).

  13. Pharmacological Stimulation of NADH Oxidation Ameliorates Obesity and Related Phenotypes in Mice

    PubMed Central

    Hwang, Jung Hwan; Kim, Dong Wook; Jo, Eun Jin; Kim, Yong Kyung; Jo, Young Suk; Park, Ji Hoon; Yoo, Sang Ku; Park, Myung Kyu; Kwak, Tae Hwan; Kho, Young Lim; Han, Jin; Choi, Hueng-Sik; Lee, Sang-Hee; Kim, Jin Man; Lee, InKyu; Kyung, Taeyoon; Jang, Cholsoon; Chung, Jongkyeong; Kweon, Gi Ryang; Shong, Minho

    2009-01-01

    OBJECTIVE Nicotinamide adenine dinucleotides (NAD+ and NADH) play a crucial role in cellular energy metabolism, and a dysregulated NAD+-to-NADH ratio is implicated in metabolic syndrome. However, it is still unknown whether a modulating intracellular NAD+-to-NADH ratio is beneficial in treating metabolic syndrome. We tried to determine whether pharmacological stimulation of NADH oxidation provides therapeutic effects in rodent models of metabolic syndrome. RESEARCH DESIGN AND METHODS We used β-lapachone (βL), a natural substrate of NADH:quinone oxidoreductase 1 (NQO1), to stimulate NADH oxidation. The βL-induced pharmacological effect on cellular energy metabolism was evaluated in cells derived from NQO1-deficient mice. In vivo therapeutic effects of βL on metabolic syndrome were examined in diet-induced obesity (DIO) and ob/ob mice. RESULTS NQO1-dependent NADH oxidation by βL strongly provoked mitochondrial fatty acid oxidation in vitro and in vivo. These effects were accompanied by activation of AMP-activated protein kinase and carnitine palmitoyltransferase and suppression of acetyl-coenzyme A (CoA) carboxylase activity. Consistently, systemic βL administration in rodent models of metabolic syndrome dramatically ameliorated their key symptoms such as increased adiposity, glucose intolerance, dyslipidemia, and fatty liver. The treated mice also showed higher expressions of the genes related to mitochondrial energy metabolism (PPARγ coactivator-1α, nuclear respiratory factor-1) and caloric restriction (Sirt1) consistent with the increased mitochondrial biogenesis and energy expenditure. CONCLUSIONS Pharmacological activation of NADH oxidation by NQO1 resolves obesity and related phenotypes in mice, opening the possibility that it may provide the basis for a new therapy for the treatment of metabolic syndrome. PMID:19136651

  14. Initial Evidence for Adaptive Selection on the NADH Subunit Two of Freshwater Dolphins by Analyses of Mitochondrial Genomes

    PubMed Central

    Caballero, Susana; Duchêne, Sebastian; Garavito, Manuel F.; Slikas, Beth; Baker, C. Scott

    2015-01-01

    A small number of cetaceans have adapted to an entirely freshwater environment, having colonized rivers in Asia and South America from an ancestral origin in the marine environment. This includes the ‘river dolphins’, early divergence from the odontocete lineage, and two species of true dolphins (Family Delphinidae). Successful adaptation to the freshwater environment may have required increased demands in energy involved in processes such as the mitochondrial osmotic balance. For this reason, riverine odontocetes provide a compelling natural experiment in adaptation of mammals from marine to freshwater habitats. Here we present initial evidence of positive selection in the NADH dehydrogenase subunit 2 of riverine odontocetes by analyses of full mitochondrial genomes, using tests of selection and protein structure modeling. The codon model with highest statistical support corresponds to three discrete categories for amino acid sites, those under positive, neutral, and purifying selection. With this model we found positive selection at site 297 of the NADH dehydrogenase subunit 2 (dN/dS>1.0,) leading to a substitution of an Ala or Val from the ancestral state of Thr. A phylogenetic reconstruction of 27 cetacean mitogenomes showed that an Ala substitution has evolved at least four times in cetaceans, once or more in the three ‘river dolphins’ (Families Pontoporidae, Lipotidae and Inidae), once in the riverine Sotalia fluviatilis (but not in its marine sister taxa), once in the riverine Orcaella brevirostris from the Mekong River (but not in its marine sister taxa) and once in two other related marine dolphins. We located the position of this amino acid substitution in an alpha-helix channel in the trans-membrane domain in both the E. coli structure and Sotalia fluviatilis model. In E. coli this position is located in a helix implicated in a proton translocation channel of respiratory complex 1 and may have a similar role in the NADH dehydrogenases of

  15. A microplate reader-based method to quantify NADH-cytochrome b5 reductase activity for diagnosis of recessive congenital methaemoglobinemia.

    PubMed

    Kedar, Prabhakar; Desai, Anand; Warang, Prashant; Colah, Roshan

    2017-05-01

    Congenital methemoglobinemia due to NADH-cytochrome b5 reductase 3 (CYB5R3) deficiencies is an autosomal recessive disorder that occurs sporadically worldwide, A sensitive, accurate, and rapid analysis of NADH-CYB5R enzyme concentrations is necessary for the diagnosis of RCM. Here we present an alternative microplate method that is based on a standard 96-well microplate format and microplate reader that simplify the quantification of NADH-CYB5R activity. TECAN (Infinite 200 PRO series) microplate reader with Tecan's proven Magellan™ software measured the NADH-CYB5R enzyme activity in 250 normal controls and previously diagnosed 25 cases of RCM due to NADH-CYB5R deficiency in the Indian population using 96-well microplates using 200 μl of total reaction mixture and also compared with standard spectrophotometric assay. We have also studied stability of the hemolysate stored at 4 and -20°C temperature. Enzyme activity in all 25 samples ranged from 6.09 to 10.07 IU/g Hb (mean ± SD: 8.08 ± 1.99 IU/g Hb) where as normal control ranged (n = 250) between 13.42 and 21.58 IU/g Hb) (mean ± SD: 17.5 ± 4.08 IU/g of Hb). Data obtained from the microplate reader were compared with standard spectrophotometer method and found 100% concordance using both methods. Microplate method allows differentiating between normal, deficient and intermediate enzyme activity. It was observed that samples had significant loss of activity when stored at 4°C and retained stable activity at -20°C for 1 week time. Our new method, incorporating a whole process of enzyme assay into a microplate format is readily applicable and allows rapid monitoring of enzyme assay. It is readily applicable to quantitative assay on pediatric sample as well as large number of samples for population screening.

  16. Syntrophomonas wolfei Uses an NADH-Dependent, Ferredoxin-Independent [FeFe]-Hydrogenase To Reoxidize NADH

    PubMed Central

    Losey, Nathaniel A.; Mus, Florence; Peters, John W.; Le, Huynh M.

    2017-01-01

    ABSTRACT Syntrophomonas wolfei syntrophically oxidizes short-chain fatty acids (four to eight carbons in length) when grown in coculture with a hydrogen- and/or formate-using methanogen. The oxidation of 3-hydroxybutyryl-coenzyme A (CoA), formed during butyrate metabolism, results in the production of NADH. The enzyme systems involved in NADH reoxidation in S. wolfei are not well understood. The genome of S. wolfei contains a multimeric [FeFe]-hydrogenase that may be a mechanism for NADH reoxidation. The S. wolfei genes for the multimeric [FeFe]-hydrogenase (hyd1ABC; SWOL_RS05165, SWOL_RS05170, SWOL_RS05175) and [FeFe]-hydrogenase maturation proteins (SWOL_RS05180, SWOL_RS05190, SWOL_RS01625) were coexpressed in Escherichia coli, and the recombinant Hyd1ABC was purified and characterized. The purified recombinant Hyd1ABC was a heterotrimer with an αβγ configuration and a molecular mass of 115 kDa. Hyd1ABC contained 29.2 ± 1.49 mol of Fe and 0.7 mol of flavin mononucleotide (FMN) per mole enzyme. The purified, recombinant Hyd1ABC reduced NAD+ and oxidized NADH without the presence of ferredoxin. The HydB subunit of the S. wolfei multimeric [FeFe]-hydrogenase lacks two iron-sulfur centers that are present in known confurcating NADH- and ferredoxin-dependent [FeFe]-hydrogenases. Hyd1ABC is a NADH-dependent hydrogenase that produces hydrogen from NADH without the need of reduced ferredoxin, which differs from confurcating [FeFe]-hydrogenases. Hyd1ABC provides a mechanism by which S. wolfei can reoxidize NADH produced during syntrophic butyrate oxidation when low hydrogen partial pressures are maintained by a hydrogen-consuming microorganism. IMPORTANCE Our work provides mechanistic understanding of the obligate metabolic coupling that occurs between hydrogen-producing fatty and aromatic acid-degrading microorganisms and their hydrogen-consuming partners in the process called syntrophy (feeding together). The multimeric [FeFe]-hydrogenase used NADH without the

  17. Stability and reactivity of liposome-encapsulated formate dehydrogenase and cofactor system in carbon dioxide gas-liquid flow.

    PubMed

    Yoshimoto, Makoto; Yamashita, Takayuki; Yamashiro, Takuya

    2010-01-01

    Formate dehydrogenase from Candida boidinii (CbFDH) is potentially applicable in reduction of CO(2) through oxidation of cofactor NADH into NAD(+). For this, the CbFDH activity needs to be maintained under practical reaction conditions, such as CO(2) gas-liquid flow. In this work, CbFDH and cofactor were encapsulated in liposomes and the liposomal enzymes were characterized in an external loop airlift bubble column. The airlift was operated at 45 degrees C with N(2) or CO(2) as gas phase at the superficial gas velocity U(G) of 2.0 or 3.0 cm/s. The activities of liposomal CbFDH/cofactor systems were highly stable in the airlift regardless of the type of gas phase because liposome membranes prevented interactions of the encapsulated enzyme and cofactor molecules with the gas-liquid interface of bubbles. On the other hand, free CbFDH was deactivated in the airlift especially at high U(G) with CO(2) bubbles. The liposomal CbFDH/NADH could catalyze reduction of CO(2) in the airlift giving the fractional oxidation of the liposomal NADH of 23% at the reaction time of 360 min. The cofactor was kept inside liposomes during the reaction operation with less than 10% of leakage. All of the results obtained demonstrate that the liposomal CbFDH/NADH functions as a stable catalyst for reduction of CO(2) in the airlift. (c) 2010 American Institute of Chemical Engineers

  18. Cloning, sequence, and disruption of the Saccharomyces diastaticus DAR1 gene encoding a glycerol-3-phosphate dehydrogenase.

    PubMed

    Wang, H T; Rahaim, P; Robbins, P; Yocum, R R

    1994-11-01

    The Saccharomyces diastaticus DAR1 gene was cloned by complementation in an Escherichia coli strain auxogrophic for glycerol-3-phosphate. DAR1 encodes an NADH-dependent dihydroxyacetone phosphate reductase (sn-glycerol-3-phosphate dehydrogenase [G3PDase; EC 1.1.1.8]) homologous to several other eukaryotic G3PDases. DAR1 is distinct from GUT2, which encodes a glucose-repressed mitochondrial G3PDase, but is identical to GPD1 from S. cerevisiae, a close relative of S. diastaticus. The level of DAR1-encoded G3PDase was increased about threefold in a medium of high osmolarity. Disruption of DAR1 in a haploid S. cerevisiae was not lethal but led to a decrease in cytoplasmic NADH-dependent G3PDase activity, an increase in osmotic sensitivity, and a 25% reduction in glycerol secretion from cells grown anaerobically on glucose.

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

    PubMed

    Beauchamp, Justin; Vieille, Claire

    2015-01-01

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

  20. Intron loss from the NADH dehydrogenase subunit 4 gene of lettuce mitochondrial DNA: evidence for homologous recombination of a cDNA intermediate.

    PubMed

    Geiss, K T; Abbas, G M; Makaroff, C A

    1994-04-01

    The mitochondrial gene coding for subunit 4 of the NADH dehydrogenase complex I (nad4) has been isolated and characterized from lettuce, Lactuca sativa. Analysis of nad4 genes in a number of plants by Southern hybridization had previously suggested that the intron content varied between species. Characterization of the lettuce gene confirms this observation. Lettuce nad4 contains two exons and one group IIA intron, whereas previously sequenced nad4 genes from turnip and wheat contain three group IIA introns. Northern analysis identified a transcript of 1600 nucleotides, which represents the mature nad4 mRNA and a primary transcript of 3200 nucleotides. Sequence analysis of lettuce and turnip nad4 cDNAs was used to confirm the intron/exon border sequences and to examine RNA editing patterns. Editing is observed at the 5' and 3' ends of the lettuce transcript, but is absent from sequences that correspond to exons two, three and the 5' end of exon four in turnip and wheat. In contrast, turnip transcripts are highly edited in this region, suggesting that homologous recombination of an edited and spliced cDNA intermediate was involved in the loss of introns two and three from an ancestral lettuce nad4 gene.

  1. Enzymatic characterization of a novel bovine liver dihydrodiol dehydrogenase--reaction mechanism and bile acid dehydrogenase activity.

    PubMed

    Nanjo, H; Adachi, H; Morihana, S; Mizoguchi, T; Nishihara, T; Terada, T

    1995-05-11

    Bovine liver cytosolic dihydrodiol dehydrogenase (DD3) has been characterized by its unique dihydrodiol dehydrogenase activity for trans-benzenedihydrodiol (trans-1,2-dihydrobenzene-1,2-diol) with the highest affinity and the greatest velocity among three multiple forms of dihydrodiol dehydrogenases (DD1-DD3). It is the first time that DD3 has shown a significant dehydrogenase activity for (S)-(+)-1-indanol with low Km value (0.33 +/- 0.022 mM) and high K(cat) value (25 +/- 0.79 min-1). The investigation of the product inhibition of (S)-(+)-1-indanol with NADP+ versus 1-indanone and NADPH clearly showed that the enzymatic reaction of DD3 may follow a typical ordered Bi Bi mechanism similar to many aldo/keto reductases. Additionally, DD3 was shown to catalyze the dehydrogenation of bile acids (lithocholic acid, taurolithocholic acid and taurochenodeoxycholic acid) having no 12-hydroxy groups with low Km values (17 +/- 0.65, 33 +/- 1.9 and 890 +/- 73 microM, respectively). In contrast, DD1, 3 alpha-hydroxysteroid dehydrogenase, shows a broad substrate specificity for many bile acids with higher affinity than those of DD3. Competitive inhibition of DD3 with androsterone against dehydrogenase activity for (S)-(+)-1-indanol, trans-benzenedihydrodiol or lithocholic acid suggests that these three substrates bind to the same substrate binding site of DD3, different from the case of human liver bile acid binder/dihydrodiol dehydrogenase (Takikawa, H., Stolz, A., Sugiyama, Y., Yoshida, H., Yamamoto, M. and Kaplowitz, N. (1990) J. Biol. Chem. 265, 2132-2136). Considering the reaction mechanism, DD3 may also play an important role in bile acids metabolism as well as the detoxication of aromatic hydrocarbons.

  2. The Na+-Translocating NADH:Quinone Oxidoreductase Enhances Oxidative Stress in the Cytoplasm of Vibrio cholerae

    PubMed Central

    Muras, Valentin; Dogaru-Kinn, Paul; Minato, Yusuke; Häse, Claudia C.

    2016-01-01

    ABSTRACT We searched for a source of reactive oxygen species (ROS) in the cytoplasm of the human pathogen Vibrio cholerae and addressed the mechanism of ROS formation using the dye 2′,7′-dichlorofluorescein diacetate (DCFH-DA) in respiring cells. By comparing V. cholerae strains with or without active Na+-translocating NADH:quinone oxidoreductase (Na+-NQR), this respiratory sodium ion redox pump was identified as a producer of ROS in vivo. The amount of cytoplasmic ROS detected in V. cholerae cells producing variants of Na+-NQR correlated well with rates of superoxide formation by the corresponding membrane fractions. Membranes from wild-type V. cholerae showed increased superoxide production activity (9.8 ± 0.6 μmol superoxide min−1 mg−1 membrane protein) compared to membranes from the mutant lacking Na+-NQR (0.18 ± 0.01 μmol min−1 mg−1). Overexpression of plasmid-encoded Na+-NQR in the nqr deletion strain resulted in a drastic increase in the formation of superoxide (42.6 ± 2.8 μmol min−1 mg−1). By analyzing a variant of Na+-NQR devoid of quinone reduction activity, we identified the reduced flavin adenine dinucleotide (FAD) cofactor of cytoplasmic NqrF subunit as the site for intracellular superoxide formation in V. cholerae. The impact of superoxide formation by the Na+-NQR on the virulence of V. cholerae is discussed. IMPORTANCE In several studies, it was demonstrated that the Na+-NQR in V. cholerae affects virulence in a yet unknown manner. We identified the reduced FAD cofactor in the NADH-oxidizing NqrF subunit of the Na+-NQR as the site of superoxide formation in the cytoplasm of V. cholerae. Our study provides the framework to understand how reactive oxygen species formed during respiration could participate in the regulated expression of virulence factors during the transition from aerobic to microaerophilic (intestinal) habitats. This hypothesis may turn out to be right for many other pathogens which, like V. cholerae, depend on

  3. Furfural reduction mechanism of a zinc-dependent alcohol dehydrogenase from Cupriavidus necator JMP134

    PubMed Central

    Kang, ChulHee; Hayes, Robert; Sanchez, Emiliano J.; Webb, Brian N.; Li, Qunrui; Hooper, Travis; Nissen, Mark S.; Xun, Luying

    2012-01-01

    Summary FurX is a tetrameric Zn-dependent alcohol dehydrogenase (ADH) from Cupriavidus necator JMP134. The enzyme rapidly reduces furfural with NADH as the reducing power. For the first time among characterized ADHs, the high-resolution structures of all reaction steps were obtained in a time-resolved manner, thereby illustrating the complete catalytic events of NADH-dependent reduction of furfural and the dynamic Zn2+ coordination among Glu66, water, substrate and product. In the fully closed conformation of the NADH complex, the catalytic turnover proved faster than observed for the partially closed conformation due to an effective proton transfer network. The domain motion triggered by NAD(H) association/dissociation appeared to facilitate dynamic interchanges in Zn2+ coordination with substrate and product molecules, ultimately increasing the enzymatic turnover rate. NAD+ dissociation appeared to be a slow process, involving multiple steps in concert with a domain opening and reconfiguration of Glu66. This agrees with the report that the cofactor is not dissociated from FurX during ethanol-dependent reduction of furfural, in which ethanol reduces NAD+ to NADH that is subsequently used for furfural reduction. PMID:22081946

  4. Activation of liver alcohol dehydrogenase by glycosylation.

    PubMed Central

    Tsai, C S; White, J H

    1983-01-01

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

  5. Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD(+).

    PubMed

    Malver, Olaf; Sebastian, Mina J; Oppenheimer, Norman J

    2014-11-01

    A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD(+) with a redox potential of -324mV and a 341nm λmax for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD(+) by primary alcohols. With HLADH 1-butanol has the highest Vmax at 49% that of β-NAD(+). The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon-hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD(+). In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD(+) by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD(+) has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Engineering of carboligase activity reaction in Candida glabrata for acetoin production.

    PubMed

    Li, Shubo; Xu, Nan; Liu, Liming; Chen, Jian

    2014-03-01

    Utilization of Candida glabrata overproducing pyruvate is a promising strategy for high-level acetoin production. Based on the known regulatory and metabolic information, acetaldehyde and thiamine were fed to identify the key nodes of carboligase activity reaction (CAR) pathway and provide a direction for engineering C. glabrata. Accordingly, alcohol dehydrogenase, acetaldehyde dehydrogenase, pyruvate decarboxylase, and butanediol dehydrogenase were selected to be manipulated for strengthening the CAR pathway. Following the rational metabolic engineering, the engineered strain exhibited increased acetoin biosynthesis (2.24 g/L). In addition, through in silico simulation and redox balance analysis, NADH was identified as the key factor restricting higher acetoin production. Correspondingly, after introduction of NADH oxidase, the final acetoin production was further increased to 7.33 g/L. By combining the rational metabolic engineering and cofactor engineering, the acetoin-producing C. glabrata was improved stepwise, opening a novel pathway for rational development of microorganisms for bioproduction. Copyright © 2013. Published by Elsevier Inc.

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

    PubMed

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

    2018-01-02

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

  8. Inhibition of 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) activity of human lung microsomes by genistein, daidzein, coumestrol and C(18)-, C(19)- and C(21)-hydroxysteroids and ketosteroids.

    PubMed

    Blomquist, Charles H; Lima, Paul H; Hotchkiss, John R

    2005-07-01

    Epidemiologic data suggest a relationship between dietary intake of phytochemicals and a lower incidence of some cancers. Modulation of steroid hormone metabolism has been proposed as a basis for this effect. It has been shown that aromatase, 3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) are inhibited by the isoflavones, genistein and daidzein, and by coumestrol. In general, the extent of inhibition has been expressed in terms of IC50-values, which do not give information as to the pattern of inhibition, i.e., competitive, non-competitive, or mixed. Less is known of the effects of these compounds on 3alpha-HSD. The human lung is known to have a high level of 17beta-HSD and 3alpha-HSD activity. During the course of studies to characterize both activities in normal and inflamed lung and lung tumors we noted that 3alpha-HSD activity with 5alpha-DHT of microsomes from normal, adult lung was particularly susceptible to inhibition by coumestrol. To clarify the pattern of inhibition, the inhibition constants Ki and K'i were evaluated from plots of 1/v versus [I] and [S]/v versus [I]. Genistein, daidzein and coumestrol gave mixed inhibition patterns versus both 5alpha-DHT and NADH. In contrast, 5alpha-androstane-3,17-dione and 5alpha-pregnane-3,20-dione were competitive with 5alpha-DHT. NAD inhibited competitively with NADH. Our findings demonstrate that phytochemicals have the potential to inhibit 5alpha-DHT metabolism and thereby affect the androgen status of the human lung. The observation of a mixed inhibition pattern suggests these compounds bind to more than one form of the enzyme within the catalytic pathway.

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

    PubMed Central

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

    1985-01-01

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

  10. Cloning, sequence, and disruption of the Saccharomyces diastaticus DAR1 gene encoding a glycerol-3-phosphate dehydrogenase.

    PubMed Central

    Wang, H T; Rahaim, P; Robbins, P; Yocum, R R

    1994-01-01

    The Saccharomyces diastaticus DAR1 gene was cloned by complementation in an Escherichia coli strain auxogrophic for glycerol-3-phosphate. DAR1 encodes an NADH-dependent dihydroxyacetone phosphate reductase (sn-glycerol-3-phosphate dehydrogenase [G3PDase; EC 1.1.1.8]) homologous to several other eukaryotic G3PDases. DAR1 is distinct from GUT2, which encodes a glucose-repressed mitochondrial G3PDase, but is identical to GPD1 from S. cerevisiae, a close relative of S. diastaticus. The level of DAR1-encoded G3PDase was increased about threefold in a medium of high osmolarity. Disruption of DAR1 in a haploid S. cerevisiae was not lethal but led to a decrease in cytoplasmic NADH-dependent G3PDase activity, an increase in osmotic sensitivity, and a 25% reduction in glycerol secretion from cells grown anaerobically on glucose. PMID:7961476

  11. Structural Insights into l-Tryptophan Dehydrogenase from a Photoautotrophic Cyanobacterium, Nostoc punctiforme.

    PubMed

    Wakamatsu, Taisuke; Sakuraba, Haruhiko; Kitamura, Megumi; Hakumai, Yuichi; Fukui, Kenji; Ohnishi, Kouhei; Ashiuchi, Makoto; Ohshima, Toshihisa

    2017-01-15

    l-Tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH), despite exhibiting high amino acid sequence identity (>30%)/homology (>50%) with NAD(P) + -dependent l-Glu/l-Leu/l-Phe/l-Val dehydrogenases, exclusively catalyzes reversible oxidative deamination of l-Trp to 3-indolepyruvate in the presence of NAD + Here, we determined the crystal structure of the apo form of NpTrpDH. The structure of the NpTrpDH monomer, which exhibited high similarity to that of l-Glu/l-Leu/l-Phe dehydrogenases, consisted of a substrate-binding domain (domain I, residues 3 to 133 and 328 to 343) and an NAD + /NADH-binding domain (domain II, residues 142 to 327) separated by a deep cleft. The apo-NpTrpDH existed in an open conformation, where domains I and II were apart from each other. The subunits dimerized themselves mainly through interactions between amino acid residues around the β-1 strand of each subunit, as was observed in the case of l-Phe dehydrogenase. The binding site for the substrate l-Trp was predicted by a molecular docking simulation and validated by site-directed mutagenesis. Several hydrophobic residues, which were located in the active site of NpTrpDH and possibly interacted with the side chain of the substrate l-Trp, were arranged similarly to that found in l-Leu/l-Phe dehydrogenases but fairly different from that of an l-Glu dehydrogenase. Our crystal structure revealed that Met-40, Ala-69, Ile-74, Ile-110, Leu-288, Ile-289, and Tyr-292 formed a hydrophobic cluster around the active site. The results of the site-directed mutagenesis experiments suggested that the hydrophobic cluster plays critical roles in protein folding, l-Trp recognition, and catalysis. Our results provide critical information for further characterization and engineering of this enzyme. In this study, we determined the three-dimensional structure of l-Trp dehydrogenase, analyzed its various site-directed substitution mutants at residues located in the active site, and obtained the

  12. Identification of a mitochondrial alcohol dehydrogenase in Schizosaccharomyces pombe: new insights into energy metabolism

    PubMed Central

    Crichton, Paul G.; Affourtit, Charles; Moore, Anthony L.

    2006-01-01

    In the present study we have shown that mitochondria isolated from Schizosaccharomyces pombe exhibit antimycin A-sensitive oxygen uptake activity that is exclusively dependent on ethanol and is inhibited by trifluoroethanol, a potent inhibitor of ADH (alcohol dehydrogenase). Ethanol-dependent respiratory activity has, to our knowledge, not been reported in S. pombe mitochondria to date, which is surprising as it has been concluded previously that only one ADH gene, encoding a cytosolic enzyme, occurs in this yeast. Spectrophotometric enzyme assays reveal that ADH activity in isolated mitochondria is increased ∼16-fold by Triton X-100, which demonstrates that the enzyme is located in the matrix. Using genetic knockouts, we show conclusively that the novel mitochondrial ADH is encoded by adh4 and, as such, is unrelated to ADH isoenzymes found in mitochondria of other yeasts. By performing a modular-kinetic analysis of mitochondrial electron transfer, we furthermore show how ethanol-dependent respiratory activity (which involves oxidation of matrix-located NADH) compares with that observed when succinate or externally added NADH are used as substrates. This analysis reveals distinct kinetic differences between substrates which fully explain the lack of respiratory control generally observed during ethanol oxidation in yeast mitochondria. PMID:16999687

  13. Substrate specificity of sheep liver sorbitol dehydrogenase.

    PubMed Central

    Lindstad, R I; Köll, P; McKinley-McKee, J S

    1998-01-01

    The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo> D-ribo > L-xylo > D-lyxo approximately L-arabino > D-arabino > L-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH > -CH2NH2 > -CH2OCH3 approximately -CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of

  14. Lactate metabolism and cytosolic NADH reducing equivalents in ovine adipocytes.

    PubMed

    Yang, Y T; White, L S; Muir, L A

    1982-01-01

    1. Isolated ovine adipocytes, unlike rat adipose tissue, could utilize lactate at a high rate. 2. When the rate of fatty acid synthesis was attenuated with 5-(tetradecyloxy)-2-furoic acid, a fatty acid synthesis inhibitor, there was a good positive correlation between the rates of lactate oxidation to CO2 and lactate incorporation into fatty acids. 3. Addition of 2,4-dinitrophenol enhanced lactate oxidation to CO2 independent of fatty acid synthesis. Under this condition, estimated cytosolic NADH formation from lactate dehydrogenation exceeded the need of NADH for cytosolic oxaloacetate reduction and for glyceride glycerol formation. 4. Mitochondria isolated from ovine adipocytes oxidized added NADH rapidly in a reconstituted alpha-glycerophosphate shuttle system. 5. It is possible that the ability of ovine adipocytes to utilize lactate may be related to the active alpha-glycerophosphate shuttle for cytosolic NADH reoxidation.

  15. High activity of mitochondrial glycerophosphate dehydrogenase and glycerophosphate-dependent ROS production in prostate cancer cell lines.

    PubMed

    Chowdhury, Subir K R; Gemin, Adam; Singh, Gurmit

    2005-08-12

    Most malignant cells are highly glycolytic and produce high levels of reactive oxygen species (ROS) compared to normal cells. Mitochondrial glycerophosphate dehydrogenase (mGPDH) participates in the reoxidation of cytosolic NADH by delivering reducing equivalents from this molecule into the electron transport chain, thus sustaining glycolysis. Here, we investigate the role of mGPDH in maintaining an increased rate of glycolysis and evaluate glycerophosphate-dependent ROS production in prostate cancer cell lines (LNCaP, DU145, PC3, and CL1). Immunoblot, polarographic, and spectrophotometric analyses revealed that mGPDH abundance and activity was significantly elevated in prostate cancer cell lines when compared to the normal prostate epithelial cell line PNT1A. Furthermore, both the glycolytic capacity and glycerophosphate-dependent ROS production was increased 1.68- to 4.44-fold and 5- to 7-fold, respectively, in prostate cancer cell lines when compared to PNT1A cells. Overall, these data demonstrate that mGPDH is involved in maintaining a high rate of glycolysis and is an important site of electron leakage leading to ROS production in prostate cancer cells.

  16. Effects of interaction with gene carrier polyethyleneimines on conformation and enzymatic activity of pig heart lactate dehydrogenase.

    PubMed

    Wang, Fan; Mo, Junyong; Huang, Aimin; Zhang, Min; Ma, Lin

    2018-06-15

    Polyethyleneimine (PEI) has long been considered as "golden standard" for polymeric gene delivery carrier, however also induces cytotoxicity. To make a further insight into the molecular basis of PEI cytotoxicity, fluorescence, absorption and circular dichroism spectroscopy were conducted to investigate the influence of PEI (average molecular weight 25,000 and 1800 Da) on the conformation of pig heart lactate dehydrogenase (LDH) and its catalytic efficiency. Zeta-potential measurement and isothermal titration calorimetry were used to reveal the interaction between PEI and LDH. PEI was found to bind onto the surface of LDH predominantly via hydrophobic interaction, inducing a more compact conformation and an increased surface hydrophobicity of the enzyme. The conformational change of LDH induced by PEI binding had little influence on the complex formation between LDH and reduced nicotinamide adenine dinucleotide (NADH, the co-enzyme). However, the nonspecific binding of PEI on the surface of LDH retarded the turnover of the enzyme. Meanwhile, the large quantity of amine groups on the polymer chain made PEI subject to form complexes with NADH and pyruvate (the substrate) via hydrogen bond and electrostatic interaction, which greatly reduced the binding efficient of LDH. The polymer size played an important role in PEI-LDH interaction. The smaller size of lower molecular weight PEI facilitated the close contact with LDH and consequential reduction of the turnover number of the enzyme. However, higher molecular weight PEI was more favorable for competitive binding with NADH and pyruvate and generally decreased the catalytic efficient of LDH. Copyright © 2018. Published by Elsevier B.V.

  17. Global Kinetic Analysis of Mammalian E3 Reveals pH-dependent NAD+/NADH Regulation, Physiological Kinetic Reversibility, and Catalytic Optimum*

    PubMed Central

    Moxley, Michael A.; Beard, Daniel A.; Bazil, Jason N.

    2016-01-01

    Mammalian E3 is an essential mitochondrial enzyme responsible for catalyzing the terminal reaction in the oxidative catabolism of several metabolites. E3 is a key regulator of metabolic fuel selection as a component of the pyruvate dehydrogenase complex (PDHc). E3 regulates PDHc activity by altering the affinity of pyruvate dehydrogenase kinase, an inhibitor of the enzyme complex, through changes in reduction and acetylation state of lipoamide moieties set by the NAD+/NADH ratio. Thus, an accurate kinetic model of E3 is needed to predict overall mammalian PDHc activity. Here, we have combined numerous literature data sets and new equilibrium spectroscopic experiments with a multitude of independently collected forward and reverse steady-state kinetic assays using pig heart E3. The latter kinetic assays demonstrate a pH-dependent transition of NAD+ activation to inhibition, shown here, to our knowledge, for the first time in a single consistent data set. Experimental data were analyzed to yield a thermodynamically constrained four-redox-state model of E3 that simulates pH-dependent activation/inhibition and active site redox states for various conditions. The developed model was used to determine substrate/product conditions that give maximal E3 rates and show that, due to non-Michaelis-Menten behavior, the maximal flux is different compared with the classically defined kcat. PMID:26644471

  18. Isolation and characterization of two cDNA clones encoding for glutamate dehydrogenase in Nicotiana plumbaginifolia.

    PubMed

    Ficarelli, A; Tassi, F; Restivo, F M

    1999-03-01

    We have isolated two full length cDNA clones encoding Nicotiana plumbaginifolia NADH-glutamate dehydrogenase. Both clones share amino acid boxes of homology corresponding to conserved GDH catalytic domains and putative mitochondrial targeting sequence. One clone shows a putative EF-hand loop. The level of the two transcripts is affected differently by carbon source.

  19. Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species

    PubMed Central

    Aguilar-Arnal, Lorena; Ranjit, Suman; Stringari, Chiara; Orozco-Solis, Ricardo; Gratton, Enrico; Sassone-Corsi, Paolo

    2016-01-01

    Sirtuin 1 (SIRT1) is an NAD+-dependent deacetylase that functions as metabolic sensor of cellular energy and modulates biochemical pathways in the adaptation to changes in the environment. SIRT1 substrates include histones and proteins related to enhancement of mitochondrial function as well as antioxidant protection. Fluctuations in intracellular NAD+ levels regulate SIRT1 activity, but how SIRT1 enzymatic activity impacts on NAD+ levels and its intracellular distribution remains unclear. Here, we show that SIRT1 determines the nuclear organization of protein-bound NADH. Using multiphoton microscopy in live cells, we show that free and bound NADH are compartmentalized inside of the nucleus, and its subnuclear distribution depends on SIRT1. Importantly, SIRT6, a chromatin-bound deacetylase of the same class, does not influence NADH nuclear localization. In addition, using fluorescence fluctuation spectroscopy in single living cells, we reveal that NAD+ metabolism in the nucleus is linked to subnuclear dynamics of active SIRT1. These results reveal a connection between NAD+ metabolism, NADH distribution, and SIRT1 activity in the nucleus of live cells and pave the way to decipher links between nuclear organization and metabolism. PMID:27791113

  20. Alcohol Dehydrogenase Activities of Wine Yeasts in Relation to Higher Alcohol Formation

    PubMed Central

    Singh, Rajendra; Kunkee, Ralph E.

    1976-01-01

    Alcohol dehydrogenase activities were examined in cell-free extracts of 10 representative wine yeast strains having various productivities of higher alcohols (fusel oil). The amount of fusel alcohols (n-propanol, isobutanol, active pentanol, and isopentanol) produced by the different yeasts and the specific alcohol dehydrogenase activities with the corresponding alcohols as substrates were found to be significantly related. No such relationship was found for ethanol. The amounts of higher alcohols formed during vinification could be predicted from the specific activities of the alcohol dehydrogenases with high accuracy. The results suggest a close relationship between the control of the activities of alcohol dehydrogenase and the formation of fusel oil alcohols. Also, new procedures for the prediction of higher alcohol formation during alcoholic beverage fermentation are suggested. PMID:16345179

  1. A water-forming NADH oxidase from Lactobacillus pentosus suitable for the regeneration of synthetic biomimetic cofactors

    PubMed Central

    Nowak, Claudia; Beer, Barbara; Pick, André; Roth, Teresa; Lommes, Petra; Sieber, Volker

    2015-01-01

    The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox). Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13% FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyze the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H2O2 instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as by-product. PMID:26441891

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

    PubMed Central

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

    2012-01-01

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

  3. Apoptosis-inducing Factor (AIF) and Its Family Member Protein, AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2)*

    PubMed Central

    Elguindy, Mahmoud M.; Nakamaru-Ogiso, Eiko

    2015-01-01

    Apoptosis-inducing factor (AIF) and AMID (AIF-homologous mitochondrion-associated inducer of death) are flavoproteins. Although AIF was originally discovered as a caspase-independent cell death effector, bioenergetic roles of AIF, particularly relating to complex I functions, have since emerged. However, the role of AIF in mitochondrial respiration and redox metabolism has remained unknown. Here, we investigated the redox properties of human AIF and AMID by comparing them with yeast Ndi1, a type 2 NADH:ubiquinone oxidoreductase (NDH-2) regarded as alternative complex I. Isolated AIF and AMID containing naturally incorporated FAD displayed no NADH oxidase activities. However, after reconstituting isolated AIF or AMID into bacterial or mitochondrial membranes, N-terminally tagged AIF and AMID displayed substantial NADH:O2 activities and supported NADH-linked proton pumping activities in the host membranes almost as efficiently as Ndi1. NADH:ubiquinone-1 activities in the reconstituted membranes were highly sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide (IC50 = ∼1 μm), a quinone-binding inhibitor. Overexpressing N-terminally tagged AIF and AMID enhanced the growth of a double knock-out Escherichia coli strain lacking complex I and NDH-2. In contrast, C-terminally tagged AIF and NADH-binding site mutants of N-terminally tagged AIF and AMID failed to show both NADH:O2 activity and the growth-enhancing effect. The disease mutant AIFΔR201 showed decreased NADH:O2 activity and growth-enhancing effect. Furthermore, we surprisingly found that the redox activities of N-terminally tagged AIF and AMID were sensitive to rotenone, a well known complex I inhibitor. We propose that AIF and AMID are previously unidentified mammalian NDH-2 enzymes, whose bioenergetic function could be supplemental NADH oxidation in cells. PMID:26063804

  4. Apoptosis-inducing Factor (AIF) and Its Family Member Protein, AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2).

    PubMed

    Elguindy, Mahmoud M; Nakamaru-Ogiso, Eiko

    2015-08-21

    Apoptosis-inducing factor (AIF) and AMID (AIF-homologous mitochondrion-associated inducer of death) are flavoproteins. Although AIF was originally discovered as a caspase-independent cell death effector, bioenergetic roles of AIF, particularly relating to complex I functions, have since emerged. However, the role of AIF in mitochondrial respiration and redox metabolism has remained unknown. Here, we investigated the redox properties of human AIF and AMID by comparing them with yeast Ndi1, a type 2 NADH:ubiquinone oxidoreductase (NDH-2) regarded as alternative complex I. Isolated AIF and AMID containing naturally incorporated FAD displayed no NADH oxidase activities. However, after reconstituting isolated AIF or AMID into bacterial or mitochondrial membranes, N-terminally tagged AIF and AMID displayed substantial NADH:O₂ activities and supported NADH-linked proton pumping activities in the host membranes almost as efficiently as Ndi1. NADH:ubiquinone-1 activities in the reconstituted membranes were highly sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide (IC₅₀ = ∼1 μm), a quinone-binding inhibitor. Overexpressing N-terminally tagged AIF and AMID enhanced the growth of a double knock-out Escherichia coli strain lacking complex I and NDH-2. In contrast, C-terminally tagged AIF and NADH-binding site mutants of N-terminally tagged AIF and AMID failed to show both NADH:O₂ activity and the growth-enhancing effect. The disease mutant AIFΔR201 showed decreased NADH:O₂ activity and growth-enhancing effect. Furthermore, we surprisingly found that the redox activities of N-terminally tagged AIF and AMID were sensitive to rotenone, a well known complex I inhibitor. We propose that AIF and AMID are previously unidentified mammalian NDH-2 enzymes, whose bioenergetic function could be supplemental NADH oxidation in cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Asymmetric reduction of ketones and β-keto esters by (S)-1-phenylethanol dehydrogenase from denitrifying bacterium Aromatoleum aromaticum.

    PubMed

    Dudzik, A; Snoch, W; Borowiecki, P; Opalinska-Piskorz, J; Witko, M; Heider, J; Szaleniec, M

    2015-06-01

    Enzyme-catalyzed enantioselective reductions of ketones and keto esters have become popular for the production of homochiral building blocks which are valuable synthons for the preparation of biologically active compounds at industrial scale. Among many kinds of biocatalysts, dehydrogenases/reductases from various microorganisms have been used to prepare optically pure enantiomers from carbonyl compounds. (S)-1-phenylethanol dehydrogenase (PEDH) was found in the denitrifying bacterium Aromatoleum aromaticum (strain EbN1) and belongs to the short-chain dehydrogenase/reductase family. It catalyzes the stereospecific oxidation of (S)-1-phenylethanol to acetophenone during anaerobic ethylbenzene mineralization, but also the reverse reaction, i.e., NADH-dependent enantioselective reduction of acetophenone to (S)-1-phenylethanol. In this work, we present the application of PEDH for asymmetric reduction of 42 prochiral ketones and 11 β-keto esters to enantiopure secondary alcohols. The high enantioselectivity of the reaction is explained by docking experiments and analysis of the interaction and binding energies of the theoretical enzyme-substrate complexes leading to the respective (S)- or (R)-alcohols. The conversions were carried out in a batch reactor using Escherichia coli cells with heterologously produced PEDH as whole-cell catalysts and isopropanol as reaction solvent and cosubstrate for NADH recovery. Ketones were converted to the respective secondary alcohols with excellent enantiomeric excesses and high productivities. Moreover, the progress of product formation was studied for nine para-substituted acetophenone derivatives and described by neural network models, which allow to predict reactor behavior and provides insight on enzyme reactivity. Finally, equilibrium constants for conversion of these substrates were derived from the progress curves of the reactions. The obtained values matched very well with theoretical predictions.

  6. Properties of lactate dehydrogenase from the isopod, Saduria entomon.

    PubMed

    Mulkiewicz, E; Zietara, M S; Stachowiak, K; Skorkowski, E F

    2000-07-01

    Saduria entomon lactate dehydrogenase (LDH-A4*) from thorax muscle was purified about 89 fold to specific activity 510 micromol NADH/min/mg using Cibacron Blue 3GA Agarose and Oxamate-Agarose chromatographies. The enzyme is a tetramer, with molecular weight of 140 kDa for the native enzyme and 36 kDa for the subunit. The isoelectric point was at pH 5.7. The enzyme possesses high heat stability (T50 = 71.5 degrees C). The optimum pH for pyruvate reduction reaction was 6.5, while for lactate oxidation one, the maximum activity was at pH 9.1. The Km for pyruvate was minimal at 5 degrees C, the average environmental temperature of the isopod. The Km values determined at 30 degrees C and optimal pH for pyruvate reduction and lactate oxidation were 0.18 and 90.04 mM, respectively. Amino acid compositional analyses showed the strongest resemblance of the isopod isoenzyme to cod (Gadus morhua) LDH-C4.

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

    USDA-ARS?s Scientific Manuscript database

    ALDH2 catalyzes oxidation of toxic aldehydes to their corresponding carboxylic acids. Magnesium ions influence enzyme activity in part by increasing NADH binding affinity. Traditional fluorescence measurements have monitored the blue shift of the NADH fluorescence spectrum to elucidate the extent of...

  8. Engineering of a functional human NADH-dependent cytochrome P450 system

    PubMed Central

    Döhr, Olaf; Paine, Mark J. I.; Friedberg, Thomas; Roberts, Gordon C. K.; Wolf, C. Roland

    2001-01-01

    A functional human NADH-dependent cytochrome P450 system has been developed by altering the cofactor preference of human NADPH cytochrome P450 reductase (CPR), the redox partner for P450s. This has been achieved by a single amino acid change of the conserved aromatic amino acid Trp-676, which covers the re-side of the FAD isoalloxazine ring in the nicotinamide-binding site. Of the mutations made, the substitution of Trp-676 with alanine (W676A) resulted in a functional NADH-dependent enzyme, which catalyzed the reduction of cytochrome c and ferricyanide as well as facilitated the metabolism of 7-ethoxyresorufin by CYP1A2. Kinetic analysis measuring cytochrome c activity revealed that the NADH-dependent kcat of W676A is equivalent (90%) to the NADPH-dependent kcat of the wild-type enzyme, with W676A having an approximately 1,000-fold higher specificity for NADH. The apparent KMNADPH and KMNADH values of W676A are 80- and 150-fold decreased, respectively. In accordance with structural data, which show a bipartite binding mode of NADPH, substitution of Trp-676 does not affect 2′-AMP binding as seen by the inhibition of both wild-type CPR and the W676A mutant. Furthermore, NADPH was a potent inhibitor of the W676A NADH-dependent cytochrome c reduction and CYP1A2 activity. Overall, the results show that Trp-676 of human CPR plays a major role in cofactor discrimination, and substitution of this conserved aromatic residue with alanine results in an efficient NADH-dependent cytochrome P450 system. PMID:11136248

  9. Suppression of BRCA2 by Mutant Mitochondrial DNA in Prostate Cancer

    DTIC Science & Technology

    2011-05-01

    Briefly, the electron transfer activities of complex I/III (NADH dehydrogenase/cytochrome bc1 complex: catalyzes the electron transfer from NADH to...ferricytochrome c) and complex II/III (succinate dehydrogenase/cytochrome bc1 complex: catalyzes the electron transfer from succinate to ferricytochrome...The electron transfer activity of complex IV (cytochrome c oxidase: catalyzes the final step of the respiratory chain by transferring electrons from

  10. Gravity Responsive NADH Oxidase of the Plasma Membrane

    NASA Technical Reports Server (NTRS)

    Morre, D. James (Inventor)

    2002-01-01

    A method and apparatus for sensing gravity using an NADH oxidase of the plasma membrane which has been found to respond to unit gravity and low centrifugal g forces. The oxidation rate of NADH supplied to the NADH oxidase is measured and translated to represent the relative gravitational force exerted on the protein. The NADH oxidase of the plasma membrane may be obtained from plant or animal sources or may be produced recombinantly.

  11. Metabolic control by sirtuins and other enzymes that sense NAD+, NADH, or their ratio.

    PubMed

    Anderson, Kristin A; Madsen, Andreas S; Olsen, Christian A; Hirschey, Matthew D

    2017-12-01

    NAD + is a dinucleotide cofactor with the potential to accept electrons in a variety of cellular reduction-oxidation (redox) reactions. In its reduced form, NADH is a ubiquitous cellular electron donor. NAD + , NADH, and the NAD + /NADH ratio have long been known to control the activity of several oxidoreductase enzymes. More recently, enzymes outside those participating directly in redox control have been identified that sense these dinucleotides, including the sirtuin family of NAD + -dependent protein deacylases. In this review, we highlight examples of non-redox enzymes that are controlled by NAD + , NADH, or NAD + /NADH. In particular, we focus on the sirtuin family and assess the current evidence that the sirtuin enzymes sense these dinucleotides and discuss the biological conditions under which this might occur; we conclude that sirtuins sense NAD + , but neither NADH nor the ratio. Finally, we identify future studies that might be informative to further interrogate physiological and pathophysiological changes in NAD + and NADH, as well as enzymes like sirtuins that sense and respond to redox changes in the cell. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    1992-01-01

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

  13. 9-Hydroxyprostaglandin dehydrogenase activity in the adult rat kidney. Regional distribution and sub-fractionation.

    PubMed

    Asciak, C P; Domazet, Z

    1975-02-20

    1. Catabolism of prostaglandin F2alpha in the adult rat kidney takes place by the following sequence of enzymatic steps: (1) 15-hydroxyprostaglandin dehydrogenase; (2) prostaglandin delta13-reductase; and (3) 9-hydroxyprostaglandin dehydrogenase. 2. 9-Hydroxyprostaglandin dehydrogenase activity was highest in the cortex with lesser amounts in the medulla and negligible activity detected in the papilla. A similar distribution was observed for 15-hydroxyprostaglandin dehydrogenase and prostaglandin delta13-reductase. 3. Most of the 9-hydroxyprostaglandin dehydrogenase activity in the homogenate was found in the high-speed supernatant as also observed for 15-hydroxyprostaglandin dehydrogenase and prostaglandin delta13-reductase. 4. These observations indicate that the rat kidney contains an abundance of prostaglandin-catabolising enzymes which favour formation of metabolites of the E-type.

  14. Determination of ammonium ion using a reagentless amperometric biosensor based on immobilized alanine dehydrogenase.

    PubMed

    Tan, Ling Ling; Musa, Ahmad; Lee, Yook Heng

    2011-01-01

    The use of the enzyme alanine dehydrogenase (AlaDH) for the determination of ammonium ion (NH(4)(+)) usually requires the addition of pyruvate substrate and reduced nicotinamide adenine dinucleotide (NADH) simultaneously to effect the reaction. This addition of reagents is inconvenient when an enzyme biosensor based on AlaDH is used. To resolve the problem, a novel reagentless amperometric biosensor using a stacked methacrylic membrane system coated onto a screen-printed carbon paste electrode (SPE) for NH(4)(+) ion determination is described. A mixture of pyruvate and NADH was immobilized in low molecular weight poly(2-hydroxyethyl methacrylate) (pHEMA) membrane, which was then deposited over a photocured pHEMA membrane (photoHEMA) containing alanine dehydrogenase (AlaDH) enzyme. Due to the enzymatic reaction of AlaDH and the pyruvate substrate, NH(4)(+) was consumed in the process and thus the signal from the electrocatalytic oxidation of NADH at an applied potential of +0.55 V was proportional to the NH(4)(+) ion concentration under optimal conditions. The stacked methacrylate membranes responded rapidly and linearly to changes in NH(4)(+) ion concentrations between 10-100 mM, with a detection limit of 0.18 mM NH(4)(+) ion. The reproducibility of the amperometrical NH(4)(+) biosensor yielded low relative standard deviations between 1.4-4.9%. The stacked membrane biosensor has been successfully applied to the determination of NH(4)(+) ion in spiked river water samples without pretreatment. A good correlation was found between the analytical results for NH(4)(+) obtained from the biosensor and the Nessler spectrophotometric method.

  15. Influence of oxygen on NADH recycling and oxidative stress resistance systems in Lactobacillus panis PM1

    PubMed Central

    2013-01-01

    Lactobacillus panis strain PM1 is an obligatory heterofermentative and aerotolerant microorganism that also produces 1,3-propanediol from glycerol. This study investigated the metabolic responses of L. panis PM1 to oxidative stress under aerobic conditions. Growth under aerobic culture triggered an early entrance of L. panis PM1 into the stationary phase along with marked changes in end-product profiles. A ten-fold higher concentration of hydrogen peroxide was accumulated during aerobic culture compared to microaerobic culture. This H2O2 level was sufficient for the complete inhibition of L. panis PM1 cell growth, along with a significant reduction in end-products typically found during anaerobic growth. In silico analysis revealed that L. panis possessed two genes for NADH oxidase and NADH peroxidase, but their expression levels were not significantly affected by the presence of oxygen. Specific activities for these two enzymes were observed in crude extracts from L. panis PM1. Enzyme assays demonstrated that the majority of the H2O2 in the culture media was the product of NADH: H2O2 oxidase which was constitutively-active under both aerobic and microaerobic conditions; whereas, NADH peroxidase was positively-activated by the presence of oxygen and had a long induction time in contrast to NADH oxidase. These observations indicated that a coupled NADH oxidase - NADH peroxidase system was the main oxidative stress resistance mechanism in L. panis PM1, and was regulated by oxygen availability. Under aerobic conditions, NADH is mainly reoxidized by the NADH oxidase - peroxidase system rather than through the production of ethanol (or 1,3-propanediol or succinic acid production if glycerol or citric acid is available). This system helped L. panis PM1 directly use oxygen in its energy metabolism by producing extra ATP in contrast to homofermentative lactobacilli. PMID:23369580

  16. High activity of mitochondrial glycerophosphate dehydrogenase and glycerophosphate-dependent ROS production in prostate cancer cell lines

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

    Chowdhury, Subir K.R.; Department of Pathology and Molecular Medicine, McMaster University, 1200 Main St. West, Hamilton, Ont., L8N 3Z5; Gemin, Adam

    Most malignant cells are highly glycolytic and produce high levels of reactive oxygen species (ROS) compared to normal cells. Mitochondrial glycerophosphate dehydrogenase (mGPDH) participates in the reoxidation of cytosolic NADH by delivering reducing equivalents from this molecule into the electron transport chain, thus sustaining glycolysis. Here, we investigate the role of mGPDH in maintaining an increased rate of glycolysis and evaluate glycerophosphate-dependent ROS production in prostate cancer cell lines (LNCaP, DU145, PC3, and CL1). Immunoblot, polarographic, and spectrophotometric analyses revealed that mGPDH abundance and activity was significantly elevated in prostate cancer cell lines when compared to the normal prostate epithelialmore » cell line PNT1A. Furthermore, both the glycolytic capacity and glycerophosphate-dependent ROS production was increased 1.68- to 4.44-fold and 5- to 7-fold, respectively, in prostate cancer cell lines when compared to PNT1A cells. Overall, these data demonstrate that mGPDH is involved in maintaining a high rate of glycolysis and is an important site of electron leakage leading to ROS production in prostate cancer cells.« less

  17. Identification of proteins capable of metal reduction from the proteome of the Gram-positive bacterium Desulfotomaculum reducens MI-1 using an NADH-based activity assay

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

    Otwell, Annie E.; Sherwood, Roberts; Zhang, Sheng

    Metal reduction capability has been found in numerous species of environmentally abundant Gram-positive bacteria. However, understanding of microbial metal reduction is based almost solely on studies of Gram-negative organisms. In this study, we focus on Desulfotomaculum reducens MI-1, a Gram-positive metal reducer whose genome lacks genes with similarity to any characterized metal reductase. D. reducens has been shown to reduce not only Fe(III), but also the environmentally important contaminants U(VI) and Cr(VI). By extracting, separating, and analyzing the functional proteome of D. reducens, using a ferrozine-based assay in order to screen for chelated Fe(III)-NTA reduction with NADH as electron donor,more » we have identified proteins not previously characterized as iron reductases. Their function was confirmed by heterologous expression in E. coli. These are the protein NADH:flavin oxidoreductase (Dred_2421) and a protein complex composed of oxidoreductase FAD/NAD(P)-binding subunit (Dred_1685) and dihydroorotate dehydrogenase 1B (Dred_1686). Dred_2421 was identified in the soluble proteome and is predicted to be a cytoplasmic protein. Dred_1685 and Dred_1686 were identified in both the soluble as well as the insoluble (presumably membrane) protein fraction, suggesting a type of membrane-association, although PSORTb predicts both proteins are cytoplasmic. Furthermore, we show that these proteins have the capability to reduce soluble Cr(VI) and U(VI) with NADH as electron donor. This study is the first functional proteomic analysis of D. reducens, and one of the first analyses of metal and radionuclide reduction in an environmentally relevant Gram-positive bacterium.« less

  18. Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence.

    PubMed

    Li, Dong; Zheng, Wei; Qu, Jianan Y

    2008-10-15

    A time-resolved spectroscopic imaging system is built to study the fluorescence characteristics of nicotinamide adenine dinucleotide (NADH), an important metabolic coenzyme and endogenous fluorophore in cells. The system provides a unique approach to measure fluorescence signals in different cellular organelles and cytoplasm. The ratios of free over protein-bound NADH signals in cytosol and nucleus are slightly higher than those in mitochondria. The mitochondrial fluorescence contributes about 70% of overall cellular fluorescence and is not a completely dominant signal. Furthermore, NADH signals in mitochondria, cytosol, and the nucleus respond to the changes of cellular activity differently, suggesting that cytosolic and nuclear fluorescence may complicate the well-known relationship between mitochondrial fluorescence and cellular metabolism.

  19. Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis.

    PubMed

    Watanabe, Seiya; Abu Saleh, Ahmed; Pack, Seung Pil; Annaluru, Narayana; Kodaki, Tsutomu; Makino, Keisuke

    2007-09-01

    A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis (PsXR and PsXDH, respectively) has the ability to convert xylose to ethanol together with the unfavourable excretion of xylitol, which may be due to intercellular redox imbalance caused by the different coenzyme specificity between NADPH-preferring XR and NAD(+)-dependent XDH. In this study, we focused on the effect(s) of mutated NADH-preferring PsXR in fermentation. The R276H and K270R/N272D mutants were improved 52- and 146-fold, respectively, in the ratio of NADH/NADPH in catalytic efficiency [(k(cat)/K(m) with NADH)/(k(cat)/K(m) with NADPH)] compared with the wild-type (WT), which was due to decrease of k(cat) with NADPH in the R276H mutant and increase of K(m) with NADPH in the K270R/N272D mutant. Furthermore, R276H mutation led to significant thermostabilization in PsXR. The most positive effect on xylose fermentation to ethanol was found by using the Y-R276H strain, expressing PsXR R276H mutant and PsXDH WT: 20 % increase of ethanol production and 52 % decrease of xylitol excretion, compared with the Y-WT strain expressing PsXR WT and PsXDH WT. Measurement of intracellular coenzyme concentrations suggested that maintenance of the of NADPH/NADP(+) and NADH/NAD(+) ratios is important for efficient ethanol fermentation from xylose by recombinant S. cerevisiae.

  20. Structural Basis for NADH/NAD+ Redox Sensing by a Rex Family Repressor

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

    McLaughlin, K.J.; Soares, A.; Strain-Damerell, C. M.

    2010-05-28

    Nicotinamide adenine dinucleotides have emerged as key signals of the cellular redox state. Yet the structural basis for allosteric gene regulation by the ratio of reduced NADH to oxidized NAD{sup +} is poorly understood. A key sensor among Gram-positive bacteria, Rex represses alternative respiratory gene expression until a limited oxygen supply elevates the intracellular NADH:NAD{sup +} ratio. Here we investigate the molecular mechanism for NADH/NAD{sup +} sensing among Rex family members by determining structures of Thermus aquaticus Rex bound to (1) NAD{sup +}, (2) DNA operator, and (3) without ligand. Comparison with the Rex/NADH complex reveals that NADH releases Rexmore » from the DNA site following a 40{sup o} closure between the dimeric subunits. Complementary site-directed mutagenesis experiments implicate highly conserved residues in NAD-responsive DNA-binding activity. These rare views of a redox sensor in action establish a means for slight differences in the nicotinamide charge, pucker, and orientation to signal the redox state of the cell.« less

  1. Role of mannitol dehydrogenases in osmoprotection of Gluconobacter oxydans.

    PubMed

    Zahid, Nageena; Deppenmeier, Uwe

    2016-12-01

    Gluconobacter (G.) oxydans is able to incompletely oxidize various sugars and polyols for the production of biotechnologically important compound. Recently, we have shown that the organism produces and accumulates mannitol as compatible solute under osmotic stress conditions. The present study describes the role of two cytoplasmic mannitol dehydrogenases for osmotolerance of G. oxydans. It was shown that Gox1432 is a NADP + -dependent mannitol dehydrogenase (EC 1.1.1.138), while Gox0849 uses NAD + as cofactor (EC 1.1.1.67). The corresponding genes were deleted and the mutants were analyzed for growth under osmotic stress and non-stress conditions. A severe growth defect was detected for Δgox1432 when grown in high osmotic media, while the deletion of gox0849 had no effect when cells were exposed to 450 mM sucrose in the medium. Furthermore, the intracellular mannitol content was reduced in the mutant lacking the NADP + -dependent enzyme Gox1432 in comparison to the parental strain and the Δgox0849 mutant under stress conditions. In addition, transcriptional analysis revealed that Gox1432 is more important for mannitol production in G. oxydans than Gox0849 as the transcript abundance of gene gox1432 was 30-fold higher than of gox0849. In accordance, the activity of the NADH-dependent enzyme Gox0849 in the cell cytoplasm was 10-fold lower in comparison to the NADPH-dependent mannitol dehydrogenase Gox1432. Overexpression of gox1432 in the corresponding deletion mutant restored growth of the cells under osmotic stress, further strengthening the importance of the NADP + -dependent mannitol dehydrogenase for osmotolerance in G. oxydans. These findings provide detailed insights into the molecular mechanism of mannitol-mediated osmoprotection in G. oxydans and are helpful engineering strains with improved osmotolerance for biotechnological applications.

  2. Highly ordered mesoporous carbons as electrode material for the construction of electrochemical dehydrogenase- and oxidase-based biosensors.

    PubMed

    Zhou, Ming; Shang, Li; Li, Bingling; Huang, Lijian; Dong, Shaojun

    2008-11-15

    In this work, the excellent catalytic activity of highly ordered mesoporous carbons (OMCs) to the electrooxidation of nicotinamide adenine dinucleotide (NADH) and hydrogen peroxide (H(2)O(2)) was described for the construction of electrochemical alcohol dehydrogenase (ADH) and glucose oxidase (GOD)-based biosensors. The high density of edge-plane-like defective sites and high specific surface area of OMCs could be responsible for the electrocatalytic behavior at OMCs modified glassy carbon electrode (OMCs/GE), which induced a substantial decrease in the overpotential of NADH and H(2)O(2) oxidation reaction compared to carbon nanotubes modified glassy carbon electrode (CNTs/GE). Such ability of OMCs permits effective low-potential amperometric biosensing of ethanol and glucose, respectively, at Nafion/ADH-OMCs/GE and Nafion/GOD-OMCs/GE. Especially, as an amperometric glucose biosensor, Nafion/GOD-OMCs/GE showed large determination range (500-15,000 micromoll(-1)), high sensitivity (0.053 nA micromol(-1)), fast (9+/-1s) and stable response (amperometric response retained 90% of the initial activity after 10h stirring of 2 mmoll(-1) glucose solution) to glucose as well as the effective discrimination to the possible interferences, which may make it to readily satisfy the need for the routine clinical diagnosis of diabetes. By comparing the electrochemical performance of OMCs with that of CNTs as electrode material for the construction of ADH- and GOD-biosensors in this work, we reveal that OMCs could be a favorable and promising carbon electrode material for constructing other electrochemical dehydrogenase- and oxidase-based biosensors, which may have wide potential applications in biocatalysis, bioelectronics and biofuel cells.

  3. The structure of the yeast NADH dehydrogenase (Ndi1) reveals overlapping binding sites for water- and lipid-soluble substrates.

    PubMed

    Iwata, Momi; Lee, Yang; Yamashita, Tetsuo; Yagi, Takao; Iwata, So; Cameron, Alexander D; Maher, Megan J

    2012-09-18

    Bioenergy is efficiently produced in the mitochondria by the respiratory system consisting of complexes I-V. In various organisms, complex I can be replaced by the alternative NADH-quinone oxidoreductase (NDH-2), which catalyzes the transfer of an electron from NADH via FAD to quinone, without proton pumping. The Ndi1 protein from Saccharomyces cerevisiae is a monotopic membrane protein, directed to the matrix. A number of studies have investigated the potential use of Ndi1 as a therapeutic agent against complex I disorders, and the NDH-2 enzymes have emerged as potential therapeutic targets for treatments against the causative agents of malaria and tuberculosis. Here we present the crystal structures of Ndi1 in its substrate-free, NAD(+)- and ubiquinone- (UQ2) complexed states. The structures reveal that Ndi1 is a peripheral membrane protein forming an intimate dimer, in which packing of the monomeric units within the dimer creates an amphiphilic membrane-anchor domain structure. Crucially, the structures of the Ndi1-NAD(+) and Ndi1-UQ2 complexes show overlapping binding sites for the NAD(+) and quinone substrates.

  4. Mechanistic study of manganese-substituted glycerol dehydrogenase using a kinetic and thermodynamic analysis.

    PubMed

    Fang, Baishan; Niu, Jin; Ren, Hong; Guo, Yingxia; Wang, Shizhen

    2014-01-01

    Mechanistic insights regarding the activity enhancement of dehydrogenase by metal ion substitution were investigated by a simple method using a kinetic and thermodynamic analysis. By profiling the binding energy of both the substrate and product, the metal ion's role in catalysis enhancement was revealed. Glycerol dehydrogenase (GDH) from Klebsiella pneumoniae sp., which demonstrated an improvement in activity by the substitution of a zinc ion with a manganese ion, was used as a model for the mechanistic study of metal ion substitution. A kinetic model based on an ordered Bi-Bi mechanism was proposed considering the noncompetitive product inhibition of dihydroxyacetone (DHA) and the competitive product inhibition of NADH. By obtaining preliminary kinetic parameters of substrate and product inhibition, the number of estimated parameters was reduced from 10 to 4 for a nonlinear regression-based kinetic parameter estimation. The simulated values of time-concentration curves fit the experimental values well, with an average relative error of 11.5% and 12.7% for Mn-GDH and GDH, respectively. A comparison of the binding energy of enzyme ternary complex for Mn-GDH and GDH derived from kinetic parameters indicated that metal ion substitution accelerated the release of dioxyacetone. The metal ion's role in catalysis enhancement was explicated.

  5. Electrochemiluminescence detection of NADH and ethanol based on partial sulfonation of sol-gel network with gold nanoparticles.

    PubMed

    Deng, Liu; Zhang, Lihua; Shang, Li; Guo, Shaojun; Wen, Dan; Wang, Fuan; Dong, Shaojun

    2009-03-15

    We developed a stable, sensitive electrochemiluminescence (ECL) biosensor based on the synthesis of a new sol-gel material with the ion-exchange capacity sol-gel to coimmobilize the Ru(bpy)(3)(2+) and enzyme. The partial sulfonated (3-mercaptopropyl)-trimethoxysilane sol-gel (PSSG) film acted as both an ion exchanger for the immobilization of Ru(bpy)(3)(2+) and a matrix to immobilize gold nanoparticles (AuNPs). The AuNPs/PSSG/Ru(bpy)(3)(2+) film modified electrode allowed sensitive the ECL detection of NADH as low as 1 nM. Such an ability of AuNPs/PSSG/Ru(bpy)(3)(2+) film to promote the electron transfer between Ru(bpy)(3)(2+) and the electrode suggested a new, promising biocompatible platform for the development of dehydrogenase-based ECL biosensors. With alcohol dehydrogenase (ADH) as a model, we then constructed an ethanol biosensor, which had a linear range of 5 microM to 5.2 mM with a detection limit of 12nM.

  6. Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

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

    Brown, Steven D; Guss, Adam M; Karpinets, Tatiana V

    2011-01-01

    Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assaysmore » confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.« less

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

    PubMed

    Moummou, Hanane; Tonfack, Libert Brice; Chervin, Christian; Benichou, Mohamed; Youmbi, Emmanuel; Ginies, Christian; Latché, Alain; Pech, Jean-Claude; van der Rest, Benoît

    2012-10-15

    A tomato short-chain dehydrogenase-reductase (SlscADH1) is preferentially expressed in fruit with a maximum expression at the breaker stage while expression in roots, stems, leaves and flowers is very weak. It represents a potential candidate for the formation of aroma volatiles by interconverting alcohols and aldehydes. The SlscADH1 recombinant protein produced in Escherichia coli exhibited dehydrogenase-reductase activity towards several volatile compounds present in tomato flavour with a strong preference for the NAD/NADH co-factors. The strongest activity was observed for the reduction of hexanal (K(m)=0.175mM) and phenylacetaldehyde (K(m)=0.375mM) in the presence of NADH. The oxidation process of hexanol and 1-phenylethanol was much less efficient (K(m)s of 2.9 and 23.0mM, respectively), indicating that the enzyme preferentially acts as a reductase. However activity was observed only for hexanal, phenylacetaldehyde, (E)-2-hexenal and acetaldehyde and the corresponding alcohols. No activity could be detected for other aroma volatiles important for tomato flavour, such as methyl-butanol/methyl-butanal, 5-methyl-6-hepten-2-one/5-methyl-6-hepten-2-ol, citronellal/citronellol, neral/nerol, geraniol. In order to assess the function of the SlscADH1 gene, transgenic plants have been generated using the technique of RNA interference (RNAi). Constitutive down-regulation using the 35S promoter resulted in the generation of dwarf plants, indicating that the SlscADH1 gene, although weakly expressed in vegetative tissues, had a function in regulating plant development. Fruit-specific down-regulation using the 2A11 promoter had no morphogenetic effect and did not alter the aldehyde/alcohol balance of the volatiles compounds produced by the fruit. Nevertheless, SlscADH1-inhibited fruit unexpectedly accumulated higher concentrations of C5 and C6 volatile compounds of the lipoxygenase pathway, possibly as an indirect effect of the suppression of SlscADH1 on the catabolism of

  8. Imaging the NADH:NAD+ Homeostasis for Understanding the Metabolic Response of Mycobacterium to Physiologically Relevant Stresses.

    PubMed

    Bhat, Shabir A; Iqbal, Iram K; Kumar, Ashwani

    2016-01-01

    The NADH:NAD + ratio is the primary indicator of the metabolic state of bacteria. NAD(H) homeostasis is critical for Mycobacterium tuberculosis (Mtb) survival and is thus considered an important drug target, but the spatio-temporal measurements of NAD(H) remain a challenge. Genetically encoded fluorescent biosensors of the NADH:NAD + ratios were recently described, paving the way for investigations of the metabolic state of pathogens during infection. Here we have adapted the genetically encoded biosensor Peredox for measurement of the metabolic state of Mtb in vitro and during infection of macrophage cells. Using Peredox, here we show that inhibition of the electron transport chain, disruption of the membrane potential and proton gradient, exposure to reactive oxygen species and treatment with antimycobacterial drugs led to the accumulation of NADH in mycobacterial cells. We have further demonstrated that Mtb residing in macrophages displays higher NADH:NAD + ratios, that may indicate a metabolic stress faced by the intracellular Mtb. We also demonstrate that the Mtb residing in macrophages display a metabolic heterogeneity, which may perhaps explain the tolerance displayed by intracellular Mtb. Next we studied the effect of immunological modulation by interferon gamma on metabolism of intracellular Mtb, since macrophage activation is known to restrict mycobacterial growth. We observed that activation of resting macrophages with interferon-gamma results in higher NADH:NAD + levels in resident Mtb cells. We have further demonstrated that exposure of Isoniazid, Bedaquiline, Rifampicin, and O-floxacin results in higher NADH:NAD + ratios in the Mtb residing in macrophages. However, intracellular Mtb displays lower NADH:NAD + ratio upon exposure to clofazimine. In summary, we have generated reporter strains capable of measuring the metabolic state of Mtb cells in vitro and in vivo with spatio-temporal resolution. We believe that this tool will facilitate further

  9. Characterisation of the two malate dehydrogenases from Phytomonas sp. Purification of the glycosomal isoenzyme.

    PubMed

    Uttaro, A D; Opperdoes, F R

    1997-10-01

    Two NAD(H)-dependent malate dehydrogenase (MDH) isoenzymes were detected in Phytomonas isolated from the lactiferous tubes of Euphorbia characias. The total specific activity in crude extracts using oxaloacetate as substrate was 3.3 U mg-1 of protein. The two isoenzymes had isoelectric points of 6.0 and 7.2, respectively. The acidic isoform represented 80% of the total activity in the cell and was present in the glycosome. It was purified to homogeneity by a method involving hydrophobic interaction chromatography on Phenyl-Sepharose followed by ionic exchange on CM-Sepharose and affinity chromatography on Blue-Sepharose. The purified glycosomal MDH is a homodimeric protein with a subunit molecular mass of 37 kDa and it has a low substrate specificity, since it was able to reduce both aromatic and aliphatic alpha-ketoacids as substrate including oxaloacetate, phenyl pyruvate, alpha-keto iso-caproate and pyruvate. The apparent K(m)s for oxaloacetate and NADH were 166 and 270 microM, respectively and for L-malate and NAD+, 3000 and 246 microM, respectively. The basic isoform was present in the mitochondrion. It has a high substrate specificity and an apparent K(m) of 132 and 63 microM for oxaloacetate and NADH, respectively, and of 450 and 91 microM, respectively, with L-malate and NAD+.

  10. Deproteinization is Necessary for the Accurate Determination of Ammonia Levels by Glutamate Dehydrogenase Assay in Blood Plasma From Subjects With Liver Injury.

    PubMed

    Vodenicarovova, Melita; Skalska, Hana; Holecek, Milan

    2017-11-08

    To determine the effect of presence of high concentrations of nicotinamide adenine dinucleotide (NADH)- and nicotinamide adenine dinucleotide phosphate (NADPH)-consuming enzymes on the accuracy of glutamate dehydrogenase (GLDH) assay for ammonia. We measured ammonia concentrations using GLDH and NADH or NADPH in blood-plasma specimens and specimens deproteinized by sulfosalicylic acid from CCl4-treated or control rats. The nonspecific oxidation of NADH and NADPH was measured in mixtures without GLDH. We observed a gradual decrease (~0.5%) in absorbance in the plasma of controls after the addition of NADH but not after adding NADPH. The decrease in absorbance in plasma of CCl4-treated animals was 13.2% and 5.2% after the addition of NADH and NADPH, respectively. The decrease in absorbance was not detected in deproteinized specimens. The values of ammonia concentration were higher in the plasma specimens compared with the deproteinized ones. Deproteinization is necessary for accurate measurement of ammonia using GLDH assay in the blood plasma of subjects with liver injury. © American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

  11. Site-Specific S-Glutathiolation of Mitochondrial NADH Ubiquinone Reductase

    PubMed Central

    Chen, Chwen-Lih; Zhang, Liwen; Yeh, Alexander; Chen, Chun-An; Green-Church, Kari B.; Zweier, Jay L.; Chen, Yeong-Renn

    2008-01-01

    The generation of reactive oxygen species in mitochondria acts as a redox signal in triggering cellular events such as apoptosis, proliferation, and senescence. Overproduction of superoxide (O2·-) and O2·--derived oxidants change the redox status of the mitochondrial GSH pool. An electron transport protein, Mitochondrial Complex I, is the major host of reactive/regulatory protein thiols. An important response of protein thiols to oxidative stress is to reversibly form protein mixed disulfide via S-glutathiolation. Exposure of Complex I to oxidized GSH, GSSG, resulted in specific S-glutathiolation at the 51 kDa and 75 kDa subunits. Here, to investigate the molecular mechanism of S-glutathiolation of Complex I, we prepared isolated bovine Complex I under non-reducing conditions and employed the techniques of mass spectrometry and EPR spin trapping for analysis. LC/MS/MS analysis of tryptic digests of the 51 kDa and 75 kDa polypeptides from glutathiolated Complex I (GS-NQR) revealed that two specific cysteines (C206 and C187) of the 51 kDa subunit and one specific cysteine (C367) of the 75 kDa subunit were involved in redox modifications with GS binding. The electron transfer activity (ETA) of GS-NQR in catalyzing NADH oxidation by Q1 was significantly enhanced. However, O2·- generation activity (SGA) mediated by GS-NQR suffered a mild loss as measured by EPR spin trapping, suggesting the protective role of S-glutathiolation in the intact Complex I. Exposure of NADH dehydrogenase (NDH), the flavin subcomplex of Complex I, to GSSG resulted in specific S-glutathiolation on the 51 kDa subunit. Both ETA and SGA of S-glutathiolated NDH (GS-NDH) decreased in parallel as the dosage of GSSG increased. LC/MS/MS analysis of a tryptic digest of the 51 kDa subunit from GS-NDH revealed that C206, C187, and C425 were glutathiolated. C425 of the 51 kDa subunit is a ligand residue of the 4Fe-4S N3 center, suggesting that destruction of 4Fe-4S is the major mechanism involved in the

  12. Altered Kinetics Properties of Erythrocyte Lactate Dehydrogenase in Type II Diabetic Patients and Its Implications for Lactic Acidosis.

    PubMed

    Mali, Aniket V; Bhise, Sunita S; Katyare, Surendra S; Hegde, Mahabaleshwar V

    2018-01-01

    Recent studies have been noted that the erythrocytes from Type II diabetic patients show significantly altered structural and functional characteristics along with the changed intracellular concentrations of glycolytic intermediates. More recent studies from our laboratory have shown that the activities of enzymes of glycolytic pathway changed significantly in RBCs from Type II diabetic patients. In particular the levels of lactate dehydrogenase (LDH) increased significantly. Lactic acidosis is an established feature of diabetes and LDH plays a crucial role in conversion of pyruvate to lactate and reportedly, the levels of lactate are significantly high which is consistent with our observation on increased levels of LDH. Owing to this background, we examined the role of erythrocyte LDH in lactic acidosis by studying its kinetics properties in Type II diabetic patients. Km, Vmax and apparent catalytic efficiency were determined using pyruvate and NADH as the substrates. With pyruvate as the substrate the Km values were comparable but Vmax increased significantly in the diabetic group. With NADH as the substrate the enzyme activity of the diabetic group resolved in two components as against a single component in the controls. The Apparent Kcat and Kcat/Km values for pyruvate increased in the diabetic group. The Ki for pyruvate increased by two fold for the enzyme from diabetic group with a marginal decrease in Ki for NADH. The observed changes in catalytic attributes are conducive to enable the enzyme to carry the reaction in forward direction towards conversion of pyruvate to lactate leading to lactic acidosis.

  13. NAD+/NADH and skeletal muscle mitochondrial adaptations to exercise

    PubMed Central

    White, Amanda T.

    2012-01-01

    The pyridine nucleotides, NAD+ and NADH, are coenzymes that provide oxidoreductive power for the generation of ATP by mitochondria. In skeletal muscle, exercise perturbs the levels of NAD+, NADH, and consequently, the NAD+/NADH ratio, and initial research in this area focused on the contribution of redox control to ATP production. More recently, numerous signaling pathways that are sensitive to perturbations in NAD+(H) have come to the fore, as has an appreciation for the potential importance of compartmentation of NAD+(H) metabolism and its subsequent effects on various signaling pathways. These pathways, which include the sirtuin (SIRT) proteins SIRT1 and SIRT3, the poly(ADP-ribose) polymerase (PARP) proteins PARP1 and PARP2, and COOH-terminal binding protein (CtBP), are of particular interest because they potentially link changes in cellular redox state to both immediate, metabolic-related changes and transcriptional adaptations to exercise. In this review, we discuss what is known, and not known, about the contribution of NAD+(H) metabolism and these aforementioned proteins to mitochondrial adaptations to acute and chronic endurance exercise. PMID:22436696

  14. Purification, properties and immunological relationship of L (+)-lactate dehydrogenase from Lactobacillus casei.

    PubMed

    Gordon, G L; Doelle, H W

    1976-08-16

    The fructose-1,6-bisphosphate-activated L-lactate dehydrogenase (EC 1.1.1.27) from Lactobacillus casei ATCC 393 has been purified to homogenity by including affinity chromatography (cibacronblue-Sephadex-G-200) and preparative polyacrylamide gel electrophoresis into the purification procedures. The enzyme has an Mr of 132000-135000 with a subunit Mr of 34000. The pH optimum was found to be 5.4 insodium acetate buffer. Tris/maleate and citrate/phosphate buffers inhibited enzyme activity at this pH. The enzyme was completely inactivated by a temperature increase from 60 degrees C to 70 degrees C. Pyruvate saturation curves were sigmoidal in the absence of fructose 1,6-bisphosphate. In the presence of 20 muM fructose 1,6-bisphosphate a Km of 1.0 mM for pyruvate was obtained, whereas fructose 1,6-bisphosphate had no effect on the Km of 0.01 mM for NADH. The use of pyruvate analogues revealed two types of pyruvate binding sites, a catalytic and an effector site. The enzyme from L. casei appears to be subject to strict metabolic control, since ADP, ATP, dihydroxyacetone phosphate and 6-phosphogluconate are strong inhibitors. Immunodiffusion experiments with a rabbit antiserum to L. casei lactate dehydrogenase revealed that L. casei ATCC 393 L (+)-lactate dehydrogenase is probably not immunologically related to group D and group N streptococci. Of 24 lactic acid bacterial strains tested only 5 strains did cross-react: L. casei ATCC 393 = L. casei var. rhamnosus ATCC 7469 - L. casei var. alactosus NCDO 680 greater than L. casei UQM 95 greater than L. plantarum ATCC 14917.

  15. Complementation of mitochondrial electron transport chain by manipulation of the NAD+/NADH ratio.

    PubMed

    Titov, Denis V; Cracan, Valentin; Goodman, Russell P; Peng, Jun; Grabarek, Zenon; Mootha, Vamsi K

    2016-04-08

    A decline in electron transport chain (ETC) activity is associated with many human diseases. Although diminished mitochondrial adenosine triphosphate production is recognized as a source of pathology, the contribution of the associated reduction in the ratio of the amount of oxidized nicotinamide adenine dinucleotide (NAD(+)) to that of its reduced form (NADH) is less clear. We used a water-forming NADH oxidase from Lactobacillus brevis (LbNOX) as a genetic tool for inducing a compartment-specific increase of the NAD(+)/NADH ratio in human cells. We used LbNOX to demonstrate the dependence of key metabolic fluxes, gluconeogenesis, and signaling on the cytosolic or mitochondrial NAD(+)/NADH ratios. Expression of LbNOX in the cytosol or mitochondria ameliorated proliferative and metabolic defects caused by an impaired ETC. The results underscore the role of reductive stress in mitochondrial pathogenesis and demonstrate the utility of targeted LbNOX for direct, compartment-specific manipulation of redox state. Copyright © 2016, American Association for the Advancement of Science.

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

    NASA Astrophysics Data System (ADS)

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

    2009-09-01

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

  17. Non-enzymatic oxidation of NADH by quinones

    NASA Astrophysics Data System (ADS)

    Scherbak, Nikolai; Strid, Åke; Eriksson, Leif A.

    2005-10-01

    Non-enzymatic oxidation of NADH by a large number of different quinones has been explored both theoretically and experimentally. It is concluded that the smaller benzo- and naphtho-quinones are capable of oxidising NADH in aqueous solution, whereas the larger anthraquinone is not. The mechanisms of stepwise electron and proton transfers are explored, and ruled out in favour of direct hydride transfer. For menadione (2-methyl-1,4-naphthoquinone), no reaction is observed experimentally; theoretically we find that there is a very close balance between the energetic cost of hydride removal from NADH and the energy gain of formation of the menadione semiquinone radical anion.

  18. Cofactor engineering to regulate NAD+/NADH ratio with its application to phytosterols biotransformation.

    PubMed

    Su, Liqiu; Shen, Yanbing; Zhang, Wenkai; Gao, Tian; Shang, Zhihua; Wang, Min

    2017-10-30

    ) conversion ratio by MNR M3N2 was 94% in the conversion system with soybean oil as reaction media to promote the solubility of phytosterols. The ratio of NAD + /NADH is an important factor for the transformation of phytosterols. Expression of NADH: flavin oxidoreductase and water-forming NADH oxidase in MNR improved AD (D) production. Besides the manipulation of key enzyme activities, which included in phytosterols degradation pathways, maintenance the balance of redox also played an important role in promoting steroid biotransformation. The recombinant MNR strain may be useful in industrial production.

  19. Imaging the NADH:NAD+ Homeostasis for Understanding the Metabolic Response of Mycobacterium to Physiologically Relevant Stresses

    PubMed Central

    Bhat, Shabir A.; Iqbal, Iram K.; Kumar, Ashwani

    2016-01-01

    The NADH:NAD+ ratio is the primary indicator of the metabolic state of bacteria. NAD(H) homeostasis is critical for Mycobacterium tuberculosis (Mtb) survival and is thus considered an important drug target, but the spatio-temporal measurements of NAD(H) remain a challenge. Genetically encoded fluorescent biosensors of the NADH:NAD+ ratios were recently described, paving the way for investigations of the metabolic state of pathogens during infection. Here we have adapted the genetically encoded biosensor Peredox for measurement of the metabolic state of Mtb in vitro and during infection of macrophage cells. Using Peredox, here we show that inhibition of the electron transport chain, disruption of the membrane potential and proton gradient, exposure to reactive oxygen species and treatment with antimycobacterial drugs led to the accumulation of NADH in mycobacterial cells. We have further demonstrated that Mtb residing in macrophages displays higher NADH:NAD+ ratios, that may indicate a metabolic stress faced by the intracellular Mtb. We also demonstrate that the Mtb residing in macrophages display a metabolic heterogeneity, which may perhaps explain the tolerance displayed by intracellular Mtb. Next we studied the effect of immunological modulation by interferon gamma on metabolism of intracellular Mtb, since macrophage activation is known to restrict mycobacterial growth. We observed that activation of resting macrophages with interferon-gamma results in higher NADH:NAD+ levels in resident Mtb cells. We have further demonstrated that exposure of Isoniazid, Bedaquiline, Rifampicin, and O-floxacin results in higher NADH:NAD+ ratios in the Mtb residing in macrophages. However, intracellular Mtb displays lower NADH:NAD+ ratio upon exposure to clofazimine. In summary, we have generated reporter strains capable of measuring the metabolic state of Mtb cells in vitro and in vivo with spatio-temporal resolution. We believe that this tool will facilitate further studies on

  20. A light-responsive and periodic NADH oxidase activity of the cell surface of Tetrahymena and of human buffy coat cells

    NASA Technical Reports Server (NTRS)

    Peter, A. D.; Morre, D. J.; Morre, D. M.

    2000-01-01

    Oxidation of external NADH (NADH is an impermeant substrate) by cells of Tetrahymena pyriformis oscillated with a period of 24-26 min. The period length in darkness (25.6 min) appeared to be slightly longer than the period in light (approximately 24 min). When Tetrahymena were placed in darkness for 30-50 min and then returned to light, a new maximum in the rate of NADH oxidation was observed 36-38 min (13 + 24) min after the beginning of the light treatment. The cell-surface NADH oxidase of human buffy coats (a mixture of white cells and platelets) also was periodic and light responsive.

  1. Crosstalk of Signaling and Metabolism Mediated by the NAD(+)/NADH Redox State in Brain Cells.

    PubMed

    Winkler, Ulrike; Hirrlinger, Johannes

    2015-12-01

    The energy metabolism of the brain has to be precisely adjusted to activity to cope with the organ's energy demand, implying that signaling regulates metabolism and metabolic states feedback to signaling. The NAD(+)/NADH redox state constitutes a metabolic node well suited for integration of metabolic and signaling events. It is affected by flux through metabolic pathways within a cell, but also by the metabolic state of neighboring cells, for example by lactate transferred between cells. Furthermore, signaling events both in neurons and astrocytes have been reported to change the NAD(+)/NADH redox state. Vice versa, a number of signaling events like astroglial Ca(2+) signals, neuronal NMDA-receptors as well as the activity of transcription factors are modulated by the NAD(+)/NADH redox state. In this short review, this bidirectional interdependence of signaling and metabolism involving the NAD(+)/NADH redox state as well as its potential relevance for the physiology of the brain and the whole organism in respect to blood glucose regulation and body weight control are discussed.

  2. 2-Oxoglutarate dehydrogenase is a more significant source of O2(·-)/H2O2 than pyruvate dehydrogenase in cardiac and liver tissue.

    PubMed

    Mailloux, Ryan J; Gardiner, Danielle; O'Brien, Marisa

    2016-08-01

    Pyruvate dehydrogenase (Pdh) and 2-oxoglutarate dehydrogenase (Ogdh) are vital for Krebs cycle metabolism and sources of reactive oxygen species (ROS). O2(·-)/H2O2 formation by Pdh and Ogdh from porcine heart were compared when operating under forward or reverse electron transfer conditions. Comparisons were also conducted with liver and cardiac mitochondria. During reverse electron transfer (RET) from NADH, purified Ogdh generated ~3-3.5× more O2(·-)/H2O2 in comparison to Pdh when metabolizing 0.5-10µM NADH. Under forward electron transfer (FET) conditions Ogdh generated ~2-4× more O2(·-)/H2O2 than Pdh. In both liver and cardiac mitochondria, Ogdh displayed significantly higher rates of ROS formation when compared to Pdh. Ogdh was also a significant source of ROS in liver mitochondria metabolizing 50µM and 500µM pyruvate or succinate. Finally, we also observed that DTT directly stimulated O2(·-)/H2O2 formation by purified Pdh and Ogdh and in cardiac or liver mitochondria in the absence of substrates and cofactors. Taken together, Ogdh is a more potent source of ROS than Pdh in liver and cardiac tissue. Ogdh is also an important ROS generator regardless of whether pyruvate or succinate serve as the sole source of carbon. Our observations provide insight into the ROS generating capacity of either complex in cardiac and liver tissue. The evidence presented herein also indicates DTT, a reductant that is routinely added to biological samples, should be avoided when assessing mitochondrial O2(·-)/H2O2 production. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. NADH induces the generation of superoxide radicals in leaf peroxisomes. [Pisum sativum L

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

    del Rio, L.A.; Sandalio, L.M.; Palma, J.M.

    1989-03-01

    In peroxisomes isolated from pea leaves (Pisum sativum L.) the production of superoxide free radicals (O{sub 2}{sup {minus}}) by xanthine and NADH was investigated. In peroxisomal membranes, 100 micromolar NADH induced the production of O{sub 2}{sup {minus}} radicals. In the soluble fractions of peroxisomes, no generation of O{sub 2}{sup {minus}} radicals was observed by incubation with either NADH or xanthine, although xanthine oxidase was found located predominantly in the matrix of peroxisomes. The failure of xanthine to induce superoxide generation was probably due to the inability to fully suppress the endogenous Mn-superoxide dismutase activity by inhibitors which were inactive againstmore » xanthine oxidase. The generation of superoxide radicals in leaf peroxisomes together with the recently described production of these oxygen radicals in glyoxysomes suggests that O{sub 2}{sup {minus}} generation could be a common metabolic property of peroxisomes and further supports the existence of active oxygen-related roles for peroxisomes in cellular metabolism.« less

  4. Alternative Pathway of Metronidazole Activation in Trichomonas vaginalis Hydrogenosomes

    PubMed Central

    Hrdý, Ivan; Cammack, Richard; Stopka, Pavel; Kulda, Jaroslav; Tachezy, Jan

    2005-01-01

    Metronidazole and related 5-nitroimidazoles are the only available drugs in the treatment of human urogenital trichomoniasis caused by the protozoan parasite Trichomonas vaginalis. The drugs are activated to cytotoxic anion radicals by their reduction within the hydrogenosomes. It has been established that electrons required for metronidazole activation are released from pyruvate by the activity of pyruvate:ferredoxin oxidoreductase and transferred to the drug by a low-redox-potential carrier, ferredoxin. Here we describe a novel pathway involved in the drug activation within the hydrogenosome. The source of electrons is malate, another major hydrogenosomal substrate, which is oxidatively decarboxylated to pyruvate and CO2 by NAD-dependent malic enzyme. The electrons released during this reaction are transferred from NADH to ferredoxin by NADH dehydrogenase homologous to the catalytic module of mitochondrial complex I, which uses ferredoxin as electron acceptor. Trichomonads acquire high-level metronidazole resistance only after both pyruvate- and malate-dependent pathways of metronidazole activation are eliminated from the hydrogenosomes. PMID:16304169

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

    PubMed Central

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

    2016-01-01

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

  6. Mycoplasma bovis NADH oxidase functions as both a NADH oxidizing and O2 reducing enzyme and an adhesin.

    PubMed

    Zhao, Gang; Zhang, Hui; Chen, Xi; Zhu, Xifang; Guo, Yusi; He, Chenfei; Anwar Khan, Farhan; Chen, Yingyu; Hu, Changmin; Chen, Huanchun; Guo, Aizhen

    2017-03-03

    Mycoplasma bovis causes considerable economic losses in the cattle industry worldwide. In mycoplasmal infections, adhesion to the host cell is of the utmost importance. In this study, the amino acid sequence of NOX was predicted to have enzymatic domains. The nox gene was then cloned and expressed in Escherichia coli. The enzymatic activity of recombinant NOX (rNOX) was confirmed based on its capacity to oxidize NADH to NAD + and reduce O 2 to H 2 O 2 . The adherence of rNOX to embryonic bovine lung (EBL) cells was confirmed with confocal laser scanning microscopy, enzyme-linked immunosorbent assay, and flow cytometry. Both preblocking EBL cells with purified rNOX and preneutralizing M. bovis with polyclonal antiserum to rNOX significantly reduced the adherence of M. bovis to EBL cells. Mycoplasma bovis NOX- expressed a truncated NOX protein at a level 10-fold less than that of the wild type. The capacities of M. bovis NOX- for cell adhesion and H 2 O 2 production were also significantly reduced. The rNOX was further used to pan phage displaying lung cDNA library and fibronectin was determined to be potential ligand. In conclusion, M. bovis NOX functions as both an active NADH oxidase and adhesin, and is therefore a potential virulence factor.

  7. Hairpin stabilized fluorescent silver nanoclusters for quantitative detection of NAD+ and monitoring NAD+/NADH based enzymatic reactions.

    PubMed

    Jain, Priyamvada; Chakma, Babina; Patra, Sanjukta; Goswami, Pranab

    2017-03-01

    A set of 90 mer long ssDNA candidates, with different degrees of cytosine (C-levels) (% and clusters) was analyzed for their function as suitable Ag-nanocluster (AgNC) nucleation scaffolds. The sequence (P4) with highest C-level (42.2%) emerged as the only candidate supporting the nucleation process as evident from its intense fluorescence peak at λ 660 nm . Shorter DNA subsets derived from P4 with only stable hairpin structures could support the AgNC formation. The secondary hairpin structures were confirmed by PAGE, and CD studies. The number of base pairs in the stem region also contributes to the stability of the hairpins. A shorter 29 mer sequence (Sub 3) (ΔG = -1.3 kcal/mol) with 3-bp in the stem of a 7-mer loop conferred highly stable AgNC. NAD + strongly quenched the fluorescence of Sub 3-AgNC in a concentration dependent manner. Time resolved photoluminescence studies revealed the quenching involves a combined static and dynamic interaction where the binding constant and number of binding sites for NAD + were 0.201 L mol -1 and 3.6, respectively. A dynamic NAD + detection range of 50-500 μM with a limit of detection of 22.3 μM was discerned. The NAD + mediated quenching of AgNC was not interfered by NADH, NADP + , monovalent and divalent ions, or serum samples. The method was also used to follow alcohol dehydrogenase and lactate dehydrogenase catalyzed physiological reactions in a turn-on and turn-off assay, respectively. The proposed method with ssDNA-AgNC could therefore be extended to monitor other NAD + /NADH based enzyme catalyzed reactions in a turn-on/turn-off approach. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Fiber-Optic Bio-sniffer (Biochemical Gas Sensor) Using Reverse Reaction of Alcohol Dehydrogenase for Exhaled Acetaldehyde.

    PubMed

    Iitani, Kenta; Chien, Po-Jen; Suzuki, Takuma; Toma, Koji; Arakawa, Takahiro; Iwasaki, Yasuhiko; Mitsubayashi, Kohji

    2018-02-23

    Volatile organic compounds (VOCs) exhaled in breath have huge potential as indicators of diseases and metabolisms. Application of breath analysis for disease screening and metabolism assessment is expected since breath samples can be noninvasively collected and measured. In this research, a highly sensitive and selective biochemical gas sensor (bio-sniffer) for gaseous acetaldehyde (AcH) was developed. In the AcH bio-sniffer, a reverse reaction of alcohol dehydrogenase (ADH) was employed for reducing AcH to ethanol and simultaneously consuming a coenzyme, reduced form of nicotinamide adenine dinucleotide (NADH). The concentration of AcH can be quantified by fluorescence detection of NADH that was consumed by reverse reaction of ADH. The AcH bio-sniffer was composed of an ultraviolet light-emitting diode (UV-LED) as an excitation light source, a photomultiplier tube (PMT) as a fluorescence detector, and an optical fiber probe, and these three components were connected with a bifurcated optical fiber. A gas-sensing region of the fiber probe was developed with a flow-cell and an ADH-immobilized membrane. In the experiment, after optimization of the enzyme reaction conditions, the selectivity and dynamic range of the AcH bio-sniffer were investigated. The AcH bio-sniffer showed a short measurement time (within 2 min) and a broad dynamic range for determination of gaseous AcH, 0.02-10 ppm, which encompassed a typical AcH concentration in exhaled breath (1.2-6.0 ppm). Also, the AcH bio-sniffer exhibited a high selectivity to gaseous AcH based on the specificity of ADH. The sensor outputs were observed only from AcH-contained standard gaseous samples. Finally, the AcH bio-sniffer was applied to measure the concentration of AcH in exhaled breath from healthy subjects after ingestion of alcohol. As a result, a significant difference of AcH concentration between subjects with different aldehyde dehydrogenase type 2 (ALDH2) phenotypes was observed. The AcH bio-sniffer can be

  9. NADH/NADPH bi-cofactor-utilizing and thermoactive ketol-acid reductoisomerase from Sulfolobus acidocaldarius.

    PubMed

    Chen, Chin-Yu; Ko, Tzu-Ping; Lin, Kuan-Fu; Lin, Bo-Lin; Huang, Chun-Hsiang; Chiang, Cheng-Hung; Horng, Jia-Cherng

    2018-05-08

    Ketol-acid reductoisomerase (KARI) is a bifunctional enzyme in the second step of branched-chain amino acids biosynthetic pathway. Most KARIs prefer NADPH as a cofactor. However, KARI with a preference for NADH is desirable in industrial applications including anaerobic fermentation for the production of branched-chain amino acids or biofuels. Here, we characterize a thermoacidophilic archaeal Sac-KARI from Sulfolobus acidocaldarius and present its crystal structure at a 1.75-Å resolution. By comparison with other holo-KARI structures, one sulphate ion is observed in each binding site for the 2'-phosphate of NADPH, implicating its NADPH preference. Sac-KARI has very high affinity for NADPH and NADH, with K M values of 0.4 μM for NADPH and 6.0 μM for NADH, suggesting that both are good cofactors at low concentrations although NADPH is favoured over NADH. Furthermore, Sac-KARI can catalyze 2(S)-acetolactate (2S-AL) with either cofactor from 25 to 60 °C, but the enzyme has higher activity by using NADPH. In addition, the catalytic activity of Sac-KARI increases significantly with elevated temperatures and reaches an optimum at 60 °C. Bi-cofactor utilization and the thermoactivity of Sac-KARI make it a potential candidate for use in metabolic engineering or industrial applications under anaerobic or harsh conditions.

  10. Renewable Molecular Flasks with NADH Models: Combination of Light-Driven Proton Reduction and Biomimetic Hydrogenation of Benzoxazinones.

    PubMed

    Zhao, Liang; Wei, Jianwei; Lu, Junhua; He, Cheng; Duan, Chunying

    2017-07-17

    Using small molecules with defined pockets to catalyze chemical transformations resulted in attractive catalytic syntheses that echo the remarkable properties of enzymes. By modulating the active site of a nicotinamide adenine dinucleotide (NADH) model in a redox-active molecular flask, we combined biomimetic hydrogenation with in situ regeneration of the active site in a one-pot transformation using light as a clean energy source. This molecular flask facilitates the encapsulation of benzoxazinones for biomimetic hydrogenation of the substrates within the inner space of the flask using the active sites of the NADH models. The redox-active metal centers provide an active hydrogen source by light-driven proton reduction outside the pocket, allowing the in situ regeneration of the NADH models under irradiation. This new synthetic platform, which offers control over the location of the redox events, provides a regenerating system that exhibits high selectivity and efficiency and is extendable to benzoxazinone and quinoxalinone systems. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Structural and Functional Studies of WlbA: A Dehydrogenase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

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

    Thoden, James B.; Holden, Hazel M.

    2010-09-08

    2,3-Diacetamido-2,3-dideoxy-D-mannuronic acid (ManNAc3NAcA) is an unusual dideoxy sugar first identified nearly 30 years ago in the lipopolysaccharide of Pseudomonas aeruginosa O:3a,d. It has since been observed in other organisms, including Bordetella pertussis, the causative agent of whooping cough. Five enzymes are required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetyl-D-glucosamine. Here we describe a structural study of WlbA, the NAD-dependent dehydrogenase that catalyzes the second step in the pathway, namely, the oxidation of the C-3{prime} hydroxyl group on the UDP-linked sugar to a keto moiety and the reduction of NAD{sup +} to NADH. This enzyme has been shown to usemore » {alpha}-ketoglutarate as an oxidant to regenerate the oxidized dinucleotide. For this investigation, three different crystal structures were determined: the enzyme with bound NAD(H), the enzyme in a complex with NAD(H) and {alpha}-ketoglutarate, and the enzyme in a complex with NAD(H) and its substrate (UDP-N-acetyl-D-glucosaminuronic acid). The tetrameric enzyme assumes an unusual quaternary structure with the dinucleotides positioned quite closely to one another. Both {alpha}-ketoglutarate and the UDP-linked sugar bind in the WlbA active site with their carbon atoms (C-2 and C-3{prime}, respectively) abutting the re face of the cofactor. They are positioned {approx}3 {angstrom} from the nicotinamide C-4. The UDP-linked sugar substrate adopts a highly unusual curved conformation when bound in the WlbA active site cleft. Lys 101 and His 185 most likely play key roles in catalysis.« less

  12. Genetically encoded probes for NAD+/NADH monitoring.

    PubMed

    Bilan, Dmitry S; Belousov, Vsevolod V

    2016-11-01

    NAD + and NADH participate in many metabolic reactions. The NAD + /NADH ratio is an important parameter reflecting the general metabolic and redox state of different types of cells. For a long time, in situ and in vivo NAD + /NADH monitoring has been hampered by the lack of suitable tools. The recent development of genetically encoded indicators based on fluorescent proteins linked to specific nucleotide-binding domains has already helped to address this monitoring problem. In this review, we will focus on four available indicators: Peredox, Frex family probes, RexYFP and SoNar. Each indicator has advantages and limitations. We will also discuss the most important points that should be considered when selecting a suitable indicator for certain experimental conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. NAD(H) and NADP(H) Redox Couples and Cellular Energy Metabolism.

    PubMed

    Xiao, Wusheng; Wang, Rui-Sheng; Handy, Diane E; Loscalzo, Joseph

    2018-01-20

    The nicotinamide adenine dinucleotide (NAD + )/reduced NAD + (NADH) and NADP + /reduced NADP + (NADPH) redox couples are essential for maintaining cellular redox homeostasis and for modulating numerous biological events, including cellular metabolism. Deficiency or imbalance of these two redox couples has been associated with many pathological disorders. Recent Advances: Newly identified biosynthetic enzymes and newly developed genetically encoded biosensors enable us to understand better how cells maintain compartmentalized NAD(H) and NADP(H) pools. The concept of redox stress (oxidative and reductive stress) reflected by changes in NAD(H)/NADP(H) has increasingly gained attention. The emerging roles of NAD + -consuming proteins in regulating cellular redox and metabolic homeostasis are active research topics. The biosynthesis and distribution of cellular NAD(H) and NADP(H) are highly compartmentalized. It is critical to understand how cells maintain the steady levels of these redox couple pools to ensure their normal functions and simultaneously avoid inducing redox stress. In addition, it is essential to understand how NAD(H)- and NADP(H)-utilizing enzymes interact with other signaling pathways, such as those regulated by hypoxia-inducible factor, to maintain cellular redox homeostasis and energy metabolism. Additional studies are needed to investigate the inter-relationships among compartmentalized NAD(H)/NADP(H) pools and how these two dinucleotide redox couples collaboratively regulate cellular redox states and cellular metabolism under normal and pathological conditions. Furthermore, recent studies suggest the utility of using pharmacological interventions or nutrient-based bioactive NAD + precursors as therapeutic interventions for metabolic diseases. Thus, a better understanding of the cellular functions of NAD(H) and NADP(H) may facilitate efforts to address a host of pathological disorders effectively. Antioxid. Redox Signal. 28, 251-272.

  14. Mitochondrial Respiratory Defect Causes Dysfunctional Lactate Turnover via AMP-activated Protein Kinase Activation in Human-induced Pluripotent Stem Cell-derived Hepatocytes*

    PubMed Central

    Im, Ilkyun; Jang, Mi-jin; Park, Seung Ju; Lee, Sang-Hee; Choi, Jin-Ho; Yoo, Han-Wook; Kim, Seyun; Han, Yong-Mahn

    2015-01-01

    A defective mitochondrial respiratory chain complex (DMRC) causes various metabolic disorders in humans. However, the pathophysiology of DMRC in the liver remains unclear. To understand DMRC pathophysiology in vitro, DMRC-induced pluripotent stem cells were generated from dermal fibroblasts of a DMRC patient who had a homoplasmic mutation (m.3398T→C) in the mitochondrion-encoded NADH dehydrogenase 1 (MTND1) gene and that differentiated into hepatocytes (DMRC hepatocytes) in vitro. DMRC hepatocytes showed abnormalities in mitochondrial characteristics, the NAD+/NADH ratio, the glycogen storage level, the lactate turnover rate, and AMPK activity. Intriguingly, low glycogen storage and transcription of lactate turnover-related genes in DMRC hepatocytes were recovered by inhibition of AMPK activity. Thus, AMPK activation led to metabolic changes in terms of glycogen storage and lactate turnover in DMRC hepatocytes. These data demonstrate for the first time that energy depletion may lead to lactic acidosis in the DMRC patient by reduction of lactate uptake via AMPK in liver. PMID:26491018

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

    Tam, Tsz Kin; Chen, Baowei; Lei, Chenghong

    NAD/NADH is a coenzyme found in all living cells, carrying electrons from one reaction to another. We report on characterizations of in situ regeneration of NADH via lipoamide dehydrogenase (LD)-catalyzed electron transfer reaction to regenerate NADH using UV-vis spectroelectrochemistry. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) of NADH regeneration were measured as 0.80 {+-} 0.15 mM and 1.91 {+-} 0.09 {micro}M s-1 in a 1-mm thin-layer spectroelectrochemical cell using gold gauze as the working electrode at the applied potential -0.75 V (vs. Ag/AgCl). The electrocatalytic reduction of the NAD system was further coupled with the enzymatic conversion of pyruvatemore » to lactate by lactate dehydrogenase to examine the coenzymatic activity of the regenerated NADH. Although the reproducible electrocatalytic reduction of NAD into NADH is known to be difficult compared to the electrocatalytic oxidation of NADH, our spectroelectrochemical results indicate that the in situ regeneration of NADH via LD-catalyzed electron transfer reaction is fast and sustainable and can be potentially applied to many NAD/NADH-dependent enzyme systems.« less

  16. Removal of H2O2 and generation of superoxide radical: Role of cytochrome c and NADH

    PubMed Central

    Velayutham, Murugesan; Hemann, Craig; Zweier, Jay L.

    2011-01-01

    In cells, mitochondria, endoplasmic reticulum, and peroxisomes are the major sources of reactive oxygen species (ROS) under physiological and pathophysiological conditions. Cytochrome c (cyt c) is known to participate in mitochondrial electron transport and has antioxidant and peroxidase activities. Under oxidative or nitrative stress, the peroxidase activity of Fe3+cyt c is increased. The level of NADH is also increased under pathophysiological conditions such as ischemia and diabetes and a concurrent increase in hydrogen peroxide (H2O2) production occurs. Studies were performed to understand the related mechanisms of radical generation and NADH oxidation by Fe3+cyt c in the presence of H2O2. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with NADH, Fe3+cyt c, and H2O2 in the presence of methyl-β-cyclodextrin. An EPR spectrum corresponding to the superoxide radical adduct of DMPO encapsulated in methyl-β-cyclodextrin was obtained. This EPR signal was quenched by the addition of the superoxide scavenging enzyme Cu,Zn-superoxide dismutase (SOD1). The amount of superoxide radical adduct formed from the oxidation of NADH by the peroxidase activity of Fe3+cyt c increased with NADH and H2O2 concentration. From these results, we propose a mechanism in which the peroxidase activity of Fe3+cyt c oxidizes NADH to NAD•, which in turn donates an electron to O2 resulting in superoxide radical formation. A UV-visible spectroscopic study shows that Fe3+cyt c is reduced in the presence of both NADH and H2O2. Our results suggest that Fe3+cyt c could have a novel role in the deleterious effects of ischemia/reperfusion and diabetes due to increased production of superoxide radical. In addition, Fe3+cyt c may play a key role in the mitochondrial “ROS-induced ROS-release (RIRR)” signaling and in mitochondrial and cellular injury/death. The increased oxidation of NADH and generation of superoxide radical

  17. Study the oxidative injury of yeast cells by NADH autofluorescence

    NASA Astrophysics Data System (ADS)

    Liang, Ju; Wu, Wen-Lan; Liu, Zhi-Hong; Mei, Yun-Jun; Cai, Ru-Xiu; Shen, Ping

    2007-06-01

    Autofluorescence has an advantage over the extrinsic fluorescence of an unperturbed environment during investigation, especially in complex system such as biological cells and tissues. NADH is an important fluorescent substance in living cells. The time courses of intracellular NADH autofluorescence in the process of yeast cells exposed to H 2O 2 and ONOO - have been recorded in detail in this work. In the presence of different amounts of H 2O 2 and ONOO -, necrosis, apoptosis and reversible injury are initiated in yeast cells, which are confirmed by acridine orange/ethidum bromide and Annexin V/propidium iodide staining. It is found that intracellular NADH content increases momently in the beginning of the apoptotic process and then decreases continually till the cell dies. The most remarkable difference between the apoptotic and the necrotic process is that the NADH content in the latter case changes much more sharply. Further in the case of reversible injury, the time course of intracellular NADH content is completely different from the above two pathways of cell death. It just decreases to some degree firstly and then resumes to the original level. Based on the role of NADH in mitochondrial respiratory chain, the time course of intracellular NADH content is believed to have reflected the response of mitochondrial redox state to oxidative stress. Thus, it is found that the mitochondrial redox state changes differently in different pathways of oxidative injury in yeast cells.

  18. A continuous spectrophotometric assay method for peptidylarginine deiminase type 4 activity.

    PubMed

    Liao, Ya-Fan; Hsieh, Hui-Chieh; Liu, Guang-Yaw; Hung, Hui-Chih

    2005-12-15

    A simple, continuous spectrophotometric assay for peptidylarginine deiminase (PAD) is described. Deimination of peptidylarginine results in the formation of peptidylcitrulline and ammonia. The ammonia released during peptidylarginine hydrolysis is coupled to the glutamate-dehydrogenase-catalyzed reductive amination of alpha-ketoglutarate to glutamate and reduced nicotinamide adenine dinucleotide (NADH) oxidation. The disappearance of absorbance at 340nm due to NADH oxidation is continuously measured. The specific activity obtained by this new protocol for highly purified human PAD is comparable to that obtained by a commonly used colorimetric procedure, which measures the ureido group of peptidylcitrulline by coupling with diacetyl monoxime. The present continuous spectrophotometric method is highly sensitive and accurate and is thus suitable for enzyme kinetic analysis of PAD. The Ca(2+) concentration for half-maximal activity of PAD obtained by this method is comparable to that previously obtained by the colorimetric procedure.

  19. The steady-state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K 12. Nitrite and hydroxylamine reduction.

    PubMed Central

    Jackson, R H; Cole, J A; Cornish-Bowden, A

    1981-01-01

    The reduction of both NO2- and hydroxylamine by the NADH-dependent nitrite reductase of Escherichia coli K 12 (EC 1.6.6.4) appears to follow Michaelis-Menten kinetics over a wide range of NADH concentrations. Substrate inhibition can, however, be detected at low concentrations of the product NAD+. In addition, NAD+ displays mixed product inhibition with respect to NADH and mixed or uncompetitive inhibition with respect to hydroxylamine. These inhibition characteristics are consistent with a mechanism in which hydroxylamine binds during catalysis to a different enzyme form from that generated when NAD+ is released. The apparent maximum velocity with NADH as varied substrate increases as the NAD+ concentration increases from 0.05 to 0.7 mM with 1 mM-NO2- or 100 mM-hydroxylamine as oxidized substrate. This increase is more marked for hydroxylamine reduction than for NO2- reduction. Models incorporating only one binding site for NAD can account for the variation in the Michaelis-Menten parameters for both NADH and hydroxylamine with [NAD+] for hydroxylamine reduction. According to these models, activation of the reaction occurs by reversal of an over-reduction of the enzyme by NADH. If the observed activation of the enzyme by NAD+ derives both from activation of the generation of the enzyme-hydroxylamine complex from the enzyme-NO2- complex during NO2- reduction and from activation of the reduction of the enzyme-hydroxylamine complex to form NH4+, then the variation of Vapp. for NO2- or hydroxylamine with [NAD+] is consistent with the occurrence of the same enzyme-hydroxylamine complex as an intermediate in both reactions. PMID:6279095

  20. Mutational analysis of the hyc-operon determining the relationship between hydrogenase-3 and NADH pathway in Enterobacter aerogenes.

    PubMed

    Pi, Jian; Jawed, Muhammad; Wang, Jun; Xu, Li; Yan, Yunjun

    2016-01-01

    In this study, the hydrogenase-3 gene cluster (hycDEFGH) was isolated and identified from Enterobacter aerogenes CCTCC AB91102. All gene products were highly homologous to the reported bacterial hydrogenase-3 (Hyd-3) proteins. The genes hycE, hycF, hycG encoding the subunits of hydrogenase-3 were targeted for genetic knockout to inhibit the FHL hydrogen production pathway via the Red recombination system, generating three mutant strains AB91102-E (ΔhycE), AB91102-F (ΔhycF) and AB91102-G (ΔhycG). Deletion of the three genes affected the integrity of hydrogenase-3. The hydrogen production experiments with the mutant strains showed that no hydrogen was detected compared with the wild type (0.886 mol/mol glucose), demonstrating that knocking out any of the three genes could inhibit NADH hydrogen production pathway. Meanwhile, the metabolites of the mutant strains were significantly changed in comparison with the wild type, indicating corresponding changes in metabolic flux by mutation. Additionally, the activity of NADH-mediated hydrogenase was found to be nil in the mutant strains. The chemostat experiments showed that the NADH/NAD(+) ratio of the mutant strains increased nearly 1.4-fold compared with the wild type. The NADH-mediated hydrogenase activity and NADH/NAD(+) ratio analysis both suggested that NADH pathway required the involvement of the electron transport chain of hydrogenase-3. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2016-04-01

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

  2. Nitric oxide inhibits succinate dehydrogenase-driven oxygen consumption in potato tuber mitochondria in an oxygen tension-independent manner.

    PubMed

    Simonin, Vagner; Galina, Antonio

    2013-01-01

    NO (nitric oxide) is described as an inhibitor of plant and mammalian respiratory chains owing to its high affinity for COX (cytochrome c oxidase), which hinders the reduction of oxygen to water. In the present study we show that in plant mitochondria NO may interfere with other respiratory complexes as well. We analysed oxygen consumption supported by complex I and/or complex II and/or external NADH dehydrogenase in Percoll-isolated potato tuber (Solanum tuberosum) mitochondria. When mitochondrial respiration was stimulated by succinate, adding the NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine) or DETA-NONOate caused a 70% reduction in oxygen consumption rate in state 3 (stimulated with 1 mM of ADP). This inhibition was followed by a significant increase in the Km value of SDH (succinate dehydrogenase) for succinate (Km of 0.77±0.19 to 34.3±5.9 mM, in the presence of NO). When mitochondrial respiration was stimulated by external NADH dehydrogenase or complex I, NO had no effect on respiration. NO itself and DETA-NONOate had similar effects to SNAP. No significant inhibition of respiration was observed in the absence of ADP. More importantly, SNAP inhibited PTM (potato tuber mitochondria) respiration independently of oxygen tensions, indicating a different kinetic mechanism from that observed in mammalian mitochondria. We also observed, in an FAD reduction assay, that SNAP blocked the intrinsic SDH electron flow in much the same way as TTFA (thenoyltrifluoroacetone), a non-competitive SDH inhibitor. We suggest that NO inhibits SDH in its ubiquinone site or its Fe-S centres. These data indicate that SDH has an alternative site of NO action in plant mitochondria.

  3. Interaction between NADH and electron-transferring flavoprotein from Megasphaera elsdenii.

    PubMed

    Sato, Kyosuke; Nishina, Yasuzo; Shiga, Kiyoshi

    2013-06-01

    Electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii is a heterodimer containing two FAD cofactors. Isolated ETF contains only one FAD molecule, FAD-1, because the other, FAD-2, is lost during purification. FAD-2 is recovered by adding FAD to the isolated ETF. The two FAD molecules in holoETF were characterized using NADH. Spectrophotometric titration of isolated ETF with NADH showed a two-electron reduction of FAD-1 according to a monophasic profile indicating that FAD-1 receives electrons from NADH without involvement of FAD-2. When holoETF was titrated with NADH, FAD-2 was reduced to an anionic semiquinone and then was fully reduced before the reduction of FAD-1. The midpoint potential values at pH 7 were +81, -136 and -279 mV for the reduction of oxidized FAD-2 to semiquinone, semiquinone to the fully reduced FAD-2 and the two-electron reduction of FAD-1, respectively. Both FAD-1 and FAD-2 in holoETF were reduced by excess NADH very rapidly. The reduction of FAD-2 was slowed by replacement of FAD-1 with 8-cyano-FAD indicating that FAD-2 receives electrons from FAD-1 but not from NADH directly. The present results suggest that FAD-2 is the counterpart of the FAD in human ETF, which contains one FAD and one AMP.

  4. Overexpression, crystallization and preliminary X-ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa.

    PubMed

    Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won

    2006-02-01

    The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-alpha-ketobutyrate. It belongs to the D-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B6 (pyridoxine). Erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 A from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 A. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (VM) of 3.64 A3 Da(-1) and a solvent content of 66%.

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

    PubMed

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

    2015-01-02

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

  6. The MTL1 Pentatricopeptide Repeat Protein Is Required for Both Translation and Splicing of the Mitochondrial NADH DEHYDROGENASE SUBUNIT7 mRNA in Arabidopsis.

    PubMed

    Haïli, Nawel; Planchard, Noelya; Arnal, Nadège; Quadrado, Martine; Vrielynck, Nathalie; Dahan, Jennifer; des Francs-Small, Catherine Colas; Mireau, Hakim

    2016-01-01

    Mitochondrial translation involves a complex interplay of ancient bacteria-like features and host-derived functionalities. Although the basic components of the mitochondrial translation apparatus have been recognized, very few protein factors aiding in recruiting ribosomes on mitochondria-encoded messenger RNA (mRNAs) have been identified in higher plants. In this study, we describe the identification of the Arabidopsis (Arabidopsis thaliana) MITOCHONDRIAL TRANSLATION FACTOR1 (MTL1) protein, a new member of the Pentatricopeptide Repeat family, and show that it is essential for the translation of the mitochondrial NADH dehydrogenase subunit7 (nad7) mRNA. We demonstrate that mtl1 mutant plants fail to accumulate the Nad7 protein, even though the nad7 mature mRNA is produced and bears the same 5' and 3' extremities as in wild-type plants. We next observed that polysome association of nad7 mature mRNA is specifically disrupted in mtl1 mutants, indicating that the absence of Nad7 results from a lack of translation of nad7 mRNA. These findings illustrate that mitochondrial translation requires the intervention of gene-specific nucleus-encoded PPR trans-factors and that their action does not necessarily involve the 5' processing of their target mRNA, as observed previously. Interestingly, a partial decrease in nad7 intron 2 splicing was also detected in mtl1 mutants, suggesting that MTL1 is also involved in group II intron splicing. However, this second function appears to be less essential for nad7 expression than its role in translation. MTL1 will be instrumental to understand the multifunctionality of PPR proteins and the mechanisms governing mRNA translation and intron splicing in plant mitochondria. © 2016 American Society of Plant Biologists. All Rights Reserved.

  7. Photoelectrochemical NADH Regeneration using Pt-Modified p -GaAs Semiconductor Electrodes

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

    Stufano, Paolo; Paris, Aubrey R.; Bocarsly, Andrew

    Cofactor regeneration in enzymatic reductions is crucial for the application of enzymes to both biological and energy-related catalysis. Specifically, regenerating NADH from NAD + is of great interest, and using electrochemistry to achieve this end is considered a promising option. Here in this paper, we report the first example of photoelectrochemical NADH regeneration at the illuminated (λ >600 nm), metal-modified p-type semiconductor electrode Pt/p-GaAs. Although bare p-GaAs electrodes produce only enzymatically inactive NAD 2, NADH was produced at the illuminated Pt-modified p-GaAs surface. At low overpotential (–0.75 V vs. Ag/AgCl), Pt/p-GaAs exhibited a seven-fold greater Faradaic efficiency for the formationmore » of NADH than Pt alone, with reduced competition from the hydrogen evolution reaction. Improved Faradaic efficiency and low overpotential suggest the possible utility of Pt/p-GaAs in energy-related NADH-dependent enzymatic processes.« less

  8. Photoelectrochemical NADH Regeneration using Pt-Modified p -GaAs Semiconductor Electrodes

    DOE PAGES

    Stufano, Paolo; Paris, Aubrey R.; Bocarsly, Andrew

    2017-02-22

    Cofactor regeneration in enzymatic reductions is crucial for the application of enzymes to both biological and energy-related catalysis. Specifically, regenerating NADH from NAD + is of great interest, and using electrochemistry to achieve this end is considered a promising option. Here in this paper, we report the first example of photoelectrochemical NADH regeneration at the illuminated (λ >600 nm), metal-modified p-type semiconductor electrode Pt/p-GaAs. Although bare p-GaAs electrodes produce only enzymatically inactive NAD 2, NADH was produced at the illuminated Pt-modified p-GaAs surface. At low overpotential (–0.75 V vs. Ag/AgCl), Pt/p-GaAs exhibited a seven-fold greater Faradaic efficiency for the formationmore » of NADH than Pt alone, with reduced competition from the hydrogen evolution reaction. Improved Faradaic efficiency and low overpotential suggest the possible utility of Pt/p-GaAs in energy-related NADH-dependent enzymatic processes.« less

  9. Lactate dehydrogenase activity drives hair follicle stem cell activation

    PubMed Central

    Aimee, Flores; John, Schell; Abby, Krall; David, Jelinek; Matilde, Miranda; Melina, Grigorian; Daniel, Braas; White Andrew, C; Jessica, Zhou; Nick, Graham; Thomas, Graeber; Pankaj, Seth; Denis, Evseenko; Hilary, Coller; Jared, Rutter; Heather, Christofk; Lowry William, E

    2017-01-01

    Summary While normally dormant, Hair Follicle Stem Cells (HFSCs) quickly become activated to divide during a new hair cycle. The quiescence of HFSCs is known to be regulated by a number of intrinsic and extrinsic mechanisms. Here we provide several lines of evidence to demonstrate that HFSCs utilize glycolytic metabolism and produce significantly more lactate than other cells in the epidermis. Furthermore, lactate generation appears to be critical for the activation of HFSCs as deletion of lactate dehydrogenase (Ldha) prevented their activation. Conversely, genetically promoting lactate production in HFSCs through mitochondrial pyruvate carrier (Mpc1) deletion accelerated their activation and the hair cycle. Finally, we identify small molecules that increase lactate production by stimulating Myc levels or inhibiting Mpc1 carrier activity and can topically induce the hair cycle. These data suggest that HFSCs maintain a metabolic state that allow them to remain dormant and yet quickly respond to appropriate proliferative stimuli. PMID:28812580

  10. Nicotinamide pre-treatment ameliorates NAD(H) hyperoxidation and improves neuronal function after severe hypoxia

    PubMed Central

    Shetty, Pavan K; Galeffi, Francesca; Turner, Dennis A.

    2014-01-01

    Prolonged hypoxia leads to irreversible loss of neuronal function and metabolic impairment of nicotinamide adenine dinucleotide recycling (between NAD+ and NADH) immediately after reoxygenation, resulting in NADH hyperoxidation. We test whether addition of nicotinamide (to enhance NAD+ levels) or PARP-1 inhibition (to prevent consumption of NAD+) can be effective in improving either loss of neuronal function or hyperoxidation following severe hypoxic injury in hippocampal slices. After severe, prolonged hypoxia (maintained for 3 min after spreading depression) there was hyperoxidation of NADH following reoxygenation, an increased soluble NAD+/NADH ratio, loss of neuronal field excitatory post-synaptic potential (fEPSP) and decreased ATP content. Nicotinamide incubation (5 mM) 2 hr prior to hypoxia significantly increased total NAD(H) content, improved neuronal recovery, enhanced ATP content, and prevented NADH hyperoxidation. The nicotinamide-induced increase in total soluble NAD(H) was more significant in the cytosolic compartment than within mitochondria. Prolonged incubation with PJ-34 (>1hr) led to enhanced baseline NADH fluorescence prior to hypoxia, as well as improved neuronal recovery, NADH hyperoxidation and ATP content on recovery from severe hypoxia and reoxygenation. In this acute model of severe neuronal dysfunction prolonged incubation with either nicotinamide or PJ-34 prior to hypoxia improved recovery of neuronal function, enhanced NADH reduction and ATP content, but neither treatment restored function when administered during or after prolonged hypoxia and reoxygenation. PMID:24184921

  11. Monoclonal antibody to a cancer-specific and drug-responsive hydroquinone (NADH) oxidase from the sera of cancer patients

    NASA Technical Reports Server (NTRS)

    Cho, NaMi; Chueh, Pin-Ju; Kim, Chinpal; Caldwell, Sara; Morre, Dorothy M.; Morre, D. James

    2002-01-01

    Monoclonal antibodies were generated in mice to a 34-kDa circulating form of a drug-responsive hydroquinone (NADH) oxidase with a protein disulfide-thiol interchange activity specific to the surface of cancer cells and the sera of cancer patients. Screening used Western blots with purified 34-kDa tNOX from HeLa cells and the sera of cancer patients. Epitopes were sought that inhibited the drug-responsive oxidation of NADH with the sera of cancer patients, but which had no effect on NADH oxidation with the sera of healthy volunteers. Two such antisera were generated. One, designated monoclonal antibody (mAb) 12.1, was characterized extensively. The NADH oxidase activity inhibited by mAb 12.1 also was inhibited by the quinone site inhibitor capsaicin (8-methyl- N-vanillyl-6-noneamide). The inhibition was competitive for the drug-responsive protein disulfide-thiol interchange activity assayed either by restoration of activity to scrambled RNase or by cleavage of a dithiodipyridine substrate, and was uncompetitive for NADH oxidation. Both the mAb 12.1 and the postimmune antisera immunoprecipitated drug-responsive NOX activity and identified the same 34-kDa tNOX protein in the sera of cancer patients that was absent from sera of healthy volunteers, and was utilized as immunogen. Preimmune sera from the same mouse as the postimmune antisera was without effect. Both mouse ascites containing mAb 12.1 and postimmune sera (but not preimmune sera) slowed the growth of human cancer cell lines in culture, but did not affect the growth of non-cancerous cell lines. Immunocytochemical and histochemical findings showed that mAb 12.1 reacted with the surface membranes of human carcinoma cells and tissues.

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

    PubMed

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

    2011-11-22

    Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(-)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L(-1) of optically pure D(-)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 μmoles min(-1) (mg protein)(-1). By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50 °C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(-) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates.

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

    PubMed Central

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

    2011-01-01

    Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(−)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L-1 of optically pure D(−)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 μmoles min-1 (mg protein)-1. By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50 °C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(−) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates. PMID:22065761

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

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

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

    PubMed Central

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

    1996-01-01

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

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

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

    Malik, Radhika; Viola, Ronald E.

    2010-10-28

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

  17. Electrophoretically mediated microanalysis of a nicotinamide adenine dinucleotide-dependent enzyme and its facile multiplexing using an active pixel sensor UV detector.

    PubMed

    Urban, Pawel L; Goodall, David M; Bergström, Edmund T; Bruce, Neil C

    2007-08-31

    An electrophoretically mediated microanalysis (EMMA) method has been developed for yeast alcohol dehydrogenase and quantification of reactant and product cofactors, NAD and NADH. The enzyme substrate ethanol (1% (v/v)) was added to the buffer (50 mM borate, pH 8.8). Results are presented for parallel capillary electrophoresis with a novel miniature UV area detector, with an active pixel sensor imaging an array of two or six parallel capillaries connected via a manifold to a single output capillary in a commercial CE instrument, allowing conversions with five different yeast alcohol dehydrogenase concentrations to be quantified in a single experiment.

  18. DFT-based prediction of reactivity of short-chain alcohol dehydrogenase

    NASA Astrophysics Data System (ADS)

    Stawoska, I.; Dudzik, A.; Wasylewski, M.; Jemioła-Rzemińska, M.; Skoczowski, A.; Strzałka, K.; Szaleniec, M.

    2017-06-01

    The reaction mechanism of ketone reduction by short chain dehydrogenase/reductase, ( S)-1-phenylethanol dehydrogenase from Aromatoleum aromaticum, was studied with DFT methods using cluster model approach. The characteristics of the hydride transfer process were investigated based on reaction of acetophenone and its eight structural analogues. The results confirmed previously suggested concomitant transfer of hydride from NADH to carbonyl C atom of the substrate with proton transfer from Tyr to carbonyl O atom. However, additional coupled motion of the next proton in the proton-relay system, between O2' ribose hydroxyl and Tyr154 was observed. The protonation of Lys158 seems not to affect the pKa of Tyr154, as the stable tyrosyl anion was observed only for a neutral Lys158 in the high pH model. The calculated reaction energies and reaction barriers were calibrated by calorimetric and kinetic methods. This allowed an excellent prediction of the reaction enthalpies (R2 = 0.93) and a good prediction of the reaction kinetics (R2 = 0.89). The observed relations were validated in prediction of log K eq obtained for real whole-cell reactor systems that modelled industrial synthesis of S-alcohols.

  19. Mycophenolic acid exposure and complement fraction C3 influence inosine 5'-monophosphate dehydrogenase activity in systemic lupus erythematosus.

    PubMed

    Mino, Yasuaki; Naito, Takafumi; Shimoyama, Kumiko; Ogawa, Noriyoshi; Kawakami, Junichi

    2017-07-01

    Background Mycophenolate mofetil has recently been reported to be effective against systemic lupus erythematosus. The influence of the pharmacokinetics of mycophenolic acid, the active form of mycophenolate mofetil and the major inactive mycophenolic acid phenolic glucuronide on the activity of the target enzyme inosine 5'-monophosphate dehydrogenase, is expected to be revealed. The aim of this study was to identify the factors associated with inosine 5'-monophosphate dehydrogenase activity in systemic lupus erythematosus patients. Methods Fifty systemic lupus erythematosus patients in remission maintenance phase (29 received mycophenolate mofetil [MMF+] and 21 did not [MMF-]) were enrolled. Median and interquartile range of dose of mycophenolate mofetil were 1500 and 1000-1500 mg/day, respectively. Stepwise multiple linear regression analysis was performed to assess the dependence between inosine 5'-monophosphate dehydrogenase activity and 25 predictor values including predose plasma concentrations of free mycophenolic acid and mycophenolic acid phenolic glucuronide. Results Median and interquartile range of predose total plasma concentrations of mycophenolic acid and mycophenolic acid phenolic glucuronide were 2.73 and 1.43-5.73 and 25.5 and 13.1-54.7  µg/mL, respectively. Predose inosine 5'-monophosphate dehydrogenase activity was significantly higher in MMF+ than MMF- patients (median 38.3 and 20.6 nmoL xanthosine 5'-monophosphate/g haemoglobin/h, P<0.01). The plasma concentration of free mycophenolic acid phenolic glucuronide, complement fraction C3 and body weight were significant predictors accounting for interindividual variability in the inosine 5'-monophosphate dehydrogenase activity (adjusted R 2  = 0.52, P < 0.01) in a multivariate analysis. Conclusions Predose inosine 5'-monophosphate dehydrogenase activity was higher in systemic lupus erythematosus patients receiving mycophenolate mofetil therapy. Inosine 5'-monophosphate dehydrogenase

  20. Water-insoluble material from apple pomace makes changes in intracellular NAD⁺/NADH ratio and pyrophosphate content and stimulates fermentative production of hydrogen.

    PubMed

    Sato, Osamu; Suzuki, Yuma; Sato, Yuki; Sasaki, Shinsuke; Sonoki, Tomonori

    2015-05-01

    Apple pomace is one of the major agricultural residues in Aomori prefecture, Japan, and it would be useful to develop effective applications for it. As apple pomace contains easily fermentable sugars such as glucose, fructose and sucrose, it can be used as a feedstock for the fermentation of fuels and chemicals. We previously isolated a new hydrogen-producing bacterium, Clostridium beijerinckii HU-1, which could produce H2 at a production rate of 14.5 mmol of H2/L/h in a fed-batch culture at 37 °C, pH 6.0. In this work we found that the HU-1 strain produces H2 at an approximately 20% greater rate when the fermentation medium contains the water-insoluble material from apple pomace. The water-insoluble material from apple pomace caused a metabolic shift that stimulated H2 production. HU-1 showed a decrease of lactate production, which consumes NADH, accompanied by an increase of the intracellular pyrophosphate content, which is an inhibitor of lactate dehydrogenase. The intracellular NAD(+)/NADH ratios of HU-1 during H2 fermentation were maintained in a more reductive state than those observed without the addition of the water insoluble material. To correct the abnormal intracellular redox balance, caused by the repression of lactate production, H2 production with NADH oxidation must be stimulated. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  1. Regulation by magnesium of potato tuber mitochondrial respiratory activities.

    PubMed

    Vicente, Joaquim A F; Madeira, Vítor M C; Vercesi, Anibal E

    2004-12-01

    Dehydrogenase activities of potato tuber mitochondria and corresponding phosphorylation rates were measured for the dependence on external and mitochondrial matrix Mg2+. Magnesium stimulated state 3 and state 4 respiration, with significantly different concentrations of matrix Mg2+ required for optimal activities of the several substrates. Maximal stimulation of respiration with all substrates was obtained at 2-mM external Mg2+. However, respiration of malate, citrate, and alpha-ketoglutarate requires at least 4-mM Mg2+ inside mitochondria for maximization of dehydrogenase activities. The phosphorylation system, requires a low level of internal Mg2+ (0.25 mM) to reach high activity, as judged by succinate-dependent respiration. However, mitochondria respiring on citrate or alpha-ketoglutarate only sustain high levels of phosphorylation with at least 4-mM matrix Mg2+. Respiration of succinate is active without external and matrix Mg2+, although stimulated by the cation. Respiration of alpha-ketoglutarate was strictly dependent on external Mg2+ required for substrate transport into mitochondria, and internal Mg2+ is required for dehydrogenase activity. Respiration of citrate and malate also depend on internal Mg2+ but, unlike alpha-ketoglutarate, some activity still remains without external Mg2+. All the substrates revealed insensitive to external and internal mitochondrial Ca2+, except the exogenous NADH dehydrogenase, which requires either external Ca2+ or Mg2+ for detectable activity. Calcium is more efficient than Mg2+, both having cumulative stimulation. Unlike Ca2+, Mn2+ could substitute for Mg2+, before and after addition of A23, showing its ability to regulate phosphorylation and succinate dehydrogenase activities, with almost the same efficiency as Mg2+.

  2. Redox imbalance stress in diabetes mellitus: Role of the polyol pathway.

    PubMed

    Yan, Liang-Jun

    2018-03-01

    In diabetes mellitus, the polyol pathway is highly active and consumes approximately 30% glucose in the body. This pathway contains 2 reactions catalyzed by aldose reductase (AR) and sorbitol dehydrogenase, respectively. AR reduces glucose to sorbitol at the expense of NADPH, while sorbitol dehydrogenase converts sorbitol to fructose at the expense of NAD + , leading to NADH production. Consumption of NADPH, accumulation of sorbitol, and generation of fructose and NADH have all been implicated in the pathogenesis of diabetes and its complications. In this review, the roles of this pathway in NADH/NAD + redox imbalance stress and oxidative stress in diabetes are highlighted. A potential intervention using nicotinamide riboside to restore redox balance as an approach to fighting diabetes is also discussed.

  3. Studies on the regulation of the human E1 subunit of the 2-oxoglutarate dehydrogenase complex, including the identification of a novel calcium-binding site.

    PubMed

    Armstrong, Craig T; Anderson, J L Ross; Denton, Richard M

    2014-04-15

    The regulation of the 2-oxoglutarate dehydrogenase complex is central to intramitochondrial energy metabolism. In the present study, the active full-length E1 subunit of the human complex has been expressed and shown to be regulated by Ca2+, adenine nucleotides and NADH, with NADH exerting a major influence on the K0.5 value for Ca2+. We investigated two potential Ca2+-binding sites on E1, which we term site 1 (D114ADLD) and site 2 (E139SDLD). Comparison of sequences from vertebrates with those from Ca2+-insensitive non-vertebrate complexes suggest that site 1 may be the more important. Consistent with this view, a mutated form of E1, D114A, shows a 6-fold decrease in sensitivity for Ca2+, whereas variant ∆site1 (in which the sequence of site 1 is replaced by A114AALA) exhibits an almost complete loss of Ca2+ activation. Variant ∆site2 (in which the sequence is replaced with A139SALA) shows no measurable change in Ca2+ sensitivity. We conclude that site 1, but not site 2, forms part of a regulatory Ca2+-binding site, which is distinct from other previously described Ca2+-binding sites.

  4. Stabilized NADH as a Countermeasure for Jet Lag

    NASA Technical Reports Server (NTRS)

    Kay, Gary G.; Viirre, Erik; Clark, Jonathan

    2001-01-01

    Current remedies for jet lag (phototherapy, melatonin, stimulant, and sedative medications) are limited in efficacy and practicality. The efficacy of a stabilized, sublingual form of reduced nicotin amide adenine dinucleotide (NADH, ENADAlert, Menuco Corp.) as a countermeasure for jet lag was examined. Because NADH increases cellular production of ATP and facilitates dopamine synthesis, it may counteract the effects of jet lag on cognitive functioning and sleepiness. Thirty-five healthy, employed subjects participated in this double-blind, placebo-controlled study. Training and baseline testing were conducted on the West Coast before subjects flew overnight to the East Coast, where they would experience a 3-hour time difference. Upon arrival, individuals were randomly assigned to receive either 20 mg of sublingual stabilized ADH (n=18) or identical placebo tablets (n=17). All participants completed computer-administered tests (including CogScreen7) to assess changes in cognitive functioning, mood, and sleepiness in the morning and afternoon. Jet lag resulted in increased sleepiness for over half the participants and deterioration of cognitive functioning for approximately one third. The morning following the flight, subjects experienced lapses of attention in addition to disruptions in working memory, divided attention, and visual perceptual speed. Individuals who received NADH performed significantly better on 5 of 8 cognitive and psychomotor test measures (P less than or equal to 0.5) and showed a trend for better performance on the other three measures (P less than or equal to .l0). Subjects also reported less sleepiness compared with those who received placebo. No adverse effects were observed with NADH treatment. Stabilized NADH significantly reduced jet lag-induced disruptions of cognitive functioning, was easily administered, and was found to have no adverse side effects.

  5. Columnar alterations of NADH fluorescence during hypoxia-ischemia in immature rat brain.

    PubMed

    Welsh, F A; Vannucci, R C; Brierley, J B

    1982-01-01

    Cerebral hypoxia-ischemia was produced in 7-day postnatal rats by unilateral carotid artery ligation combined with systemic hypoxia (8% O2). Levels of high energy phosphates, which were only slightly altered in the contralateral hemisphere, were nearly depleted in the ipsilateral hemisphere during the 3-h hypoxic insult. With hypoxia of between 1 and 3 hours' duration, columnar alterations of cortical NADH fluorescence occurred in the same location and regional pattern as did histologic damage demonstrated previously (Rice et al., 1981). In regions exhibiting columns of NADH fluorescence, there was no evidence of a columnar reduction of high energy phosphates as levels of ATP and phosphocreatine were nearly zero. Recovery from 3 h of hypoxia was accompanied by partial and regionally heterogeneous restoration of ATP within the ipsilateral hemisphere. Columnar variations of NADH fluorescence were not detected in the recovery period; rather, regions with impaired restitution of high energy phosphates exhibited NADH fluorescence that was diminished diffusely compared to the contralateral hemisphere. The correlation between depressed NADH fluorescence and depleted ATP, present as cortical columns during hypoxia and as larger regions during recovery, suggests that decreased formation of NADH may be limiting the resynthesis of high energy phosphates.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  8. Efficient synthesis of a (S)-fluoxetine intermediate using carbonyl reductase coupled with glucose dehydrogenase.

    PubMed

    Tang, Yunping; Zhang, Guomei; Wang, Zheng; Liu, Dan; Zhang, Linglu; Zhou, Yafeng; Huang, Ju; Yu, Fangmiao; Yang, Zuisu; Ding, Guofang

    2018-02-01

    (S)-3-chloro-1-phenyl-1-propanol ((S)-CPPO) is an important chiral intermediate predominantly used in the synthesis of the chiral side chain of (S)-fluoxetine. In this study, carbonyl reductase (CBR) from Novosphingobium aromaticivorans was successfully expressed in recombinant E. coli. The enzymatic activity of the recombinant CBR was significantly increased to 1875 U/mL in the fed-batch fermentation in a 10 L fermenter and recombinant CBR was then purified and characterized. By regenerating NADH with glucose dehydrogenase, 100 g/L 3-chloro-1-phenyl-1-propanone (3-CPP) was successfully converted to (S)-CPPO with a conversion of 100% and ee value of 99.6% after 12 h at 30 °C in PBS buffer (pH 7.0), which are the highest reported to date for the bio-production of (S)-CPPO and presented great potential for green production of (S)-CPPO at industrial scale. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Metabolic evolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia coli.

    PubMed

    Zhu, Xinna; Tan, Zaigao; Xu, Hongtao; Chen, Jing; Tang, Jinlei; Zhang, Xueli

    2014-07-01

    Reducing equivalents are an important cofactor for efficient synthesis of target products. During metabolic evolution to improve succinate production in Escherichia coli strains, two reducing equivalent-conserving pathways were activated to increase succinate yield. The sensitivity of pyruvate dehydrogenase to NADH inhibition was eliminated by three nucleotide mutations in the lpdA gene. Pyruvate dehydrogenase activity increased under anaerobic conditions, which provided additional NADH. The pentose phosphate pathway and transhydrogenase were activated by increased activities of transketolase and soluble transhydrogenase SthA. These data suggest that more carbon flux went through the pentose phosphate pathway, thus leading to production of more reducing equivalent in the form of NADPH, which was then converted to NADH through soluble transhydrogenase for succinate production. Reverse metabolic engineering was further performed in a parent strain, which was not metabolically evolved, to verify the effects of activating these two reducing equivalent-conserving pathways for improving succinate yield. Activating pyruvate dehydrogenase increased succinate yield from 1.12 to 1.31mol/mol, whereas activating the pentose phosphate pathway and transhydrogenase increased succinate yield from 1.12 to 1.33mol/mol. Activating these two pathways in combination led to a succinate yield of 1.5mol/mol (88% of theoretical maximum), suggesting that they exhibited a synergistic effect for improving succinate yield. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  10. Site and significance of chemically modifiable cysteine residues in glutamate dehydrogenase of Clostridium symbiosum and the use of protection studies to measure coenzyme binding.

    PubMed Central

    Syed, S E; Hornby, D P; Brown, P E; Fitton, J E; Engel, P C

    1994-01-01

    Protein chemical studies of NAD(+)-dependent glutamate dehydrogenase (GDH; EC 1.4.1.2) from Clostridium symbiosum indicate only two cysteine residues/subunit, in good agreement with the gene sequence. Experiments with various thiol-modifying reagents reveal that in native clostridial GDH only one of these two cysteines is accessible for reaction. This residue does not react with iodoacetate, iodoacetamide, N-ethylmaleimide or N-phenylmaleimide, but reaction with either p-chloromercuribenzene sulphonate or 5,5'-dithiobis(2-nitrobenzoic acid) causes complete inactivation, preventable by NAD+ or NADH but not by glutamate or 2-oxoglutarate. Protection studies with combinations of substrates show that glutamate enhances protection by NADH, whereas 2-oxoglutarate diminishes it. These studies were also used to determine a dissociation constant (0.69 mM) for the enzyme-NAD+ complex. Similar data for NADH indicated mildly cooperative binding with a Hill coefficient of 1.32. The significance of these results is discussed in the light of the high-resolution crystallographic structure for clostridial GDH and in relation to information for GDH from other sources. PMID:8129708

  11. Fecal hydroxysteroid dehydrogenase activities in vegetarian Seventh-Day Adventists, control subjects, and bowel cancer patients.

    PubMed

    Macdonald, I A; Webb, G R; Mahony, D E

    1978-10-01

    Cell-free extracts were prepared from mixed fecal anaerobic bacteria grown from stools of 14 vegetarian Seventh-Day Adventists, 16 omnivorous control subjects, and eight patients recently diagnosed with cancer of the large bowel. Preparations were assayed for NAD- and NADP-dependent 3alpha-, 7alpha- and 12alpha-hydroxysteroid dehydrogenases with bile salts and androsterone as substrates (eight substrate-cofactor combinations were tested). A significant intergroup difference was observed in the amounts of NAD- and NADP-dependent 7alpha-hydroxysteroid dehydrogenase produced: bowel cancer patients exceeded controls, and controls exceeded Seventh-Day Adventists. Other enzyme activity comparisons were not significant. The pH values of the stools were significantly higher in cancer patients compared to Seventh-Day Adventists; values were 7.03 +/- 0.60 and 6.46 +/- 0.58 respectively. The pH value for controls was 6.66 +/- 0.62. A plot of pH value versus NADP-dependent 7alpha-hydroxysteroid dehydrogenase tended to separate the cancer patients from the other groups. Comparative data suggest that much of the 3alpha-hydroxysteroid dehydrogenase active against bile salt is also active against androsterone.

  12. Determining In Vivo Regulation of Cardiac Pyruvate Dehydrogenase Based on Label Flux from Hyperpolarized [1-13C]Pyruvate

    PubMed Central

    Heather, Lisa C.; Griffin, Julian L.; Clarke, Kieran; Radda, George K.; Tyler, Damian J.

    2015-01-01

    Background Pyruvate dehydrogenase (PDH) is a key regulator of cardiac substrate selection and is regulated by both pyruvate dehydrogenase kinase (PDK)-mediated phosphorylation and feedback inhibition. The extent to which chronic upregulation of PDK protein levels, acutely increased PDK activity and acute feedback inhibition limit PDH flux remains unclear because existing in vitro assessment methods inherently disrupt the enzyme complex. We have previously demonstrated that hyperpolarized 13C-labelled metabolic tracers with magnetic resonance spectroscopy (MRS) can monitor flux through PDH in vivo. The aim of this study was to determine the relative contributions of acute and chronic changes in PDK and PDH activities to in vivo myocardial PDH flux. Methodology/Principal Findings We examined both fed and fasted rats with either hyperpolarized [1-13C]pyruvate alone or hyperpolarized [1-13C]pyruvate co-infused with malate (to modulate mitochondrial NADH/NAD+ and acetyl-CoA/CoA ratios, which alter both PDH activity and flux). To confirm the metabolic fate of infused malate, we performed in vitro 1H NMR spectroscopy on cardiac tissue extracts. We observed that in fed rats, where PDH activity was high, the presence of malate increased PDH flux by 27%, whereas in the fasted state, malate infusion had no effect on PDH flux. Conclusions/Significance These observations suggest that pyruvate oxidation is limited by feedback inhibition from acetyl-CoA only when PDH activity is high. Therefore, in the case of PDH, and potentially other enzymes, hyperpolarized 13C MR can be used to non-invasively assess enzymatic regulation. PMID:21387444

  13. Sensitive electrochemical detection of NADH and ethanol at low potential based on pyrocatechol violet electrodeposited on single walled carbon nanotubes-modified pencil graphite electrode.

    PubMed

    Zhu, Jun; Wu, Xiao-Yan; Shan, Dan; Yuan, Pei-Xin; Zhang, Xue-Ji

    2014-12-01

    In this work, the electrodeposition of pyrocatechol violet (PCV) was initially investigated by the electrochemical surface plasmon resonance (ESPR) technique. Subsequently, PCV was used as redox-mediator and was electrodeposited on the surface of pencil graphite electrode (PGE) modified with single-wall carbon nanotubes (SWCNTs). Owing to the remarkable synergistic effect of SWCNTs and PCV, PGE/SWCNTs/PCV exhibited excellent electrocatalytic activity towards dihydronicotinamide adenine dinucleotide (NADH) oxidation at low potential (0.2V vs. SCE) with fast amperometric response (<10s), broad linear range (1.3-280 μM), good sensitivity (146.2 μA mM(-1)cm(-2)) and low detection limit (1.3 μM) at signal-to-noise ratio of 3. Thus, this PGE/SWCNTs/PCV could be further used to fabricate a sensitive and economic ethanol biosensor using alcohol dehydrogenase (ADH) via a glutaraldehyde/BSA cross-linking procedure. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    2002-01-01

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

  15. Growth- and substrate-dependent transcription of formate dehydrogenase and hydrogenase coding genes in Syntrophobacter fumaroxidans and Methanospirillum hungatei.

    PubMed

    Worm, Petra; Stams, Alfons J M; Cheng, Xu; Plugge, Caroline M

    2011-01-01

    Transcription of genes coding for formate dehydrogenases (fdh genes) and hydrogenases (hyd genes) in Syntrophobacter fumaroxidans and Methanospirillum hungatei was studied following growth under different conditions. Under all conditions tested, all fdh and hyd genes were transcribed. However, transcription levels of the individual genes varied depending on the substrate and growth conditions. Our results strongly suggest that in syntrophically grown S. fumaroxidans cells, the [FeFe]-hydrogenase (encoded by Sfum_844-46), FDH1 (Sfum_2703-06) and Hox (Sfum_2713-16) may confurcate electrons from NADH and ferredoxin to protons and carbon dioxide to produce hydrogen and formate, respectively. Based on bioinformatic analysis, a membrane-integrated energy-converting [NiFe]-hydrogenase (Mhun_1741-46) of M. hungatei might be involved in the energy-dependent reduction of CO(2) to formylmethanofuran. The best candidates for F(420)-dependent N(5),N(10)-methyl-H(4) MPT and N(5),N(10),-methylene-H(4)MPT reduction are the cytoplasmic [NiFe]-hydrogenase and FDH1. 16S rRNA ratios indicate that in one of the triplicate co-cultures of S. fumaroxidans and M. hungatei, less energy was available for S. fumaroxidans. This led to enhanced transcription of genes coding for the Rnf-complex (Sfum_2694-99) and of several fdh and hyd genes. The Rnf-complex probably reoxidized NADH with ferredoxin reduction, followed by ferredoxin oxidation by the induced formate dehydrogenases and hydrogenases.

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

    PubMed Central

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

    2013-01-01

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

  17. Effective immobilization of alcohol dehydrogenase on carbon nanoscaffolds for ethanol biofuel cell.

    PubMed

    Umasankar, Yogeswaran; Adhikari, Bal-Ram; Chen, Aicheng

    2017-12-01

    An efficient approach for immobilizing alcohol dehydrogenase (ADH) while enhancing its electron transfer ability has been developed using poly(2-(trimethylamino)ethyl methacrylate) (MADQUAT) cationic polymer and carbon nanoscaffolds. The carbon nanoscaffolds were comprised of single-walled carbon nanotubes (SWCNTs) wrapped with reduced graphene oxide (rGO). The ADH entrapped within the MADQUAT that was present on the carbon nanoscaffolds exhibited a high electron exchange capability with the electrode through its cofactor β-nicotinamide adenine dinucleotide hydrate and β-nicotinamide adenine dinucleotide reduced disodium salt hydrate (NAD + /NADH) redox reaction. The advantages of the carbon nanoscaffolds used as the support matrix and the MADQUAT employed for the entrapment of ADH versus physisorption were demonstrated via cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Our experimental results showed a higher electron transfer, electrocatalytic activity, and rate constant for MADQUAT entrapped ADH on the carbon nanoscaffolds. The immobilization of ADH using both MADQUAT and carbon nanoscaffolds exhibited strong potential for the development of an efficient bio-anode for ethanol powered biofuel cells. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    1990-01-01

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

  19. [Activity of liver mitochondrial NAD+-dependent dehydrogenases of the krebs cycle in rats with acetaminophen-induced hepatitis developed under conditions of alimentary protein deficiency].

    PubMed

    Voloshchuk, O N; Kopylchuk, G P

    2016-01-01

    Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD(+)/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deprivation. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD(+)/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein caused a more pronounced decrease in the activity of NAD(+)-dependent dehydrogenases studied and a 2.2-fold increase of the mitochondrial NAD(+)/NADН ratio. This suggests that alimentary protein deprivation potentiated drug-induced liver damage.

  20. NADH oxidase activity (NOX) and enlargement of HeLa cells oscillate with two different temperature-compensated period lengths of 22 and 24 minutes corresponding to different NOX forms

    NASA Technical Reports Server (NTRS)

    Wang, S.; Pogue, R.; Morre, D. M.; Morre, D. J.

    2001-01-01

    NOX proteins are cell surface-associated and growth-related hydroquinone (NADH) oxidases with protein disulfide-thiol interchange activity. A defining characteristic of NOX proteins is that the two enzymatic activities alternate to generate a regular period length of about 24 min. HeLa cells exhibit at least two forms of NOX. One is tumor-associated (tNOX) and is inhibited by putative quinone site inhibitors (e.g., capsaicin or the antitumor sulfonylurea, LY181984). Another is constitutive (CNOX) and refractory to inhibition. The periodic alternation of activities and drug sensitivity of the NADH oxidase activity observed with intact HeLa cells was retained in isolated plasma membranes and with the solubilized and partially purified enzyme. At least two activities were present. One had a period length of 24 min and the other had a period length of 22 min. The lengths of both the 22 and the 24 min periods were temperature compensated (approximately the same when measured at 17, 27 or 37 degrees C) whereas the rate of NADH oxidation approximately doubled with each 10 degrees C rise in temperature. The rate of increase in cell area of HeLa cells when measured by video-enhanced light microscopy also exhibited a complex period of oscillations reflective of both 22 and 24 min period lengths. The findings demonstrate the presence of a novel oscillating NOX activity at the surface of cancer cells with a period length of 22 min in addition to the constitutive NOX of non-cancer cells and tissues with a period length of 24 min.

  1. Ethanol Metabolism by HeLa Cells Transduced with Human Alcohol Dehydrogenase Isoenzymes: Control of the Pathway by Acetaldehyde Concentration†

    PubMed Central

    Matsumoto, Michinaga; Cyganek, Izabela; Sanghani, Paresh C.; Cho, Won Kyoo; Liangpunsakul, Suthat; Crabb, David W.

    2010-01-01

    Background Human class I alcohol dehydrogenase 2 isoenzymes (encoded by the ADH1B locus) have large differences in kinetic properties; however, individuals inheriting the alleles for the different isoenzymes exhibit only small differences in alcohol elimination rates. This suggests that other cellular factors must regulate the activity of the isoenzymes. Methods The activity of the isoenzymes expressed from ADH1B*1, ADH1B*2, and ADH1B*3 cDNAs was examined in stably transduced HeLa cell lines, including lines which expressed human low Km aldehyde dehydrogenase (ALDH2). The ability of the cells to metabolize ethanol was compared with that of HeLa cells expressing rat class I ADH (HeLa-rat ADH cells), rat hepatoma (H4IIEC3) cells, and rat hepatocytes. Results The isoenzymes had similar protein half-lives in the HeLa cells. Rat hepatocytes, H4IIEC3 cells, and HeLa-rat ADH cells oxidized ethanol much faster than the cells expressing the ADH1B isoenzymes. This was not explained by high cellular NADH levels or endogenous inhibitors; but rather because the activity of the β1 and β2 ADHs were constrained by the accumulation of acetaldehyde, as shown by the increased rate of ethanol oxidation by cell lines expressing β2 ADH plus ALDH2. Conclusion The activity of the human β2 ADH isoenzyme is sensitive to inhibition by acetaldehyde, which likely limits its activity in vivo. This study emphasizes the importance of maintaining a low steady–state acetaldehyde concentration in hepatocytes during ethanol metabolism. PMID:21166830

  2. Multiphoton fluorescence imaging of NADH to quantify metabolic changes in epileptic tissue in vitro

    NASA Astrophysics Data System (ADS)

    Chia, Thomas H.; Zinter, Joseph; Spencer, Dennis D.; Williamson, Anne; Levene, Michael J.

    2007-02-01

    A powerful advantage of multiphoton microscopy is its ability to image endogenous fluorophores such as the ubiquitous coenzyme NADH in discrete cellular populations. NADH is integral in both oxidative and non-oxidative cellular metabolism. NADH loses fluorescence upon oxidation to NAD +; thus changes in NADH fluorescence can be used to monitor metabolism. Recent studies have suggested that hypo metabolic astrocytes play an important role in cases of temporal lobe epilepsy (TLE). Current theories suggest this may be due to defective and/or a reduced number of mitochondria or dysfunction of the neuronal-astrocytic metabolic coupling. Measuring NADH fluorescence changes following chemical stimulation enables the quantification of the cellular distribution of metabolic anomalies in epileptic brain tissue compared to healthy tissue. We present what we believe to be the first multiphoton microscopy images of NADH from the human brain. We also present images of NADH fluorescence from the hippocampus of the kainate-treated rat TLE model. In some experiments, human and rat astrocytes were selectively labeled with the fluorescent dye sulforhodamine 101 (SR101). Our results demonstrate that multiphoton microscopy is a powerful tool for assaying the metabolic pathologies associated with temporal lobe epilepsy in humans and in rodent models.

  3. Effects of NADH-preferring xylose reductase expression on ethanol production from xylose in xylose-metabolizing recombinant Saccharomyces cerevisiae.

    PubMed

    Lee, Sung-Haeng; Kodaki, Tsutomu; Park, Yong-Cheol; Seo, Jin-Ho

    2012-04-30

    Efficient conversion of xylose to ethanol is an essential factor for commercialization of lignocellulosic ethanol. To minimize production of xylitol, a major by-product in xylose metabolism and concomitantly improve ethanol production, Saccharomyces cerevisiae D452-2 was engineered to overexpress NADH-preferable xylose reductase mutant (XR(MUT)) and NAD⁺-dependent xylitol dehydrogenase (XDH) from Pichia stipitis and endogenous xylulokinase (XK). In vitro enzyme assay confirmed the functional expression of XR(MUT), XDH and XK in recombinant S. cerevisiae strains. The change of wild type XR to XR(MUT) along with XK overexpression led to reduction of xylitol accumulation in microaerobic culture. More modulation of the xylose metabolism including overexpression of XR(MUT) and transaldolase, and disruption of the chromosomal ALD6 gene encoding aldehyde dehydrogenase (SX6(MUT)) improved the performance of ethanol production from xylose remarkably. Finally, oxygen-limited fermentation of S. cerevisiae SX6(MUT) resulted in 0.64 g l⁻¹ h⁻¹ xylose consumption rate, 0.25 g l⁻¹ h⁻¹ ethanol productivity and 39% ethanol yield based on the xylose consumed, which were 1.8, 4.2 and 2.2 times higher than the corresponding values of recombinant S. cerevisiae expressing XR(MUT), XDH and XK only. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. In Silico Discovery of a Substituted 6-Methoxy-quinalidine with Leishmanicidal Activity in Leishmania infantum.

    PubMed

    Stevanović, Strahinja; Perdih, Andrej; Senćanski, Milan; Glišić, Sanja; Duarte, Margarida; Tomás, Ana M; Sena, Filipa V; Sousa, Filipe M; Pereira, Manuela M; Solmajer, Tom

    2018-03-27

    There is an urgent need for the discovery of new antileishmanial drugs with a new mechanism of action. Type 2 NADH dehydrogenase from Leishmania infantum ( Li NDH2) is an enzyme of the parasite's respiratory system, which catalyzes the electron transfer from NADH to ubiquinone without coupled proton pumping. In previous studies of the related NADH: ubiquinone oxidoreductase crystal structure from Saccharomyces cerevisiae , two ubiquinone-binding sites (UQ I and UQ II ) were identified and shown to play an important role in the NDH-2-catalyzed oxidoreduction reaction. Based on the available structural data, we developed a three-dimensional structural model of Li NDH2 using homology detection methods and performed an in silico virtual screening campaign to search for potential inhibitors targeting the Li NDH2 ubiquinone-binding site 1-UQ I . Selected compounds displaying favorable properties in the computational screening experiments were assayed for inhibitory activity in the structurally similar recombinant NDH-2 from S. aureus and leishmanicidal activity was determined in the wild-type axenic amastigotes and promastigotes of L. infantum . The identified compound, a substituted 6-methoxy-quinalidine, showed promising nanomolar leishmanicidal activity on wild-type axenic promastigotes and amastigotes of L. infantum and the potential for further development.

  5. Kinetic mechanism and quaternary structure of Aminobacter aminovorans NADH:flavin oxidoreductase: an unusual flavin reductase with bound flavin.

    PubMed

    Russell, Thomas R; Demeler, Borries; Tu, Shiao-Chun

    2004-02-17

    The homodimeric NADH:flavin oxidoreductase from Aminobacter aminovorans is an NADH-specific flavin reductase herein designated FRD(Aa). FRD(Aa) was characterized with respect to purification yields, thermal stability, isoelectric point, molar absorption coefficient, and effects of phosphate buffer strength and pH on activity. Evidence from this work favors the classification of FRD(Aa) as a flavin cofactor-utilizing class I flavin reductase. The isolated native FRD(Aa) contained about 0.5 bound riboflavin-5'-phosphate (FMN) per enzyme monomer, but one bound flavin cofactor per monomer was obtainable in the presence of excess FMN or riboflavin. In addition, FRD(Aa) holoenzyme also utilized FMN, riboflavin, or FAD as a substrate. Steady-state kinetic results of substrate titrations, dead-end inhibition by AMP and lumichrome, and product inhibition by NAD(+) indicated an ordered sequential mechanism with NADH as the first binding substrate and reduced FMN as the first leaving product. This is contrary to the ping-pong mechanism shown by other class I flavin reductases. The FMN bound to the native FRD(Aa) can be fully reduced by NADH and subsequently reoxidized by oxygen. No NADH binding was detected using 90 microM FRD(Aa) apoenzyme and 300 microM NADH. All results favor the interpretation that the bound FMN was a cofactor rather than a substrate. It is highly unusual that a flavin reductase using a sequential mechanism would require a flavin cofactor to facilitate redox exchange between NADH and a flavin substrate. FRD(Aa) exhibited a monomer-dimer equilibrium with a K(d) of 2.7 microM. Similarities and differences between FRD(Aa) and certain flavin reductases are discussed.

  6. Overexpression, crystallization and preliminary X-­ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa

    PubMed Central

    Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won

    2006-01-01

    The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-α-ketobutyrate. It belongs to the d-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B6 (pyridoxine). Erythronate-4-­phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 Å from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 Å. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (V M) of 3.64 Å3 Da−1 and a solvent content of 66%. PMID:16511285

  7. Stimulation of NADH-dependent microsomal DNA strand cleavage by rifamycin SV.

    PubMed

    Kukiełka, E; Cederbaum, A I

    1995-04-15

    Rifamycin SV is an antibiotic anti-bacterial agent used in the treatment of tuberculosis. This drug can autoxidize, especially in the presence of metals, and generate reactive oxygen species. A previous study indicated that rifamycin SV can increase NADH-dependent microsomal production of reactive oxygen species. The current study evaluated the ability of rifamycin SV to interact with iron and increase microsomal production of hydroxyl radical, as detected by conversion of supercoiled plasmid DNA into the relaxed open circular state. The plasmid used was pBluescript II KS(-), and the forms of DNA were separated by agarose-gel electrophoresis. Incubation of rat liver microsomes with plasmid plus NADH plus ferric-ATP caused DNA strand cleavage. The addition of rifamycin SV produced a time- and concentration-dependent increase in DNA-strand cleavage. No stimulation by rifamycin SV occurred in the absence of microsomes, NADH or ferric-ATP. Stimulation occurred with other ferric complexes besides ferric-ATP, e.g. ferric-histidine, ferric-citrate, ferric-EDTA, and ferric-(NH4)2SO4. Rifamycin SV did not significantly increase the high rates of DNA strand cleavage found with NADPH as the microsomal reductant. The stimulation of NADH-dependent microsomal DNA strand cleavage was completely blocked by catalase, superoxide dismutase, GSH and a variety of hydroxyl-radical-scavenging agents, but not by anti-oxidants that prevent microsomal lipid peroxidation. Redox cycling agents, such as menadione and paraquat, in contrast with rifamycin SV, stimulated the NADPH-dependent reaction; menadione and rifamycin SV were superior to paraquat in stimulating the NADH-dependent reaction. These results indicate that rifamycin SV can, in the presence of an iron catalyst, increase microsomal production of reactive oxygen species which can cause DNA-strand cleavage. In contrast with other redox cycling agents, the stimulation by rifamycin SV is more pronounced with NADH than with NADPH as the

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

    PubMed Central

    2012-01-01

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

  9. Cloning, expression, and characterization of a novel (S)-specific alcohol dehydrogenase from Lactobacillus kefir.

    PubMed

    Chen, Qilei; Hu, Youjia; Zhao, Wenjie; Zhu, Chunbao; Zhu, Baoquan

    2010-01-01

    A gene encoding a novel (S)-specific NADH-dependent alcohol dehydrogenase (LK-ADH) was isolated from the genomic DNA of Lactobacillus kefir DSM 20587 by thermal asymmetric interlaced-polymerase chain reaction. The nucleotide sequence of (S)-LK-ADH gene (adhS) was determined, which consists of an open reading frame of 1,044 bp, coding for 347 amino acids with a molecular mass of 37.065 kDa. After a BLAST similarity search in GenBank database, the amino acid sequence of (S)-LK-ADH showed some homologies to several zinc containing medium-chain alcohol dehydrogenases. This novel gene was deposited into GenBank with the accession number of EU877965. adhS gene was subcloned into plasmid pET-28a(+), and recombinant (S)-LK-ADH was successfully expressed in E. coli BL21(DE3) by isopropyl-beta-D-1-thiogalactopyranoside induction. Purified enzyme showed a high enantioselectivity in the reduction of acetophenone to (S)-phenylethanol with an ee value of 99.4%. The substrate specificity and cofactor preference of recombinant (S)-LK-ADH were also tested.

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

    PubMed

    Miyazaki, Kentaro

    2005-05-27

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

  11. [Effects of Light Near-Infrared Radiation on Rats Assessed by Succinate Dehydrogenase Activity in Lymphocytes on Blood Smears].

    PubMed

    Khunderyakova, N V; Zakharchenko, A V; Zakharchenko, M V; Muller, H; Fedotcheva, I; Kondrashova, M N

    2015-01-01

    Biological effects of light near infrared radiation (850 nm), with modulation acoustic frequency of 101 Hz, was studied. The study was conducted on rats, the effect was recorded by succinate dehydrogenase activity in lymphocytes on the blood smear after administration of the activating dose of adrenaline, which simulates the state of the organism in the early stages of the pathogenic effects (stress). A pronounced regulating effect of infrared radiation on the activity of succinate dehydrogenase in animals activated by adrenaline was shown. Infrared radiation has a normalizing effect reducing the degree of inhibition or activation of the enzyme induced by adrenaline and had no effect on the control animals. Thus, by modulating the activity of succinate dehydrogenase infrared radiation regulates energy production in the mitochondria supported by the most powerful oxidation substrate--succinic acid, which is especially pronounced under stress.

  12. Effect of feeding and of DDT on the activity of hepatic glucose 6- phosphate dehydrogenase in two salmonids

    USGS Publications Warehouse

    Buhler, Donald R.; Benville, P.

    1969-01-01

    The specific activity of liver glucose 6-phosphate dehydrogenase in yearling rainbow trout remained unchanged when the fish were starved for periods as long as 8 weeks and when starved animals were fed diets of various compositions. Injection of insulin concurrently with refeeding also failed to alter the specific activity of the enzyme in trout. The absence of a dietary or insulin influence on the teleost enzyme system is to be contrasted with studies in mammals in which the activity of hepatic glucose 6-P dehydrogenase was markedly stimulated after refeeding starved animals or injection of insulin.Ingestion of the pesticide DDT by juvenile coho salmon or adult rainbow trout also had no effect on the specific activity of liver glucose 6-P dehydrogenase and DDT failed to inhibit the rainbow trout enzyme in vitro. These results also differ considerably from those found in higher animals.These results suggest that the glucose 6-P dehydrogenase enzyme in teleosts may be under a different type of regulatory control from that found in mammals.

  13. UVB induces epidermal 11β-hydroxysteroid dehydrogenase type 1 activity in vivo.

    PubMed

    Tiganescu, Ana; Hupe, Melanie; Jiang, Yan J; Celli, Anna; Uchida, Yoshikazu; Mauro, Theodora M; Bikle, Daniel D; Elias, Peter M; Holleran, Walter M

    2015-05-01

    Detrimental consequences of ultraviolet radiation (UVR) in skin include photoageing, immunosuppression and photocarcinogenesis, processes also significantly regulated by local glucocorticoid (GC) availability. In man, the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) generates the active GC cortisol from cortisone (or corticosterone from 11-dehydrocorticosterone in rodents). 11β-HSD1 oxo-reductase activity requires the cofactor NADPH, generated by hexose-6-phosphate dehydrogenase. We previously demonstrated increased 11β-HSD1 levels in skin obtained from photoexposed versus photoprotected anatomical regions. However, the direct effect of UVR on 11β-HSD1 expression remains to be elucidated. To investigate the cutaneous regulation of 11β-HSD1 following UVR in vivo, the dorsal skin of female SKH1 mice was irradiated with 50, 100, 200 and 400 mJ/cm(2) UVB. Measurement of transepidermal water loss, 11β-HSD1 activity, mRNA/protein expression and histological studies was taken at 1, 3 and 7 days postexposure. 11β-HSD1 and hexose-6-phosphate dehydrogenase mRNA expression peaked 1 day postexposure to 400 mJ/cm(2) UVB before subsequently declining (days 3 and 7). Corresponding increases in 11β-HSD1 protein and enzyme activity were observed 3 days postexposure coinciding with reduced GC receptor mRNA expression. Immunofluorescence studies revealed 11β-HSD1 localization to hyperproliferative epidermal keratinocytes in UVB-exposed skin. 11β-HSD1 expression and activity were also induced by 200 and 100 (but not 50) mJ/cm(2) UVB and correlated with increased transepidermal water loss (indicative of barrier disruption). UVB-induced 11β-HSD1 activation represents a novel mechanism that may contribute to the regulation of cutaneous responses to UVR exposure. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Changes in cinnamyl alcohol dehydrogenase activities from sugarcane cultivars inoculated with Sporisorium scitamineum sporidia.

    PubMed

    Santiago, Rocío; Alarcón, Borja; de Armas, Roberto; Vicente, Carlos; Legaz, María Estrella

    2012-06-01

    This study describes a method for determining cinnamyl alcohol dehydrogenase activity in sugarcane stems using reverse phase (RP) high-performance liquid chromatography to elucidate their possible lignin origin. Activity is assayed using the reverse mode, the oxidation of hydroxycinnamyl alcohols into hydroxycinnamyl aldehydes. Appearance of the reaction products, coniferaldehyde and sinapaldehyde is determined by measuring absorbance at 340 and 345 nm, respectively. Disappearance of substrates, coniferyl alcohol and sinapyl alcohol is measured at 263 and 273 nm, respectively. Isocratic elution with acetonitrile:acetic acid through an RP Mediterranea sea C18 column is performed. As case examples, we have examined two different cultivars of sugarcane; My 5514 is resistant to smut, whereas B 42231 is susceptible to the pathogen. Inoculation of sugarcane stems elicits lignification and produces significant increases of coniferyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD). Production of lignin increases about 29% in the resistant cultivar and only 13% in the susceptible cultivar after inoculation compared to uninoculated plants. Our results show that the resistance of My 5514 to smut is likely derived, at least in part, to a marked increase of lignin concentration by the activation of CAD and SAD. Copyright © Physiologia Plantarum 2012.

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

    PubMed

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

    2008-09-15

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

  16. 21 CFR 862.1670 - Sorbitol dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  17. Novel characteristics of UDP-glucose dehydrogenase activities in maize: non-involvement of alcohol dehydrogenases in cell wall polysaccharide biosynthesis.

    PubMed

    Kärkönen, Anna; Fry, Stephen C

    2006-03-01

    UDP-glucose dehydrogenase (UDPGDH) activity was detected in extracts of maize cell-cultures and developing leaves. The reaction product was confirmed as UDP-glucuronate. Leaf extracts from null mutants defective in one or both of the ethanol dehydrogenase genes, ADH1 and ADH2, had similar UDPGDH activities to wild-type, showing that UDPGDH activity is not primarily due to ADH proteins. The mutants showed no defect in their wall matrix pentose:galactose ratios, or matrix:cellulose ratio, showing that ADHs were not required for normal wall biosynthesis. The majority of maize leaf UDPGDH activity had K (m) (for UDP-glucose) 0.5-1.0 mM; there was also a minor activity with an unusually high K (m) of >50 mM. In extracts of cultured cells, kinetic data indicated at least three UDPGDHs, with K (m) values (for UDP-glucose) of roughly 0.027, 2.8 and >50 mM (designated enzymes E(L), E(M) and E(H) respectively). E(M) was the single major contributor to extractable UDPGDH activity when assayed at 0.6-9.0 mM UDP-Glc. Most studies, in other plant species, had reported only E(L)-like isoforms. Ethanol (100 mM) partially inhibited UDPGDH activity assayed at low, but not high, UDP-glucose concentrations, supporting the conclusion that at least E(H) activity is not due to ADH. At 30 microM UDP-glucose, 20-150 microM UDP-xylose inhibited UDPGDH activity, whereas 5-15 microM UDP-xylose promoted it. In conclusion, several very different UDPGDH isoenzymes contribute to UDP-glucuronate and hence wall matrix biosynthesis in maize, but ADHs are not responsible for these activities.

  18. Inhibitors of type II NADH:menaquinone oxidoreductase represent a class of antitubercular drugs

    PubMed Central

    Weinstein, Edward A.; Yano, Takahiro; Li, Lin-Sheng; Avarbock, David; Avarbock, Andrew; Helm, Douglas; McColm, Andrew A.; Duncan, Ken; Lonsdale, John T.; Rubin, Harvey

    2005-01-01

    Mycobacterium tuberculosis (Mtb) is an obligate aerobe that is capable of long-term persistence under conditions of low oxygen tension. Analysis of the Mtb genome predicts the existence of a branched aerobic respiratory chain terminating in a cytochrome bd system and a cytochrome aa3 system. Both chains can be initiated with type II NADH:menaquinone oxidoreductase. We present a detailed biochemical characterization of the aerobic respiratory chains from Mtb and show that phenothiazine analogs specifically inhibit NADH:menaquinone oxidoreductase activity. The emergence of drug-resistant strains of Mtb has prompted a search for antimycobacterial agents. Several phenothiazines analogs are highly tuberculocidal in vitro, suppress Mtb growth in a mouse model of acute infection, and represent lead compounds that may give rise to a class of selective antibiotics. PMID:15767566

  19. Furaldehyde substrate specificity and kinetics of Saccharomyces cerevisiae alcohol dehydrogenase 1 variants.

    PubMed

    Laadan, Boaz; Wallace-Salinas, Valeria; Carlsson, Åsa Janfalk; Almeida, João Rm; Rådström, Peter; Gorwa-Grauslund, Marie F

    2014-08-09

    A previously discovered mutant of Saccharomyces cerevisiae alcohol dehydrogenase 1 (Adh1p) was shown to enable a unique NADH-dependent reduction of 5-hydroxymethylfurfural (HMF), a well-known inhibitor of yeast fermentation. In the present study, site-directed mutagenesis of both native and mutated ADH1 genes was performed in order to identify the key amino acids involved in this substrate shift, resulting in Adh1p-variants with different substrate specificities. In vitro activities of the Adh1p-variants using two furaldehydes, HMF and furfural, revealed that HMF reduction ability could be acquired after a single amino acid substitution (Y295C). The highest activity, however, was reached with the double mutation S110P Y295C. Kinetic characterization with both aldehydes and the in vivo primary substrate acetaldehyde also enabled to correlate the alterations in substrate affinity with the different amino acid substitutions. We demonstrated the key role of Y295C mutation in HMF reduction by Adh1p. We generated and kinetically characterized a group of protein variants using two furaldehyde compounds of industrial relevance. Also, we showed that there is a threshold after which higher in vitro HMF reduction activities do not correlate any more with faster in vivo rates of HMF conversion, indicating other cell limitations in the conversion of HMF.

  20. Fluorophores advanced glycation end products (AGEs)-to-NADH ratio is predictor for diabetic chronic kidney and cardiovascular disease.

    PubMed

    Ciobanu, Dana M; Olar, Loredana E; Stefan, Razvan; Veresiu, Ioan A; Bala, Cornelia G; Mircea, Petru A; Roman, Gabriela

    2015-01-01

    An imbalance in advanced glycation end products (AGEs) and NADH formation has been associated with diabetic chronic kidney disease (CKD) and cardiovascular disease (CVD). No data have been reported on simultaneous measurement of AGEs and NADH in type 2 diabetes (T2DM) patients. We aimed to compare AGEs, NADH and the AGEs-to-NADH ratio in T2DM and controls, and to assess its relationship with diabetic CKD and CVD. In this cross-sectional study, we measured serum AGEs (370/435nm) and NADH (370/460nm) in T2DM patients (n=63) and controls (n=25) using fluorescence spectroscopy. The AGEs-to-NADH ratio was analyzed according to diabetic CKD and CVD. We found significantly higher AGEs-to-NADH ratio in T2DM compared to controls. The AGEs-to-NADH ratio was significantly associated with triglycerides, blood glucose, HDL-cholesterol, estimated glomerular filtration rate. The AGEs-to-NADH ratio was a significant predictor for the presence of diabetic CKD and CVD when using ROC curves. Multivariate analysis showed that triglycerides and the presence of T2DM were predictors for the AGEs-to-NADH ratio. These findings suggest that the fluorophores AGEs-to-NADH ratio could be a new biomarker for the presence of diabetic CKD and CVD. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Requirement for Coenzyme Q in Plasma Membrane Electron Transport

    NASA Astrophysics Data System (ADS)

    Sun, I. L.; Sun, E. E.; Crane, F. L.; Morre, D. J.; Lindgren, A.; Low, H.

    1992-12-01

    Coenzyme Q is required in the electron transport system of rat hepatocyte and human erythrocyte plasma membranes. Extraction of coenzyme Q from the membrane decreases NADH dehydrogenase and NADH:oxygen oxidoreductase activity. Addition of coenzyme Q to the extracted membrane restores the activity. Partial restoration of activity is also found with α-tocopherylquinone, but not with vitamin K_1. Analogs of coenzyme Q inhibit NADH dehydrogenase and oxidase activity and the inhibition is reversed by added coenzyme Q. Ferricyanide reduction by transmembrane electron transport from HeLa cells is inhibited by coenzyme Q analogs and restored with added coenzyme Q10. Reduction of external ferricyanide and diferric transferrin by HeLa cells is accompanied by proton release from the cells. Inhibition of the reduction by coenzyme Q analogs also inhibits the proton release, and coenzyme Q10 restores the proton release activity. Trans-plasma membrane electron transport stimulates growth of serum-deficient cells, and added coenzyme Q10 increases growth of HeLa (human adenocarcinoma) and BALB/3T3 (mouse fibroblast) cells. The evidence is consistent with a function for coenzyme Q in a trans-plasma membrane electron transport system which influences cell growth.

  2. Reconstitution of the Escherichia coli pyruvate dehydrogenase complex.

    PubMed Central

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

    1975-01-01

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

  3. NADH Electrooxidation Using Bis(1,10-phenanthroline-5,6-dione) (2,2′-bipyridine)ruthenium(II)-Exchanged Zirconium Phosphate Modified Carbon Paste Electrodes

    PubMed Central

    Santiago, Mitk’El B.; Vélez, Meredith M.; Borrero, Solmarie; Díaz, Agustín; Casillas, Craig A.; Hofmann, Cristina; Guadalupe, Ana R.; Colón, Jorge L.

    2007-01-01

    We present a carbon paste electrode (CPE) modified using the electron mediator bis(1,10-phenanthroline-5,6-dione) (2,2′-bipyridine)ruthenium(II) ([Ru(phend)2bpy]2+) exchanged into the inorganic layered material zirconium phosphate (ZrP). X-Ray powder diffraction showed that the interlayer distance of ZrP increases upon [Ru(phend)2bpy]2+ intercalation from 10.3 Å to 14.2 Å. The UV-vis and IR spectroscopies results showed the characteristic peaks expected for [Ru(phend)2bpy]2+. The UV-vis spectrophotometric results indicate that the [Ru(phend)2bpy]2+ concentration inside the ZrP layers increased as a function of the loading level. The exchanged [Ru(phend)2bpy]2+ exhibited luminescence even at low concentration. Modified CPEs were constructed and analyzed using cyclic voltammetry. The intercalated mediator remained electroactive within the layers (E°′ = −38.5 mV vs. Ag/AgCl, 3.5 M NaCl) and electrocatalysis of NADH oxidation was observed. The kinetics of the modified CPE shows a Michaelis –Menten behavior. This CPE was used for the oxidation of NADH in the presence of Bakers’ yeast alcohol dehydrogenase. A calibration plot for ethanol is presented. PMID:18516242

  4. Involvement of NADH Oxidase in Competition and Endocarditis Virulence in Streptococcus sanguinis

    PubMed Central

    Ge, Xiuchun; Yu, Yang; Zhang, Min; Chen, Lei; Chen, Weihua; Elrami, Fadi; Kong, Fanxiang; Kitten, Todd

    2016-01-01

    Here, we report for the first time that the Streptococcus sanguinis nox gene encoding NADH oxidase is involved in both competition with Streptococcus mutans and virulence for infective endocarditis. An S. sanguinis nox mutant was found to fail to inhibit the growth of Streptococcus mutans under microaerobic conditions. In the presence of oxygen, the recombinant Nox protein of S. sanguinis could reduce oxygen to water and oxidize NADH to NAD+. The oxidation of NADH to NAD+ was diminished in the nox mutant. The nox mutant exhibited decreased levels of extracellular H2O2; however, the intracellular level of H2O2 in the mutant was increased. Furthermore, the virulence of the nox mutant was attenuated in a rabbit endocarditis model. The nox mutant also was shown to be more sensitive to blood killing, oxidative and acid stresses, and reduced growth in serum. Thus, NADH oxidase contributes to multiple phenotypes related to competitiveness in the oral cavity and systemic virulence. PMID:26930704

  5. Involvement of NADH Oxidase in Competition and Endocarditis Virulence in Streptococcus sanguinis.

    PubMed

    Ge, Xiuchun; Yu, Yang; Zhang, Min; Chen, Lei; Chen, Weihua; Elrami, Fadi; Kong, Fanxiang; Kitten, Todd; Xu, Ping

    2016-05-01

    Here, we report for the first time that the Streptococcus sanguinis nox gene encoding NADH oxidase is involved in both competition with Streptococcus mutans and virulence for infective endocarditis. An S. sanguinis nox mutant was found to fail to inhibit the growth of Streptococcus mutans under microaerobic conditions. In the presence of oxygen, the recombinant Nox protein of S. sanguinis could reduce oxygen to water and oxidize NADH to NAD(+) The oxidation of NADH to NAD(+) was diminished in the nox mutant. The nox mutant exhibited decreased levels of extracellular H2O2; however, the intracellular level of H2O2 in the mutant was increased. Furthermore, the virulence of the nox mutant was attenuated in a rabbit endocarditis model. The nox mutant also was shown to be more sensitive to blood killing, oxidative and acid stresses, and reduced growth in serum. Thus, NADH oxidase contributes to multiple phenotypes related to competitiveness in the oral cavity and systemic virulence. Copyright © 2016 Ge et al.

  6. Characterization of Truncated Tumor-Associated NADH Oxidase (ttNOX)

    NASA Technical Reports Server (NTRS)

    Karr, Laurel J.; Malone, Christine C.; Burk, Melissa; Moore, Blake P.; Achari, Aniruddha; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    Bacterial, plant and animal cells possess novel surface proteins that exhibit both NADH oxidation (NOX) or hydroquinone and protein disulfide-thiol interchange. These enzymatic activities alternate to yield oscillating patterns wjth period lengths of approximately 24 minutes. The catalytic period of NOX proteins are temperature compensated and gravity responsive. We report the cloning, expression and characterization of truncated tumor-associated NADH oxidase (ttNOX), in which the membrane spanning region has been deleted. The cDNA (originated from HeLa cells) was cloned into pET-34b and pET-14b (Novagen) vectors for E. coli expression. Optimized expression and purification protocols yielded greater than 300mg per liter of culture with greater than 95% purity. Circular dichroism data was collected from a 2.7mg/ml solution in a 0.1mm cuvette with variable scanning using an Olis RSM CD spectrophotometer. The ellipticity values were scanned from 190 to 260nm. The spectra recorded have characteristics for alpha proteins with band maxima at 216nm and a possible shoulder at 212nm at 12OC and 250 C. Protein crystal screens are in progress and, to date, only small crystals have been observed. The regular periodic oscillatory change in the ttNOX protein is indicative of a possible time-keeping functional role. A single protein possessing alternating catalytic activities, with a potential biological clock function, is unprecedented and structural determination is paramount to understanding this role.

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

    PubMed

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

    2018-05-26

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

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

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

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

    1995-09-10

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

  9. Disruption of key NADH-binding pocket residues of the Mycobacterium tuberculosis InhA affects DD-CoA binding ability.

    PubMed

    Shaw, Daniel J; Robb, Kirsty; Vetter, Beatrice V; Tong, Madeline; Molle, Virginie; Hunt, Neil T; Hoskisson, Paul A

    2017-07-05

    Tuberculosis (TB) is a global health problem that affects over 10 million people. There is an urgent need to develop novel antimicrobial therapies to combat TB. To achieve this, a thorough understanding of key validated drug targets is required. The enoyl reductase InhA, responsible for synthesis of essential mycolic acids in the mycobacterial cell wall, is the target for the frontline anti-TB drug isoniazid. To better understand the activity of this protein a series of mutants, targeted to the NADH co-factor binding pocket were created. Residues P193 and W222 comprise a series of hydrophobic residues surrounding the cofactor binding site and mutation of both residues negatively affect InhA function. Construction of an M155A mutant of InhA results in increased affinity for NADH and DD-CoA turnover but with a reduction in V max for DD-CoA, impairing overall activity. This suggests that NADH-binding geometry of InhA likely permits long-range interactions between residues in the NADH-binding pocket to facilitate substrate turnover in the DD-CoA binding region of the protein. Understanding the precise details of substrate binding and turnover in InhA and how this may affect protein-protein interactions may facilitate the development of improved inhibitors enabling the development of novel anti-TB drugs.

  10. Purification, characterization and function of dihydrolipoamide dehydrogenase from the cyanobacterium Anabaena sp. strain P.C.C. 7119.

    PubMed Central

    Serrano, A

    1992-01-01

    A dihydrolipoamide dehydrogenase (dihydrolipoamide: NAD+ oxidoreductase, EC 1.8.1.4) (DLD) has been found in the soluble fraction of cells of both unicellular (Synechococcus sp. strain P.C.C. 6301) and filamentous (Calothrix sp. strain P.C.C. 7601 and Anabaena sp. strain P.C.C. 7119) cyanobacteria. DLD from Anabaena sp. was purified 3000-fold to electrophoretic homogeneity. The purified enzyme exhibited a specific activity of 190 units/mg and was characterized as a dimeric FAD-containing protein with a native molecular mass of 104 kDa, a Stokes' radius of 4.28 nm and a very acidic pI value of about 3.7. As is the case with the same enzyme from other sources, cyanobacterial DLD showed specificity for NADH and lipoamide, or lipoic acid, as substrates. Nevertheless, the strong acidic character of the Anabaena DLD is a distinctive feature with respect to the same enzyme from other organisms. The presence of essential thiol groups was suggested by the inactivation produced by thiol-group-reactive reagents and heavy-metal ions, with lipoamide, but not NAD+, behaving as a protective agent. The function and physiological significance of Anabaena DLD are discussed in relation to the fact that 2-oxoacid dehydrogenase complexes have not been detected so far in filamentous cyanobacteria. Glycine decarboxylase activity, which might be involved in photorespiratory metabolism, has been found, however, in cell extracts of Anabaena sp. strain P.C.C. 7119 as the present study demonstrates. Images Fig. 2. PMID:1471997

  11. Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis

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

    Plapp, Bryce V.; Savarimuthu, Baskar Raj; Ferraro, Daniel J.

    During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5'-diphosphoribose have an open protein conformation with the catalytic zinc in one position, tetracoordinated by Cys-46, His-67, Cys-174, and a water molecule. The bidentatemore » chelators 2,2'-bipyridine and 1,10-phenanthroline displace the water and form a pentacoordinated zinc. The enzyme–NADH complex has a closed conformation similar to that of ternary complexes with coenzyme and substrate analogues; the coordination of the catalytic zinc is similar to that found in the apoenzyme, except that a minor, alternative position for the catalytic zinc is ~1.3 Å from the major position and closer to Glu-68, which could form the alternative coordination to the catalytic zinc. Complexes with NADH and N-1-methylhexylformamide or N-benzylformamide (or with NAD+ and fluoro alcohols) have the classical tetracoordinated zinc, and no water is bound to the zinc or the nicotinamide rings. The major forms of the enzyme in the mechanism have a tetracoordinated zinc, where the carboxylate group of Glu-68 could participate in the exchange of water and substrates on the zinc. Hydride transfer in the Michaelis complexes does not involve a nearby water.« less

  12. Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis

    PubMed Central

    2017-01-01

    During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5′-diphosphoribose have an open protein conformation with the catalytic zinc in one position, tetracoordinated by Cys-46, His-67, Cys-174, and a water molecule. The bidentate chelators 2,2′-bipyridine and 1,10-phenanthroline displace the water and form a pentacoordinated zinc. The enzyme–NADH complex has a closed conformation similar to that of ternary complexes with coenzyme and substrate analogues; the coordination of the catalytic zinc is similar to that found in the apoenzyme, except that a minor, alternative position for the catalytic zinc is ∼1.3 Å from the major position and closer to Glu-68, which could form the alternative coordination to the catalytic zinc. Complexes with NADH and N-1-methylhexylformamide or N-benzylformamide (or with NAD+ and fluoro alcohols) have the classical tetracoordinated zinc, and no water is bound to the zinc or the nicotinamide rings. The major forms of the enzyme in the mechanism have a tetracoordinated zinc, where the carboxylate group of Glu-68 could participate in the exchange of water and substrates on the zinc. Hydride transfer in the Michaelis complexes does not involve a nearby water. PMID:28640600

  13. In vitro activation of NAD-dependent alcohol dehydrogenases by Nudix hydrolases is more widespread than assumed.

    PubMed

    Ochsner, Andrea M; Müller, Jonas E N; Mora, Carlos A; Vorholt, Julia A

    2014-08-25

    In the Gram-positive methylotroph Bacillus methanolicus, methanol oxidation is catalyzed by an NAD-dependent methanol dehydrogenase (Mdh) that belongs to the type III alcohol dehydrogenase (Adh) family. It was previously shown that the in vitro activity of B. methanolicus Mdh is increased by the endogenous activator protein Act, a Nudix hydrolase. Here we show that this feature is not unique, but more widespread among type III Adhs in combination with Act or other Act-like Nudix hydrolases. In addition, we studied the effect of site directed mutations in the predicted active site of Mdh and two other type III Adhs with regard to activity and activation by Act. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  14. Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes*

    PubMed Central

    Christensen, Caspar Elo; Karlsson, Magnus; Winther, Jakob R.; Jensen, Pernille Rose; Lerche, Mathilde H.

    2014-01-01

    Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD+]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD+]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD+]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD+]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD+]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments. PMID:24302737

  15. Fed-batch control based upon the measurement of intracellular NADH

    NASA Technical Reports Server (NTRS)

    Armiger, W. B.; Lee, J. F.; Montalvo, L. M.; Forro, J. R.

    1987-01-01

    A series of experiments demonstrating that on-line measurements of intracellular NADH by culture fluorescence can be used to monitor and control the fermentation process are described. A distinct advantage of intercellular NADH measurements over other monitoring techniques such as pH and dissolved oxygen is that it directly measures real time events occurring within the cell rather than changes in the environment. When coupled with other measurement parameters, it can provide a finer degree of sophistication in process control.

  16. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  17. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  18. Role of tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase.

    PubMed

    Pennacchio, Angela; Esposito, Luciana; Zagari, Adriana; Rossi, Mosè; Raia, Carlo A

    2009-09-01

    A mutant of the thermostable NAD(+)-dependent (S)-stereospecific alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) which has a single substitution, Trp95Leu, located at the substrate binding pocket, was fully characterized to ascertain the role of Trp95 in discriminating between chiral secondary alcohols suggested by the wild-type SsADH crystallographic structure. The Trp95Leu mutant displays no apparent activity with short-chain primary and secondary alcohols and poor activity with aromatic substrates and coenzyme. Moreover, the Trp --> Leu substitution affects the structural stability of the archaeal ADH, decreasing its thermal stability without relevant changes in secondary structure. The double mutant Trp95Leu/Asn249Tyr was also purified to assist in crystallographic analysis. This mutant exhibits higher activity but decreased affinity toward aliphatic alcohols, aldehydes as well as NAD(+) and NADH compared to the wild-type enzyme. The crystal structure of the Trp95Leu/Asn249Tyr mutant apo form, determined at 2.0 A resolution, reveals a large local rearrangement of the substrate site with dramatic consequences. The Leu95 side-chain conformation points away from the catalytic metal center and the widening of the substrate site is partially counteracted by a concomitant change of Trp117 side chain conformation. Structural changes at the active site are consistent with the reduced activity on substrates and decreased coenzyme binding.

  19. Isolation, Oxygen Sensitivity, and Virulence of NADH Oxidase Mutants of the Anaerobic Spirochete Brachyspira (Serpulina) hyodysenteriae, Etiologic Agent of Swine Dysentery

    PubMed Central

    Stanton, Thad B.; Rosey, Everett L.; Kennedy, Michael J.; Jensen, Neil S.; Bosworth, Brad T.

    1999-01-01

    Brachyspira (Serpulina) hyodysenteriae, the etiologic agent of swine dysentery, uses the enzyme NADH oxidase to consume oxygen. To investigate possible roles for NADH oxidase in the growth and virulence of this anaerobic spirochete, mutant strains deficient in oxidase activity were isolated and characterized. The cloned NADH oxidase gene (nox; GenBank accession no. U19610) on plasmid pER218 was inactivated by replacing 321 bp of coding sequence with either a gene for chloramphenicol resistance (cat) or a gene for kanamycin resistance (kan). The resulting plasmids, respectively, pCmΔNOX and pKmΔNOX, were used to transform wild-type B. hyodysenteriae B204 cells and generate the antibiotic-resistant strains Nox-Cm and Nox-Km. PCR and Southern hybridization analyses indicated that the chromosomal wild-type nox genes in these strains had been replaced, through allelic exchange, by the inactivated nox gene containing cat or kan. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis revealed that both nox mutant cell lysates were missing the 48-kDa Nox protein. Soluble NADH oxidase activity levels in cell lysates of Nox-Cm and Nox-Km were reduced 92 to 96% compared to the activity level in parent strain B204. In an aerotolerance test, cells of both nox mutants were at least 100-fold more sensitive to oxygen exposure than were cells of the wild-type parent strain B204. In swine experimental infections, both nox mutants were less virulent than strain B204 in that fewer animals were colonized by the mutant cells and infected animals displayed mild, transient signs of disease, with no deaths. These results provide evidence that NADH oxidase serves to protect B. hyodysenteriae cells against oxygen toxicity and that the enzyme, in that role, contributes to the pathogenic ability of the spirochete. PMID:10543819

  20. 21 CFR 862.1670 - Sorbitol dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  1. NADH Dehydrogenase Subunit-2 237 Leu/Met Polymorphism Modulates the Effects of Coffee Consumption on the Risk of Hypertension in Middle-Aged Japanese Men

    PubMed Central

    Kokaze, Akatsuki; Ishikawa, Mamoru; Matsunaga, Naomi; Karita, Kanae; Yoshida, Masao; Ohtsu, Tadahiro; Shirasawa, Takako; Sekii, Hideaki; Ito, Taku; Kawamoto, Teruyoshi; Takashima, Yutaka

    2009-01-01

    Background Habitual coffee consumption has been reported to lower blood pressure in the Japanese population. The NADH dehydrogenase subunit-2 237 leucine/methionine (ND2-237 Leu/Met) polymorphism is associated with longevity and modifies the effects of alcohol consumption on blood pressure in the Japanese population. The objective of this study was to determine whether this polymorphism also modifies the effects of coffee consumption on blood pressure or the risk of hypertension in middle-aged Japanese men. Methods A total of 398 men (mean age ± standard deviation, 53.8 ± 7.8 years) were selected from among individuals visiting the hospital for regular medical check-ups. Hypertension was defined as a systolic blood pressure ≥140 mm Hg, diastolic blood pressure ≥90 mm Hg, or antihypertensive drug treatment. Polymerase chain reaction-restriction fragment length polymorphism using the restriction enzyme AluI was performed to determine ND2-237 Leu/Met genotype. Results In subjects with ND2-237Leu, coffee consumption was significantly and negatively associated with diastolic blood pressure (P = 0.007). The odds ratio (OR) for hypertension was significantly lower in subjects with ND2-237Leu who consumed 2 or 3 cups of coffee per day than in those who consumed less than 1 cup of coffee per day (OR, 0.517; 95% confidence interval [CI], 0.276 to 0.968; P = 0.039). After adjustment, the OR remained significant (OR = 0.399; 95% CI, 0.184 to 0.869; P = 0.020). Moreover, after adjustment, the OR was significantly lower in subjects with ND2-237Leu who consumed more than 4 cups of coffee per day than in those who consumed less than 1 cup of coffee per day (OR, 0.246; 95% CI, 0.062 to 0.975; P = 0.046). However, the association between ND2-237Met genotype and hypertension did not depend on coffee consumption. Conclusions The present results suggest that the ND2-237 Leu/Met polymorphism modulates the effects of coffee consumption on hypertension risk in middle-aged Japanese

  2. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  3. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  4. Pyruvate dehydrogenase complex and nicotinamide nucleotide transhydrogenase constitute an energy consuming redox circuit

    PubMed Central

    Fisher-Wellman, Kelsey H.; Lin, Chien-Te; Ryan, Terence E.; Reese, Lauren R.; Gilliam, Laura A. A.; Cathey, Brook L.; Lark, Daniel S.; Smith, Cody D.; Muoio, Deborah M.; Neufer, P. Darrell

    2015-01-01

    SUMMARY Cellular proteins rely on reversible redox reactions to establish and maintain biological structure and function. How redox catabolic (NAD+:NADH) and anabolic (NADP+:NADPH) processes integrate during metabolism to maintain cellular redox homeostasis however is unknown. The present work identifies a continuously cycling, mitochondrial membrane potential-dependent redox circuit between the pyruvate dehydrogenase complex (PDHC) and nicotinamide nucleotide transhydrogenase (NNT). PDHC is shown to produce H2O2 in relation to reducing pressure within the complex. The H2O2 produced however is effectively masked by a continuously cycling redox circuit that links, via glutathione/thioredoxin, to NNT, which catalyzes the regeneration of NADPH from NADH at the expense of the mitochondrial membrane potential. The net effect is an automatic fine tuning of NNT-mediated energy expenditure to metabolic balance at the level of PDHC. In mitochondria, genetic or pharmacological disruptions in the PDHC-NNT redox circuit negate counterbalance changes in energy expenditure. At the whole animal level, mice lacking functional NNT (C57BL/6J) are characterized by lower energy expenditure rates, consistent with their well known susceptibility to diet-induced obesity. These findings suggest the integration of redox sensing of metabolic balance with compensatory changes in energy expenditure provides a potential mechanism by which cellular redox homeostasis is maintained and body weight is defended during periods of positive and negative energy balance. PMID:25643703

  5. Pyruvate dehydrogenase complex and nicotinamide nucleotide transhydrogenase constitute an energy-consuming redox circuit.

    PubMed

    Fisher-Wellman, Kelsey H; Lin, Chien-Te; Ryan, Terence E; Reese, Lauren R; Gilliam, Laura A A; Cathey, Brook L; Lark, Daniel S; Smith, Cody D; Muoio, Deborah M; Neufer, P Darrell

    2015-04-15

    Cellular proteins rely on reversible redox reactions to establish and maintain biological structure and function. How redox catabolic (NAD+/NADH) and anabolic (NADP+/NADPH) processes integrate during metabolism to maintain cellular redox homoeostasis, however, is unknown. The present work identifies a continuously cycling mitochondrial membrane potential (ΔΨm)-dependent redox circuit between the pyruvate dehydrogenase complex (PDHC) and nicotinamide nucleotide transhydrogenase (NNT). PDHC is shown to produce H2O2 in relation to reducing pressure within the complex. The H2O2 produced, however, is effectively masked by a continuously cycling redox circuit that links, via glutathione/thioredoxin, to NNT, which catalyses the regeneration of NADPH from NADH at the expense of ΔΨm. The net effect is an automatic fine-tuning of NNT-mediated energy expenditure to metabolic balance at the level of PDHC. In mitochondria, genetic or pharmacological disruptions in the PDHC-NNT redox circuit negate counterbalance changes in energy expenditure. At the whole animal level, mice lacking functional NNT (C57BL/6J) are characterized by lower energy-expenditure rates, consistent with their well-known susceptibility to diet-induced obesity. These findings suggest the integration of redox sensing of metabolic balance with compensatory changes in energy expenditure provides a potential mechanism by which cellular redox homoeostasis is maintained and body weight is defended during periods of positive and negative energy balance.

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

    PubMed

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

    2016-04-01

    In plants, the last step in the biosynthesis of the osmoprotectant glycine betaine (GB) is the NAD(+)-dependent oxidation of betaine aldehyde (BAL) catalysed by some aldehyde dehydrogenase (ALDH) 10 enzymes that exhibit betaine aldehyde dehydrogenase (BADH) activity. Given the irreversibility of the reaction, the short-term regulation of these enzymes is of great physiological relevance to avoid adverse decreases in the NAD(+):NADH ratio. In the present study, we report that the Spinacia oleracea BADH (SoBADH) is reversibly and partially inactivated by BAL in the absence of NAD(+)in a time- and concentration-dependent mode. Crystallographic evidence indicates that the non-essential Cys(450)(SoBADH numbering) forms a thiohemiacetal with BAL, totally blocking the productive binding of the aldehyde. It is of interest that, in contrast to Cys(450), the catalytic cysteine (Cys(291)) did not react with BAL in the absence of NAD(+) The trimethylammonium group of BAL binds in the same position in the inactivating or productive modes. Accordingly, BAL does not inactivate the C(450)SSoBADH mutant and the degree of inactivation of the A(441)I and A(441)C mutants corresponds to their very different abilities to bind the trimethylammonium group. Cys(450)and the neighbouring residues that participate in stabilizing the thiohemiacetal are strictly conserved in plant ALDH10 enzymes with proven or predicted BADH activity, suggesting that inactivation by BAL is their common feature. Under osmotic stress conditions, this novel partial and reversible covalent regulatory mechanism may contribute to preventing NAD(+)exhaustion, while still permitting the synthesis of high amounts of GB and avoiding the accumulation of the toxic BAL. © 2016 Authors; published by Portland Press Limited.

  7. Changes in Oxidative Damage, Inflammation and [NAD(H)] with Age in Cerebrospinal Fluid

    PubMed Central

    Guest, Jade; Grant, Ross; Mori, Trevor A.; Croft, Kevin D.

    2014-01-01

    An extensive body of evidence indicates that oxidative stress and inflammation play a central role in the degenerative changes of systemic tissues in aging. However a comparatively limited amount of data is available to verify whether these processes also contribute to normal aging within the brain. High levels of oxidative damage results in key cellular changes including a reduction in available nicotinamide adenine dinucleotide (NAD+), an essential molecule required for a number of vital cellular processes including DNA repair, immune signaling and epigenetic processing. In this study we quantified changes in [NAD(H)] and markers of inflammation and oxidative damage (F2-isoprostanes, 8-OHdG, total antioxidant capacity) in the cerebrospinal fluid (CSF) of healthy humans across a wide age range (24–91 years). CSF was collected from consenting patients who required a spinal tap for the administration of anesthetic. CSF of participants aged >45 years was found to contain increased levels of lipid peroxidation (F2-isoprostanes) (p = 0.04) and inflammation (IL-6) (p = 0.00) and decreased levels of both total antioxidant capacity (p = 0.00) and NAD(H) (p = 0.05), compared to their younger counterparts. A positive association was also observed between plasma [NAD(H)] and CSF NAD(H) levels (p = 0.03). Further analysis of the data identified a relationship between alcohol intake and CSF [NAD(H)] and markers of inflammation. The CSF of participants who consumed >1 standard drink of alcohol per day contained lower levels of NAD(H) compared to those who consumed no alcohol (p<0.05). An increase in CSF IL-6 was observed in participants who reported drinking >0–1 (p<0.05) and >1 (p<0.05) standard alcoholic drinks per day compared to those who did not drink alcohol. Taken together these data suggest a progressive age associated increase in oxidative damage, inflammation and reduced [NAD(H)] in the brain which may be exacerbated by alcohol intake. PMID

  8. SIRT3 deacetylates and increases pyruvate dehydrogenase activity in cancer cells.

    PubMed

    Ozden, Ozkan; Park, Seong-Hoon; Wagner, Brett A; Song, Ha Yong; Zhu, Yueming; Vassilopoulos, Athanassios; Jung, Barbara; Buettner, Garry R; Gius, David

    2014-11-01

    Pyruvate dehydrogenase E1α (PDHA1) is the first component enzyme of the pyruvate dehydrogenase (PDH) complex 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 (PDHA1(K321R)) increases PDH activity, compared to the K321 acetylation mimic (PDHA1(K321Q)) or wild-type PDHA1. Finally, PDHA1(K321Q) exhibited a more transformed in vitro cellular phenotype compared to PDHA1(K321R). These results suggest that the acetylation of PDHA1 provides another layer of enzymatic regulation, in addition to phosphorylation, involving a reversible acetyllysine, suggesting that the acetylome, as well as the kinome, links glycolysis to respiration. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Some Lactobacillus l-Lactate Dehydrogenases Exhibit Comparable Catalytic Activities for Pyruvate and Oxaloacetate

    PubMed Central

    Arai, Kazuhito; Kamata, Takeo; Uchikoba, Hiroyuki; Fushinobu, Shinya; Matsuzawa, Hiroshi; Taguchi, Hayao

    2001-01-01

    The nonallosteric and allosteric l-lactate dehydrogenases of Lactobacillus pentosus and L. casei, respectively, exhibited broad substrate specificities, giving virtually the same maximal reaction velocity and substrate Km values for pyruvate and oxaloacetate. Replacement of Pro101 with Asn reduced the activity of the L. pentosus enzyme toward these alternative substrates to a greater extent than the activity toward pyruvate. PMID:11114942

  10. Sirtuin 3 (SIRT3) Protein Regulates Long-chain Acyl-CoA Dehydrogenase by Deacetylating Conserved Lysines Near the Active Site

    PubMed Central

    Bharathi, Sivakama S.; Zhang, Yuxun; Mohsen, Al-Walid; Uppala, Radha; Balasubramani, Manimalha; Schreiber, Emanuel; Uechi, Guy; Beck, Megan E.; Rardin, Matthew J.; Vockley, Jerry; Verdin, Eric; Gibson, Bradford W.; Hirschey, Matthew D.; Goetzman, Eric S.

    2013-01-01

    Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases. PMID:24121500

  11. Comparative 13C Metabolic Flux Analysis of Pyruvate Dehydrogenase Complex-Deficient, l-Valine-Producing Corynebacterium glutamicum▿†

    PubMed Central

    Bartek, Tobias; Blombach, Bastian; Lang, Siegmund; Eikmanns, Bernhard J.; Wiechert, Wolfgang; Oldiges, Marco; Nöh, Katharina; Noack, Stephan

    2011-01-01

    l-Valine can be formed successfully using C. glutamicum strains missing an active pyruvate dehydrogenase enzyme complex (PDHC). Wild-type C. glutamicum and four PDHC-deficient strains were compared by 13C metabolic flux analysis, especially focusing on the split ratio between glycolysis and the pentose phosphate pathway (PPP). Compared to the wild type, showing a carbon flux of 69% ± 14% through the PPP, a strong increase in the PPP flux was observed in PDHC-deficient strains with a maximum of 113% ± 22%. The shift in the split ratio can be explained by an increased demand of NADPH for l-valine formation. In accordance, the introduction of the Escherichia coli transhydrogenase PntAB, catalyzing the reversible conversion of NADH to NADPH, into an l-valine-producing C. glutamicum strain caused the PPP flux to decrease to 57% ± 6%, which is below the wild-type split ratio. Hence, transhydrogenase activity offers an alternative perspective for sufficient NADPH supply, which is relevant for most amino acid production systems. Moreover, as demonstrated for l-valine, this bypass leads to a significant increase of product yield due to a concurrent reduction in carbon dioxide formation via the PPP. PMID:21784914

  12. A novel archaeal alanine dehydrogenase homologous to ornithine cyclodeaminase and mu-crystallin.

    PubMed

    Schröder, Imke; Vadas, Alexander; Johnson, Eric; Lim, Sierin; Monbouquette, Harold G

    2004-11-01

    A novel alanine dehydrogenase (AlaDH) showing no significant amino acid sequence homology with previously known bacterial AlaDHs was purified to homogeneity from the soluble fraction of the hyperthermophilic archaeon Archaeoglobus fulgidus. AlaDH catalyzed the reversible, NAD+-dependent deamination of L-alanine to pyruvate and NH4+. NADP(H) did not serve as a coenzyme. The enzyme is a homodimer of 35 kDa per subunit. The Km values for L-alanine, NAD+, pyruvate, NADH, and NH4+ were estimated at 0.71, 0.60, 0.16, 0.02, and 17.3 mM, respectively. The A. fulgidus enzyme exhibited its highest activity at about 82 degrees C (203 U/mg for reductive amination of pyruvate) yet still retained 30% of its maximum activity at 25 degrees C. The thermostability of A. fulgidus AlaDH was increased by more than 10-fold by 1.5 M KCl to a half-life of 55 h at 90 degrees C. At 25 degrees C in the presence of this salt solution, the enzyme was approximately 100% stable for more than 3 months. Closely related A. fulgidus AlaDH homologues were found in other archaea. On the basis of its amino acid sequence, A. fulgidus AlaDH is a member of the ornithine cyclodeaminase-mu-crystallin family of enzymes. Similar to the mu-crystallins, A. fulgidus AlaDH did not exhibit any ornithine cyclodeaminase activity. The recombinant human mu-crystallin was assayed for AlaDH activity, but no activity was detected. The novel A. fulgidus gene encoding AlaDH, AF1665, is designated ala.

  13. Crystallization and preliminary crystallographic study of 3 alpha, 20 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans.

    PubMed

    Fitzgerald, P M; Duax, W L; Punzi, J S; Orr, J C

    1984-05-15

    3 alpha, 20 beta-Hydroxysteroid dehydrogenase, an NADH-dependent oxidoreductase isolated from Streptomyces hydrogenans , is a tetramer containing four subunits each of Mr 25,000. The enzyme has been crystallized by the vapor diffusion technique using either phosphate or borate buffered ammonium sulfate (pH between 6.0 and 8.7) as the precipitant. The crystals are hexagonal bipyramids ; they have the symmetry of space group P6(4)22 (or P6(2)22), with unit cell dimensions a = 127.3 A, c = 112.2 A. Volume and density considerations imply that the crystallographic asymmetric unit contains two monomers, and therefore that the tetramer possesses a 2-fold axis of symmetry that is coincident with a crystallographic 2-fold symmetry element.

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

    PubMed

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

    2016-09-25

    Plant NAD(+)-dependent aminoaldehyde dehydrogenases (AMADHs, EC 1.2.1.19) belong to the family 10 of aldehyde dehydrogenases. They participate in the metabolism of polyamines or osmoprotectants. The enzymes are characterized by their broad substrate specificity covering ω-aminoaldehydes, aliphatic and aromatic aldehydes as well as nitrogen-containing heterocyclic aldehydes. The isoenzyme 1 from tomato (Solanum lycopersicum; SlAMADH1) oxidizes aliphatic aldehydes very efficiently and converts also furfural, its derivatives or benzaldehyde, which are present at low concentrations in alcoholic distillates such as fruit brandy. In this work, SlAMADH1 was examined as a bioanalytical tool for their detection. These aldehydes arise from fermentation processes or thermal degradation of sugars and their presence is related to health complications after consumption including nausea, emesis, sweating, decrease in blood pressure, hangover headache, among others. Sixteen samples of slivovitz (plum brandy) from local producers in Moravia, Czech Republic, were analyzed for their aldehyde content using a spectrophotometric activity assay with SlAMADH1. In all cases, there were oxidative responses observed when monitoring NADH production in the enzymatic reaction. Aldehydes in the distillate samples were also subjected to a standard determination using reversed-phase HPLC with spectrophotometric and tandem mass spectrometric detection after a derivatization with 2,4-dinitrophenylhydrazine. Results obtained by both methods were found to correlate well for a majority of the analyzed samples. The possible applicability of SlAMADH1 for the evaluation of aldehyde content in food and beverages has now been demonstrated. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD+-Auxotrophic Mutant ▿

    PubMed Central

    Zhou, Yongjin; Wang, Lei; Yang, Fan; Lin, Xinping; Zhang, Sufang; Zhao, Zongbao K.

    2011-01-01

    NAD (NAD+) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD+ level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels in Escherichia coli cells that were genetically engineered to be NAD+ auxotrophic. First, we expressed the ntt4 gene encoding the NAD(H) transporter in the E. coli mutant YJE001, which had a deletion of the nadC gene responsible for NAD+ de novo biosynthesis, and we showed NTT4 conferred on the mutant strain better growth in the presence of exogenous NAD+. We then constructed the NAD+-auxotrophic mutant YJE003 by disrupting the essential gene nadE, which is responsible for the last step of NAD+ biosynthesis in cells harboring the ntt4 gene. The minimal NAD+ level was determined in M9 medium in proliferating YJE003 cells that were preloaded with NAD+, while the maximal NAD(H) level was determined by exposing the cells to high concentrations of exogenous NAD(H). Compared with supplementation of NADH, cells grew faster and had a higher intracellular NAD(H) level when NAD+ was fed. The intracellular NAD(H) level increased with the increase of exogenous NAD+ concentration, until it reached a plateau. Thus, a minimal NAD(H) level of 0.039 mM and a maximum of 8.49 mM were determined, which were 0.044× and 9.6× those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed. PMID:21742902

  16. Defining NADH-Driven Allostery Regulating Apoptosis-Inducing Factor

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

    Brosey, Chris A.; Ho, Chris; Long, Winnie Z.

    Apoptosis-inducing factor (AIF) is critical for mitochondrial respiratory complex biogenesis and for mediating necroptotic parthanatos; these functions are seemingly regulated by enigmatic allosteric switching driven by NADH charge-transfer complex (CTC) formation. In this paper, we define molecular pathways linking AIF's active site to allosteric switching regions by characterizing dimer-permissive mutants using small-angle X-ray scattering (SAXS) and crystallography and by probing AIF-CTC communication networks using molecular dynamics simulations. Collective results identify two pathways propagating allostery from the CTC active site: (1) active-site H454 links to S480 of AIF's central β-strand to modulate a hydrophobic border at the dimerization interface, and (2)more » an interaction network links AIF's FAD cofactor, central β-strand, and Cβ-clasp whereby R529 reorientation initiates C-loop release during CTC formation. Finally, this knowledge of AIF allostery and its flavoswitch mechanism provides a foundation for biologically understanding and biomedically controlling its participation in mitochondrial homeostasis and cell death.« less

  17. Defining NADH-Driven Allostery Regulating Apoptosis-Inducing Factor

    DOE PAGES

    Brosey, Chris A.; Ho, Chris; Long, Winnie Z.; ...

    2016-11-03

    Apoptosis-inducing factor (AIF) is critical for mitochondrial respiratory complex biogenesis and for mediating necroptotic parthanatos; these functions are seemingly regulated by enigmatic allosteric switching driven by NADH charge-transfer complex (CTC) formation. In this paper, we define molecular pathways linking AIF's active site to allosteric switching regions by characterizing dimer-permissive mutants using small-angle X-ray scattering (SAXS) and crystallography and by probing AIF-CTC communication networks using molecular dynamics simulations. Collective results identify two pathways propagating allostery from the CTC active site: (1) active-site H454 links to S480 of AIF's central β-strand to modulate a hydrophobic border at the dimerization interface, and (2)more » an interaction network links AIF's FAD cofactor, central β-strand, and Cβ-clasp whereby R529 reorientation initiates C-loop release during CTC formation. Finally, this knowledge of AIF allostery and its flavoswitch mechanism provides a foundation for biologically understanding and biomedically controlling its participation in mitochondrial homeostasis and cell death.« less

  18. Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

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

    Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.

    Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

  19. Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

    DOE PAGES

    Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.; ...

    2017-07-07

    Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

  20. Effects of folic acid deficiency in pregnant Wistar rats on the activities of D5-3 beta hydroxysteroid dehydrogenase and glucose-6 phosphate dehydrogenase in the ovaries of their litters.

    PubMed

    Uche-Nwachi, E O; Caxton-Martins, A E

    1997-06-01

    Histochemical studies of the activities of glucose-6-phosphate dehydrogenase (G-6-PD) and D5-3 beta-hydroxysteroid dehydrogenase (D5-3 beta-HSD) in the ovaries of 40 day old litters of Wistar rats whose mothers were folic acid deficient from the 13th day of gestation showed very weak or no enzyme activity. Biochemical estimations of these enzymes showed that the specific activity of 3 beta-HSD in the experimental animal was 20% that of control while that of G-6-PD in the experimental animals was 14% that of control. This implies that folic acid deficiency instituted at a critical period in gestation in Wistar rats adversely affects steroidogenesis in the ovaries of their litters.

  1. The investigation of plasma glucose-6-phosphate dehydrogenase, 6-phoshogluconate dehydrogenase, glutathione reductase in premenauposal patients with iron deficiency anemia.

    PubMed

    Ozcicek, Fatih; Aktas, Mehmet; Türkmen, Kultigin; Coban, T Abdulkadir; Cankaya, Murat

    2014-07-01

    Iron is an essential element that is necessary for all cells in the body. Iron deficiency anemia (IDA) is one of the most common nutritional disorders in both developed and developing countries. The glutathione pathway is paramount to antioxidant defense and glucose-6-phosphate dehydrogenase (G6PD)-deficient cells do not cope well with oxidative damage. The goal of this study was to check the activities of G6PD, 6-phosphogluconate dehydrogenase, glutathione reductase in patients with IDA. We analyzed the plasma samples of 102 premenopausal women with IDA and 88 healthy control subjects. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activity as compared to the reduction of NADP +, glutathione reductase activity was performed based on the oxidation of NADPH. 2 ml of plasma were used in all analyzes. SPSS program was used for all of the statistical analysis. Diagnosis of iron deficiency in patients belonging to the analysis of blood were ferritin 3.60 ± 2.7 ng / mL, hemoglobin 9.4 ± 1.5 mg / dl and hematocrit 30.7 ± 4.1% ratio; in healthy subjects ferritin 53.5 ± 41.7 ng/ml, hemoglobin level 13.9 ± 1.3 mg / dl and hematocrit ratio 42 ± 3.53%. When compared to healthy subjects the glutathione reductase level (P<0.001) was found to be significantly higher in patients with IDA. IDA patients with moderate and severe anemia had lower GR activity when compared to IDA patients with mild anemia. But the plasma levels of glucose-6-phosphate dehydrogenase (P<0,600) and 6-phosphogluconate dehydrogenase (P<0,671) did not show any differences between healthy subjects and in patients with IDA. It was shown that Glucose-6-Phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase have no effect on iron-deficiency anemia in patients. The plasma GR levels of premenopausal women with IDA were found to be higher compared to healthy subjects, which could be secondary to erythrocyte protection against oxidative stress being commonly seen in IDA.

  2. Identification of mitochondrial electron transport chain-mediated NADH radical formation by EPR spin-trapping techniques.

    PubMed

    Matsuzaki, Satoshi; Kotake, Yashige; Humphries, Kenneth M

    2011-12-20

    The mitochondrial electron transport chain (ETC) is a major source of free radical production. However, due to the highly reactive nature of radical species and their short lifetimes, accurate detection and identification of these molecules in biological systems is challenging. The aim of this investigation was to determine the free radical species produced from the mitochondrial ETC by utilizing EPR spin-trapping techniques and the recently commercialized spin-trap, 5-(2,2-dimethyl-1,3-propoxycyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO). We demonstrate that this spin-trap has the preferential quality of having minimal mitochondrial toxicity at concentrations required for radical detection. In rat heart mitochondria and submitochondrial particles supplied with NADH, the major species detected under physiological pH was a carbon-centered radical adduct, indicated by markedly large hyperfine coupling constant with hydrogen (a(H) > 2.0 mT). In the presence of the ETC inhibitors, the carbon-centered radical formation was increased and exhibited NADH concentration dependency. The same carbon-centered radical could also be produced with the NAD biosynthesis precursor, nicotinamide mononucleotide, in the presence of a catalytic amount of NADH. The results support the conclusion that the observed species is a complex I derived NADH radical. The formation of the NADH radical could be blocked by hydroxyl radical scavengers but not SOD. In vitro experiments confirmed that an NADH-radical is readily formed by hydroxyl radical but not superoxide anion, further implicating hydroxyl radical as an upstream mediator of NADH radical production. These findings demonstrate the identification of a novel mitochondrial radical species with potential physiological significance and highlight the diverse mechanisms and sites of production within the ETC.

  3. Determination of NAD + and NADH level in a Single Cell Under H 2O 2 Stress by Capillary Electrophoresis

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

    Xi, Wenjun

    2008-01-01

    A capillary electrophoresis (CE) method is developed to determine both NAD + and NADH levels in a single cell, based on an enzymatic cycling reaction. The detection limit can reach down to 0.2 amol NAD + and 1 amol NADH on a home-made CE-LIF setup. The method showed good reproducibility and specificity. After an intact cell was injected into the inlet of a capillary and lysed using a Tesla coil, intracellular NAD + and NADH were separated, incubated with the cycling buffer, and quantified by the amount of fluorescent product generated. NADH and NAD + levels of single cells ofmore » three cell lines and primary astrocyte culture were determined using this method. Comparing cellular NAD + and NADH levels with and without exposure to oxidative stress induced by H 2O 2, it was found that H9c2 cells respond to the stress by reducing both cellular NAD + and NADH levels, while astrocytes respond by increasing cellular NADH/NAD + ratio.« less

  4. Caloric restriction counteracts age-related changes in the activities of sorbitol metabolizing enzymes from mouse liver

    PubMed Central

    Hagopian, Kevork; Ramsey, Jon J.; Weindruch, Richard

    2009-01-01

    The influence of caloric restriction (CR) on hepatic sorbitol-metabolizing enzyme activities was investigated in young and old mice. Aldose reductase and sorbitol dehydrogenase activities were significantly lower in old CR mice than in old controls. Young CR mice showed decreased aldose reductase activity and a trend towards decreased sorbitol dehydrogenase when compared to controls. Metabolites of the pathway, namely sorbitol, glucose and fructose were decreased by CR in young and old mice. Pyruvate levels were decreased by CR in both young and old mice, while lactate decreased only in old CR. Malate levels increased in old CR but remained unchanged in young CR, when compared with controls. Accordingly, the lactae/pyruvate and malate/pyruvate ratios in young and old CR mice were increased, indicating increased NADH/NAD and NADPH/NADP redox couples, respectively. The results indicate that decreased glucose levels under CR conditions lead to decreased sorbitol pathway enzyme activities and metabolite levels, and could contribute to the beneficial effects of long-term CR through decreased sorbitol levels and NADPH sparing. PMID:18953666

  5. Structure and Function of the Catalytic Domain of the Dihydrolipoyl Acetyltransferase Component in Escherichia coli Pyruvate Dehydrogenase Complex*

    PubMed Central

    Wang, Junjie; Nemeria, Natalia S.; Chandrasekhar, Krishnamoorthy; Kumaran, Sowmini; Arjunan, Palaniappa; Reynolds, Shelley; Calero, Guillermo; Brukh, Roman; Kakalis, Lazaros; Furey, William; Jordan, Frank

    2014-01-01

    The Escherichia coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components: E1p, E2p, and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LDs), a peripheral subunit binding domain (PSBD), and a catalytic domain (E2pCD). Herein are reported the following. 1) The x-ray structure of E2pCD revealed both intra- and intertrimer interactions, similar to those reported for other E2pCDs. 2) Reconstitution of recombinant LD and E2pCD with E1p and E3p into PDHc could maintain at least 6.4% activity (NADH production), confirming the functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD, and E3 even in the absence of PSBD and of a covalent link between domains within E2p. 3) Direct acetyl transfer between LD and coenzyme A catalyzed by E2pCD was observed with a rate constant of 199 s−1, comparable with the rate of NADH production in the PDHc reaction. Hence, neither reductive acetylation of E2p nor acetyl transfer within E2p is rate-limiting. 4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by hydrogen/deuterium exchange mass spectrometry. The inclusion of each additional domain of E2p strengthened the interaction with E1p, and the interaction was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifested by the appearance of a circular dichroism band characteristic of the canonical 4′-aminopyrimidine tautomer of bound thiamin diphosphate (AP). PMID:24742683

  6. Improvement of exopolysaccharide production in Lactobacillus casei LC2W by overexpression of NADH oxidase gene.

    PubMed

    Li, Nan; Wang, Yuanlong; Zhu, Ping; Liu, Zhenmin; Guo, Benheng; Ren, Jing

    2015-02-01

    Lactobacillus casei LC2W is an exopolysaccharide (EPS)-producing strain with probiotic effects. To investigate the regulation mechanism of EPS biosynthesis and to improve EPS production through cofactor engineering, a H₂O-forming NADH oxidase gene was cloned from Streptococcus mutans and overexpressed in L. casei LC2W under the control of constitutive promoter P₂₃. The recombinant strain LC-nox exhibited 0.854 U/mL of NADH oxidase activity, which was elevated by almost 20-fold in comparison with that of wild-type strain. As a result, overexpression of NADH oxidase resulted in a reduction in growth rate. In addition, lactate production was decreased by 22% in recombinant strain. It was proposed that more carbon source was saved and used for the biosynthesis of EPS, the production of which was reached at 219.4 mg/L, increased by 46% compared to that of wild-type strain. This work provided a novel and convenient genetic approach to manipulate metabolic flux and to increase EPS production. To the best of our knowledge, this is the first report which correlates cofactor engineering with EPS production. Copyright © 2015 Elsevier GmbH. All rights reserved.

  7. Microbial metabolic activity in soil as measured by dehydrogenase determinations

    NASA Technical Reports Server (NTRS)

    Casida, L. E., Jr.

    1977-01-01

    The dehydrogenase technique for measuring the metabolic activity of microorganisms in soil was modified to use a 6-h, 37 C incubation with either glucose or yeast extract as the electron-donating substrate. The rate of formazan production remained constant during this time interval, and cellular multiplication apparently did not occur. The technique was used to follow changes in the overall metabolic activities of microorganisms in soil undergoing incubation with a limiting concentration of added nutrient. The sequence of events was similar to that obtained by using the Warburg respirometer to measure O2 consumption. However, the major peaks of activity occurred earlier with the respirometer. This possibly is due to the lack of atmospheric CO2 during the O2 consumption measurements.

  8. Trehalose Mediated Inhibition of Lactate Dehydrogenase from Rabbit Muscle. The Application of Kramers' Theory in Enzyme Catalysis.

    PubMed

    Hernández-Meza, Juan M; Sampedro, José G

    2018-04-19

    Lactate dehydrogenase (LDH) catalyzes the reduction of pyruvate to lactate by using NADH. LDH kinetics has been proposed to be dependent on the dynamics of a loop over the active site. Kramers' theory has been useful in the study of enzyme catalysis dependent on large structural dynamics. In this work, LDH kinetics was studied in the presence of trehalose and at different temperatures. In the absence of trehalose, temperature increase raised exponentially the LDH V max and revealed a sigmoid transition of K m toward a low-affinity state similar to protein unfolding. Notably, LDH V max diminished when in the presence of trehalose, while pyruvate affinity increased and the temperature-mediated binding site transition was hindered. The effect of trehalose on k cat was viscosity dependent as described by Kramers' theory since V max correlated inversely with the viscosity of the medium. As a result, activation energy ( E a ) for pyruvate reduction was dramatically increased by trehalose presence. This work provides experimental evidence that the dynamics of a structural component in LDH is essential for catalysis, i.e., the closing of the loop on the active site. While the trehalose mediated-increased of pyruvate affinity is proposed to be due to the compaction and/or increase of structural order at the binding site.

  9. Alterations in cerebral metabolism observed in living rodents using fluorescence lifetime microscopy of intrinsic NADH (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Yaseen, Mohammad A.; Sakadžić, Sava; Sutin, Jason; Wu, Weicheng; Fu, Buyin; Boas, David A.

    2017-02-01

    Monitoring cerebral energy metabolism at a cellular level is essential to improve our understanding of healthy brain function and its pathological alterations. In this study, we resolve specific alterations in cerebral metabolism utilizing minimally-invasive 2-Photon fluorescence lifetime imaging (2P-FLIM) measurements of reduced nicotinamide adenine dinucleotide (NADH) fluorescence, collected in vivo from anesthetized rats and mice. Time-resolved lifetime measurements enables distinction of different components contributing to NADH autofluorescence. These components reportedly represent different enzyme-bound formulations of NADH. Our observations from this study confirm the hypothesis that NADH FLIM can identify specific alterations in cerebral metabolism. Using time-correlated single photon counting (TCSPC) equipment and a custom-built multimodal imaging system, 2-photon fluorescence lifetime imaging (FLIM) was performed in cerebral tissue with high spatial and temporal resolution. Multi-exponential fits for NADH fluorescence lifetimes indicate 4 distinct components, or 'species.' We observed distinct variations in the relative proportions of these components before and after pharmacological-induced impairments to several reactions involved in anaerobic glycolysis and aerobic oxidative metabolism. Classification models developed with experimental data correctly predict the metabolic impairments associated with bicuculline-induced focal seizures in separate experiments. Compared to traditional intensity-based NADH measurements, lifetime imaging of NADH is less susceptible to the adverse effects of overlying blood vessels. Evaluating NADH measurements will ultimately lead to a deeper understanding of cerebral energetics and its pathology-related alterations. Such knowledge will likely aid development of therapeutic strategies for neurodegenerative diseases such as Alzheimer's Disease, Parkinson's disease, and stroke.

  10. A novel NADP(+)-dependent dehydrogenase activity for 7alpha/beta- and 11beta-hydroxysteroids in human liver nuclei: A third 11beta-hydroxysteroid dehydrogenase.

    PubMed

    Robinzon, B; Prough, R A

    2009-06-15

    Human tissue from uninvolved liver of cancer patients was fractionated using differential centrifugation and characterized for 11betaHSD enzyme activity against corticosterone, dehydrocorticosterone, 7alpha- and 7beta-hydroxy-dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. An enzyme activity was observed in nuclear protein fractions that utilized either NADP(+) or NAD(+), but not NADPH and NADH, as pyridine nucleotide cofactor with K(m) values of 12+/-2 and 390+/-2microM, compared to the K(m) for microsomal 11betaHSD1 of 43+/-8 and 264+/-24microM, respectively. The K(m) for corticosterone in the NADP(+)-dependent nuclear oxidation reaction was 102+/-16nM, compared to 4.3+/-0.8microM for 11betaHSD1. The K(cat) values for nuclear activity with NADP(+) was 1687nmol/min/mg/micromol, compared to 755nmol/min/mg/micromol for microsomal 11betaHSD1 activity. Inhibitors of 11betaHSD1 decreased both nuclear and microsomal enzyme activities, suggesting that the nuclear activity may be due to an enzyme similar to 11betaHSD Type 1 and 2.

  11. A mediator-adapted diaphorase variant for a glucose dehydrogenase-diaphorase biocatalytic system.

    PubMed

    Sugiyama, Taiki; Goto, Yoshio; Matsumoto, Ryuhei; Sakai, Hideki; Tokita, Yuichi; Hatazawa, Tsuyonobu

    2010-10-15

    Biofuel cell is an energy conversion device of the next generation which enables use of safer and higher energy-density fuels such as glucose. We have been developing a biofuel cell that comprises the three enzymes: glucose dehydrogenase (GDH) and diaphorase (DI) on anode, and bilirubin oxidase (BOD) on cathode. In this work, we have developed a DI variant suitable for our biofuel cell by using directed molecular evolution method. A gene library of DI variants was constructed by using error-prone PCR and the variant proteins were expressed in an Escherichia coli system. 8000 isolated variants have been screened with activity against 2-amino-1,4-naphthoquinone (ANQ), and 10 of them have been qualified which were then purified and examined their activities against ANQ. A highest activity was observed in G122D variant of which glycine residue at position 122 is substituted to aspartate. Enzymatic kinetic analyses show that KM for ANQ in G122D is 1/3 of that in wild type (G122D: 356 μM, wild type: 1.08 mM), whereas kcat and KM for NADH is almost the same, clearly showing that G122D mutation has given DI an improvement in enzymatic activity at lower ANQ concentration. The effect of this mutation was considered electrochemically in solution and in immobilized layer. The results show that G122D variant DI gave a higher current at lower ANQ concentration in solution, as well as in immobilized condition where GDH is co-immobilized within. Copyright © 2010 Elsevier B.V. All rights reserved.

  12. Unexpected combined effects of NADH dehydrogenase subunit-2 237 Leu/Met polymorphism and green tea consumption on renal function in male Japanese health check-up examinees: a cross-sectional study.

    PubMed

    Kokaze, Akatsuki; Ishikawa, Mamoru; Matsunaga, Naomi; Karita, Kanae; Yoshida, Masao; Ohtsu, Tadahiro; Ochiai, Hirotaka; Shirasawa, Takako; Nanri, Hinako; Hoshino, Hiromi; Takashima, Yutaka

    2013-11-20

    NADH dehydrogenase subunit-2 237 leucine/methionine (ND2-237 Leu/Met) polymorphism is associated with longevity in Japanese. A previous study has shown that ND2-237 Leu/Met polymorphism modulates the effects of green tea consumption on risk of hypertension. For men with ND2-237Leu, habitual green tea consumption may reduce the risk of hypertension. Moreover, there is a combined effect of ND2-237 Leu/Met polymorphism and alcohol consumption on risk of mildly decreased estimated glomerular filtration rate (eGFR) (<90 ml/min/1.73 m2). Several beneficial effects of green tea on the kidney have been reported. The objective of this study was to investigate whether ND2-237 Leu/Met polymorphism modifies the effects of green tea consumption on risk of mildly decreased eGFR in male Japanese health check-up examinees. For ND2-237Leu genotypic men, after adjustment for confounding factors, green tea consumption may increase the risk of mildly decreased eGFR (P for trend = 0.016). The adjusted odds ratio (OR) for mildly decreased eGFR was significantly higher in subjects with ND2-237Leu who consume ≥6 cups of green tea per day than those who consume ≤1 cup of green tea per day (adjusted OR = 5.647, 95% confidence interval: 1.528-20.88, P = 0.009). On the other hand, for ND2-237Met genotypic men, green tea consumption does not appear to determine the risk of mildly decreased eGFR. The present results suggest that ND2-237 Leu/Met polymorphism unexpectedly modifies the effects of green tea consumption on eGFR and the risk of mildly decreased eGFR in male Japanese subjects.

  13. Unexpected combined effects of NADH dehydrogenase subunit-2 237 Leu/Met polymorphism and green tea consumption on renal function in male Japanese health check-up examinees: a cross-sectional study

    PubMed Central

    2013-01-01

    Background NADH dehydrogenase subunit-2 237 leucine/methionine (ND2-237 Leu/Met) polymorphism is associated with longevity in Japanese. A previous study has shown that ND2-237 Leu/Met polymorphism modulates the effects of green tea consumption on risk of hypertension. For men with ND2-237Leu, habitual green tea consumption may reduce the risk of hypertension. Moreover, there is a combined effect of ND2-237 Leu/Met polymorphism and alcohol consumption on risk of mildly decreased estimated glomerular filtration rate (eGFR) (<90 ml/min/1.73 m2). Several beneficial effects of green tea on the kidney have been reported. The objective of this study was to investigate whether ND2-237 Leu/Met polymorphism modifies the effects of green tea consumption on risk of mildly decreased eGFR in male Japanese health check-up examinees. Results For ND2-237Leu genotypic men, after adjustment for confounding factors, green tea consumption may increase the risk of mildly decreased eGFR (P for trend = 0.016). The adjusted odds ratio (OR) for mildly decreased eGFR was significantly higher in subjects with ND2-237Leu who consume ≥6 cups of green tea per day than those who consume ≤1 cup of green tea per day (adjusted OR = 5.647, 95% confidence interval: 1.528-20.88, P = 0.009). On the other hand, for ND2-237Met genotypic men, green tea consumption does not appear to determine the risk of mildly decreased eGFR. Conclusion The present results suggest that ND2-237 Leu/Met polymorphism unexpectedly modifies the effects of green tea consumption on eGFR and the risk of mildly decreased eGFR in male Japanese subjects. PMID:24252463

  14. Reversible inactivation of CO dehydrogenase with thiol compounds

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

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

    2014-05-09

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

  15. Localization and Function of the Membrane-bound Riboflavin in the Na+-translocating NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae*

    PubMed Central

    Casutt, Marco S.; Huber, Tamara; Brunisholz, René; Tao, Minli; Fritz, Günter; Steuber, Julia

    2010-01-01

    The sodium ion-translocating NADH:quinone oxidoreductase (Na+-NQR) from the human pathogen Vibrio cholerae is a respiratory membrane protein complex that couples the oxidation of NADH to the transport of Na+ across the bacterial membrane. The Na+-NQR comprises the six subunits NqrABCDEF, but the stoichiometry and arrangement of these subunits are unknown. Redox-active cofactors are FAD and a 2Fe-2S cluster on NqrF, covalently attached FMNs on NqrB and NqrC, and riboflavin and ubiquinone-8 with unknown localization in the complex. By analyzing the cofactor content and NADH oxidation activity of subcomplexes of the Na+-NQR lacking individual subunits, the riboflavin cofactor was unequivocally assigned to the membrane-bound NqrB subunit. Quantitative analysis of the N-terminal amino acids of the holo-complex revealed that NqrB is present in a single copy in the holo-complex. It is concluded that the hydrophobic NqrB harbors one riboflavin in addition to its covalently attached FMN. The catalytic role of two flavins in subunit NqrB during the reduction of ubiquinone to ubiquinol by the Na+-NQR is discussed. PMID:20558724

  16. Glutathionylation regulates cytosolic NADP+-dependent isocitrate dehydrogenase activity.

    PubMed

    Shin, Seoung Woo; Oh, Chang Joo; Kil, In Sup; Park, Jeen-Woo

    2009-04-01

    Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) is susceptible to inactivation by numerous thiol-modifying reagents. This study now reports that Cys269 of IDPc is a target for S-glutathionylation and that this modification is reversed by dithiothreitol as well as enzymatically by cytosolic glutaredoxin in the presence of GSH. Glutathionylated IDPc was significantly less susceptible than native protein to peptide fragmentation by reactive oxygen species and proteolytic digestion. Glutathionylation may play a protective role in the degradation of protein through the structural alterations of IDPc. HEK293 cells treated with diamide displayed decreased IDPc activity and accumulated glutathionylated enzyme. Using immunoprecipitation with an anti-IDPc IgG and immunoblotting with an anti-GSH IgG, we purified and positively identified glutathionylated IDPc from the kidneys of mice subjected to ischemia/reperfusion injury and from the livers of ethanol-administered rats. These results suggest that IDPc activity is modulated through enzymatic glutathionylation and deglutathionylation during oxidative stress.

  17. Detection of ATP and NADH: A Bioluminescent Experience.

    ERIC Educational Resources Information Center

    Selig, Ted C.; And Others

    1984-01-01

    Described is a bioluminescent assay for adenosine triphosphate (ATP) and reduced nicotineamide-adenine dinucleotide (NADH) that meets the requirements of an undergraduate biochemistry laboratory course. The 3-hour experiment provides students with experience in bioluminescence and analytical biochemistry yet requires limited instrumentation,…

  18. Aluminum and its effect in the equilibrium between folded/unfolded conformation of NADH.

    PubMed

    Formoso, Elena; Mujika, Jon I; Grabowski, Slawomir J; Lopez, Xabier

    2015-11-01

    Nicotinamide adenine dinucleotide (NADH) is one of the most abundant cofactor employed by proteins and enzymes. The molecule is formed by two nucleotides that can lead to two main conformations: folded/closed and unfolded/open. Experimentally, it has been determined that the closed form is about 2 kcal/mol more stable than the open formed. Computationally, a correct description of the NADH unfolding process is challenging due to different reasons: 1) The unfolding process shows a very low energy difference between the two conformations 2) The molecule can form a high number of internal hydrogen bond interactions 3) Subtle effects such as dispersion may be important. In order to tackle all these effects, we have employed a number of different state of the art computational techniques, including: a) well-tempered metadynamics, b) geometry optimizations, and c) Quantum Theory of Atoms in Molecules (QTAIM) calculations, to investigate the conformational change of NADH in solution and interacting with aluminum. All the results indicate that aluminum indeed favors the closed conformation of NADH, due mainly to the formation of a more rigid structure through key hydrogen bond interactions. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Crystallization and preliminary crystallographic analysis of a flavoprotein NADH oxidase from Lactobacillus brevis

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

    Kuzu, Mutlu; Niefind, Karsten; Hummel, Werner

    2005-05-01

    The water-forming flavoenzyme NADH oxidase was crystallized successfully for the first time. The crystals diffract X-rays to at least 4.0 Å resolution. NADH oxidase (NOX) from Lactobacillus brevis is a homotetrameric flavoenzyme composed of 450 amino acids per subunit. The molecular weight of each monomer is 48.8 kDa. The enzyme catalyzes the oxidation of two equivalents of NADH and reduces one equivalent of oxygen to yield two equivalents of water, without releasing hydrogen peroxide after the reduction of the first equivalent of NADH. Crystals of this protein were grown in the presence of 34% polyethylene glycol monomethyl ether 2000, 0.1more » M sodium acetate and 0.2 M ammonium sulfate at pH 5.4. They belong to the tetragonal space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = 74.8, b = 95.7, c = 116.9 Å, α = γ = 90, β = 103.8°. The current diffraction limit is 4.0 Å. The self-rotation function of the native data set is consistent with a NOX tetramer in the asymmetric unit.« less

  20. Mitochondria from the left heart ventricles of both normotensive and spontaneously hypertensive rats oxidize externally added NADH mostly via a novel malate/oxaloacetate shuttle as reconstructed in vitro.

    PubMed

    Atlante, Anna; Seccia, Teresa M; De Bari, Lidia; Marra, Ersilia; Passarella, Salvatore

    2006-07-01

    A substantial increase in NADH production, arising from accelerated glycolysis, occurs in cardiac hypertrophy and this raises the question of how the NADH is oxidised. We have addressed this problem by reconstructing appropriate mitochondrial shuttles in vitro, using mitochondria from the left ventricles of both normotensive and spontaneously hypertensive rats at 5 and 24 weeks of age as model systems for left ventricle hypertrophy and hypertrophy/hypertension respectively. We found that most NADH oxidation occurs via a novel malate/oxaloacetate shuttle, the activity of which increases with time and with the progression of hypertrophy and development of hypertension as judged by statistical ANOVA analysis. In contrast, alpha-glycerol-phosphate and the malate/aspartate shuttles were shown to make only a minor contribution to NADH oxidation in a manner essentially independent of age and progression of hypertrophy/hypertension. The rate of malate transport in exchange with oxaloacetate proved to limit the rate of NADH oxidation via this malate/oxaloacetate shuttle.

  1. Succinate dehydrogenase activity and soma size of motoneurons innervating different portions of the rat tibialis anterior

    NASA Technical Reports Server (NTRS)

    Ishihara, A.; Roy, R. R.; Edgerton, V. R.

    1995-01-01

    The spatial distribution, soma size and oxidative enzyme activity of gamma and alpha motoneurons innervating muscle fibres in the deep (away from the surface of the muscle) and superficial (close to the surface of the muscle) portions of the tibialis anterior in normal rats were determined. The deep portion had a higher percentage of high oxidative fibres than the superficial portion of the muscle. Motoneurons were labelled by retrograde neuronal transport of fluorescent tracers: Fast Blue and Nuclear Yellow were injected into the deep portion and Nuclear Yellow into the superficial portion of the muscle. Therefore, motoneurons innervating the deep portion were identified by both a blue fluorescent cytoplasm and a golden-yellow fluorescent nucleus, while motoneurons innervating the superficial portion were identified by only a golden-yellow fluorescent nucleus. After staining for succinate dehydrogenase activity on the same section used for the identification of the motoneurons, soma size and succinate dehydrogenase activity of the motoneurons were measured. The gamma and alpha motoneurons innervating both the deep and superficial portions were located primarily at L4 and were intermingled within the same region of the dorsolateral portion of the ventral horn in the spinal cord. Mean soma size was similar for either gamma or alpha motoneurons in the two portions of the muscle. The alpha motoneurons innervating the superficial portion had a lower mean succinate dehydrogenase activity than those innervating the deep portion of the muscle. An inverse relationship between soma size and succinate dehydrogenase activity of alpha, but not gamma, motoneurons innervating both the deep and superficial portions was observed. Based on three-dimensional reconstructions within the spinal cord, there were no apparent differences in the spatial distribution of the motoneurons, either gamma or alpha, associated with the deep and superficial compartments of the muscle. The data

  2. Variants of glycerol dehydrogenase having D-lactate dehydrogenase activity and uses thereof

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

    Wang, Qingzhao; Shanmugam, Keelnatham T.; Ingram, Lonnie O'Neal

    The present invention provides methods of designing and generating glycerol dehydrogenase (GlyDH) variants that have altered function as compared to a parent polypeptide. The present invention further provides nucleic acids encoding GlyDH polypeptide variants having altered function as compared to the parent polypeptide. Host cells comprising polynucleotides encoding GlyDH variants and methods of producing lactic acids are also provided in various aspects of the invention.

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

    PubMed

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

    2017-03-01

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

  4. Single sample extraction and HPLC processing for quantification of NAD and NADH levels in Saccharomyces cerevisiae

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

    Sporty, J; Kabir, M M; Turteltaub, K

    A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, approximately 10{sup 8} yeast cells were harvested by centrifugation and then lysed under non-oxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50-mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH{sub 3}CN + 50-mM ammonium acetate (3:1; v:v) was added to the cell lysates. After sample centrifugation to pellet precipitated proteins, organic solvent removal was performed on supernatants by chloroform extraction. Themore » remaining aqueous phase was dried and resuspended in 50-mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV-VIS absorbance detection. Applicability of this procedure for quantifying NAD and NADH levels was evaluated by culturing yeast under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. NAD and NADH contents are similar to previously reported levels in yeast obtained using enzymatic assays performed separately on acid (for NAD) and alkali (for NADH) extracts. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (1) applicable to quantification of these metabolites in mammalian and bacterial cell cultures; and (2) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures.« less

  5. Directed modification of L-LcLDH1, an L-lactate dehydrogenase from Lactobacillus casei, to improve its specific activity and catalytic efficiency towards phenylpyruvic acid.

    PubMed

    Li, Jian-Fang; Li, Xue-Qing; Liu, Yan; Yuan, Feng-Jiao; Zhang, Ting; Wu, Min-Chen; Zhang, Ji-Ru

    2018-05-22

    To improve the specific activity and catalytic efficiency of L-LcLDH1, an NADH-dependent allosteric L-lactate dehydrogenase from L. casei, towards phenylpyruvic acid (PPA), its directed modification was conducted based on the semi-rational design. The three variant genes, Lcldh1 Q88R , Lcldh1 I229A and Lcldh1 T235G , were constructed by whole-plasmid PCR as designed theoretically, and expressed in E. coli BL21(DE3), respectively. The purified mutant, L-LcLDH1 Q88R or L-LcLDH1 I229A , displayed the specific activity of 451.5 or 512.4 U/mg towards PPA, by which the asymmetric reduction of PPA afforded L-phenyllactic acid (PLA) with an enantiomeric excess (ee p ) more than 99%. Their catalytic efficiencies (k cat /K m ) without D-fructose-1,6-diphosphate (D-FDP) were 4.8- and 5.2-fold that of L-LcLDH1. Additionally, the k cat /K m values of L-LcLDH1 Q88R and L-LcLDH1 I229A with D-FDP were 168.4- and 8.5-fold higher than those of the same enzymes without D-FDP, respectively. The analysis of catalytic mechanisms by molecular docking (MD) simulation indicated that substituting I229 in L-LcLDH1 with Ala enlarges the space of substrate-binding pocket, and that the replacement of Q88 with Arg makes the inlet of pocket larger than that of L-LcLDH1. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Ethanol-induced alcohol dehydrogenase E (AdhE) potentiates pneumolysin in Streptococcus pneumoniae.

    PubMed

    Luong, Truc Thanh; Kim, Eun-Hye; Bak, Jong Phil; Nguyen, Cuong Thach; Choi, Sangdun; Briles, David E; Pyo, Suhkneung; Rhee, Dong-Kwon

    2015-01-01

    Alcohol impairs the host immune system, rendering the host more vulnerable to infection. Therefore, alcoholics are at increased risk of acquiring serious bacterial infections caused by Streptococcus pneumoniae, including pneumonia. Nevertheless, how alcohol affects pneumococcal virulence remains unclear. Here, we showed that the S. pneumoniae type 2 D39 strain is ethanol tolerant and that alcohol upregulates alcohol dehydrogenase E (AdhE) and potentiates pneumolysin (Ply). Hemolytic activity, colonization, and virulence of S. pneumoniae, as well as host cell myeloperoxidase activity, proinflammatory cytokine secretion, and inflammation, were significantly attenuated in adhE mutant bacteria (ΔadhE strain) compared to D39 wild-type bacteria. Therefore, AdhE might act as a pneumococcal virulence factor. Moreover, in the presence of ethanol, S. pneumoniae AdhE produced acetaldehyde and NADH, which subsequently led Rex (redox-sensing transcriptional repressor) to dissociate from the adhE promoter. An increase in AdhE level under the ethanol condition conferred an increase in Ply and H2O2 levels. Consistently, S. pneumoniae D39 caused higher cytotoxicity to RAW 264.7 cells than the ΔadhE strain under the ethanol stress condition, and ethanol-fed mice (alcoholic mice) were more susceptible to infection with the D39 wild-type bacteria than with the ΔadhE strain. Taken together, these data indicate that AdhE increases Ply under the ethanol stress condition, thus potentiating pneumococcal virulence. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. Bacterial inactivation of the anticancer drug doxorubicin.

    PubMed

    Westman, Erin L; Canova, Marc J; Radhi, Inas J; Koteva, Kalinka; Kireeva, Inga; Waglechner, Nicholas; Wright, Gerard D

    2012-10-26

    Microbes are exposed to compounds produced by members of their ecological niche, including molecules with antibiotic or antineoplastic activities. As a result, even bacteria that do not produce such compounds can harbor the genetic machinery to inactivate or degrade these molecules. Here, we investigated environmental actinomycetes for their ability to inactivate doxorubicin, an aminoglycosylated anthracycline anticancer drug. One strain, Streptomyces WAC04685, inactivates doxorubicin via a deglycosylation mechanism. Activity-based purification of the enzymes responsible for drug inactivation identified the NADH dehydrogenase component of respiratory electron transport complex I, which was confirmed by gene inactivation studies. A mechanism where reduction of the quinone ring of the anthracycline by NADH dehydrogenase leads to deglycosylation is proposed. This work adds anticancer drug inactivation to the enzymatic inactivation portfolio of actinomycetes and offers possibilities for novel applications in drug detoxification. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Reduction of Clofazimine by Mycobacterial Type 2 NADH:Quinone Oxidoreductase

    PubMed Central

    Yano, Takahiro; Kassovska-Bratinova, Sacha; Teh, J. Shin; Winkler, Jeffrey; Sullivan, Kevin; Isaacs, Andre; Schechter, Norman M.; Rubin, Harvey

    2011-01-01

    The mechanism of action of clofazimine (CFZ), an antimycobacterial drug with a long history, is not well understood. The present study describes a redox cycling pathway that involves the enzymatic reduction of CFZ by NDH-2, the primary respiratory chain NADH:quinone oxidoreductase of mycobacteria and nonenzymatic oxidation of reduced CFZ by O2 yielding CFZ and reactive oxygen species (ROS). This pathway was demonstrated using isolated membranes and purified recombinant NDH-2. The reduction and oxidation of CFZ was measured spectrally, and the production of ROS was measured using a coupled assay system with Amplex Red. Supporting the ROS-based killing mechanism, bacteria grown in the presence of antioxidants are more resistant to CFZ. CFZ-mediated increase in NADH oxidation and ROS production were not observed in membranes from three different Gram-negative bacteria but was observed in Staphylococcus aureus and Saccharomyces cerevisiae, which is consistent with the known antimicrobial specificity of CFZ. A more soluble analog of CFZ, KS6, was synthesized and was shown to have the same activities as CFZ. These studies describe a pathway for a continuous and high rate of reactive oxygen species production in Mycobacterium smegmatis treated with CFZ and a CFZ analog as well as evidence that cell death produced by these agents are related to the production of these radical species. PMID:21193400

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

    PubMed

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

    2013-06-01

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

  10. Trimethylamine-N-oxide counteracts urea effects on rabbit muscle lactate dehydrogenase function: a test of the counteraction hypothesis.

    PubMed Central

    Baskakov, I; Wang, A; Bolen, D W

    1998-01-01

    Trimethylamine-N-oxide (TMAO) in the cells of sharks and rays is believed to counteract the deleterious effects of the high intracellular concentrations of urea in these animals. It has been hypothesized that TMAO has the generic ability to counteract the effects of urea on protein structure and function, regardless of whether that protein actually evolved in the presence of these two solutes. Rabbit muscle lactate dehydrogenase (LDH) did not evolve in the presence of either solute, and it is used here to test the validity of the counteraction hypothesis. With pyruvate as substrate, results show that its Km and the combined Km of pyruvate and NADH are increased by urea, decreased by TMAO, and in 1:1 and 2:1 mixtures of urea:TMAO the Km values are essentially equivalent to the Km values obtained in the absence of the two solutes. In contrast, values of k(cat) and the Km for NADH as a substrate are unperturbed by urea, TMAO, or urea:TMAO mixtures. All of these effects are consistent with TMAO counteraction of the effects of urea on LDH kinetic parameters, supporting the premise that counteraction is a property of the solvent system and is independent of the evolutionary history of the protein. PMID:9591690

  11. Utilization of xylitol dehydrogenase in a combined microbial/enzymatic process for production of xylitol from D-glucose.

    PubMed

    Mayer, Gerhard; Kulbe, Klaus D; Nidetzky, Bernd

    2002-01-01

    The production of xylitol from D-glucose occurs through a three-step process in which D-arabitol and D-xylulose are formed as the first and second intermediate product, respectively, and both are obtained via microbial bioconversion reactions. Catalytic hydrogenation of D-xylulose yields xylitol; however, it is contaminated with D-arabitol. The aim of this study was to increase the stereoselectivity of the D-xylulose reduction step by using enzymatic catalysis. Recombinant xylitol dehydrogenase from the yeast Galactocandida mastotermitis was employed to catalyze xylitol formation from D-xylulose in an NADH-dependent reaction, and coenzyme regeneration was achieved by means of formate dehydrogenase-catalyzed oxidation of formate into carbon dioxide. The xylitol yield from D-xylulose was close to 100%. Optimal productivity was found for initial coenzyme concentrations of between 0.5 and 0.75 mM. In the presence of 0.30 M (45 g/L) D-xylulose and 2000 U/L of both dehydrogenases, exhaustive substrate turnover was achieved typically in a 4-h reaction time. The enzymes were recovered after the reaction in yields of approx 90% by means of ultrafiltration and could be reused for up to six cycles of D-xylulose reduction. The advantages of incorporating the enzyme-catalyzed step in a process for producing xylitol from D-glucose are discussed, and strategies for downstream processing are proposed by which the observed coenzyme turnover number of approx 600 could be increased significantly.

  12. Independent modulation of the activity of alpha-ketoglutarate dehydrogenase complex by Ca2+ and Mg2+.

    PubMed

    Panov, A; Scarpa, A

    1996-01-16

    The activity of alpha-ketoglutarate dehydrogenase complex (KGDHC), an important enzyme regulating several metabolic pathways, could be regulated by changes in the environment within the mitochondrial matrix. It has been postulated that the activity of this and other dehydrogenases in vivo could be modulated by changes in the intramitochondrial concentrations of Ca2+ or Mg2+. Using a purified alpha-ketoglutarate dehydrogenase from pig hearts, the effect of Ca2+ and/or Mg2+ on the enzyme activity was investigated. Either Ca2+ or Mg2+ increased enzyme activity, and the effects were additive if the concentrations of free divalent cations were below 0.1 and 1 mM for Ca2+ and Mg2+, respectively. In the presence of 1 mM alpha-ketoglutarate and other cofactors, the KM for Mg2+ was 25 microM and less than 1 microM for Ca2+. The KM for alpha-ketoglutarate was a function of the divalent cation(s) present: 4 +/- 1.1 mM in the absence of Ca2+, with or without Mg2+; 2.2 mM in the presence of 1.8 microM Ca2+ alone; and 0.3 mM in the presence of both Ca2+ and Mg2+. Mg2+ increased KGDHC activity only in the presence of thiamine pyrophosphate (TPP) indicating that KGDHC requires both TPP and Mg2+ for enzyme's maximal activity. The affinity of KGDHC for NAD+ is significantly changed by either Mg2+ or Ca2+. The conclusions are that changes in both Ca2+ and Mg2+, in concentrations possibly occurring within mitochondria, could control KGDHC activity and that thiamine pyrophosphate is required for maximal enzyme activity.

  13. A Novel NADP+- Dependent Dehydrogenase Activity for 7 α/β and 11 β-hydroxysteroids in human liver nuclei: A Third 11 β-Hydroxysteroid Dehydrogenase

    PubMed Central

    Robinzon, B.; Prough, R.A.

    2009-01-01

    Human tissue from uninvolved liver of cancer patients was fractionated using differential centrifugation and characterized for 11βHSD enzyme activity against corticosterone, dehydrocorticosterone, 7α and 7β-hydroxy-dehydroepiandrosterone, and 7-oxodehydroepiandrosterone. An enzyme activity was observed in nuclear protein fractions that utilized either NADP+ or NAD+, but not NADPH and NADH, as pyridine nucleotide cofactor with Km values of 12 ± 2 and 390 ± 2 μM, compared to the Km for microsomal 11βHSD1 of 43 ± 8 and 264 ± 24 μM, respectively. The Km for corticosterone in the NADP+-dependent nuclear oxidation reaction was 102 ± 16 nM, compared to 4.3 ± 0.8 μM for 11βHSD1. The Kcat values for nuclear activity with NADP+ was 1,687 nmol/min/mg/μmol, compared to 755 nmol/min/mg/μmol for microsomal 11βHSD1 activity. Inhibitors of 11βHSD1 decreased both nuclear and microsomal enzyme activities, suggesting that the nuclear activity may be due to an enzyme similar to 11βHSD Type 1 and 2. PMID:19416720

  14. Human sperm NADH and NADPH diaphorase cytochemistry: correlation with sperm motility.

    PubMed

    Zini, A; O'Bryan, M K; Israel, L; Schlegel, P N

    1998-03-01

    We have examined the correlation between the retention of residual sperm cytoplasm and sperm motility in semen from men presenting for infertility evaluation. Semen samples (n = 12) were obtained from nonazoospermic men presenting for infertility evaluation at our institution. Samples were fractionated into high-, intermediate-, and low-density subpopulations by Percoll gradients in order to examine the correlation between the retention of residual sperm cytoplasm and sperm motility. Residual sperm cytoplasm retention was detected by cytochemical staining of sperm for nicotinamide adenine dinucleotide (NADH)- or nicotinamide adenine dinucleotide phosphate (NADPH)-dependent diaphorase activity. The different sperm subpopulations (low, intermediate, and high density) had significantly different percentages of sperm with droplet retention (analysis of variance, P < 0.05). Using either NADH or NADPH diaphorase staining as a marker of the cytoplasmic space, a significant negative correlation was observed between the percentage of sperm with residual cytoplasmic droplets and the percentage of motile sperm (r = -0.58 and -0.61, respectively, P < 0.05). Assessment of residual sperm cytoplasm retention is a simple diagnostic test. Although this test is of unproven value in the management of infertile men, this and other studies suggest that it may provide useful data on sperm function.

  15. Phasor Fluorescence Lifetime Microscopy of Free and Protein-Bound NADH Reveals Neural Stem Cell Differentiation Potential

    PubMed Central

    Stringari, Chiara; Nourse, Jamison L.; Flanagan, Lisa A.; Gratton, Enrico

    2012-01-01

    In the stem cell field there is a lack of non invasive and fast methods to identify stem cell’s metabolic state, differentiation state and cell-lineage commitment. Here we describe a label-free method that uses NADH as an intrinsic biomarker and the Phasor approach to Fluorescence Lifetime microscopy to measure the metabolic fingerprint of cells. We show that different metabolic states are related to different cell differentiation stages and to stem cell bias to neuronal and glial fate, prior the expression of lineage markers. Our data demonstrate that the NADH FLIM signature distinguishes non-invasively neurons from undifferentiated neural progenitor and stem cells (NPSCs) at two different developmental stages (E12 and E16). NPSCs follow a metabolic trajectory from a glycolytic phenotype to an oxidative phosphorylation phenotype through different stages of differentiation. NSPCs are characterized by high free/bound NADH ratio, while differentiated neurons are characterized by low free/bound NADH ratio. We demonstrate that the metabolic signature of NPSCs correlates with their differentiation potential, showing that neuronal progenitors and glial progenitors have a different free/bound NADH ratio. Reducing conditions in NPSCs correlates with their neurogenic potential, while oxidative conditions correlate with glial potential. For the first time we show that FLIM NADH metabolic fingerprint provides a novel, and quantitative measure of stem cell potential and a label-free and non-invasive means to identify neuron- or glial- biased progenitors. PMID:23144844

  16. The blood perfusion and NADH/FAD content combined analysis in patients with diabetes foot

    NASA Astrophysics Data System (ADS)

    Dremin, Victor V.; Sidorov, Victor V.; Krupatkin, Alexander I.; Galstyan, Gagik R.; Novikova, Irina N.; Zherebtsova, Angelina I.; Zherebtsov, Evgeny A.; Dunaev, Andrey V.; Abdulvapova, Zera N.; Litvinova, Karina S.; Rafailov, Ilya E.; Sokolovski, Sergei G.; Rafailov, Edik U.

    2016-03-01

    Skin blood microcirculation and the metabolism activity of tissue were examined on the patients with type 2 diabetes. Laser Doppler flowmetry (LDF) with 1064 nm laser light source and fluorescence spectroscopy (FS) with excitation light of 365 nm and 450 nm have been used to monitor the blood perfusion and the content of coenzymes NADH and FAD. Concluding, the proposed combined LDF and tissue FS approach allows to identify the significant violations in the blood microcirculation and metabolic activity for type 2 diabetes patients.

  17. Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase.

    PubMed

    Van Noorden, C J

    1984-01-01

    Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase has found many applications in biomedical research. However, up to several years ago, the methods used often appeared to be unreliable because many artefacts occurred during processing and staining of tissue sections or cells. The development of histochemical methods preventing loss or redistribution of the enzyme by using either polyvinyl alcohol as a stabilizer or a semipermeable membrane interposed between tissue section and incubation medium, has lead to progress in the topochemical localization of glucose-6-phosphate dehydrogenase. Optimization of incubation conditions has further increased the precision of histochemical methods. Precise cytochemical methods have been developed either by the use of a polyacrylamide carrier in which individual cells have been incorporated before staining or by including polyvinyl alcohol in the incubation medium. In the present text, these methods for the histochemical and cytochemical localization of glucose-6-phosphate dehydrogenase for light microscopical and electron microscopical purposes are extensively discussed along with immunocytochemical techniques. Moreover, the validity of the staining methods is considered both for the localization of glucose-6-phosphate dehydrogenase activity in cells and tissues and for cytophotometric analysis. Finally, many applications of the methods are reviewed in the fields of functional heterogeneity of tissues, early diagnosis of carcinoma, effects of xenobiotics on cellular metabolism, diagnosis of inherited glucose-6-phosphate dehydrogenase deficiency, analysis of steroid-production in reproductive organs, and quality control of oocytes of mammals. It is concluded that the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase is of highly significant value in the study of diseased tissues. In many cases, the first pathological change is an increase in glucose-6-phosphate dehydrogenase activity

  18. Physiological and fermentation properties of Bacillus coagulans and a mutant lacking fermentative lactate dehydrogenase activity.

    PubMed

    Su, Yue; Rhee, Mun Su; Ingram, Lonnie O; Shanmugam, K T

    2011-03-01

    Bacillus coagulans, a sporogenic lactic acid bacterium, grows optimally at 50-55 °C and produces lactic acid as the primary fermentation product from both hexoses and pentoses. The amount of fungal cellulases required for simultaneous saccharification and fermentation (SSF) at 55 °C was previously reported to be three to four times lower than for SSF at the optimum growth temperature for Saccharomyces cerevisiae of 35 °C. An ethanologenic B. coagulans is expected to lower the cellulase loading and production cost of cellulosic ethanol due to SSF at 55 °C. As a first step towards developing B. coagulans as an ethanologenic microbial biocatalyst, activity of the primary fermentation enzyme L-lactate dehydrogenase was removed by mutation (strain Suy27). Strain Suy27 produced ethanol as the main fermentation product from glucose during growth at pH 7.0 (0.33 g ethanol per g glucose fermented). Pyruvate dehydrogenase (PDH) and alcohol dehydrogenase (ADH) acting in series contributed to about 55% of the ethanol produced by this mutant while pyruvate formate lyase and ADH were responsible for the remainder. Due to the absence of PDH activity in B. coagulans during fermentative growth at pH 5.0, the l-ldh mutant failed to grow anaerobically at pH 5.0. Strain Suy27-13, a derivative of the l-ldh mutant strain Suy27, that produced PDH activity during anaerobic growth at pH 5.0 grew at this pH and also produced ethanol as the fermentation product (0.39 g per g glucose). These results show that construction of an ethanologenic B. coagulans requires optimal expression of PDH activity in addition to the removal of the LDH activity to support growth and ethanol production.

  19. A mitochondrial NADH-dependent fumarate reductase involved in the production of succinate excreted by procyclic Trypanosoma brucei.

    PubMed

    Coustou, Virginie; Besteiro, Sébastien; Rivière, Loïc; Biran, Marc; Biteau, Nicolas; Franconi, Jean-Michel; Boshart, Michael; Baltz, Théo; Bringaud, Frédéric

    2005-04-29

    Trypanosoma brucei is a parasitic protist responsible for sleeping sickness in humans. The procyclic stage of T. brucei expresses a soluble NADH-dependent fumarate reductase (FRDg) in the peroxisome-like organelles called glycosomes. This enzyme is responsible for the production of about 70% of the excreted succinate, the major end product of glucose metabolism in this form of the parasite. Here we functionally characterize a new gene encoding FRD (FRDm1) expressed in the procyclic stage. FRDm1 is a mitochondrial protein, as evidenced by immunolocalization, fractionation of digitonin-permeabilized cells, and expression of EGFP-tagged FRDm1 in the parasite. RNA interference was used to deplete FRDm1, FRDg, or both together. The analysis of the resulting mutant cell lines showed that FRDm1 is responsible for 30% of the cellular NADH-FRD activity, which solves a long standing debate regarding the existence of a mitochondrial FRD in trypanosomatids. FRDg and FRDm1 together account for the total NADH-FRD activity in procyclics, because no activity was measured in the double mutant lacking expression of both proteins. Analysis of the end products of 13C-enriched glucose excreted by these mutant cell lines showed that FRDm1 contributes to the production of between 14 and 44% of the succinate excreted by the wild type cells. In addition, depletion of one or both FRD enzymes results in up to 2-fold reduction of the rate of glucose consumption. We propose that FRDm1 is involved in the maintenance of the redox balance in the mitochondrion, as proposed for the ancestral soluble FRD presumably present in primitive anaerobic cells.

  20. Lowering effect of firefly squid powder on triacylglycerol content and glucose-6-phosphate dehydrogenase activity in rat liver.

    PubMed

    Takeuchi, Hiroyuki; Morita, Ritsuko; Shirai, Yoko; Nakagawa, Yoshihisa; Terashima, Teruya; Ushikubo, Shun; Matsuo, Tatsuhiro

    2014-01-01

    Effects of dietary firefly squid on serum and liver lipid levels were investigated. Male Wistar rats were fed a diet containing 5% freeze-dried firefly squid or Japanese flying squid for 2 weeks. There was no significant difference in the liver triacylglycerol level between the control and Japanese flying squid groups, but the rats fed the firefly squid diet had a significantly lower liver triacylglycerol content than those fed the control diet. No significant difference was observed in serum triacylglycerol levels between the control and firefly squid groups. The rats fed the firefly squid had a significantly lower activity of liver glucose-6-phosphate dehydrogenase compared to the rats fed the control diet. There was no significant difference in liver fatty acid synthetase activity among the three groups. Hepatic gene expression and lipogenic enzyme activity were investigated; a DNA microarray showed that the significantly enriched gene ontology category of down-regulated genes in the firefly squid group was "lipid metabolic process". The firefly squid group had lower mRNA level of glucose-6-phosphate dehydrogenase compared to the controls. These results suggest that an intake of firefly squid decreases hepatic triacylglycerol in rats, and the reduction of mRNA level and enzyme activity of glucose-6-phosphate dehydrogenase might be related to the mechanisms.

  1. Lactate Dehydrogenase Undergoes a Substantial Structural Change to Bind its Substrate

    PubMed Central

    Qiu, Linlin; Gulotta, Miriam; Callender, Robert

    2007-01-01

    Employing temperature-jump relaxation spectroscopy, we investigate the kinetics and thermodynamics of the formation of a very early ternary binding intermediate formed when lactate dehydrogenase (LDH) binds a substrate mimic on its way to forming the productive LDH/NADH·substrate Michaelis complex. Temperature-jump scans show two distinct submillisecond processes are involved in the formation of this ternary binding intermediate, called the encounter complex here. The on-rate of the formation of the encounter complex from LDH/NADH with oxamate (a substrate mimic) is determined as a function of temperature and in the presence of small concentrations of a protein destabilizer (urea) and protein stabilizer (TMAO). It shows a strong temperature dependence with inverse Arrhenius behavior and a temperature-dependent enthalpy (heat capacity of 610 ± 84 cal/Mol K), is slowed in the presence of TMAO and speeded up in the presence of urea. These results suggest that LDH/NADH occupies a range of conformations, some competent to bind substrate (open structure; a minority population) and others noncompetent (closed), in fast equilibrium with each other in accord with a select fit model of binding. From the thermodynamic results, the two species differ in the rearrangement of low energy hydrogen bonds as would arise from changes in internal hydrogen bonding and/or increases in the solvation of the protein structure. The binding-competent species can bind ligand at or very near diffusion-limited speeds, suggesting that the binding pocket is substantially exposed to solvent in these species. This would be in contrast to the putative closed structure where the binding pocket resides deep within the protein interior. PMID:17483169

  2. Investigation of the NADH/NAD+ ratio in Ralstonia eutropha using the fluorescence reporter protein Peredox.

    PubMed

    Tejwani, Vijay; Schmitt, Franz-Josef; Wilkening, Svea; Zebger, Ingo; Horch, Marius; Lenz, Oliver; Friedrich, Thomas

    2017-01-01

    Ralstonia eutropha is a hydrogen-oxidizing ("Knallgas") bacterium that can easily switch between heterotrophic and autotrophic metabolism to thrive in aerobic and anaerobic environments. Its versatile metabolism makes R. eutropha an attractive host for biotechnological applications, including H 2 -driven production of biodegradable polymers and hydrocarbons. H 2 oxidation by R. eutropha takes place in the presence of O 2 and is mediated by four hydrogenases, which represent ideal model systems for both biohydrogen production and H 2 utilization. The so-called soluble hydrogenase (SH) couples reversibly H 2 oxidation with the reduction of NAD + to NADH and has already been applied successfully in vitro and in vivo for cofactor regeneration. Thus, the interaction of the SH with the cellular NADH/NAD + pool is of major interest. In this work, we applied the fluorescent biosensor Peredox to measure the [NADH]:[NAD + ] ratio in R. eutropha cells under different metabolic conditions. The results suggest that the sensor operates close to saturation level, indicating a rather high [NADH]:[NAD + ] ratio in aerobically grown R. eutropha cells. Furthermore, we demonstrate that multicomponent analysis of spectrally-resolved fluorescence lifetime data of the Peredox sensor response to different [NADH]:[NAD + ] ratios represents a novel and sensitive tool to determine the redox state of cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Human 2-Oxoglutarate Dehydrogenase Complex E1 Component Forms a Thiamin-derived Radical by Aerobic Oxidation of the Enamine Intermediate*

    PubMed Central

    Nemeria, Natalia S.; Ambrus, Attila; Patel, Hetalben; Gerfen, Gary; Adam-Vizi, Vera; Tretter, Laszlo; Zhou, Jieyu; Wang, Junjie; Jordan, Frank

    2014-01-01

    Herein are reported unique properties of the human 2-oxoglutarate dehydrogenase multienzyme complex (OGDHc), a rate-limiting enzyme in the Krebs (citric acid) cycle. (a) Functionally competent 2-oxoglutarate dehydrogenase (E1o-h) and dihydrolipoyl succinyltransferase components have been expressed according to kinetic and spectroscopic evidence. (b) A stable free radical, consistent with the C2-(C2α-hydroxy)-γ-carboxypropylidene thiamin diphosphate (ThDP) cation radical was detected by electron spin resonance upon reaction of the E1o-h with 2-oxoglutarate (OG) by itself or when assembled from individual components into OGDHc. (c) An unusual stability of the E1o-h-bound C2-(2α-hydroxy)-γ-carboxypropylidene thiamin diphosphate (the “ThDP-enamine”/C2α-carbanion, the first postdecarboxylation intermediate) was observed, probably stabilized by the 5-carboxyl group of OG, not reported before. (d) The reaction of OG with the E1o-h gave rise to superoxide anion and hydrogen peroxide (reactive oxygen species (ROS)). (e) The relatively stable enzyme-bound enamine is the likely substrate for oxidation by O2, leading to the superoxide anion radical (in d) and the radical (in b). (f) The specific activity assessed for ROS formation compared with the NADH (overall complex) activity, as well as the fraction of radical intermediate occupying active centers of E1o-h are consistent with each other and indicate that radical/ROS formation is an “off-pathway” side reaction comprising less than 1% of the “on-pathway” reactivity. However, the nearly ubiquitous presence of OGDHc in human tissues, including the brain, makes these findings of considerable importance in human metabolism and perhaps disease. PMID:25210035

  4. Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions

    PubMed Central

    Kasischke, Karl A; Lambert, Elton M; Panepento, Ben; Sun, Anita; Gelbard, Harris A; Burgess, Robert W; Foster, Thomas H; Nedergaard, Maiken

    2011-01-01

    Oxygen transport imposes a possible constraint on the brain's ability to sustain variable metabolic demands, but oxygen diffusion in the cerebral cortex has not yet been observed directly. We show that concurrent two-photon fluorescence imaging of endogenous nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation exposes well-defined boundaries of tissue oxygen diffusion in the mouse cortex. The NADH fluorescence increases rapidly over a narrow, very low pO2 range with a p50 of 3.4±0.6 mm Hg, thereby establishing a nearly binary reporter of significant, metabolically limiting hypoxia. The transient cortical tissue boundaries of NADH fluorescence exhibit remarkably delineated geometrical patterns, which define the limits of tissue oxygen diffusion from the cortical microcirculation and bear a striking resemblance to the ideal Krogh tissue cylinder. The visualization of microvessels and their regional contribution to oxygen delivery establishes penetrating arterioles as major oxygen sources in addition to the capillary network and confirms the existence of cortical oxygen fields with steep microregional oxygen gradients. Thus, two-photon NADH imaging can be applied to expose vascular supply regions and to localize functionally relevant microregional cortical hypoxia with micrometer spatial resolution. PMID:20859293

  5. Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions.

    PubMed

    Kasischke, Karl A; Lambert, Elton M; Panepento, Ben; Sun, Anita; Gelbard, Harris A; Burgess, Robert W; Foster, Thomas H; Nedergaard, Maiken

    2011-01-01

    Oxygen transport imposes a possible constraint on the brain's ability to sustain variable metabolic demands, but oxygen diffusion in the cerebral cortex has not yet been observed directly. We show that concurrent two-photon fluorescence imaging of endogenous nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation exposes well-defined boundaries of tissue oxygen diffusion in the mouse cortex. The NADH fluorescence increases rapidly over a narrow, very low pO(2) range with a p(50) of 3.4 ± 0.6 mm Hg, thereby establishing a nearly binary reporter of significant, metabolically limiting hypoxia. The transient cortical tissue boundaries of NADH fluorescence exhibit remarkably delineated geometrical patterns, which define the limits of tissue oxygen diffusion from the cortical microcirculation and bear a striking resemblance to the ideal Krogh tissue cylinder. The visualization of microvessels and their regional contribution to oxygen delivery establishes penetrating arterioles as major oxygen sources in addition to the capillary network and confirms the existence of cortical oxygen fields with steep microregional oxygen gradients. Thus, two-photon NADH imaging can be applied to expose vascular supply regions and to localize functionally relevant microregional cortical hypoxia with micrometer spatial resolution.

  6. Investigation of the Ionization Mechanism of NAD+/NADH-Modified Gold Electrodes in ToF-SIMS Analysis.

    PubMed

    Hua, Xin; Zhao, Li-Jun; Long, Yi-Tao

    2018-06-04

    Analysis of nicotinamide adenine dinucleotide (NAD + /NADH)-modified electrodes is important for in vitro monitoring of key biological processes. In this work, time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to analyze NAD + /NADH-modified gold electrodes. Interestingly, no obvious characteristic peaks of nicotinamide fragment could be observed in the mass spectra of NAD + /NADH in their neutral sodium pyrophosphate form. However, after acidification, the characteristic peaks for both NAD + and NADH were detected. This was due to the suppression effect of inner pyrophosphoric salts in both neutral molecules. Besides, it was proved that the suppression by inner salt was intramolecular. No obvious suppression was found between neighboring molecules. These results demonstrated the suppression effect of inner salts in ToF-SIMS analysis, providing useful evidence for the study of ToF-SIMS ionization mechanism of organic molecule-modified electrodes. Graphical Abstract ᅟ.

  7. Reductions in the mitochondrial enzyme α-ketoglutarate dehydrogenase complex in neurodegenerative disease - beneficial or detrimental?

    PubMed

    Chen, Huanlian; Denton, Travis T; Xu, Hui; Calingasan, Noel; Beal, M Flint; Gibson, Gary E

    2016-12-01

    Reductions in metabolism and excess oxidative stress are prevalent in multiple neurodegenerative diseases. The activity of the mitochondrial enzyme α-ketoglutarate dehydrogenase complex (KGDHC) appears central to these abnormalities. KGDHC is diminished in multiple neurodegenerative diseases. KGDHC can not only be rate limiting for NADH production and for substrate level phosphorylation, but is also a source of reactive oxygen species (ROS). The goal of these studies was to determine how changes in KGDHC modify baseline ROS, the ability to buffer ROS, baseline glutathionylation, calcium modulation and cell death in response to external oxidants. In vivo, reducing KGDHC with adeno virus diminished neurogenesis and increased oxidative stress. In vitro, treatments of short duration increased ROS and glutathionylation and enhanced the ability of the cells to diminish the ROS from added oxidants. However, long-term reductions lessened the ability to diminish ROS, diminished glutathionylation and exaggerated oxidant-induced changes in calcium and cell death. Increasing KGDHC enhanced the ability of the cells to diminish externally added ROS and protected against oxidant-induced changes in calcium and cell death. The results suggest that brief periods of diminished KGDHC are protective, while prolonged reductions are harmful. Furthermore, elevated KGDHC activities are protective. Thus, mitogenic therapies that increase KGDHC may be beneficial in neurodegenerative diseases. Read the Editorial Highlight for this article on Page 689. © 2016 International Society for Neurochemistry.

  8. Improved bioethanol production in an engineered Kluyveromyces lactis strain shifted from respiratory to fermentative metabolism by deletion of NDI1

    PubMed Central

    González-Siso, María Isabel; Touriño, Alba; Vizoso, Ángel; Pereira-Rodríguez, Ángel; Rodríguez-Belmonte, Esther; Becerra, Manuel; Cerdán, María Esperanza

    2015-01-01

    In this paper, we report the metabolic engineering of the respiratory yeast Kluyveromyces lactis by construction and characterization of a null mutant (Δklndi1) in the single gene encoding a mitochondrial alternative internal dehydrogenase. Isolated mitochondria of the Δklndi1 mutant show unaffected rate of oxidation of exogenous NADH, but no oxidation of matrix NADH; this confirms that KlNdi1p is the only internal NADH dehydrogenase in K. lactis mitochondria. Permeabilized cells of the Δklndi1 mutant do not show oxidation of matrix NADH, which suggests that shuttle systems to transfer the NADH from mitochondrial matrix to cytosol, for being oxidized by external dehydrogenases, are not functional. The Δklndi1 mutation decreases the chronological life span in absence of nutrients. The expression of KlNDI1 is increased by glutathione reductase depletion. The Δklndi1 mutation shifts the K. lactis metabolism from respiratory to fermentative: the Δklndi1 strain shows reduced respiration rate and increased ethanol production from glucose, while it does not grow in non-fermentable carbon sources such as lactate. The biotechnological benefit of the Δklndi1 mutant for bioethanol production from waste cheese whey lactose was proved. PMID:25186243

  9. An integrated model of cardiac mitochondrial energy metabolism and calcium dynamics.

    PubMed

    Cortassa, Sonia; Aon, Miguel A; Marbán, Eduardo; Winslow, Raimond L; O'Rourke, Brian

    2003-04-01

    We present an integrated thermokinetic model describing control of cardiac mitochondrial bioenergetics. The model describes the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and mitochondrial Ca(2+) handling. The kinetic component of the model includes effectors of the TCA cycle enzymes regulating production of NADH and FADH(2), which in turn are used by the electron transport chain to establish a proton motive force (Delta mu(H)), driving the F(1)F(0)-ATPase. In addition, mitochondrial matrix Ca(2+), determined by Ca(2+) uniporter and Na(+)/Ca(2+) exchanger activities, regulates activity of the TCA cycle enzymes isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase. The model is described by twelve ordinary differential equations for the time rate of change of mitochondrial membrane potential (Delta Psi(m)), and matrix concentrations of Ca(2+), NADH, ADP, and TCA cycle intermediates. The model is used to predict the response of mitochondria to changes in substrate delivery, metabolic inhibition, the rate of adenine nucleotide exchange, and Ca(2+). The model is able to reproduce, qualitatively and semiquantitatively, experimental data concerning mitochondrial bioenergetics, Ca(2+) dynamics, and respiratory control. Significant increases in oxygen consumption (V(O(2))), proton efflux, NADH, and ATP synthesis, in response to an increase in cytoplasmic Ca(2+), are obtained when the Ca(2+)-sensitive dehydrogenases are the main rate-controlling steps of respiratory flux. These responses diminished when control is shifted downstream (e.g., the respiratory chain or adenine nucleotide translocator). The time-dependent behavior of the model, under conditions simulating an increase in workload, closely reproduces experimentally observed mitochondrial NADH dynamics in heart trabeculae subjected to changes in pacing frequency. The steady-state and time-dependent behavior of the model support the hypothesis that mitochondrial matrix Ca(2+) plays an

  10. An Integrated Model of Cardiac Mitochondrial Energy Metabolism and Calcium Dynamics

    PubMed Central

    Cortassa, Sonia; Aon, Miguel A.; Marbán, Eduardo; Winslow, Raimond L.; O'Rourke, Brian

    2003-01-01

    We present an integrated thermokinetic model describing control of cardiac mitochondrial bioenergetics. The model describes the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and mitochondrial Ca2+ handling. The kinetic component of the model includes effectors of the TCA cycle enzymes regulating production of NADH and FADH2, which in turn are used by the electron transport chain to establish a proton motive force (ΔμH), driving the F1F0-ATPase. In addition, mitochondrial matrix Ca2+, determined by Ca2+ uniporter and Na+/Ca2+ exchanger activities, regulates activity of the TCA cycle enzymes isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. The model is described by twelve ordinary differential equations for the time rate of change of mitochondrial membrane potential (ΔΨm), and matrix concentrations of Ca2+, NADH, ADP, and TCA cycle intermediates. The model is used to predict the response of mitochondria to changes in substrate delivery, metabolic inhibition, the rate of adenine nucleotide exchange, and Ca2+. The model is able to reproduce, qualitatively and semiquantitatively, experimental data concerning mitochondrial bioenergetics, Ca2+ dynamics, and respiratory control. Significant increases in oxygen consumption (VO2), proton efflux, NADH, and ATP synthesis, in response to an increase in cytoplasmic Ca2+, are obtained when the Ca2+-sensitive dehydrogenases are the main rate-controlling steps of respiratory flux. These responses diminished when control is shifted downstream (e.g., the respiratory chain or adenine nucleotide translocator). The time-dependent behavior of the model, under conditions simulating an increase in workload, closely reproduces experimentally observed mitochondrial NADH dynamics in heart trabeculae subjected to changes in pacing frequency. The steady-state and time-dependent behavior of the model support the hypothesis that mitochondrial matrix Ca2+ plays an important role in matching energy supply with demand in

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

    PubMed Central

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

    1991-01-01

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

  12. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH

    DOE PAGES

    Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K. B.; ...

    2013-06-17

    To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymesmore » having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. As a result, high-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.« less

  13. Glucose consumption rate critically depends on redox state in Corynebacterium glutamicum under oxygen deprivation.

    PubMed

    Tsuge, Yota; Uematsu, Kimio; Yamamoto, Shogo; Suda, Masako; Yukawa, Hideaki; Inui, Masayuki

    2015-07-01

    Rapid sugar consumption is important for the microbial production of chemicals and fuels. Here, we show that overexpression of the NADH dehydrogenase gene (ndh) increased glucose consumption rate in Corynebacterium glutamicum under oxygen-deprived conditions through investigating the relationship between the glucose consumption rate and intracellular NADH/NAD(+) ratio in various mutant strains. The NADH/NAD(+) ratio was strongly repressed under oxygen deprivation when glucose consumption was accelerated by the addition of pyruvate or sodium hydrogen carbonate. Overexpression of the ndh gene in the wild-type strain under oxygen deprivation decreased the NADH/NAD(+) ratio from 0.32 to 0.13, whereas the glucose consumption rate increased by 27%. Similarly, in phosphoenolpyruvate carboxylase gene (ppc)- or malate dehydrogenase gene (mdh)-deficient strains, overexpression of the ndh gene decreased the NADH/NAD(+) ratio from 1.66 to 0.37 and 2.20 to 0.57, respectively, whereas the glucose consumption rate increased by 57 and 330%, respectively. However, in a lactate dehydrogenase gene (L-ldhA)-deficient strain, although the NADH/NAD(+) ratio decreased from 5.62 to 1.13, the glucose consumption rate was not markedly altered. In a tailored D-lactate-producing strain, which lacked ppc and L-ldhA genes, but expressed D-ldhA from Lactobacillus delbrueckii, overexpression of the ndh gene decreased the NADH/NAD(+) ratio from 1.77 to 0.56, and increased the glucose consumption rate by 50%. Overall, the glucose consumption rate was found to be inversely proportional to the NADH/NAD(+) ratio in C. glutamicum cultured under oxygen deprivation. These findings could provide an option to increase the productivity of chemicals and fuels under oxygen deprivation.

  14. Locations of ectopic beats coincide with spatial gradients of NADH in a regional model of low-flow reperfusion.

    PubMed

    Kay, Matthew; Swift, Luther; Martell, Brian; Arutunyan, Ara; Sarvazyan, Narine

    2008-05-01

    We studied the origins of ectopic beats during low-flow reperfusion after acute regional ischemia in excised rat hearts. The left anterior descending coronary artery was cannulated. Perfusate was delivered to the cannula using an high-performance liquid chromatography pump. This provided not only precise control of flow rate but also avoided mechanical artifacts associated with vessel occlusion and deocclusion. Optical mapping of epicardial transmembrane potential served to identify activation wavefronts. Imaging of NADH fluorescence was used to quantify local ischemia. Our experiments suggest that low-flow reperfusion of ischemic myocardium leads to a highly heterogeneous ischemic substrate and that the degree of ischemia between adjacent patches of tissue changes in time. In contrast to transient ectopic activity observed during full-flow reperfusion, persistent ectopic arrhythmias were observed during low-flow reperfusion. The origins of ectopic beats were traceable to areas of high spatial gradients of changes in NADH fluorescence caused by low-flow reperfusion.

  15. The Tricarboxylic Acid Cycle, an Ancient Metabolic Network with a Novel Twist

    PubMed Central

    Mailloux, Ryan J.; Bériault, Robin; Lemire, Joseph; Singh, Ranji; Chénier, Daniel R.; Hamel, Robert D.; Appanna, Vasu D.

    2007-01-01

    The tricarboxylic acid (TCA) cycle is an essential metabolic network in all oxidative organisms and provides precursors for anabolic processes and reducing factors (NADH and FADH2) that drive the generation of energy. Here, we show that this metabolic network is also an integral part of the oxidative defence machinery in living organisms and α-ketoglutarate (KG) is a key participant in the detoxification of reactive oxygen species (ROS). Its utilization as an anti-oxidant can effectively diminish ROS and curtail the formation of NADH, a situation that further impedes the release of ROS via oxidative phosphorylation. Thus, the increased production of KG mediated by NADP-dependent isocitrate dehydrogenase (NADP-ICDH) and its decreased utilization via the TCA cycle confer a unique strategy to modulate the cellular redox environment. Activities of α-ketoglutarate dehydrogenase (KGDH), NAD-dependent isocitrate dehydrogenase (NAD-ICDH), and succinate dehydrogenase (SDH) were sharply diminished in the cellular systems exposed to conditions conducive to oxidative stress. These findings uncover an intricate link between TCA cycle and ROS homeostasis and may help explain the ineffective TCA cycle that characterizes various pathological conditions and ageing. PMID:17668068

  16. Purification and Characterization of Glucose 6-Phosphate Dehydrogenase, 6-Phosphogluconate Dehydrogenase, and Glutathione Reductase from Rat Heart and Inhibition Effects of Furosemide, Digoxin, and Dopamine on the Enzymes Activities.

    PubMed

    Adem, Sevki; Ciftci, Mehmet

    2016-06-01

    The present study was aimed to investigate characterization and purification of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase from rat heart and the inhibitory effect of three drugs. The purification of the enzymes was performed using 2',5'-ADP sepharose 4B affinity material. The subunit and the natural molecular weights were analyzed by SDS-PAGE and gel filtration. Biochemical characteristics such as the optimum temperature, pH, stable pH, and salt concentration were examined for each enzyme. Types of product inhibition and Ki values with Km and Vmax values of the substrates and coenzymes were determined. According to the obtained Ki and IC50 values, furosemide, digoxin, and dopamine showed inhibitory effect on the enzyme activities at low millimolar concentrations in vitro conditions. Dopamine inhibited the activity of these enzymes as competitive, whereas furosemide and digoxin inhibited the activity of the enzyme as noncompetitive. © 2016 Wiley Periodicals, Inc.

  17. White shrimp Litopenaeus vannamei recombinant lactate dehydrogenase: Biochemical and kinetic characterization.

    PubMed

    Fregoso-Peñuñuri, Ambar A; Valenzuela-Soto, Elisa M; Figueroa-Soto, Ciria G; Peregrino-Uriarte, Alma B; Ochoa-Valdez, Manuel; Leyva-Carrillo, Lilia; Yepiz-Plascencia, Gloria

    2017-09-01

    Shrimp lactate dehydrogenase (LDH) is induced in response to environmental hypoxia. Two protein subunits deduced from different transcripts of the LDH gene from the shrimp Litopenaeus vannamei (LDHvan-1 and LDHvan-2) were identified. These subunits are expressed by alternative splicing. Since both subunits are expressed in most tissues, the purification of the enzyme from the shrimp will likely produce hetero LDH containing both subunits. Therefore, the aim of this study was to overexpress, purify and characterize only one subunit as a recombinant protein, the LDHvan-2. For this, the cDNA from muscle was cloned and overexpressed in E. coli as a fusion protein containing an intein and a chitin binding protein domain (CBD). The recombinant protein was purified by chitin affinity chromatography column that retained the CBD and released solely the full and active LDH. The active protein appears to be a tetramer with molecular mass of approximately 140 kDa and can use pyruvate or lactate as substrates, but has higher specific activity with pyruvate. The enzyme is stable between pH 7.0 to 8.5, and between 20 and 50 °C with an optimal temperature of 50 °C. Two pK a of 9.3 and 6.6, and activation energy of 44.8 kJ/mol°K were found. The kinetic constants K m for NADH was 23.4 ± 1.8 μM, and for pyruvate was 203 ± 25 μM, while V max was 7.45 μmol/min/mg protein. The shrimp LDH that is mainly expressed in shrimp muscle preferentially converts pyruvate to lactate and is an important enzyme for the response to hypoxia. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2016-03-25

    We report characterization of a member of the short-chain dehydrogenase/reductase superfamily encoded in a human gene, DHRS11. The recombinant protein (DHRS11) efficiently catalyzed the conversion of the 17-keto group of estrone, 4- and 5-androstenes and 5α-androstanes into their 17β-hydroxyl metabolites with NADPH as a coenzyme. In contrast, it exhibited reductive 3β-hydroxysteroid dehydrogenase activity toward 5β-androstanes, 5β-pregnanes, 4-pregnenes and bile acids. Additionally, DHRS11 reduced α-dicarbonyls (such as diacetyl and methylglyoxal) and alicyclic ketones (such as 1-indanone and loxoprofen). The enzyme activity was inhibited in a mixed-type manner by flavonoids, and competitively by carbenoxolone, glycyrrhetinic acid, zearalenone, curcumin and flufenamic acid. The expression of DHRS11 mRNA was observed widely in human tissues, most abundantly in testis, small intestine, colon, kidney and cancer cell lines. Thus, DHRS11 represents a novel type of 17β-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Characterization of Frex as an NADH sensor for in vivo applications in the presence of NAD+ and at various pH values.

    PubMed

    Wilkening, Svea; Schmitt, Franz-Josef; Horch, Marius; Zebger, Ingo; Lenz, Oliver; Friedrich, Thomas

    2017-09-01

    The fluorescent biosensor Frex, recently introduced as a sensitive tool to quantify the NADH concentration in living cells, was characterized by time-integrated and time-resolved fluorescence spectroscopy regarding its applicability for in vivo measurements. Based on the purified sensor protein, it is shown that the NADH dependence of Frex fluorescence can be described by a Hill function with a concentration of half-maximal sensor response of K D  ≈ 4 µM and a Hill coefficient of n ≈ 2. Increasing concentrations of NADH have moderate effects on the fluorescence lifetime of Frex, which changes by a factor of two from about 500 ps in the absence of NADH to 1 ns under fluorescence-saturating NADH concentrations. Therefore, the observed sevenfold rise of the fluorescence intensity is primarily ascribed to amplitude changes. Notably, the dynamic range of Frex sensitivity towards NADH highly depends on the NAD + concentration, while the apparent K D for NADH is only slightly affected. We found that NAD + has a strong inhibitory effect on the fluorescence response of Frex during NADH sensing, with an apparent NAD + dissociation constant of K I  ≈ 400 µM. This finding was supported by fluorescence lifetime measurements, which showed that the addition of NAD + hardly affects the fluorescence lifetime, but rather reduces the number of Frex molecules that are able to bind NADH. Furthermore, the fluorescence responses of Frex to NADH and NAD + depend critically on pH and temperature. Thus, for in vivo applications of Frex, temperature and pH need to be strictly controlled or considered during data acquisition and analysis. If all these constraints are properly met, Frex fluorescence intensity measurements can be employed to estimate the minimum NADH concentration present within the cell at sufficiently low NAD + concentrations below 100 µM.

  20. Crystallization and preliminary crystallographic analysis of a flavoprotein NADH oxidase from Lactobacillus brevis

    PubMed Central

    Kuzu, Mutlu; Niefind, Karsten; Hummel, Werner; Schomburg, Dietmar

    2005-01-01

    NADH oxidase (NOX) from Lactobacillus brevis is a homotetrameric flavoenzyme composed of 450 amino acids per subunit. The molecular weight of each monomer is 48.8 kDa. The enzyme catalyzes the oxidation of two equivalents of NADH and reduces one equivalent of oxygen to yield two equivalents of water, without releasing hydrogen peroxide after the reduction of the first equivalent of NADH. Crystals of this protein were grown in the presence of 34% polyethylene glycol monomethyl ether 2000, 0.1 M sodium acetate and 0.2 M ammonium sulfate at pH 5.4. They belong to the tetragonal space group P43212, with unit-cell parameters a = 74.8, b = 95.7, c = 116.9 Å, α = γ = 90, β = 103.8°. The current diffraction limit is 4.0 Å. The self-rotation function of the native data set is consistent with a NOX tetramer in the asymmetric unit. PMID:16511087

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

    PubMed

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

    2012-01-01

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

  2. Contribution of remote substrate binding energy to the enzymatic rate acceleration for 3α-hydroxysteroid dehydrogenase/carbonyl reductase.

    PubMed

    Hwang, Chi-Ching; Chang, Pei-Ru; Wang, Tzu-Pin

    2017-10-01

    3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) catalyzes the oxidation of androsterone with NAD + to form androstanedione and NADH with the rate limiting step being the release of NADH. In this study, we elucidate the role of remote substrate binding interactions contributing to the rate enhancement by 3α-HSD/CR through steady-state kinetic studies with the truncated substrate analogs. No enzyme activity was detected for methanol, ethanol, and 2-propanol, which lack the steroid scaffold of androsterone, implying that the steroid scaffold plays an important role in enzyme catalytic specificity. As compared to cyclohexanol, the activity for 2-decalol, androstenol, and androsterone increases by 0.9-, 90-, and 200-fold in k cat , and 37-, 1.9 × 10 6 -, and 1.8 × 10 6 -fold in k cat /K B , respectively. The rate limiting step is hydride transfer for 3α-HSD/CR catalyzing the reaction of cyclohexanol with NAD + based on the observed rapid equilibrium ordered mechanism and equal deuterium isotope effects of 3.9 on V and V/K for cyclohexanol. The k cat /K B value results in ΔG ‡ of 14.7, 12.6, 6.2, and 6.2 kcal/mol for the 3α-HSD/CR catalyzed reaction of cyclohexanol, 2-decalol, androstenol, and androsterone, respectively. Thus, the uniform binding energy from the B-ring of steroids with the active site of 3α-HSD/CR equally contributes 2.1 kcal/mol to stabilize both the transition state and ground state of the ternary complex, leading to the similarity in k cat for 2-decalol and cyclohexanol. Differential binding interactions of the remote BCD-ring and CD-ring of androsterone with the active site of 3α-HSD/CR contribute 8.5 and 6.4 kcal/mol to the stabilization of the transition state, respectively. The removal of the carbonyl group at C17 of androsterone has small effects on catalysis. Both uniform and differential binding energies from the remote sites of androsterone compared to cyclohexanol contribute to the 3α-HSD/CR catalysis

  3. Structure and function of the catalytic domain of the dihydrolipoyl acetyltransferase component in Escherichia coli pyruvate dehydrogenase complex.

    PubMed

    Wang, Junjie; Nemeria, Natalia S; Chandrasekhar, Krishnamoorthy; Kumaran, Sowmini; Arjunan, Palaniappa; Reynolds, Shelley; Calero, Guillermo; Brukh, Roman; Kakalis, Lazaros; Furey, William; Jordan, Frank

    2014-05-30

    The Escherichia coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components: E1p, E2p, and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LDs), a peripheral subunit binding domain (PSBD), and a catalytic domain (E2pCD). Herein are reported the following. 1) The x-ray structure of E2pCD revealed both intra- and intertrimer interactions, similar to those reported for other E2pCDs. 2) Reconstitution of recombinant LD and E2pCD with E1p and E3p into PDHc could maintain at least 6.4% activity (NADH production), confirming the functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD, and E3 even in the absence of PSBD and of a covalent link between domains within E2p. 3) Direct acetyl transfer between LD and coenzyme A catalyzed by E2pCD was observed with a rate constant of 199 s(-1), comparable with the rate of NADH production in the PDHc reaction. Hence, neither reductive acetylation of E2p nor acetyl transfer within E2p is rate-limiting. 4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by hydrogen/deuterium exchange mass spectrometry. The inclusion of each additional domain of E2p strengthened the interaction with E1p, and the interaction was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifested by the appearance of a circular dichroism band characteristic of the canonical 4'-aminopyrimidine tautomer of bound thiamin diphosphate (AP). © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. An efficient ribitol-specific dehydrogenase from Enterobacter aerogenes.

    PubMed

    Singh, Ranjitha; Singh, Raushan; Kim, In-Won; Sigdel, Sujan; Kalia, Vipin C; Kang, Yun Chan; Lee, Jung-Kul

    2015-05-01

    An NAD(+)-dependent ribitol dehydrogenase from Enterobacter aerogenes KCTC 2190 (EaRDH) was cloned and successfully expressed in Escherichia coli. The complete 729-bp gene was amplified, cloned, expressed, and subsequently purified in an active soluble form using nickel affinity chromatography. The enzyme had an optimal pH and temperature of 11.0 and 45°C, respectively. Among various polyols, EaRDH exhibited activity only toward ribitol, with Km, Vmax, and kcat/Km values of 10.3mM, 185Umg(-1), and 30.9s(-1)mM(-1), respectively. The enzyme showed strong preference for NAD(+) and displayed no detectable activity with NADP(+). Homology modeling and sequence analysis of EaRDH, along with its biochemical properties, confirmed that EaRDH belongs to the family of NAD(+)-dependent ribitol dehydrogenases, a member of short-chain dehydrogenase/reductase (SCOR) family. EaRDH showed the highest activity and unique substrate specificity among all known RDHs. Homology modeling and docking analysis shed light on the molecular basis of its unusually high activity and substrate specificity. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Highly selective anti-Prelog synthesis of optically active aryl alcohols by recombinant Escherichia coli expressing stereospecific alcohol dehydrogenase.

    PubMed

    Li, Ming; Nie, Yao; Mu, Xiao Qing; Zhang, Rongzhen; Xu, Yan

    2016-07-03

    Biocatalytic asymmetric synthesis has been widely used for preparation of optically active chiral alcohols as the important intermediates and precursors of active pharmaceutical ingredients. However, the available whole-cell system involving anti-Prelog specific alcohol dehydrogenase is yet limited. A recombinant Escherichia coli system expressing anti-Prelog stereospecific alcohol dehydrogenase from Candida parapsilosis was established as a whole-cell system for catalyzing asymmetric reduction of aryl ketones to anti-Prelog configured alcohols. Using 2-hydroxyacetophenone as the substrate, reaction factors including pH, cell status, and substrate concentration had obvious impacts on the outcome of whole-cell biocatalysis, and xylose was found to be an available auxiliary substrate for intracellular cofactor regeneration, by which (S)-1-phenyl-1,2-ethanediol was achieved with an optical purity of 97%e.e. and yield of 89% under the substrate concentration of 5 g/L. Additionally, the feasibility of the recombinant cells toward different aryl ketones was investigated, and most of the corresponding chiral alcohol products were obtained with an optical purity over 95%e.e. Therefore, the whole-cell system involving recombinant stereospecific alcohol dehydrogenase was constructed as an efficient biocatalyst for highly enantioselective anti-Prelog synthesis of optically active aryl alcohols and would be promising in the pharmaceutical industry.

  6. Role of alcohol dehydrogenase activity and the acetaldehyde in ethanol- induced ethane and pentane production by isolated perfused rat liver.

    PubMed Central

    Müller, A; Sies, H

    1982-01-01

    The volatile hydrocarbons ethane and n-pentane are produced at increased rates by isolated perfused rat liver during the metabolism of acutely ethanol. The effect is half-maximal at 0.5 mM-ethanol, and its is not observed when inhibitors of alcohol dehydrogenase such as 4-methyl- or 4-propyl-pyrazole are also present. Propanol, another substrate for the dehydrogenase, is also active. Increased alkane production can be initiated by adding acetaldehyde in the presence of 4-methyl- or 4-propyl-pyrazole. An antioxidant, cyanidanol, suppresses the ethanol-induced alkane production. The data obtained with the isolated organ demonstrate that products known to arise from the peroxidation of polyunsaturated fatty acids are formed in the presence of ethanol and that the activity of alcohol dehydrogenase is required for the generation of the active radical species. The mere presence of ethanol, e.g. at binding sites of special form(s) of cytochrome P-450, it not sufficient to elicit an increased production of volatile hydrocarbons by rat liver. PMID:6751324

  7. The Respiratory System and Diazotrophic Activity of Acetobacter diazotrophicus PAL5

    PubMed Central

    Flores-Encarnación, M.; Contreras-Zentella, M.; Soto-Urzua, L.; Aguilar, G. R.; Baca, B. E.; Escamilla, J. E.

    1999-01-01

    The characteristics of the respiratory system of Acetobacter diazotrophicus PAL5 were investigated. Increasing aeration (from 0.5 to 4.0 liters of air min−1 liter of medium−1) had a strong positive effect on growth and on the diazotrophic activity of cultures. Cells obtained from well-aerated and diazotrophically active cultures possessed a highly active, membrane-bound electron transport system with dehydrogenases for NADH, glucose, and acetaldehyde as the main electron donors. Ethanol, succinate, and gluconate were also oxidized but to only a minor extent. Terminal cytochrome c oxidase-type activity was poor as measured by reduced N,N,N,N′-tetramethyl-p-phenylenediamine, but quinol oxidase-type activity, as measured by 2,3,5,6-tetrachloro-1,4-benzenediol, was high. Spectral and high-pressure liquid chromatography analysis of membranes revealed the presence of cytochrome ba as a putative oxidase in cells obtained from diazotrophically active cultures. Cells were also rich in c-type cytochromes; four bands of high molecular mass (i.e., 67, 56, 52, and 45 kDa) were revealed by a peroxidase activity stain in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. KCN inhibition curves of respiratory oxidase activities were biphasic, with a highly resistant component. Treatment of membranes with 0.2% Triton X-100 solubilized c-type cytochromes and resulted in a preparation that was significantly more sensitive to cyanide. Repression of diazotrophic activity in well-aerated cultures by 40 mM (NH4)2SO4 caused a significant decrease of the respiratory activities. It is noteworthy that the levels of glucose dehydrogenase and putative oxidase ba decreased 6.8- and 10-fold, respectively. In these cells, a bd-type cytochrome seems to be the major terminal oxidase. Thus, it would seem that glucose dehydrogenase and cytochrome ba are key components of the respiratory system of A. diazotrophicus during aerobic diazotrophy. PMID:10559164

  8. Characterization and evolution of an activator-independent methanol dehydrogenase from Cupriavidus necator N-1.

    PubMed

    Wu, Tung-Yun; Chen, Chang-Ting; Liu, Jessica Tse-Jin; Bogorad, Igor W; Damoiseaux, Robert; Liao, James C

    2016-06-01

    Methanol utilization by methylotrophic or non-methylotrophic organisms is the first step toward methanol bioconversion to higher carbon-chain chemicals. Methanol oxidation using NAD-dependent methanol dehydrogenase (Mdh) is of particular interest because it uses NAD(+) as the electron carrier. To our knowledge, only a limited number of NAD-dependent Mdhs have been reported. The most studied is the Bacillus methanolicus Mdh, which exhibits low enzyme specificity to methanol and is dependent on an endogenous activator protein (ACT). In this work, we characterized and engineered a group III NAD-dependent alcohol dehydrogenase (Mdh2) from Cupriavidus necator N-1 (previously designated as Ralstonia eutropha). This enzyme is the first NAD-dependent Mdh characterized from a Gram-negative, mesophilic, non-methylotrophic organism with a significant activity towards methanol. Interestingly, unlike previously reported Mdhs, Mdh2 does not require activation by known activators such as B. methanolicus ACT and Escherichia coli Nudix hydrolase NudF, or putative native C. necator activators in the Nudix family under mesophilic conditions. This enzyme exhibited higher or comparable activity and affinity toward methanol relative to the B. methanolicus Mdh with or without ACT in a wide range of temperatures. Furthermore, using directed molecular evolution, we engineered a variant (CT4-1) of Mdh2 that showed a 6-fold higher K cat/K m for methanol and 10-fold lower K cat/K m for n-butanol. Thus, CT4-1 represents an NAD-dependent Mdh with much improved catalytic efficiency and specificity toward methanol compared with the existing NAD-dependent Mdhs with or without ACT activation.

  9. Subcellular distribution of 3 beta-hydroxysteroid dehydrogenase-isomerase in bovine and murine adrenocortical tissue: species differences in the localization of activity and immunoreactivity.

    PubMed

    Perry, J E; Ishii-Ohba, H; Stalvey, J R

    1991-06-01

    Key to the production of biologically active steroids is the enzyme 3 beta-hydroxysteroid dehydrogenase-isomerase. Some controversy has arisen concerning the subcellular distribution of this enzyme within steroidogenic cells. The distribution of 3 beta-hydroxysteroid dehydrogenase-isomerase was assessed in subcellular fractions obtained from homogenates of rat, bovine, and mouse adrenal glands in two ways. The activity of 3 beta-hydroxysteroid dehydrogenase-isomerase was quantitated by measuring the conversion of radiolabeled pregnenolone to radiolabeled progesterone in an aliquot of each of the fractions obtained. The presence of the enzyme was assessed by performing Western analyses on aliquots of each of the fractions obtained with the use of a specific polyclonal antiserum against 3 beta-hydroxysteroid dehydrogenase-isomerase, the characterization of which is described. In control experiments, the degree of contamination of the fractions was determined by assessing the presence of known subcellular fraction markers with Western analysis. In the bovine and mouse adrenal glands, 3 beta-hydroxysteroid dehydrogenase-isomerase appears to be localized solely in the microsomal fraction, while in the rat, 3 beta-hydroxysteroid dehydrogenase-isomerase appears to have dual subcellular distribution: the microsomes and the inner mitochondrial membrane. We conclude that there is a species difference in the subcellular distribution of this important steroidogenic enzyme and that this species difference may be related to the steroidogenic pathway preferred in that species.

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

    PubMed

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

    2012-08-01

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

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

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

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

    1986-08-10

    Quinate dehydrogenase was isolated from young needles of the Siberian larch and partially purified by ammonium sulfate fractionation. It was found that in conifers, in contrast to other plants, quinate dehydrogenase is active both with NAD and with NADP. The values of K/sub m/ for quinate and NADP were 1.8 and 0.18 mM. The enzyme exhibits maximum activity at pH 9.0. It was assumed that NADP-dependent quinate dehydrogenase is responsible for quinic acid synthesis. The special features of the organization and regulation of the initial stages of the shikimate pathway in conifers are discussed.

  12. In-vitro Wound Healing Effect of 15-Hydroxyprostaglandin Dehydrogenase Inhibitor from Plant

    PubMed Central

    Karna, Sandeep

    2017-01-01

    Background: Prostaglandins (PGs) have short existence in vivo because they are rapidly metabolized by NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) to 15-ketoprostaglandins. Inhibition of 15-PGDH causes elevated level of PGE2 in cellular system. It will be valuable for the therapeutic management of diseases requiring elevated PGE2 levels, like wound healing. Objective: Ninety-eight plant samples were screened for the discovery of potent 15-PGDH inhibitor. Among them, top five plant extracts as potent 15-PGDH inhibitor were chosen to determine PGE2 release from HaCaT (Keratinocyte cell line) cell line. Finally, top 15-PGDH inhibitor was selected to evaluate in vitro wound healing effect on HaCaT scratch model. Method: The inhibitory activity for 15-PGDH inhibitors was evaluated using fluorescence spectrophotometer by measuring the formation of NADH at 468 nm following excitation at 340 nm. Cell viability assay and PGE2 release was evaluated in HaCaT cell line after treatment of 15-PGDH inhibitors. Scratches were made using sterile 200 μL on HaCaT cell and wound-healing effect was evaluated after treatment of 15-PGDH inhibitor. Results: 15-PGDH inhibitors elevated PGE2 levels in concentration-dependent manner. Ethanol extract of Artocarpus heterophyllus (EEAH), the most potent 15-PGDH inhibitor (IC50 = 0.62 µg/mL) with least cytotoxicity (IC50 = 670 µg/ml), elevated both intracellular and extracellular PGE2 levels. EEAH facilitated in-vitro wound healing in a HaCaT (Keratinocyte cell line) scratch model. Conclusion: EEAH might apply to treat dermal wounds by elevating PGE2 levels via COX-1 induction and 15-PGDH inhibition. SUMMARY Biological inactivation of 15-PGDH causes elevated level of PGE2 which will be useful for the management of disease that requires elevated level of PGE2. Abbreviations used: 15-PGDH: 15-hydroxyprostaglandin dehydrogenase, COX: Cyclooxygenase, DTT: Dithiothreitol, DMEM: Dulbecco's modified Eagle's media, EEAH: Ethanol

  13. In-vitro Wound Healing Effect of 15-Hydroxyprostaglandin Dehydrogenase Inhibitor from Plant.

    PubMed

    Karna, Sandeep

    2017-01-01

    Prostaglandins (PGs) have short existence in vivo because they are rapidly metabolized by NAD + -dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) to 15-ketoprostaglandins. Inhibition of 15-PGDH causes elevated level of PGE 2 in cellular system. It will be valuable for the therapeutic management of diseases requiring elevated PGE 2 levels, like wound healing. Ninety-eight plant samples were screened for the discovery of potent 15-PGDH inhibitor. Among them, top five plant extracts as potent 15-PGDH inhibitor were chosen to determine PGE 2 release from HaCaT (Keratinocyte cell line) cell line. Finally, top 15-PGDH inhibitor was selected to evaluate in vitro wound healing effect on HaCaT scratch model. The inhibitory activity for 15-PGDH inhibitors was evaluated using fluorescence spectrophotometer by measuring the formation of NADH at 468 nm following excitation at 340 nm. Cell viability assay and PGE 2 release was evaluated in HaCaT cell line after treatment of 15-PGDH inhibitors. Scratches were made using sterile 200 μL on HaCaT cell and wound-healing effect was evaluated after treatment of 15-PGDH inhibitor. 15-PGDH inhibitors elevated PGE 2 levels in concentration-dependent manner. Ethanol extract of Artocarpus heterophyllus (EEAH), the most potent 15-PGDH inhibitor (IC 50 = 0.62 µg/mL) with least cytotoxicity (IC 50 = 670 µg/ml), elevated both intracellular and extracellular PGE 2 levels. EEAH facilitated in-vitro wound healing in a HaCaT (Keratinocyte cell line) scratch model. EEAH might apply to treat dermal wounds by elevating PGE 2 levels via COX-1 induction and 15-PGDH inhibition. Biological inactivation of 15-PGDH causes elevated level of PGE 2 which will be useful for the management of disease that requires elevated level of PGE 2 . Abbreviations used: 15-PGDH: 15-hydroxyprostaglandin dehydrogenase, COX: Cyclooxygenase, DTT: Dithiothreitol, DMEM: Dulbecco's modified Eagle's media, EEAH: Ethanol extract of Artocarpus heterophyllus, MRP4

  14. 3-cyanoindole-based inhibitors of inosine monophosphate dehydrogenase: synthesis and initial structure-activity relationships.

    PubMed

    Dhar, T G Murali; Shen, Zhongqi; Gu, Henry H; Chen, Ping; Norris, Derek; Watterson, Scott H; Ballentine, Shelley K; Fleener, Catherine A; Rouleau, Katherine A; Barrish, Joel C; Townsend, Robert; Hollenbaugh, Diane L; Iwanowicz, Edwin J

    2003-10-20

    A series of novel small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH), based upon a 3-cyanoindole core, were explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SAR), derived from in vitro studies, for this new series of inhibitors is given.

  15. 21 CFR 862.1565 - 6-Phosphogluconate dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  16. 21 CFR 862.1565 - 6-Phosphogluconate dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  17. Effects of 14 days of spaceflight and nine days of recovery on cell body size and succinate dehydrogenase activity of rat dorsal root ganglion neurons

    NASA Technical Reports Server (NTRS)

    Ishihara, A.; Ohira, Y.; Roy, R. R.; Nagaoka, S.; Sekiguchi, C.; Hinds, W. E.; Edgerton, V. R.

    1997-01-01

    The cross-sectional areas and succinate dehydrogenase activities of L5 dorsal root ganglion neurons in rats were determined after 14 days of spaceflight and after nine days of recovery. The mean and distribution of the cross-sectional areas were similar to age-matched, ground-based controls for both the spaceflight and for the spaceflight plus recovery groups. The mean succinate dehydrogenase activity was significantly lower in spaceflight compared to aged-matched control rats, whereas the mean succinate dehydrogenase activity was similar in age-matched control and spaceflight plus recovery rats. The mean succinate dehydrogenase activity of neurons with cross-sectional areas between 1000 and 2000 microns2 was lower (between 7 and 10%) in both the spaceflight and the spaceflight plus recovery groups compared to the appropriate control groups. The reduction in the oxidative capacity of a subpopulation of sensory neurons having relatively large cross-sectional areas immediately following spaceflight and the sustained depression for nine days after returning to 1 g suggest that the 0 g environment induced significant alterations in proprioceptive function.

  18. Characterization of human short chain dehydrogenase/reductase SDR16C family members related to retinol dehydrogenase 10.

    PubMed

    Adams, Mark K; Lee, Seung-Ah; Belyaeva, Olga V; Wu, Lizhi; Kedishvili, Natalia Y

    2017-10-01

    All-trans-retinoic acid (RA) is a bioactive derivative of vitamin A that serves as an activating ligand for nuclear transcription factors, retinoic acid receptors. RA biosynthesis is initiated by the enzymes that oxidize retinol to retinaldehyde. It is well established that retinol dehydrogenase 10 (RDH10, SDR16C4), which belongs to the 16C family of the short chain dehydrogenase/reductase (SDR) superfamily of proteins, is the major enzyme responsible for the oxidation of retinol to retinaldehyde for RA biosynthesis during embryogenesis. However, several lines of evidence point towards the existence of additional retinol dehydrogenases that contribute to RA biosynthesis in vivo. In close proximity to RDH10 gene on human chromosome 8 are located two genes that are phylogenetically related to RDH10. The predicted protein products of these genes, retinol dehydrogenase epidermal 2 (RDHE2, SDR16C5) and retinol dehydrogenase epidermal 2-similar (RDHE2S, SDR16C6), share 59% and 56% sequence similarity with RDH10, respectively. Previously, we showed that the single ortholog of the human RDHE2 and RDHE2S in frogs, Xenopus laevis rdhe2, oxidizes retinol to retinaldehyde and is essential for frog embryonic development. In this study, we explored the potential of each of the two human proteins to contribute to RA biosynthesis. The results of this study demonstrate that human RDHE2 exhibits a relatively low but reproducible activity when expressed in either HepG2 or HEK293 cells. Expression of the native RDHE2 is downregulated in the presence of elevated levels of RA. On the other hand, the protein encoded by the human RDHE2S gene is unstable when expressed in HEK293 cells. RDHE2S protein produced in Sf9 cells is stable but has no detectable catalytic activity towards retinol. We conclude that the human RDHE2S does not contribute to RA biosynthesis, whereas the low-activity RA-sensitive human RDHE2 may have a role in adjusting the cellular levels of RA in accord with

  19. H2O2 Production in Species of the Lactobacillus acidophilus Group: a Central Role for a Novel NADH-Dependent Flavin Reductase

    PubMed Central

    Hertzberger, Rosanne; Arents, Jos; Dekker, Henk L.; Pridmore, R. David; Gysler, Christof; Kleerebezem, Michiel

    2014-01-01

    Hydrogen peroxide production is a well-known trait of many bacterial species associated with the human body. In the presence of oxygen, the probiotic lactic acid bacterium Lactobacillus johnsonii NCC 533 excretes up to 1 mM H2O2, inducing growth stagnation and cell death. Disruption of genes commonly assumed to be involved in H2O2 production (e.g., pyruvate oxidase, NADH oxidase, and lactate oxidase) did not affect this. Here we describe the purification of a novel NADH-dependent flavin reductase encoded by two highly similar genes (LJ_0548 and LJ_0549) that are conserved in lactobacilli belonging to the Lactobacillus acidophilus group. The genes are predicted to encode two 20-kDa proteins containing flavin mononucleotide (FMN) reductase conserved domains. Reductase activity requires FMN, flavin adenine dinucleotide (FAD), or riboflavin and is specific for NADH and not NADPH. The Km for FMN is 30 ± 8 μM, in accordance with its proposed in vivo role in H2O2 production. Deletion of the encoding genes in L. johnsonii led to a 40-fold reduction of hydrogen peroxide formation. H2O2 production in this mutant could only be restored by in trans complementation of both genes. Our work identifies a novel, conserved NADH-dependent flavin reductase that is prominently involved in H2O2 production in L. johnsonii. PMID:24487531

  20. The NAD(P)H-dependent glutamate dehydrogenase activities of Prevotella ruminicola B(1)4 can be attributed to one enzyme (GdhA), and gdhA expression is regulated in response to the nitrogen source available for growth.

    PubMed Central

    Wen, Z; Morrison, M

    1996-01-01

    Prevotella ruminicola B(1)4 possesses both NADPH- and NADH-linked glutamate dehydrogenase (GDH) activities, with the greatest specific activity being measured from ammonia-limited cultures. Relative to cells grown in the presence of 1 mM ammonium chloride, the NADPH-dependent activity was decreased approximately 10-fold when peptides were provided as a nitrogen source. Nondenaturing polyacrylamide gel electrophoresis (PAGE) was used to visualize the GDH protein(s) in cell extracts of P. ruminicola. For all growth conditions tested, only one GDH protein was detectable, and its relative abundance, as well as its reactivity with either NAD(P)+ or NAD(P)H, correlated well with the specific activities measured from whole-cell assays. Consistent with the findings from enzyme assays and PAGE activity gels, Northern (RNA) blot analysis revealed that expression of a gene encoding NAD(P)H-GDH activity was greatest in ammonia-grown cultures and that GDH activity is regulated in response to nitrogen source (ammonia versus peptides), probably at the level of transcription. A gene encoding the NAD(P)H-utilizing GDH activity (gdhA) was cloned, and its nucleotide sequence was determined and shown to contain an open reading frame of 1,332 bp which would encode a polypeptide of 48.8 kDa. The deduced amino acid sequence possesses three highly conserved motifs typical of family I GDHs, but several unique amino acid substitutions within these motifs were evident. These results are discussed within the context of ruminal nitrogen metabolism and the growth efficiency of succinate- and propionate-producing anaerobic bacteria. PMID:8837439

  1. Site-directed mutagenesis of conserved cysteine residues in NqrD and NqrE subunits of Na+-translocating NADH:quinone oxidoreductase.

    PubMed

    Fadeeva, M S; Bertsova, Y V; Verkhovsky, M I; Bogachev, A V

    2008-02-01

    Each of two hydrophobic subunits of Na+-translocating NADH:quinone oxidoreductase (NQR), NqrD and NqrE, contain a pair of strictly conserved cysteine residues within their transmembrane alpha-helices. Site-directed mutagenesis showed that substitutions of these residues in NQR of Vibrio harveyi blocked the Na+-dependent and 2-n-heptyl-4-hydroxyquinoline N-oxide-sensitive quinone reductase activity of the enzyme. However, these mutations did not affect the interaction of NQR with NADH and menadione. It was demonstrated that these conserved cysteine residues are necessary for the correct folding and/or the stability of the NQR complex. Mass and EPR spectroscopy showed that NQR from V. harveyi bears only a 2Fe-2S cluster as a metal-containing prosthetic group.

  2. A novel 17β-hydroxysteroid dehydrogenase in Rhodococcus sp. P14 for transforming 17β-estradiol to estrone.

    PubMed

    Ye, Xueying; Wang, Hui; Kan, Jie; Li, Jin; Huang, Tongwang; Xiong, Guangming; Hu, Zhong

    2017-10-01

    17β-hydroxysteroid dehydrogenases (17β-HSD) are a group of oxidoreductase enzymes that exhibit high specificity for 17C reduction/oxidation. However, the mechanism of 17β-HSD in oxidizing steroid hormone 17β-estradiol to estrone in bacterium is still unclear. In this work, a functional bacterium Rhodococcus sp. P14 was identified having rapid ability to oxidize estradiol into estrone in mineral salt medium (MSM) within 6 h. The functional genes encoding NADH-dependent oxidoreductase were successfully detected with the help of bioinformatics, and it was identified that it contained two consensus regions affiliated to the short-chain dehydrogenase/reductase (SDR) superfamily. Expression of 17β-HSD could be induced by estradiol in strain P14. The 17β-HSD gene from Rhodococcus sp. P14 was expressed in Escherichia coli strain BL21. Furthermore, recombinant 17β-HSD-expressing BL21 cells showed a high transformation rate, they are capable of transforming estradiol to estrone up to 94%. The purified His-17β-HSD protein also exhibited high catalyzing efficiency. In conclusion, this study provides the first evidence that a novel 17β-HSD in Rhodococcus sp. P14 can catalyze the oxidation of estradiol. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Nitrate transport is independent of NADH and NAD(P)H nitrate reductases in barley seedlings

    NASA Technical Reports Server (NTRS)

    Warner, R. L.; Huffaker, R. C.

    1989-01-01

    Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings.

  4. Involvement of NADH Oxidase in Biofilm Formation in Streptococcus sanguinis

    PubMed Central

    Ge, Xiuchun; Shi, Xiaoli; Shi, Limei; Liu, Jinlin; Stone, Victoria; Kong, Fanxiang; Kitten, Todd; Xu, Ping

    2016-01-01

    Biofilms play important roles in microbial communities and are related to infectious diseases. Here, we report direct evidence that a bacterial nox gene encoding NADH oxidase is involved in biofilm formation. A dramatic reduction in biofilm formation was observed in a Streptococcus sanguinis nox mutant under anaerobic conditions without any decrease in growth. The membrane fluidity of the mutant bacterial cells was found to be decreased and the fatty acid composition altered, with increased palmitic acid and decreased stearic acid and vaccenic acid. Extracellular DNA of the mutant was reduced in abundance and bacterial competence was suppressed. Gene expression analysis in the mutant identified two genes with altered expression, gtfP and Idh, which were found to be related to biofilm formation through examination of their deletion mutants. NADH oxidase-related metabolic pathways were analyzed, further clarifying the function of this enzyme in biofilm formation. PMID:26950587

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

    PubMed Central

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

    1994-01-01

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

  6. Palladium alpha-lipoic acid complex formulation enhances activities of Krebs cycle dehydrogenases and respiratory complexes I-IV in the heart of aged rats.

    PubMed

    Sudheesh, N P; Ajith, T A; Janardhanan, K K; Krishnan, C V

    2009-08-01

    Age-related decline in the capacity to withstand stress, such as ischemia and reperfusion, results in congestive heart failure. Though the mechanisms underlying cardiac decay are not clear, age dependent somatic damages to mitochondrial DNA (mtDNA), loss of mitochondrial function, and a resultant increase in oxidative stress in heart muscle cells may be responsible for the increased risk for cardiovascular diseases. The effect of a safe nutritional supplement, POLY-MVA, containing the active ingredient palladium alpha-lipoic acid complex, was evaluated on the activities of the Krebs cycle enzymes such as isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase as well as mitochondrial complexes I, II, III, and IV in heart mitochondria of aged male albino rats of Wistar strain. Administration of 0.05 ml/kg of POLY-MVA (which is equivalent to 0.38 mg complexed alpha-lipoic acid/kg, p.o), once daily for 30 days, was significantly (p<0.05) effective to enhance the Krebs cycle dehydrogenases, and mitochondrial electron transport chain complexes. The unique electronic and redox properties of palladium alpha-lipoic acid complex appear to be a key to this physiological effectiveness. The results strongly suggest that this formulation might be effective to protect the aging associated risk of cardiovascular and neurodegenerative diseases.

  7. Catalysis of nitrite generation from nitroglycerin by glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

    PubMed

    Seabra, Amedea B; Ouellet, Marc; Antonic, Marija; Chrétien, Michelle N; English, Ann M

    2013-11-30

    Vascular relaxation to nitroglycerin (glyceryl trinitrate; GTN) requires its bioactivation by mechanisms that remain controversial. We report here that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the release of nitrite from GTN. In assays containing dithiothreitol (DTT) and NAD(+), the GTN reductase activity of purified GAPDH produces nitrite and 1,2-GDN as the major products. A vmax of 2.6nmolmin(-)(1)mg(-)(1) was measured for nitrite production by GAPDH from rabbit muscle and a GTN KM of 1.2mM. Reductive denitration of GTN in the absence of DTT results in dose- and time-dependent inhibition of GAPDH dehydrogenase activity. Disulfiram, a thiol-modifying drug, inhibits both the dehydrogenase and GTN reductase activity of GAPDH, while DTT or tris(2-carboxyethyl)phosphine reverse the GTN-induced inhibition. Incubation of intact human erythrocytes or hemolysates with 2mM GTN for 60min results in 50% inhibition of GAPDH's dehydrogenase activity, indicating that GTN is taken up by these cells and that the dehydrogenase is a target of GTN. Thus, erythrocyte GAPDH may contribute to GTN bioactivation. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

  8. Polarized fluorescence in NADH under two-photon excitation with femtosecond laser pulses

    NASA Astrophysics Data System (ADS)

    Vasyutinskii, O. S.; Smolin, A. G.; Oswald, C.; Gericke, K. H.

    2017-04-01

    Polarized fluorescence decay in NADH molecules in aqueous solution under two-photon excitation by femtosecond laser pulses has been studied. The excitation was carried out by linear and circularly polarized radiation at four wavelengths: 720, 730, 740, and 750 nm. Time-dependent polarized fluorescence signals were recorded as a function of the excitation light polarization and used for determination of a set of molecular parameters, two lifetimes characterizing the molecular excited states, and the rotation correlation time τrot. The results obtained can be used to create and prove theoretical models describing the intensity and polarization of fluorescence in NADH involved in the regulation of the redox reactions in cells and tissues of living organisms.

  9. Stereospecificity of NAD+/NADH Reactions: A Project Experiment for Advanced Undergraduates.

    ERIC Educational Resources Information Center

    Lowrey, Jonathan S.; And Others

    1981-01-01

    Presents background information, materials needed, and experimental procedures to study enzymes dependent on pyridine nucleotide coenzymes (NAD/NADH). The experiments, suitable for advanced organic or biochemistry courses, require approximately 10-15 hours to complete. (SK)

  10. Comparison of oral nicotinamide adenine dinucleotide (NADH) versus conventional therapy for chronic fatigue syndrome.

    PubMed

    Santaella, María L; Font, Ivonne; Disdier, Orville M

    2004-06-01

    To compare effectiveness of oral therapy with reduced nicotinamide adenine dinucleotide (NADH) to conventional modalities of treatment in patients with chronic fatigue syndrome (CFS). CFS is a potentially disabling condition of unknown etiology. Although its clinical presentation is associated to a myriad of symptoms, fatigue is a universal and essential finding for its diagnosis. No therapeutic regimen has proven effective for this condition. A total of 31 patients fulfilling the Centers for Disease Control criteria for CFS, were randomly assigned to either NADH or nutritional supplements and psychological therapy for 24 months. A thorough medical history, physical examination and completion of a questionnaire on the severity of fatigue and other symptoms were performed each trimester of therapy. In addition, all of them underwent evaluation in terms of immunological parameters and viral antibody titers. Statistical analysis was applied to the demographic data, as well as to symptoms scores at baseline and at each trimester of therapy. The twelve patients who received NADH had a dramatic and statistically significant reduction of the mean symptom score in the first trimester (p < 0.001). However, symptom scores in the subsequent trimesters of therapy were similar in both treatment groups. Elevated IgG and Ig E antibody levels were found in a significant number of patients. Observed effectiveness of NADH over conventional treatment in the first trimester of the trial and the trend of improvement of that modality in the subsequent trimesters should be further assessed in a larger patient sample.

  11. Alterations in the activities of three dehydrogenases in the digestive system of two teleost fishes exposed to mercuric chloride

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

    Gupta, P.K.; Sastry, K.V.

    1981-02-01

    The effect of the 50% lethal concentration and of a sublethal concentration (0.3 mg/liter) of mercuric chloride on the activities of succinic, lactic, and pyruvic dehydrogenases in the digestive system of two teleost fishes, Ophiocephalus punctatus and Heteropneustes fossilis, respectively, has been studied at intervals of 96 h and 7, 15, and 30 days. The results show that dehydrogenases are not affected much by short-term exposure. However, the activities of all three enzymes are inhibited by chronic exposure to mercury and maximum inhibition is observed after 15 days of exposure. Among the different parts of the digestive system, the livermore » is the most affected organ, and of the two fishes, Heteropneustes is more sensitive to mercury treatment.« less

  12. Digitalis metabolism and human liver alcohol dehydrogenase.

    PubMed Central

    Frey, W A; Vallee, B L

    1980-01-01

    Human liver alcohol dehydrogenase (alcohol: NAD" oxidoreductase, EC 1.1.1.1) catalyzes the oxidation of the 3 beta-OH group of digitoxigenin, digoxigenin, and gitoxigenin to their 3-keto derivatives, which have been characterized by high performance liquid chromatography and mass spectrometry. These studies have identified human liver alcohol dehydrogenase as the unknown NAD(H)-dependent liver enzyme specific for the free hydroxyl group at C3 of the cardiac genins; this hydroxyl is the critical site of the genins' enzymatic oxidation and concomitant pharmacological inactivation in humans. Several kinetic approaches have demonstrated that ethanol and the pharmacologically active components of the digitalis glycosides are oxidized with closely similar kcat/Km values at the same site on human liver alcohol dehydrogenase, for which they compete. Human liver alcohol dehydrogenase thereby becomes an important biochemical link in the metabolism, pharmacology, and toxicology of ethanol and these glycosides, structurally unrelated agents that are both used widely. Both the competition of ethanol with these cardiac sterols and the narrow margin of safety in the therapeutic use of digitalis derivatives would seem to place at increased risk those individuals who receive digitalis and simultaneously consume large amounts of ethanol or whose alcohol dehydrogenase function is impaired. PMID:6987673

  13. FLIM data analysis of NADH and Tryptophan autofluorescence in prostate cancer cells

    NASA Astrophysics Data System (ADS)

    O'Melia, Meghan J.; Wallrabe, Horst; Svindrych, Zdenek; Rehman, Shagufta; Periasamy, Ammasi

    2016-03-01

    Fluorescence lifetime imaging microscopy (FLIM) is one of the most sensitive techniques to measure metabolic activity in living cells, tissues and whole animals. We used two- and three-photon fluorescence excitation together with time-correlated single photon counting (TCSPC) to acquire FLIM signals from normal and prostate cancer cell lines. FLIM requires complex data fitting and analysis; we explored different ways to analyze the data to match diverse cellular morphologies. After non-linear least square fitting of the multi-photon TCSPC images by the SPCImage software (Becker & Hickl), all image data are exported and further processed in ImageJ. Photon images provide morphological, NAD(P)H signal-based autofluorescent features, for which regions of interest (ROIs) are created. Applying these ROIs to all image data parameters with a custom ImageJ macro, generates a discrete, ROI specific database. A custom Excel (Microsoft) macro further analyzes the data with charts and statistics. Applying this highly automated assay we compared normal and cancer prostate cell lines with respect to their glycolytic activity by analyzing the NAD(P)H-bound fraction (a2%), NADPH/NADH ratio and efficiency of energy transfer (E%) for Tryptophan (Trp). Our results show that this assay is able to differentiate the effects of glucose stimulation and Doxorubicin in these prostate cell lines by tracking the changes in a2% of NAD(P)H, NADPH/NADH ratio and the changes in Trp E%. The ability to isolate a large, ROI-based data set, reflecting the heterogeneous cellular environment and highlighting even subtle changes -- rather than whole cell averages - makes this assay particularly valuable.

  14. Postdate pregnancy: changes of placental/membranes 11β-hydroxysteroid dehydrogenase mRNA and activity.

    PubMed

    Novembri, R; Voltolini, C; Torricelli, M; Severi, F M; Marcolongo, P; Benedetti, A; Challis, J R; Petraglia, F

    2013-11-01

    11β-Hydroxysteroid dehydrogenase 1 and 2 (11β-HSD1 and 11β-HSD2) are involved in the complex mechanism of human parturition. The present study examined mRNA expression and activity of membrane 11β-HSD1 and placental 11β-HSD2 in postdate pregnancies according to response of labor induction. In comparison to postdate women who had spontaneous delivery or after induction the non-responders showed significantly low c and high 11β-HSD2 expression and activity These data suggest that disrupted expression and activity of 11β-HSDs may occur in some postdate pregnancies. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Catalytic Mechanism of Short Ethoxy Chain Nonylphenol Dehydrogenase Belonging to a Polyethylene Glycol Dehydrogenase Group in the GMC Oxidoreductase Family

    PubMed Central

    Liu, Xin; Ohta, Takeshi; Kawabata, Takeshi; Kawai, Fusako

    2013-01-01

    Ethoxy (EO) chain nonylphenol dehydrogenase (NPEO-DH) from Ensifer sp. AS08 and EO chain octylphenol dehydrogenase from Pseudomonas putida share common molecular characteristics with polyethylene glycol (PEG) dehydrogenases (PEG-DH) and comprise a PEG-DH subgroup in the family of glucose-methanol-choline (GMC) oxidoreductases that includes glucose/alcohol oxidase and glucose/choline dehydrogenase. Three-dimensional (3D) molecular modeling suggested that differences in the size, secondary structure and hydropathy in the active site caused differences in their substrate specificities toward EO chain alkylphenols and free PEGs. Based on 3D molecular modeling, site-directed mutagenesis was utilized to introduce mutations into potential catalytic residues of NPEO-DH. From steady state and rapid kinetic characterization of wild type and mutant NPEO-DHs, we can conclude that His465 and Asn507 are directly involved in the catalysis. Asn507 mediates the transfer of proton from a substrate to FAD and His465 transfers the same proton from the reduced flavin to an electron acceptor. PMID:23306149

  16. Catalytic mechanism of short ethoxy chain nonylphenol dehydrogenase belonging to a polyethylene glycol dehydrogenase group in the GMC oxidoreductase family.

    PubMed

    Liu, Xin; Ohta, Takeshi; Kawabata, Takeshi; Kawai, Fusako

    2013-01-10

    Ethoxy (EO) chain nonylphenol dehydrogenase (NPEO-DH) from Ensifer sp. AS08 and EO chain octylphenol dehydrogenase from Pseudomonas putida share common molecular characteristics with polyethylene glycol (PEG) dehydrogenases (PEG-DH) and comprise a PEG-DH subgroup in the family of glucose-methanol-choline (GMC) oxidoreductases that includes glucose/alcohol oxidase and glucose/choline dehydrogenase. Three-dimensional (3D) molecular modeling suggested that differences in the size, secondary structure and hydropathy in the active site caused differences in their substrate specificities toward EO chain alkylphenols and free PEGs. Based on 3D molecular modeling, site-directed mutagenesis was utilized to introduce mutations into potential catalytic residues of NPEO-DH. From steady state and rapid kinetic characterization of wild type and mutant NPEO-DHs, we can conclude that His465 and Asn507 are directly involved in the catalysis. Asn507 mediates the transfer of proton from a substrate to FAD and His465 transfers the same proton from the reduced flavin to an electron acceptor.

  17. Light Driven CO2 Fixation by Using Cyanobacterial Photosystem I and NADPH-Dependent Formate Dehydrogenase

    PubMed Central

    Ihara, Masaki; Kawano, Yusuke; Urano, Miho; Okabe, Ayako

    2013-01-01

    The ultimate goal of this research is to construct a new direct CO2 fixation system using photosystems in living algae. Here, we report light-driven formate production from CO2 by using cyanobacterial photosystem I (PS I). Formate, a chemical hydrogen carrier and important industrial material, can be produced from CO2 by using the reducing power and the catalytic function of formate dehydrogenase (FDH). We created a bacterial FDH mutant that experimentally switched the cofactor specificity from NADH to NADPH, and combined it with an in vitro-reconstituted cyanobacterial light-driven NADPH production system consisting of PS I, ferredoxin (Fd), and ferredoxin-NADP+-reductase (FNR). Consequently, light-dependent formate production under a CO2 atmosphere was successfully achieved. In addition, we introduced the NADPH-dependent FDH mutant into heterocysts of the cyanobacterium Anabaena sp. PCC 7120 and demonstrated an increased formate concentration in the cells. These results provide a new possibility for photo-biological CO2 fixation. PMID:23936519

  18. Light driven CO2 fixation by using cyanobacterial photosystem I and NADPH-dependent formate dehydrogenase.

    PubMed

    Ihara, Masaki; Kawano, Yusuke; Urano, Miho; Okabe, Ayako

    2013-01-01

    The ultimate goal of this research is to construct a new direct CO2 fixation system using photosystems in living algae. Here, we report light-driven formate production from CO2 by using cyanobacterial photosystem I (PS I). Formate, a chemical hydrogen carrier and important industrial material, can be produced from CO2 by using the reducing power and the catalytic function of formate dehydrogenase (FDH). We created a bacterial FDH mutant that experimentally switched the cofactor specificity from NADH to NADPH, and combined it with an in vitro-reconstituted cyanobacterial light-driven NADPH production system consisting of PS I, ferredoxin (Fd), and ferredoxin-NADP(+)-reductase (FNR). Consequently, light-dependent formate production under a CO2 atmosphere was successfully achieved. In addition, we introduced the NADPH-dependent FDH mutant into heterocysts of the cyanobacterium Anabaena sp. PCC 7120 and demonstrated an increased formate concentration in the cells. These results provide a new possibility for photo-biological CO2 fixation.

  19. Engineered microorganisms capable of producing target compounds under anaerobic conditions

    DOEpatents

    Buelter, Thomas [Denver, CO; Meinhold, Peter [Denver, CO; Feldman, Reid M. Renny [San Francisco, CA; Hawkins, Andrew C [Parker, CO; Urano, Jun [Irvine, CA; Bastian, Sabine [Pasadena, CA; Arnold, Frances [La Canada, CA

    2012-01-17

    The present invention is generally provides recombinant microorganisms comprising engineered metabolic pathways capable of producing C3-C5 alcohols under aerobic and anaerobic conditions. The invention further provides ketol-acid reductoisomerase enzymes which have been mutated or modified to increase their NADH-dependent activity or to switch the cofactor preference from NADPH to NADH and are expressed in the modified microorganisms. In addition, the invention provides isobutyraldehyde dehydrogenase enzymes expressed in modified microorganisms. Also provided are methods of producing beneficial metabolites under aerobic and anaerobic conditions by contacting a suitable substrate with the modified microorganisms of the present invention.

  20. Glyceraldehyde-3-phosphate dehydrogenase from Chironomidae showed differential activity towards metals.

    PubMed

    Chong, Isaac K W; Ho, Wing S

    2013-09-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known to interact with different biomolecules and was implicated in many novel cellular activities including programmed cell death, nuclear RNA transport unrelated to the commonly known carbohydrate metabolism. We reported here the purification of GAPDH from Chironomidae larvae (Insecta, Diptera) that showed different biologic activity towards heavy metals. It was inhibited by copper, cobalt nickel, iron and lead but was activated by zinc. The GAPDH was purified by ammonium sulphate fractionation and Chelating Sepharose CL-6B chromatography followed by Blue Sepharose CL-6B chromatography. The 150-kDa tetrameric GAPDH showed optimal activity at pH 8.5 and 37°C. The multiple alignment of sequence of the Chironomidae GAPDH with other known species showed 78 - 88% identity to the conserved regions of the GADPH. Bioinformatic analysis unveils substantial N-terminal sequence similarity of GAPDH of Chironomidae larvae to mammalian GADPHs. However, the GADPH of Chironomidae larvae showed different biologic activities and cytotoxicity towards heavy metals. The GAPDH enzyme would undergo adaptive molecular changes through binding at the active site leading to higher tolerance to heavy metals.

  1. New approach to biosensing of co-enzyme nicotinamide adenine dinucleotide (NADH) by incorporation of neutral red in aluminum doped nanostructured ZnO thin films.

    PubMed

    V T, Fidal; T S, Chandra

    2017-06-01

    Biosensing of NADH on bare electrodes has drawbacks such as high over-potential and poisoning during the oxidation reaction. To overcome this challenge a different approach has been undertaken by incorporating neutral red (NR) in Al doped ZnO (AZO) thin films using one-pot chemical bath deposition (CBD). The surface morphology of the films was hexagonal nanorods along the c-axis, perpendicular to the substrate. The thickness of the thin films were ranging from 400 to 3000nm varying dependent on time of deposition (30 to 150min). The average diameter of the nanorods was larger in the presence of neutral red (NR-AZO) with ~300nm in contrast to its absence (AZO) with ~200nm. The density of the packing of nanorods was dependent on the citrate concentration used during deposition. Control over the dopant concentration in the films was achieved by varying the area of Al foil used in the deposition solution. The selected area diffraction (SAED) and X-ray diffraction (XRD) indicated 002 plane of orientation in the nanorods. FTIR and FT-Raman analysis revealed conserved structure of NR and AZO. Chronoamperometric (CA) analysis showed a sensitivity of 0.45μAcm -2 mM -1 and LoD of 22μM within the range 0.075-4mM of NADH. The biological sensing of NADH was validated by physical adsorption of NAD + dependent-lactate dehydrogenase (LDH) on NR-AZO. CA showed sensitivity of 0.56μAcm -2 mM -1 and LoD for lactate was 27μM in the range of 0.1-1mM of lactate. Further validation with real-time serum sample shows that LDH/NR-AZO correlates with the clinical values. The distinction in this study is that the organic mediator like neutral red has been incorporated into the grain structure of the ZnO thin film whereas other study with the mediators have only attempted surface functionalization. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader. Copyright © 2017 Elsevier B.V. All rights

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-04-01

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

  4. Leukocyte glutamate dehydrogenase activity in patients with degenerative neurological disorders.

    PubMed Central

    Aubby, D; Saggu, H K; Jenner, P; Quinn, N P; Harding, A E; Marsden, C D

    1988-01-01

    Leukocyte glutamate dehydrogenase (GDH) activity was measured in 39 normal subjects, 32 neurological controls, 66 patients with progressive ataxic disorders, 32 with multiple system atrophy, 40 with Parkinson's disease, eight with Steele-Richardson-Olszewski syndrome, eight with juvenile Parkinsonism and four with the dystonia-Parkinsonism syndrome. GDH activity was reproducible to within 10% in leukocyte pellets stored at -70 degrees C for up to 9 months, and did not vary with sex or age in control subjects. There was marked variation in the relative proportions of heat stable and heat labile forms of GDH between control subjects and on repeated assay in the same subject. Total leukocyte GDH activity was similar in normal subjects and neurological controls. Mean total GDH activity was reduced in all patient groups by between 15 to 29% compared with controls. Fourteen patients had total GDH activity below 50% of the control mean, but low values were not specific for any one disease (five had ataxic disorders, four Parkinson's disease, three multiple system atrophy, one juvenile Parkinsonism, and one dystonia-Parkinsonism). The heat labile fraction of GDH represented about 20% of total activity in control subjects, and 27% in the patients with reduced total GDH activity. Thus low GDH activity was not disease-specific in this study, and the heat-labile GDH fraction was not selectively affected. "Reduced" leucocyte GDH activity in some patients may represent no more than the lower end of a normal distribution. PMID:3204397

  5. Nitrate Transport Is Independent of NADH and NAD(P)H Nitrate Reductases in Barley Seedlings 1

    PubMed Central

    Warner, Robert L.; Huffaker, Ray C.

    1989-01-01

    Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings. PMID:11537465

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

    PubMed

    Bomati, Erin K; Noel, Joseph P

    2005-05-01

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

  7. Heat-resistant cytosolic malate dehydrogenases (cMDHs) of thermophilic intertidal snails (genus Echinolittorina): protein underpinnings of tolerance to body temperatures reaching 55°C.

    PubMed

    Liao, Ming-Ling; Zhang, Shu; Zhang, Guang-Ya; Chu, Yun-Meng; Somero, George N; Dong, Yun-Wei

    2017-06-01

    Snails of the genus Echinolittorina are among the most heat-tolerant animals; they experience average body temperatures near 41-44°C in summer and withstand temperatures up to at least 55°C. Here, we demonstrate that heat stability of function (indexed by the Michaelis-Menten constant of the cofactor NADH, K M NADH ) and structure (indexed by rate of denaturation) of cytosolic malate dehydrogenases (cMDHs) of two congeners ( E. malaccana and E. radiata ) exceeds values previously found for orthologs of this protein from less thermophilic species. The ortholog of E. malaccana is more heat stable than that of E. radiata , in keeping with the congeners' thermal environments. Only two inter-congener differences in amino acid sequence in these 332 residue proteins were identified. In both cases (positions 48 and 114), a glycine in the E. malaccana ortholog is replaced by a serine in the E. radiata protein. To explore the relationship between structure and function and to characterize how amino acid substitutions alter stability of different regions of the enzyme, we used molecular dynamics simulation methods. These computational methods allow determination of thermal effects on fine-scale movements of protein components, for example, by estimating the root mean square deviation in atom position over time and the root mean square fluctuation for individual residues. The minor changes in amino acid sequence favor temperature-adaptive change in flexibility of regions in and around the active sites. Interspecific differences in effects of temperature on fine-scale protein movements are consistent with the differences in thermal effects on binding and rates of heat denaturation. © 2017. Published by The Company of Biologists Ltd.

  8. An investigation into the enzyme histochemistry of adenocarcinomas of human large intestine and of the transitional epithelium immediately adjacent to them

    PubMed Central

    Marsden, J. R.; Dawson, I. M. P.

    1974-01-01

    Histochemical enzymatic studies were performed on 30 freshly resected large bowel carcinomas, 30 samples of normal colonic epithelium, and six samples of the histologically normal epithelium (so-called transitional epithelium) immediately adjacent to a carcinoma. Five enzymes were studied: nicotine adenine dinucleotide tetrazolium reductase (NADH-TR), glucose-6-phosphate dehydrogenase, succinate dehydrogenase, monoamine oxidase, and acid phosphatase. Quantitative and qualitative differences in enzyme activity were observed between normal, transitional, and carcinomatous mucosa as follows: monoamine oxidase activity was moderate in normal mucosa, high in transitional mucosa, and low in carcinoma. Succinate dehydrogenase activity was high in transitional mucosa and low or moderate in normal and carcinomatous mucosa. Glucose-6-phosphate dehydrogenase activity showed a gradation from low in normal mucosa to high in carcinoma while acid phosphatase showed the reverse of this pattern. The tetrazolium reductase activity was low or moderate in normal and transitional mucosa and high in carcinoma. These differences in enzyme activity and their possible clinical and metabolic significance are discussed. ImagesFig 2Fig 3 PMID:4154840

  9. Roles of the Sodium-Translocating NADH:Quinone Oxidoreductase (Na+-NQR) on Vibrio cholerae Metabolism, Motility and Osmotic Stress Resistance

    PubMed Central

    Minato, Yusuke; Halang, Petra; Quinn, Matthew J.; Faulkner, Wyatt J.; Aagesen, Alisha M.; Steuber, Julia; Stevens, Jan F.; Häse, Claudia C.

    2014-01-01

    The Na+ translocating NADH:quinone oxidoreductase (Na+-NQR) is a unique respiratory enzyme catalyzing the electron transfer from NADH to quinone coupled with the translocation of sodium ions across the membrane. Typically, Vibrio spp., including Vibrio cholerae, have this enzyme but lack the proton-pumping NADH:ubiquinone oxidoreductase (Complex I). Thus, Na+-NQR should significantly contribute to multiple aspects of V. cholerae physiology; however, no detailed characterization of this aspect has been reported so far. In this study, we broadly investigated the effects of loss of Na+-NQR on V. cholerae physiology by using Phenotype Microarray (Biolog), transcriptome and metabolomics analyses. We found that the V. cholerae ΔnqrA-F mutant showed multiple defects in metabolism detected by Phenotype Microarray. Transcriptome analysis revealed that the V. cholerae ΔnqrA-F mutant up-regulates 31 genes and down-regulates 55 genes in both early and mid-growth phases. The most up-regulated genes included the cadA and cadB genes, encoding a lysine decarboxylase and a lysine/cadaverine antiporter, respectively. Increased CadAB activity was further suggested by the metabolomics analysis. The down-regulated genes include sialic acid catabolism genes. Metabolomic analysis also suggested increased reductive pathway of TCA cycle and decreased purine metabolism in the V. cholerae ΔnqrA-F mutant. Lack of Na+-NQR did not affect any of the Na+ pumping-related phenotypes of V. cholerae suggesting that other secondary Na+ pump(s) can compensate for Na+ pumping activity of Na+-NQR. Overall, our study provides important insights into the contribution of Na+-NQR to V. cholerae physiology. PMID:24811312

  10. Inhibition of Cancer-Associated Mutant Isocitrate Dehydrogenases: Synthesis, Structure–Activity Relationship, and Selective Antitumor Activity

    PubMed Central

    2015-01-01

    Mutations of isocitrate dehydrogenase 1 (IDH1) are frequently found in certain cancers such as glioma. Different from the wild-type (WT) IDH1, the mutant enzymes catalyze the reduction of α-ketoglutaric acid to d-2-hydroxyglutaric acid (D2HG), leading to cancer initiation. Several 1-hydroxypyridin-2-one compounds were identified to be inhibitors of IDH1(R132H). A total of 61 derivatives were synthesized, and their structure–activity relationships were investigated. Potent IDH1(R132H) inhibitors were identified with Ki values as low as 140 nM, while they possess weak or no activity against WT IDH1. Activities of selected compounds against IDH1(R132C) were found to be correlated with their inhibitory activities against IDH1(R132H), as well as cellular production of D2HG, with R2 of 0.83 and 0.73, respectively. Several inhibitors were found to be permeable through the blood–brain barrier in a cell-based model assay and exhibit potent and selective activity (EC50 = 0.26–1.8 μM) against glioma cells with the IDH1 R132H mutation. PMID:25271760

  11. Bioelectrocatalytic NAD+/NADH inter-conversion: transformation of an enzymatic fuel cell into an enzymatic redox flow battery.

    PubMed

    Quah, Timothy; Milton, Ross D; Abdellaoui, Sofiene; Minteer, Shelley D

    2017-07-25

    Diaphorase and a benzylpropylviologen redox polymer were combined to create a bioelectrode that can both oxidize NADH and reduce NAD + . We demonstrate how bioelectrocatalytic NAD + /NADH inter-conversion can transform a glucose/O 2 enzymatic fuel cell (EFC) with an open circuit potential (OCP) of 1.1 V into an enzymatic redox flow battery (ERFB), which can be rapidly recharged by operation as an EFC.

  12. Crystallization of the Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae

    PubMed Central

    Casutt, Marco S.; Wendelspiess, Severin; Steuber, Julia; Fritz, Günter

    2010-01-01

    The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from the human pathogen Vibrio cholerae couples the exergonic oxidation of NADH by membrane-bound quinone to Na+ translocation across the membrane. Na+-NQR consists of six different subunits (NqrA–NqrF) and contains a [2Fe–2S] cluster, a noncovalently bound FAD, a noncovalently bound riboflavin, two covalently bound FMNs and potentially Q8 as cofactors. Initial crystallization of the entire Na+-NQR complex was achieved by the sitting-drop method using a nanolitre dispenser. Optimization of the crystallization conditions yielded flat yellow-coloured crystals with dimensions of up to 200 × 80 × 20 µm. The crystals diffracted to 4.0 Å resolution and belonged to space group P21, with unit-cell parameters a = 94, b = 146, c = 105 Å, α = γ = 90, β = 111°. PMID:21139223

  13. A New Insight into the Mechanism of NADH Model Oxidation by Metal Ions in Non-Alkaline Media.

    PubMed

    Yang, Jin-Dong; Chen, Bao-Long; Zhu, Xiao-Qing

    2018-06-11

    For a long time, it has been controversial that the three-step (e-H+-e) or two-step (e-H•) mechanism was used for the oxidations of NADH and its models by metal ions in non-alkaline media. The latter mechanism has been accepted by the majority of researchers. In this work, 1-benzyl-1,4-dihydronicotinamide (BNAH) and 1-phenyl-l,4-dihydronicotinamide (PNAH) are used as NADH models, and ferrocenium (Fc+) metal ion as an electron acceptor. The kinetics for oxidations of the NADH models by Fc+ in pure acetonitrile were monitored by using UV-Vis absorption and quadratic relationship between of kobs and the concentrations of NADH models were found for the first time. The rate expression of the reactions developed according to the three-step mechanism is quite consistent with the quadratic curves. The rate constants, thermodynamic driving forces and KIEs of each elementary step for the reactions were estimated. All the results supported the three-step mechanism. The intrinsic kinetic barriers of the proton transfer from BNAH+• to BNAH and the hydrogen atom transfer from BNAH+• to BNAH+• were estimated, the results showed that the former is 11.8 kcal/mol, and the latter is larger than 24.3 kcal/mol. It is the large intrinsic kinetic barrier of the hydrogen atom transfer that makes the reactions choose the three-step rather than two-step mechanism. Further investigation of the factors affecting the intrinsic kinetic barrier of chemical reactions indicated that the large intrinsic kinetic barrier of the hydrogen atom transfer originated from the repulsion of positive charges between BNAH+• and BNAH+•. The greatest contribution of this work is the discovery of the quadratic dependence of kobs on the concentrations of the NADH models, which is inconsistent with the conventional viewpoint of the "two-step mechanism" on the oxidations of NADH and its models by metal ions in the non-alkaline media.

  14. Evidence for the identity and some comparative properties of alpha-ketoglutarate and 2-keto-4-hydroxyglutarate dehydrogenase activity.

    PubMed

    Gupta, S C; Dekker, E E

    1980-02-10

    Enzyme preparations of pig heart and Escherichia coli are shown to catalyze a NAD+- and CoASH-dependent oxidation of 2-keto-4-hydroxyglutarate. Several independent lines of evidence support the conclusion that this hydroxyketo acid is a substrate for the well known alpha-ketoglutarate dehydrogenase complex of the citric acid cycle. The evidence includes (a) a constant ratio of specific activity values for the two substrates through several steps of purification, (b) identical elution profiles from a calcium phosphate gel-cellulose column and a constant ratio of specific activity toward the two substrates throughout the activity peak, (c) identical inactivation curves in controlled heat denaturation studies, (d) the same pH activity curves, (e) no effect on the oxidation of either keto acid by repeated freezing and thawing of dehydrogenase preparations, and (f) the same activity pattern when the E. coli complex is distributed into several fractions by sucrose density gradient centrifugation. Additionally, the same cofactors are required for maximal activity and glyoxylate inhibits the oxidation of either substrate noncompetitively. Ferricyanide-linked oxidation of 2-keto-4-hydroxyglutarate yields malate as the product and a 1:2:1 stoichiometric relationship is obtained between the amount of hydroxyketo acid oxidized, ferricyanide reduced, and malate formed.

  15. Role of Microsomal Retinol/Sterol Dehydrogenase-Like Short-Chain Dehydrogenases/Reductases in the Oxidation and Epimerization of 3α-Hydroxysteroids in Human Tissues

    PubMed Central

    Belyaeva, Olga V.; Chetyrkin, Sergei V.; Clark, Amy L.; Kostereva, Natalia V.; SantaCruz, Karen S.; Chronwall, Bibie M.; Kedishvili, Natalia Y.

    2008-01-01

    Allopregnanolone (ALLO) and androsterone (ADT) are naturally occurring 3α-hydroxysteroids that act as positive allosteric regulators of γ-aminobutyric acid type A receptors. In addition, ADT activates nuclear farnesoid X receptor and ALLO activates pregnane X receptor. At least with respect to γ-aminobutyric acid type A receptors, the biological activity of ALLO and ADT depends on the 3α-hydroxyl group and is lost upon its conversion to either 3-ketosteroid or 3β-hydroxyl epimer. Such strict structure-activity relationships suggest that the oxidation or epimerization of 3α-hydroxysteroids may serve as physiologically relevant mechanisms for the control of the local concentrations of bioactive 3α-hydroxysteroids. The exact enzymes responsible for the oxidation and epimerization of 3α-hydroxysteroids in vivo have not yet been identified, but our previous studies showed that microsomal nicotinamide adenine dinucleotide-dependent short-chain dehydrogenases/reductases (SDRs) with dual retinol/sterol dehydrogenase substrate specificity (RoDH-like group of SDRs) can oxidize and epimerize 3α-hydroxysteroids in vitro. Here, we present the first evidence that microsomal nicotinamide adenine dinucleotide-dependent 3α-hydroxysteroid dehydrogenase/epimerase activities are widely distributed in human tissues with the highest activity levels found in liver and testis and lower levels in lung, spleen, brain, kidney, and ovary. We demonstrate that RoDH-like SDRs contribute to the oxidation and epimerization of ALLO and ADT in living cells, and show that RoDH enzymes are expressed in tissues that have microsomal 3α-hydroxysteroid dehydrogenase/epimerase activities. Together, these results provide further support for the role of RoDH-like SDRs in human metabolism of 3α-hydroxysteroids and offer a new insight into the enzymology of ALLO and ADT inactivation. PMID:17289849

  16. Role of microsomal retinol/sterol dehydrogenase-like short-chain dehydrogenases/reductases in the oxidation and epimerization of 3alpha-hydroxysteroids in human tissues.

    PubMed

    Belyaeva, Olga V; Chetyrkin, Sergei V; Clark, Amy L; Kostereva, Natalia V; SantaCruz, Karen S; Chronwall, Bibie M; Kedishvili, Natalia Y

    2007-05-01

    Allopregnanolone (ALLO) and androsterone (ADT) are naturally occurring 3alpha-hydroxysteroids that act as positive allosteric regulators of gamma-aminobutyric acid type A receptors. In addition, ADT activates nuclear farnesoid X receptor and ALLO activates pregnane X receptor. At least with respect to gamma-aminobutyric acid type A receptors, the biological activity of ALLO and ADT depends on the 3alpha-hydroxyl group and is lost upon its conversion to either 3-ketosteroid or 3beta-hydroxyl epimer. Such strict structure-activity relationships suggest that the oxidation or epimerization of 3alpha-hydroxysteroids may serve as physiologically relevant mechanisms for the control of the local concentrations of bioactive 3alpha-hydroxysteroids. The exact enzymes responsible for the oxidation and epimerization of 3alpha-hydroxysteroids in vivo have not yet been identified, but our previous studies showed that microsomal nicotinamide adenine dinucleotide-dependent short-chain dehydrogenases/reductases (SDRs) with dual retinol/sterol dehydrogenase substrate specificity (RoDH-like group of SDRs) can oxidize and epimerize 3alpha-hydroxysteroids in vitro. Here, we present the first evidence that microsomal nicotinamide adenine dinucleotide-dependent 3alpha-hydroxysteroid dehydrogenase/epimerase activities are widely distributed in human tissues with the highest activity levels found in liver and testis and lower levels in lung, spleen, brain, kidney, and ovary. We demonstrate that RoDH-like SDRs contribute to the oxidation and epimerization of ALLO and ADT in living cells, and show that RoDH enzymes are expressed in tissues that have microsomal 3alpha-hydroxysteroid dehydrogenase/epimerase activities. Together, these results provide further support for the role of RoDH-like SDRs in human metabolism of 3alpha-hydroxysteroids and offer a new insight into the enzymology of ALLO and ADT inactivation.

  17. KETONES INHIBIT MITOCHONDRIAL PRODUCTION OF REACTIVE OXYGEN SPECIES PRODUCTION FOLLOWING GLUTAMATE EXCITOTOXICITY BY INCREASING NADH OXIDATION

    PubMed Central

    Maalouf, Marwan; Sullivan, Patrick G.; Davis, Laurie; Kim, Do Young; Rho, Jong M.

    2007-01-01

    Dietary protocols that increase serum levels of ketones, such as calorie restriction and the ketogenic diet, offer robust protection against a multitude of acute and chronic neurological diseases. The underlying mechanisms, however, remain unclear. Previous studies have suggested that the ketogenic diet may reduce free radical levels in the brain. Thus, one possibility is that ketones may mediate neuroprotection through antioxidant activity. In the present study, we examined the effects of the ketones β-hydroxybutyrate and acetoacetate on acutely dissociated rat neocortical neurons subjected to glutamate excitotoxicity using cellular electrophysiological and single-cell fluorescence imaging techniques. Further, we explored the effects of ketones on acutely isolated mitochondria exposed to high levels of calcium. A combination of β-hydroxybutyrate and acetoacetate (1 mM each) decreased neuronal death and prevented changes in neuronal membrane properties induced by 10 μM glutamate. Ketones also significantly decreased mitochondrial production of reactive oxygen species and the associated excitotoxic changes by increasing NADH oxidation in the mitochondrial respiratory chain, but did not affect levels of the endogenous antioxidant glutathione. In conclusion, we demonstrate that ketones reduce glutamate-induced free radical formation by increasing the NAD+/NADH ratio and enhancing mitochondrial respiration in neocortical neurons. This mechanism may, in part, contribute to the neuroprotective activity of ketones by restoring normal bioenergetic function in the face of oxidative stress. PMID:17240074

  18. Decursin and decursinol angelate selectively inhibit NADH-fumarate reductase of Ascaris suum.

    PubMed

    Shiomi, Kazuro; Hatano, Hiroko; Morimoto, Hiromi; Ui, Hideaki; Sakamoto, Kimitoshi; Kita, Kiyoshi; Tomoda, Hiroshi; Lee, Eun Woo; Heo, Tae Ryeon; Kawagishi, Hirokazu; Omura, Satoshi

    2007-11-01

    NADH-fumarate reductase (NFRD) is a key enzyme in many anaerobic helminths. Decursin and decursinol angelate have been isolated from the roots of ANGELICA GIGAS Nakai (Apiaceae) as NFRD inhibitors. They inhibited ASCARIS SUUM NFRD with IC (50) values of 1.1 and 2.7 microM, respectively. Their target is the electron transport enzyme complex I. Since the inhibitory activities of decursin against bovine heart complexes are weak, it is a selective inhibitor of the nematode complex I. In contrast, decursinol angelate moderately inhibits bovine heart complexes II and III. Decursinol inhibits A. SUUM NFRD to a similar extent, but its target is complex II. It also inhibits bovine heart complexes II and III.

  19. Aldehyde dehydrogenase inhibition combined with phenformin treatment reversed NSCLC through ATP depletion.

    PubMed

    Kang, Joon Hee; Lee, Seon-Hyeong; Lee, Jae-Seon; Nam, Boas; Seong, Tae Wha; Son, Jaekyoung; Jang, Hyonchol; Hong, Kyeong Man; Lee, Cheolju; Kim, Soo-Youl

    2016-08-02

    Among ALDH isoforms, ALDH1L1 in the folate pathway showed highly increased expression in non-small-cell lung cancer cells (NSCLC). Based on the basic mechanism of ALDH converting aldehyde to carboxylic acid with by-product NADH, we suggested that ALDH1L1 may contribute to ATP production using NADH through oxidative phosphorylation. ALDH1L1 knockdown reduced ATP production by up to 60% concomitantly with decrease of NADH in NSCLC. ALDH inhibitor, gossypol, also reduced ATP production in a dose dependent manner together with decrease of NADH level in NSCLC. A combination treatment of gossypol with phenformin, mitochondrial complex I inhibitor, synergized ATP depletion, which efficiently induced cell death. Pre-clinical xenograft model using human NSCLC demonstrated a remarkable therapeutic response to the combined treatment of gossypol and phenformin.

  20. Structural and Kinetic Basis for Substrate Selectivity in Populus tremuloides Sinapyl Alcohol Dehydrogenase

    PubMed Central

    Bomati, Erin K.; Noel, Joseph P.

    2005-01-01

    We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities. PMID:15829607

  1. A soluble NADH-dependent fumarate reductase in the reductive tricarboxylic acid cycle of Hydrogenobacter thermophilus TK-6.

    PubMed

    Miura, Akane; Kameya, Masafumi; Arai, Hiroyuki; Ishii, Masaharu; Igarashi, Yasuo

    2008-11-01

    Fumarate reductase (FRD) is an enzyme that reduces fumarate to succinate. In many organisms, it is bound to the membrane and uses electron donors such as quinol. In this study, an FRD from a thermophilic chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6, was purified and characterized. FRD activity using NADH as an electron donor was not detected in the membrane fraction but was found in the soluble fraction. The purified enzyme was demonstrated to be a novel type of FRD, consisting of five subunits. One subunit showed high sequence identity to the catalytic subunits of known FRDs. Although the genes of typical FRDs are assembled in a cluster, the five genes encoding the H. thermophilus FRD were distant from each other in the genome. Furthermore, phylogenetic analysis showed that the H. thermophilus FRD was located in a distinct position from those of known soluble FRDs. This is the first report of a soluble NADH-dependent FRD in Bacteria and of the purification of a FRD that operates in the reductive tricarboxylic acid cycle.

  2. Tricarboxylic acid cycle without malate dehydrogenase in Streptomyces coelicolor M-145.

    PubMed

    Takahashi-Íñiguez, Tóshiko; Barrios-Hernández, Joana; Rodríguez-Maldonado, Marion; Flores, María Elena

    2018-06-23

    The oxidation of malate to oxaloacetate is catalysed only by a nicotinamide adenine dinucleotide-dependent malate dehydrogenase encoded by SCO4827 in Streptomyces coelicolor. A mutant lacking the malate dehydrogenase gene was isolated and no enzymatic activity was detected. As expected, the ∆mdh mutant was unable to grow on malate as the sole carbon source. However, the mutant grew less in minimal medium with glucose and there was a delay of 36 h. The same behaviour was observed when the mutant was grown on minimal medium with casamino acids or glycerol. For unknown reasons, the mutant was not able to grow in YEME medium with glucose. The deficiency of malate dehydrogenase affected the expression of the isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase genes, decreasing the expression of both genes by approximately two- to threefold.

  3. Dual utilization of NADPH and NADH cofactors enhances xylitol production in engineered Saccharomyces cerevisiae.

    PubMed

    Jo, Jung-Hyun; Oh, Sun-Young; Lee, Hyeun-Soo; Park, Yong-Cheol; Seo, Jin-Ho

    2015-12-01

    Xylitol, a natural sweetener, can be produced by hydrogenation of xylose in hemicelluloses. In microbial processes, utilization of only NADPH cofactor limited commercialization of xylitol biosynthesis. To overcome this drawback, Saccharomyces cerevisiae D452-2 was engineered to express two types of xylose reductase (XR) with either NADPH-dependence or NADH-preference. Engineered S. cerevisiae DWM expressing both the XRs exhibited higher xylitol productivity than the yeast strain expressing NADPH-dependent XR only (DWW) in both batch and glucose-limited fed-batch cultures. Furthermore, the coexpression of S. cerevisiae ZWF1 and ACS1 genes in the DWM strain increased intracellular concentrations of NADPH and NADH and improved maximum xylitol productivity by 17%, relative to that for the DWM strain. Finally, the optimized fed-batch fermentation of S. cerevisiae DWM-ZWF1-ACS1 resulted in 196.2 g/L xylitol concentration, 4.27 g/L h productivity and almost the theoretical yield. Expression of the two types of XR utilizing both NADPH and NADH is a promising strategy to meet the industrial demands for microbial xylitol production. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Inhibitory effects of Aphanizomenon flos-aquae constituents on human UDP-glucose dehydrogenase activity.

    PubMed

    Scoglio, Stefano; Lo Curcio, Valeria; Catalani, Simona; Palma, Francesco; Battistelli, Serafina; Benedetti, Serena

    2016-12-01

    The purpose of this study was to investigate the in vitro inhibitory effects of the edible microalga Aphanizomenon flos-aquae (AFA) on human UDP-α-d-glucose 6-dehydrogenase (UGDH) activity, a cytosolic enzyme involved both in tumor progression and in phytochemical bioavailability. Both the hydrophilic and ethanolic AFA extracts as well as the constitutive active principles phycocyanin (PC), phycocyanobilin (PCB) and mycosporine-like amino acids (MAAs) were tested. Among AFA components, PCB presented the strongest inhibitory effect on UGDH activity, acting as a competitive inhibitor with respect to UDP-glucose and a non-competitive inhibitor with respect to NAD(+). In preliminary experiments, AFA PCB was also effective in reducing the colony formation capacity of PC-3 prostate cancer cells and FTC-133 thyroid cancer cells. Overall, these findings confirmed that AFA and its active principles are natural compounds with high biological activity. Further studies evaluating the effects of AFA PCB in reducing tumor cell growth and phytochemical glucuronidation are encouraged.

  5. Inhibition of mitochondrial calcium efflux by clonazepam in intact single rat cardiomyocytes and effects on NADH production.

    PubMed

    Griffiths, E J; Wei, S K; Haigney, M C; Ocampo, C J; Stern, M D; Silverman, H S

    1997-04-01

    The aims of this study were to determine: (i) whether clonazepam and CGP37157, which inhibit the Na+/Ca2+ exchanger of isolated mitochondria, could inhibit mitochondrial Ca2+ efflux in intact cells; and (ii) whether any sustained increase in mitochondrial [Ca2+] ([Ca2+]m) could alter mitochondrial NADH levels. [Ca2+]m was measured in Indo-1/AM loaded rat ventricular myocytes where the cytosolic fluorescence signal was quenched by superfusion with Mn2+. NADH levels were determined from cell autofluorescence. Upon exposure of myocytes to 50 nM norepinephrine (NE) and a stimulation rate of 3 Hz, [Ca2+]m increased from 59 +/- 3 nM to a peak of 517 +/- 115 nM (n = 8) which recovered rapidly upon return to low stimulation rate (0.2 Hz) and washout of NE. In the presence of clonazepam, the peak increase in [Ca2+]m was 937 +/- 192 nM (n = 5) which remained elevated at 652 +/- 131 nM upon removal of the stimulus. CGP37157 in some cells did give the same inhibition of mitochondrial Ca2+ efflux as clonazepam, but the effect was inconsistent since not all cells were capable of following the stimulation rate in presence of this compound. NADH levels increased upon exposure to rapid stimulation in the presence of NE alone and recovered upon return to low stimulation rates, whereas in clonazepam treated cells the recovery of NADH was prevented. We conclude that clonazepam is an effective inhibitor of mitochondrial [Ca2+] efflux in intact cells and also maintains the increase in NADH levels which occurs upon rapid stimulation of cells.

  6. Bio-sniffer (gas-phase biosensor) with secondary alcohol dehydrogenase (S-ADH) for determination of isopropanol in exhaled air as a potential volatile biomarker.

    PubMed

    Chien, Po-Jen; Suzuki, Takuma; Tsujii, Masato; Ye, Ming; Toma, Koji; Arakawa, Takahiro; Iwasaki, Yasuhiko; Mitsubayashi, Kohji

    2017-05-15

    Exhaled breath analysis has attracted lots of researchers attention in the past decades due to its advantages such as its non-invasive property and the possibility of continuous monitoring. In addition, several volatile organic compounds in breath have been identified as biomarkers for some diseases. Particularly, studies have pointed out that concentration of isopropanol (IPA) in exhaled air might relate with certain illnesses such as liver disease, chronic obstructive pulmonary (COPD), and lung cancer. In this study, a highly sensitive and selective biochemical gas sensor (bio-sniffer) for the breath IPA concentration determination was constructed and optimized. This bio-sniffer measures the concentration of IPA according to the fluorescence intensity of oxidized nicotinamide adenine dinucleotide (NADH), which was produced by an enzymatic reaction of secondary alcohol dehydrogenase (S-ADH). The NADH detection system employed an UV-LED as the excitation light, and a highly sensitive photomultiplier tube (PMT) as a fluorescence intensity detector. A gas-sensing region was developed using an optical fiber probe equipped with a flow-cell and enzyme immobilized membrane, and connected to the NADH measurement system. The calibration range of the IPA bio-sniffer was confirmed from 1ppb to 9060ppb that was comparable to other IPA analysis methods. The results of the analysis of breath IPA concentration in healthy subjects using the bio-sniffer showed a mean concentration of 16.0ppb, which was similar to other studies. These results have demonstrated that this highly sensitive and selective bio-sniffer could be used to measure the IPA in exhaled air, and it is expected to apply for breath IPA research and investigation of biomarkers for clinical diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Similar potential ATP-P production and enzymatic activities in the microplankton community off Concepción (Chile) under oxic and suboxic conditions

    NASA Astrophysics Data System (ADS)

    González, Rodrigo R.; Gutiérrez, Marcelo H.; Quiñones, Renato A.

    2007-11-01

    The effects of the oxygen minimum zone on the metabolism of the heterotrophic microplankton community (0.22-100 μm) in the Humboldt Current System, as well as the factors controlling its biomass production, remain unknown. Here we compare the effect of four sources of dissolved organic carbon (glucose, oxaloacetate, glycine, leucine) on microbial biomass production (such as ATP-P) and the potential enzymatic activities involved in catabolic pathways under oxic and suboxic conditions. Our results show significant differences ( p < 0.05) in the ATP-P production when induced by the different substrates that are used as dissolved organic carbon herein. The induction of ATP-P production is enhanced from glucose < oxaloacetate < glycine < leucine. Nevertheless, for individual substrates, no significant differences were found between incubation under oxic and suboxic conditions except in the case of leucine. For this amino acid, the induction of ATP-P synthesis was higher under suboxic than oxic conditions. The data sets of all the substrates used showed greater potential ATP-P production under suboxic than oxic conditions. The results of the potential enzymatic activities suggest that malate dehydrogenase has the highest signal of NADH oxidization activity in the microbial assemblage. Furthermore, for all experiments, the malate dehydrogenase activity data set had a significant relationship with ATP-P production. These findings suggest that the microbial community inhabiting the oxygen minimum zone has the same or greater potential growth than the community inhabiting more oxygenated strata of the water column and that malate dehydrogenase is the activity that best represents the metabolic potential of the community.

  8. Plasma lactic dehydrogenase activities in men during bed rest with exercise training

    NASA Technical Reports Server (NTRS)

    Greenleaf, J. E.; Juhos, L. T.; Young, H. L.

    1985-01-01

    Peak oxygen uptake and the activity of lactic dehydrogenase (LDH-T) and its five isoenzymes were measured by spectrophotometer in seven men before, during, and after bed rest and exercise training. Exercise training consisted of isometric leg exercises of 250 kcal/hr for a period of one hour per day. It is found that LDH-T was reduced by 0.05 percent in all three regimens by day 10 of bed rest, and that the decrease occurred at different rates. The earliest reduction in LDH-T activity in the no-exercise regimen was associated with a decrease in peak oxygen uptake of 12.3 percent. It is concluded that isometric (aerobic) muscular strength training appear to maintain skeletal muscle integrity better during bed rest than isotonic exercise training. Reduced hydrostatic pressure during bed rest, however, ultimately counteracts the effects of both moderate isometric and isotonic exercise training, and may result in decreased LDH-T activity.

  9. Aldehyde dehydrogenase inhibition combined with phenformin treatment reversed NSCLC through ATP depletion

    PubMed Central

    Lee, Jae-Seon; Nam, Boas; Seong, Tae Wha; Son, Jaekyoung; Jang, Hyonchol; Hong, Kyeong Man; Lee, Cheolju; Kim, Soo-Youl

    2016-01-01

    Among ALDH isoforms, ALDH1L1 in the folate pathway showed highly increased expression in non-small-cell lung cancer cells (NSCLC). Based on the basic mechanism of ALDH converting aldehyde to carboxylic acid with by-product NADH, we suggested that ALDH1L1 may contribute to ATP production using NADH through oxidative phosphorylation. ALDH1L1 knockdown reduced ATP production by up to 60% concomitantly with decrease of NADH in NSCLC. ALDH inhibitor, gossypol, also reduced ATP production in a dose dependent manner together with decrease of NADH level in NSCLC. A combination treatment of gossypol with phenformin, mitochondrial complex I inhibitor, synergized ATP depletion, which efficiently induced cell death. Pre-clinical xenograft model using human NSCLC demonstrated a remarkable therapeutic response to the combined treatment of gossypol and phenformin. PMID:27384481

  10. Development of a simple and efficient method for assaying cytidine monophosphate sialic acid synthetase activity using an enzymatic reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide converting system.

    PubMed

    Fujita, Akiko; Sato, Chihiro; Münster-Kühnel, Anja-K; Gerardy-Schahn, Rita; Kitajima, Ken

    2005-02-01

    A new reliable method to assay the activity of cytidine monophosphate sialic acid (CMP-Sia) synthetase (CSS) has been developed. The activation of sialic acids (Sia) to CMP-Sia is a prerequisite for the de novo synthesis of sialoglycoconjugates. In vertebrates, CSS has been cloned from human, mouse, and rainbow trout, and the crystal structure has been resolved for the mouse enzyme. The mouse and rainbow trout enzyme have been compared with respect to substrate specificity, demonstrating that the mouse enzyme exhibits a pronounced specificity for N-acetylneuraminic acid (Neu5Ac), while the rainbow trout CSS is equally active with either of three Sia species, Neu5Ac, N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (KDN). However, molecular details that explain the pronounced substrate specificities are unknown. Understanding the catalytic mechanisms of these enzymes is of major importance, since CSSs play crucial roles in cellular sialylation patterns and thus are potential drug targets in a number of pathophysiological situations. The availability of the cDNAs and the obtained structural data enable rational approaches; however, these efforts are limited by the lack of a reliable high-throughput assay system. Here we describe a new assay system that allows product quantification in a reduced nicotinamide adenine dinucleotide (NADH)-dependent color reaction. The activation reaction catalyzed by CSS, CTP+Sia-->CMP-Sia+pyrophosphate, was evaluated by a consumption of Sia, which corresponds to that of NADH on the following two successive reactions: (i) Sia-->pyruvate+ManNAc (or Man), catalyzed by a sialic acid lyase (SAL), and (ii) pyruvate+NADH-->lactate+oxidized nicotinamide adenine dinucleotide (NAD+), catalyzed by a lactate dehydrogenase (LDH). Consumption of NADH can be photometrically monitored on a microtiter plate reader for a number of test samples at the same time. Furthermore, based on the quantification of CSS used in the SAL/LDH assay

  11. [Glutamate dehydrogenase activity in the pancreatic tissue in acute experimental pancreatitis and under the action of sodium thiosulphate].

    PubMed

    Simavorian, P S; Saakian, I L; Gevorkian, D A

    1991-04-01

    It has been established that the development of acute pancreatitis is accompanied by the reduced activity of glutamate dehydrogenase in the mitochondrial fraction of pancreas, pronounced in the focus of tissue necrosis and less expressed in the reactive inflammation focus. Besides this in the pancreas redistribution of enzyme, activity in the subcellular organelles takes place and enzyme activity emerges in the cytosol and further--in the blood and peritoneum liquid. Sodium thiosulfate has a marked correlation effect.

  12. Inhibition of fumarate reductase in Leishmania major and L. donovani by chalcones.

    PubMed

    Chen, M; Zhai, L; Christensen, S B; Theander, T G; Kharazmi, A

    2001-07-01

    Our previous studies have shown that chalcones exhibit potent antileishmanial and antimalarial activities in vitro and in vivo. Preliminary studies showed that these compounds destroyed the ultrastructure of Leishmania parasite mitochondria and inhibited the respiration and the activity of mitochondrial dehydrogenases of Leishmania parasites. The present study was designed to further investigate the mechanism of action of chalcones, focusing on the parasite respiratory chain. The data show that licochalcone A inhibited the activity of fumarate reductase (FRD) in the permeabilized Leishmania major promastigote and in the parasite mitochondria, and it also inhibited solubilized FRD and a purified FRD from L. donovani. Two other chalcones, 2,4-dimethoxy-4'-allyloxychalcone (24m4ac) and 2,4-dimethoxy-4'-butoxychalcone (24mbc), also exhibited inhibitory effects on the activity of solubilized FRD in L. major promastigotes. Although licochalcone A inhibited the activities of succinate dehydrogenase (SDH), NADH dehydrogenase (NDH), and succinate- and NADH-cytochrome c reductases in the parasite mitochondria, the 50% inhibitory concentrations (IC(50)) of licochalcone A for these enzymes were at least 20 times higher than that for FRD. The IC(50) of licochalcone A for SDH and NDH in human peripheral blood mononuclear cells were at least 70 times higher than that for FRD. These findings indicate that FRD, one of the enzymes of the parasite respiratory chain, might be the specific target for the chalcones tested. Since FRD exists in the Leishmania parasite and does not exist in mammalian cells, it could be an excellent target for antiprotozoal drugs.

  13. Inhibition of Fumarate Reductase in Leishmania major and L. donovani by Chalcones

    PubMed Central

    Chen, Ming; Zhai, Lin; Christensen, Søren Brøgger; Theander, Thor G.; Kharazmi, Arsalan

    2001-01-01

    Our previous studies have shown that chalcones exhibit potent antileishmanial and antimalarial activities in vitro and in vivo. Preliminary studies showed that these compounds destroyed the ultrastructure of Leishmania parasite mitochondria and inhibited the respiration and the activity of mitochondrial dehydrogenases of Leishmania parasites. The present study was designed to further investigate the mechanism of action of chalcones, focusing on the parasite respiratory chain. The data show that licochalcone A inhibited the activity of fumarate reductase (FRD) in the permeabilized Leishmania major promastigote and in the parasite mitochondria, and it also inhibited solubilized FRD and a purified FRD from L. donovani. Two other chalcones, 2,4-dimethoxy-4′-allyloxychalcone (24m4ac) and 2,4-dimethoxy-4′-butoxychalcone (24mbc), also exhibited inhibitory effects on the activity of solubilized FRD in L. major promastigotes. Although licochalcone A inhibited the activities of succinate dehydrogenase (SDH), NADH dehydrogenase (NDH), and succinate- and NADH-cytochrome c reductases in the parasite mitochondria, the 50% inhibitory concentrations (IC50) of licochalcone A for these enzymes were at least 20 times higher than that for FRD. The IC50 of licochalcone A for SDH and NDH in human peripheral blood mononuclear cells were at least 70 times higher than that for FRD. These findings indicate that FRD, one of the enzymes of the parasite respiratory chain, might be the specific target for the chalcones tested. Since FRD exists in the Leishmania parasite and does not exist in mammalian cells, it could be an excellent target for antiprotozoal drugs. PMID:11408218

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

    PubMed Central

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

    2000-01-01

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

  15. Optically measured NADH concentrations are unaffected by propofol induced EEG silence during transient cerebral hypoperfusion in anesthetized rabbits☆

    PubMed Central

    Wang, Mei; Agarwal, Sachin; Mayevsky, Avraham; Joshi, Shailendra

    2014-01-01

    The neuroprotective benefit of intra-operative anesthetics is widely described and routinely aimed to invoke electroencephalographic (EEG) silence in anticipation of transient cerebral ischemia. Previous rat survival studies have questioned an additional benefit from achieving EEG silence during transient global cerebral hypoperfusion. Surgical preparation on twelve New Zealand white rabbits under ketamine–propofol anesthesia, included placement of skull screws for bilateral EEG monitoring, skull shaving for laser Doppler probes, and a 5 mm diameter right temporal craniotomy for the NADH probe. Transient global cerebral hypoperfusion was achieved with bilateral internal carotid artery occlusion and pharmacologically induced systemic hypotension. All animals acted as controls, and had cerebral hypoperfusion under baseline propofol anesthesia with an active EEG. Thereafter, animals were randomized to receive bolus injection of intracarotid (3–5 mg) or intravenous (10–20 mg) 1% propofol to create EEG silence for 1–2 min. The data collected at baseline, peak hypoperfusion, and 5 and 10 min post hypoperfusion was analyzed by repeated measures ANOVA with post hoc Bonferroni–Dunn test. Eleven of the twelve rabbits completed the protocol. Hemodynamics and cerebral blood flow changes were comparable in all the animals. Compared to controls, the increase in NADH during ischemia was unaffected by EEG silence with either intravenous or intraarterial propofol. We failed to observe any significant additional attenuation of the elevation in NADH levels with propofol induced EEG silence during transient global cerebral hypoperfusion. This is consistent with previous rat survival studies showing that EEG silence was not required for full neuroprotective effects of pentothal anesthesia. PMID:21570061

  16. Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. I. Introduction of a six-residue ion-pair network in the hinge region.

    PubMed

    Lebbink, J H; Knapp, S; van der Oost, J; Rice, D; Ladenstein, R; de Vos, W M

    1998-07-10

    Comparison of the recently determined three-dimensional structures of several glutamate dehydrogenases allowed for the identification of a five-residue ion-pair network in the hinge region of Pyrococcus furiosus glutamate dehydrogenase (melting temperature 113 degrees C), that is not present in the homologous glutamate dehydrogenase from Thermotoga maritima (melting temperature 93 degrees C). In order to study the role of this ion-pair network, we introduced it into the T. maritima enzyme using a site-directed mutagenesis approach. The resulting T. maritima glutamate dehydrogenases N97D, G376 K and N97D/G376 K as well as the wild-type enzyme were overproduced in Escherichia coli and subsequently purified. Elucidation of the three-dimensional structure of the double mutant N97D/G376 K at 3.0 A, showed that the designed ion-pair interactions were indeed formed. Moreover, because of interactions with an additional charged residue, a six-residue network is present in this double mutant. Melting temperatures of the mutant enzymes N97D, G376 K and N97D/G376 K, as determined by differential scanning calorimetry, did not differ significantly from that of the wild-type enzyme. Identical transition midpoints in guanidinium chloride-induced denaturation experiments were found for the wild-type and all mutant enzymes. Thermal inactivation at 85 degrees C occured more than twofold faster for all mutant enzymes than for the wild-type glutamate dehydrogenase. At temperatures of 65 degrees C and higher, the wild-type and the three mutant enzymes showed identical specific activities. However, at 58 degrees C the specific activity of N97D/G376 K and G376 K was found to be significantly higher than that of the wild-type and N97D enzymes. These results suggest that the engineered ion-pair interactions in the hinge region do not affect the stability towards temperature or guanidinium chloride-induced denaturation but rather affect the specific activity of the enzyme and the temperature

  17. Glucose-6-phosphate dehydrogenase

    MedlinePlus

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

  18. Plasma membrane NADH oxidase of maize roots responds to gravity and imposed centrifugal forces

    NASA Technical Reports Server (NTRS)

    Bacon, E.; Morre, D. J.

    2001-01-01

    NADH oxidase activities measured with excised roots of dark-grown maize (Zea mays) seedlings and with isolated plasma membrane vesicles from roots of dark-grown maize oscillated with a regular period length of 24 min and were inhibited by the synthetic auxin 2,4-dichlorophenoxyacetic [correction of dichorophenoxyacetic] acid. The activities also responded to orientation with respect to gravity and to imposed centrifugal forces. Turning the roots upside down resulted in stimulation of the activity with a lag of about 10 min. Returning the sections to the normal upright position resulted in a return to initial rates. The activity was stimulated reversibly to a maximum of about 2-fold with isolated plasma membrane vesicles, when subjected to centrifugal forces of 25 to 250 x g for 1 to 4 min duration. These findings are the first report of a gravity-responsive enzymatic activity of plant roots inhibited by auxin and potentially related to the gravity-induced growth response. c2001 Editions scientifiques et medicales Elsevier SAS.

  19. Mitochondrial energy-dissipating systems (alternative oxidase, uncoupling proteins, and external NADH dehydrogenase) are involved in development of frost-resistance of winter wheat seedlings.

    PubMed

    Grabelnych, O I; Borovik, O A; Tauson, E L; Pobezhimova, T P; Katyshev, A I; Pavlovskaya, N S; Koroleva, N A; Lyubushkina, I V; Bashmakov, V Yu; Popov, V N; Borovskii, G B; Voinikov, V K

    2014-06-01

    Gene expression, protein synthesis, and activities of alternative oxidase (AOX), uncoupling proteins (UCP), adenine nucleotide translocator (ANT), and non-coupled NAD(P)H dehydrogenases (NDex, NDPex, and NDin) were studied in shoots of etiolated winter wheat (Triticum aestivum L.) seedlings after exposure to hardening low positive (2°C for 7 days) and freezing (-2°C for 2 days) temperatures. The cold hardening efficiently increased frost-resistance of the seedlings and decreased the generation of reactive oxygen species (ROS) during further cold shock. Functioning of mitochondrial energy-dissipating systems can represent a mechanism responsible for the decrease in ROS under these conditions. These systems are different in their response to the action of the hardening low positive and freezing temperatures. The functioning of the first system causes induction of AOX and UCP synthesis associated with an increase in electron transfer via AOX in the mitochondrial respiratory chain and also with an increase in the sensitivity of mitochondrial non-phosphorylating respiration to linoleic and palmitic acids. The increase in electron transfer via AOX upon exposure of seedlings to hardening freezing temperature is associated with retention of a high activity of NDex. It seems that NDex but not the NDPex and NDin can play an important role in maintaining the functional state of mitochondria in heterotrophic tissues of plants under the influence of freezing temperatures. The involvement of the mitochondrial energy-dissipating systems and their possible physiological role in the adaptation of winter crops to cold and frost are discussed.

  20. Molecular cloning and characterization of a tumor-associated, growth-related, and time-keeping hydroquinone (NADH) oxidase (tNOX) of the HeLa cell surface

    NASA Technical Reports Server (NTRS)

    Chueh, Pin-Ju; Kim, Chinpal; Cho, NaMi; Morre, Dorothy M.; Morre, D. James

    2002-01-01

    NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit prion-like properties. The two enzymatic activities alternate to generate a regular period length of about 24 min. Here we report the expression, cloning, and characterization of a tumor-associated NADH oxidase (tNOX). The cDNA sequence of 1830 bp is located on gene Xq25-26 with an open reading frame encoding 610 amino acids. The activities of the bacterially expressed tNOX oscillate with a period length of 22 min as is characteristic of tNOX activities in situ. The activities are inhibited completely by capsaicin, which represents a defining characteristic of tNOX activity. Functional motifs identified by site-directed mutagenesis within the C-terminal portion of the tNOX protein corresponding to the processed plasma membrane-associated form include quinone (capsaicin), copper and adenine nucleotide binding domains, and two cysteines essential for catalytic activity. Four of the six cysteine to alanine replacements retained enzymatic activity, but the period lengths of the oscillations were increased. A single protein with two alternating enzymatic activities indicative of a time-keeping function is unprecedented in the biochemical literature.

  1. Maturation of the [Ni-4Fe-4S] active site of carbon monoxide dehydrogenases.

    PubMed

    Merrouch, Mériem; Benvenuti, Martino; Lorenzi, Marco; Léger, Christophe; Fourmond, Vincent; Dementin, Sébastien

    2018-02-14

    Nickel-containing enzymes are diverse in terms of function and active site structure. In many cases, the biosynthesis of the active site depends on accessory proteins which transport and insert the Ni ion. We review and discuss the literature related to the maturation of carbon monoxide dehydrogenases (CODH) which bear a nickel-containing active site consisting of a [Ni-4Fe-4S] center called the C-cluster. The maturation of this center has been much less studied than that of other nickel-containing enzymes such as urease and NiFe hydrogenase. Several proteins present in certain CODH operons, including the nickel-binding proteins CooT and CooJ, still have unclear functions. We question the conception that the maturation of all CODH depends on the accessory protein CooC described as essential for nickel insertion into the active site. The available literature reveals biological variations in CODH active site biosynthesis.

  2. Streptococcus mutans NADH oxidase lies at the intersection of overlapping regulons controlled by oxygen and NAD+ levels.

    PubMed

    Baker, J L; Derr, A M; Karuppaiah, K; MacGilvray, M E; Kajfasz, J K; Faustoferri, R C; Rivera-Ramos, I; Bitoun, J P; Lemos, J A; Wen, Z T; Quivey, R G

    2014-06-01

    NADH oxidase (Nox, encoded by nox) is a flavin-containing enzyme used by the oral pathogen Streptococcus mutans to reduce diatomic oxygen to water while oxidizing NADH to NAD(+). The critical nature of Nox is 2-fold: it serves to regenerate NAD(+), a carbon cycle metabolite, and to reduce intracellular oxygen, preventing formation of destructive reactive oxygen species (ROS). As oxygen and NAD(+) have been shown to modulate the activity of the global transcription factors Spx and Rex, respectively, Nox is potentially poised at a critical junction of two stress regulons. In this study, microarray data showed that either addition of oxygen or loss of nox resulted in altered expression of genes involved in energy metabolism and transport and the upregulation of genes encoding ROS-metabolizing enzymes. Loss of nox also resulted in upregulation of several genes encoding transcription factors and signaling molecules, including the redox-sensing regulator gene rex. Characterization of the nox promoter revealed that nox was regulated by oxygen, through SpxA, and by Rex. These data suggest a regulatory loop in which the roles of nox in reduction of oxygen and regeneration of NAD(+) affect the activity levels of Spx and Rex, respectively, and their regulons, which control several genes, including nox, crucial to growth of S. mutans under conditions of oxidative stress. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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

    PubMed

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

    1989-12-01

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

  4. A Soluble NADH-Dependent Fumarate Reductase in the Reductive Tricarboxylic Acid Cycle of Hydrogenobacter thermophilus TK-6▿

    PubMed Central

    Miura, Akane; Kameya, Masafumi; Arai, Hiroyuki; Ishii, Masaharu; Igarashi, Yasuo

    2008-01-01

    Fumarate reductase (FRD) is an enzyme that reduces fumarate to succinate. In many organisms, it is bound to the membrane and uses electron donors such as quinol. In this study, an FRD from a thermophilic chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6, was purified and characterized. FRD activity using NADH as an electron donor was not detected in the membrane fraction but was found in the soluble fraction. The purified enzyme was demonstrated to be a novel type of FRD, consisting of five subunits. One subunit showed high sequence identity to the catalytic subunits of known FRDs. Although the genes of typical FRDs are assembled in a cluster, the five genes encoding the H. thermophilus FRD were distant from each other in the genome. Furthermore, phylogenetic analysis showed that the H. thermophilus FRD was located in a distinct position from those of known soluble FRDs. This is the first report of a soluble NADH-dependent FRD in Bacteria and of the purification of a FRD that operates in the reductive tricarboxylic acid cycle. PMID:18757546

  5. A Sulfurtransferase Is Essential for Activity of Formate Dehydrogenases in Escherichia coli*

    PubMed Central

    Thomé, Rémi; Gust, Alexander; Toci, René; Mendel, Ralf; Bittner, Florian; Magalon, Axel; Walburger, Anne

    2012-01-01

    l-Cysteine desulfurases provide sulfur to several metabolic pathways in the form of persulfides on specific cysteine residues of an acceptor protein for the eventual incorporation of sulfur into an end product. IscS is one of the three Escherichia coli l-cysteine desulfurases. It interacts with FdhD, a protein essential for the activity of formate dehydrogenases (FDHs), which are iron/molybdenum/selenium-containing enzymes. Here, we address the role played by this interaction in the activity of FDH-H (FdhF) in E. coli. The interaction of IscS with FdhD results in a sulfur transfer between IscS and FdhD in the form of persulfides. Substitution of the strictly conserved residue Cys-121 of FdhD impairs both sulfur transfer from IscS to FdhD and FdhF activity. Furthermore, inactive FdhF produced in the absence of FdhD contains both metal centers, albeit the molybdenum cofactor is at a reduced level. Finally, FdhF activity is sulfur-dependent, as it shows reversible sensitivity to cyanide treatment. Conclusively, FdhD is a sulfurtransferase between IscS and FdhF and is thereby essential to yield FDH activity. PMID:22194618

  6. Pyruvate Dehydrogenase Kinase-4 Structures Reveal a Metastable Open Conformation Fostering Robust Core-free Basal Activity

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

    Wynn, R. Max; Kato, Masato; Chuang, Jacinta L.

    2008-10-21

    Human pyruvate dehydrogenase complex (PDC) is down-regulated by pyruvate dehydrogenase kinase (PDK) isoforms 1-4. PDK4 is overexpressed in skeletal muscle in type 2 diabetes, resulting in impaired glucose utilization. Here we show that human PDK4 has robust core-free basal activity, which is considerably higher than activity levels of other PDK isoforms stimulated by the PDC core. PDK4 binds the L3 lipoyl domain, but its activity is not significantly stimulated by any individual lipoyl domains or the core of PDC. The 2.0-{angstrom} crystal structures of the PDK4 dimer with bound ADP reveal an open conformation with a wider active-site cleft, comparedmore » with that in the closed conformation epitomized by the PDK2-ADP structure. The open conformation in PDK4 shows partially ordered C-terminal cross-tails, in which the conserved DW (Asp{sup 394}-Trp{sup 395}) motif from one subunit anchors to the N-terminal domain of the other subunit. The open conformation fosters a reduced binding affinity for ADP, facilitating the efficient removal of product inhibition by this nucleotide. Alteration or deletion of the DW-motif disrupts the C-terminal cross-tail anchor, resulting in the closed conformation and the nearly complete inactivation of PDK4. Fluorescence quenching and enzyme activity data suggest that compounds AZD7545 and dichloroacetate lock PDK4 in the open and the closed conformational states, respectively. We propose that PDK4 with bound ADP exists in equilibrium between the open and the closed conformations. The favored metastable open conformation is responsible for the robust basal activity of PDK4 in the absence of the PDC core.« less

  7. Pyruvate dehydrogenase kinase-4 structures reveal a metastable open conformation fostering robust core-free basal activity.

    PubMed

    Wynn, R Max; Kato, Masato; Chuang, Jacinta L; Tso, Shih-Chia; Li, Jun; Chuang, David T

    2008-09-12

    Human pyruvate dehydrogenase complex (PDC) is down-regulated by pyruvate dehydrogenase kinase (PDK) isoforms 1-4. PDK4 is overexpressed in skeletal muscle in type 2 diabetes, resulting in impaired glucose utilization. Here we show that human PDK4 has robust core-free basal activity, which is considerably higher than activity levels of other PDK isoforms stimulated by the PDC core. PDK4 binds the L3 lipoyl domain, but its activity is not significantly stimulated by any individual lipoyl domains or the core of PDC. The 2.0-A crystal structures of the PDK4 dimer with bound ADP reveal an open conformation with a wider active-site cleft, compared with that in the closed conformation epitomized by the PDK2-ADP structure. The open conformation in PDK4 shows partially ordered C-terminal cross-tails, in which the conserved DW (Asp(394)-Trp(395)) motif from one subunit anchors to the N-terminal domain of the other subunit. The open conformation fosters a reduced binding affinity for ADP, facilitating the efficient removal of product inhibition by this nucleotide. Alteration or deletion of the DW-motif disrupts the C-terminal cross-tail anchor, resulting in the closed conformation and the nearly complete inactivation of PDK4. Fluorescence quenching and enzyme activity data suggest that compounds AZD7545 and dichloroacetate lock PDK4 in the open and the closed conformational states, respectively. We propose that PDK4 with bound ADP exists in equilibrium between the open and the closed conformations. The favored metastable open conformation is responsible for the robust basal activity of PDK4 in the absence of the PDC core.

  8. Physiological Regulation of Isocitrate Dehydrogenase and the Role of 2-Oxoglutarate in Prochlorococcus sp. Strain PCC 9511

    PubMed Central

    Diez, Jesús; Gómez-Baena, Guadalupe; Rangel-Zúñiga, Oriol Alberto; García-Fernández, José Manuel

    2014-01-01

    The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42) catalyzes the oxidative decarboxylation of isocitrate, to produce 2-oxoglutarate. The incompleteness of the tricarboxylic acids cycle in marine cyanobacteria confers a special importance to isocitrate dehydrogenase in the C/N balance, since 2-oxoglutarate can only be metabolized through the glutamine synthetase/glutamate synthase pathway. The physiological regulation of isocitrate dehydrogenase was studied in cultures of Prochlorococcus sp. strain PCC 9511, by measuring enzyme activity and concentration using the NADPH production assay and Western blotting, respectively. The enzyme activity showed little changes under nitrogen or phosphorus starvation, or upon addition of the inhibitors DCMU, DBMIB and MSX. Azaserine, an inhibitor of glutamate synthase, induced clear increases in the isocitrate dehydrogenase activity and icd gene expression after 24 h, and also in the 2-oxoglutarate concentration. Iron starvation had the most significant effect, inducing a complete loss of isocitrate dehydrogenase activity, possibly mediated by a process of oxidative inactivation, while its concentration was unaffected. Our results suggest that isocitrate dehydrogenase responds to changes in the intracellular concentration of 2-oxoglutarate and to the redox status of the cells in Prochlorococcus. PMID:25061751

  9. A highly sensitive NADH sensor based on a mycelium-like nanocomposite using graphene oxide and multi-walled carbon nanotubes to co-immobilize poly(luminol) and poly(neutral red) hybrid films.

    PubMed

    Chiang Lin, Kuo; Yu Lai, Szu; Ming Chen, Shen

    2014-08-21

    Hybridization of poly(luminol) (PLM) and poly(neutral red) (PNR) has been successfully performed and further enhanced by a conductive and steric hybrid nanotemplate using graphene oxide (GO) and multi-walled carbon nanotubes (MWCNTs). The morphology of the PLM-PNR-MWCNT-GO mycelium-like nanocomposite is studied by SEM and AFM and it is found to be electroactive, pH-dependent, and stable in the electrochemical system. It shows electrocatalytic activity towards NADH with a high current response and low overpotential. Using amperometry, it has been shown to have a high sensitivity of 288.9 μA mM(-1) cm(-2) to NADH (Eapp. = +0.1 V). Linearity is estimated in a concentration range of 1.33 × 10(-8) to 1.95 × 10(-4) M with a detection limit of 1.33 × 10(-8) M (S/N = 3). Particularly, it also shows another linear range of 2.08 × 10(-4) to 5.81 × 10(-4) M with a sensitivity of 151.3 μA mM(-1) cm(-2). The hybridization and activity of PLM and PNR can be effectively enhanced by MWCNTs and GO, resulting in an active hybrid nanocomposite for determination of NADH.

  10. Modulation of alcohol dehydrogenase and ethanol metabolism by sex hormones in the spontaneously hypertensive rat. Effect of chronic ethanol administration

    PubMed Central

    Rachamin, Gloria; Macdonald, J. Alain; Wahid, Samina; Clapp, Jeremy J.; Khanna, Jatinder M.; Israel, Yedy

    1980-01-01

    In young (4-week-old) male and female spontaneously hypertensive (SH) rats, ethanol metabolic rate in vivo and hepatic alcohol dehydrogenase activity in vitro are high and not different in the two sexes. In males, ethanol metabolic rate falls markedly between 4 and 10 weeks of age, which coincides with the time of development of sexual maturity in the rat. Alcohol dehydrogenase activity is also markedly diminished in the male SH rat and correlates well with the changes in ethanol metabolism. There is virtually no influence of age on ethanol metabolic rate and alcohol dehydrogenase activity in the female SH rat. Castration of male SH rats prevents the marked decrease in ethanol metabolic rate and alcohol dehydrogenase activity, whereas ovariectomy has no effect on these parameters in female SH rats. Chronic administration of testosterone to castrated male SH rats and to female SH rats decreases ethanol metabolic rate and alcohol dehydrogenase activity to values similar to those found in mature males. Chronic administration of oestradiol-17β to male SH rats results in marked stimulation of ethanol metabolic rate and alcohol dehydrogenase activity to values similar to those found in female SH rats. Chronic administration of ethanol to male SH rats from 4 to 11 weeks of age prevents the marked age-dependent decreases in ethanol metabolic rate and alcohol dehydrogenase activity, but has virtually no effect in castrated rats. In the intoxicated chronically ethanol-fed male SH rats, serum testosterone concentrations are significantly depressed. In vitro, testosterone has no effect on hepatic alcohol dehydrogenase activity of young male and female SH rats. In conclusion, in the male SH rat, ethanol metabolic rate appears to be limited by alcohol dehydrogenase activity and is modulated by testosterone. Testosterone has an inhibitory effect and oestradiol has a testosterone-dependent stimulatory effect on alcohol dehydrogenase activity and ethanol metabolic rate in these

  11. Efficient reduction of the formation of by-products and improvement of production yield of 2,3-butanediol by a combined deletion of alcohol dehydrogenase, acetate kinase-phosphotransacetylase, and lactate dehydrogenase genes in metabolically engineered Klebsiella oxytoca in mineral salts medium.

    PubMed

    Jantama, Kaemwich; Polyiam, Pattharasedthi; Khunnonkwao, Panwana; Chan, Sitha; Sangproo, Maytawadee; Khor, Kirin; Jantama, Sirima Suvarnakuta; Kanchanatawee, Sunthorn

    2015-07-01

    Klebsiella oxytoca KMS005 (∆adhE∆ackA-pta∆ldhA) was metabolically engineered to improve 2,3-butanediol (BDO) yield. Elimination of alcohol dehydrogenase E (adhE), acetate kinase A-phosphotransacetylase (ackA-pta), and lactate dehydrogenase A (ldhA) enzymes allowed BDO production as a primary pathway for NADH re-oxidation, and significantly reduced by-products. KMS005 was screened for the efficient glucose utilization by metabolic evolution. KMS005-73T improved BDO production at a concentration of 23.5±0.5 g/L with yield of 0.46±0.02 g/g in mineral salts medium containing 50 g/L glucose in a shake flask. KMS005-73T also exhibited BDO yields of about 0.40-0.42 g/g from sugarcane molasses, cassava starch, and maltodextrin. During fed-batch fermentation, KMS005-73T produced BDO at a concentration, yield, and overall and specific productivities of 117.4±4.5 g/L, 0.49±0.02 g/g, 1.20±0.05 g/Lh, and 27.2±1.1 g/gCDW, respectively. No acetoin, lactate, and formate were detected, and only trace amounts of acetate and ethanol were formed. The strain also produced the least by-products and the highest BDO yield among other Klebsiella strains previously developed. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  12. In vivo monitoring of cellular energy metabolism using SoNar, a highly responsive sensor for NAD(+)/NADH redox state.

    PubMed

    Zhao, Yuzheng; Wang, Aoxue; Zou, Yejun; Su, Ni; Loscalzo, Joseph; Yang, Yi

    2016-08-01

    NADH and its oxidized form NAD(+) have a central role in energy metabolism, and their concentrations are often considered to be among the most important readouts of metabolic state. Here, we present a detailed protocol to image and monitor NAD(+)/NADH redox state in living cells and in vivo using a highly responsive, genetically encoded fluorescent sensor known as SoNar (sensor of NAD(H) redox). The chimeric SoNar protein was initially developed by inserting circularly permuted yellow fluorescent protein (cpYFP) into the NADH-binding domain of Rex protein from Thermus aquaticus (T-Rex). It functions by binding to either NAD(+) or NADH, thus inducing protein conformational changes that affect its fluorescent properties. We first describe steps for how to establish SoNar-expressing cells, and then discuss how to use the system to quantify the intracellular redox state. This approach is sensitive, accurate, simple and able to report subtle perturbations of various pathways of energy metabolism in real time. We also detail the application of SoNar to high-throughput chemical screening of candidate compounds targeting cell metabolism in a microplate-reader-based assay, along with in vivo fluorescence imaging of tumor xenografts expressing SoNar in mice. Typically, the approximate time frame for fluorescence imaging of SoNar is 30 min for living cells and 60 min for living mice. For high-throughput chemical screening in a 384-well-plate assay, the whole procedure generally takes no longer than 60 min to assess the effects of 380 compounds on cell metabolism.

  13. Subcellular distribution of delta 5-3 beta-hydroxy steroid dehydrogenase in the granulosa cells of the domestic fowl (Gallus domesticus).

    PubMed Central

    Armstrong, D G

    1979-01-01

    1. The distribution of 3 beta-hydroxy steroid dehydrogenase was examined in the subcellular fractions of granulosa cells collected from the ovary of the domestic fowl. 2. 3 beta-hydroxy steroid dehydrogenase activity was observed in the mitochondrial (4000g for 20min) and microsomal (105 000g for 120min) fractions. 3. Approximately three times more 3 beta-hydroxy steroid dehydrogenase activity was associated with the cytochrome oxidase activity (a mitochondrial marker enzyme) in anteovulatory-follicle granulosa cells than with that of the postovulatory follicle. 4. Comparison of the latent properties of mitochondrial 3 beta-hydroxy steroid dehydrogenase with those of cytochrome oxidase and isocitrate dehydrogenase indicated that 3 beta-hydroxy steroid dehydrogenase is located extramitochondrially. 5. This apparent distribution of 3 beta-hydroxy steroid dehydrogenase is explained on the basis that the mitochondrial activity is either an artefact caused by a redistribution in the subcellular location of the enzyme, occurring during homogenization, or by the existence of a functionally heterogeneous endoplasmic reticulum that yields particles of widely differing sedimentation properties. PMID:518548

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

    PubMed

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

    2013-07-01

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

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

    PubMed Central

    Pavlova, Sylvia I.; Jin, Ling; Gasparovich, Stephen R.

    2013-01-01

    Ethanol consumption and poor oral hygiene are risk factors for oral and oesophageal cancers. Although oral streptococci have been found to produce excessive acetaldehyde from ethanol, little is known about the mechanism by which this carcinogen is produced. By screening 52 strains of diverse oral streptococcal species, we identified Streptococcus gordonii V2016 that produced the most acetaldehyde from ethanol. We then constructed gene deletion mutants in this strain and analysed them for alcohol and acetaldehyde dehydrogenases by zymograms. The results showed that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol and ethanol, respectively. Two additional dehydrogenases, S-AdhA and TdhA, were identified with specificities to the secondary alcohol 2-propanol and threonine, respectively, but not to ethanol. S. gordonii V2016 did not show a detectable acetaldehyde dehydrogenase even though its adhE gene encodes a putative bifunctional acetaldehyde/alcohol dehydrogenase. Mutants with adhE deletion showed greater tolerance to ethanol in comparison with the wild-type and mutant with adhA or adhB deletion, indicating that AdhE is the major alcohol dehydrogenase in S. gordonii. Analysis of 19 additional strains of S. gordonii, S. mitis, S. oralis, S. salivarius and S. sanguinis showed expressions of up to three alcohol dehydrogenases, but none showed detectable acetaldehyde dehydrogenase, except one strain that showed a novel ALDH. Therefore, expression of multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase may contribute to excessive production of acetaldehyde from ethanol by certain oral streptococci. PMID:23637459

  16. NAD+/NADH and/or CoQ/CoQH2 ratios from plasma membrane electron transport may determine ceramide and sphingosine-1-phosphate levels accompanying G1 arrest and apoptosis.

    PubMed

    De Luca, Thomas; Morré, Dorothy M; Zhao, Haiyun; Morré, D James

    2005-01-01

    To elucidate possible biochemical links between growth arrest from antiproliferative chemotherapeutic agents and apoptosis, our work has focused on agents (EGCg, capsaicin, cis platinum, adriamycin, anti-tumor sulfonylureas, phenoxodiol) that target tNOX. tNOX is a cancer-specific cell surface NADH oxidase (ECTO-NOX protein), that functions in cancer cells as the terminal oxidase for plasma membrane electron transport. When tNOX is active, coenzyme Q(10) (ubiquinone) of the plasma membrane is oxidized and NADH is oxidized at the cytosolic surface of the plasma membrane. However, when tNOX is inhibited and plasma membrane electron transport is diminished, both reduced coenzyme Q(10) (ubiquinol) and NADH would be expected to accumulate. To relate inhibition of plasma membrane redox to increased ceramide levels and arrest of cell proliferation in G(1) and apoptosis, we show that neutral sphingomyelinase, a major contributor to plasma membrane ceramide, is inhibited by reduced glutathione and ubiquinone. Ubiquinol is without effect or stimulates. In contrast, sphingosine kinase, which generates anti-apoptotic sphingosine-1-phosphate, is stimulated by ubiquinone but inhibited by ubiquinol and NADH. Thus, the quinone and pyridine nucleotide products of plasma membrane redox, ubiquinone and ubiquinol, as well as NAD(+) and NADH, may directly modulate in a reciprocal manner two key plasma membrane enzymes, sphingomyelinase and sphingosine kinase, potentially leading to G(1) arrest (increase in ceramide) and apoptosis (loss of sphingosine-1-phosphate). As such, the findings provide potential links between coenzyme Q(10)-mediated plasma membrane electron transport and the anticancer action of several clinically-relevant anticancer agents.

  17. Fluoxetine elevates allopregnanolone in female rat brain but inhibits a steroid microsomal dehydrogenase rather than activating an aldo-keto reductase

    PubMed Central

    Fry, J P; Li, K Y; Devall, A J; Cockcroft, S; Honour, J W; Lovick, T A

    2014-01-01

    Background and Purpose Fluoxetine, a selective serotonin reuptake inhibitor, elevates brain concentrations of the neuroactive progesterone metabolite allopregnanolone, an effect suggested to underlie its use in the treatment of premenstrual dysphoria. One report showed fluoxetine to activate the aldo-keto reductase (AKR) component of 3α-hydroxysteroid dehydrogenase (3α-HSD), which catalyses production of allopregnanolone from 5α-dihydroprogesterone. However, this action was not observed by others. The present study sought to clarify the site of action for fluoxetine in elevating brain allopregnanolone. Experimental Approach Adult male rats and female rats in dioestrus were treated with fluoxetine and their brains assayed for allopregnanolone and its precursors, progesterone and 5α-dihydroprogesterone. Subcellular fractions of rat brain were also used to investigate the actions of fluoxetine on 3α-HSD activity in both the reductive direction, producing allopregnanolone from 5α-dihydroprogesterone, and the reverse oxidative direction. Fluoxetine was also tested on these recombinant enzyme activities expressed in HEK cells. Key Results Short-term treatment with fluoxetine increased brain allopregnanolone concentrations in female, but not male, rats. Enzyme assays on native rat brain fractions and on activities expressed in HEK cells showed fluoxetine did not affect the AKR producing allopregnanolone from 5α-dihydroprogesterone but did inhibit the microsomal dehydrogenase oxidizing allopregnanolone to 5α-dihydroprogesterone. Conclusions and Implications Fluoxetine elevated allopregnanolone in female rat brain by inhibiting its oxidation to 5α-dihydroprogesterone by a microsomal dehydrogenase. This is a novel site of action for fluoxetine, with implications for the development of new agents and/or dosing regimens to raise brain allopregnanolone. PMID:25161074

  18. Selection of an endogenous 2,3-butanediol pathway in Escherichia coli by fermentative redox balance.

    PubMed

    Liang, Keming; Shen, Claire R

    2017-01-01

    Fermentative redox balance has long been utilized as a metabolic evolution platform to improve efficiency of NADH-dependent pathways. However, such system relies on the complete recycling of NADH and may become limited when the target pathway results in excess NADH stoichiometrically. In this study, endogenous capability of Escherichia coli for 2,3-butanediol (2,3-BD) synthesis was explored using the anaerobic selection platform based on redox balance. To address the issue of NADH excess associated with the 2,3-BD pathway, we devised a substrate-decoupled system where a pathway intermediate is externally supplied in addition to the carbon source to decouple NADH recycling ratio from the intrinsic pathway stoichiometry. In this case, feeding of the 2,3-BD precursor acetoin effectively restored anaerobic growth of the mixed-acid fermentation mutant that remained otherwise inhibited even in the presence of a functional 2,3-BD pathway. Using established 2,3-BD dehydrogenases as model enzyme, we verified that the redox-based selection system is responsive to NADPH-dependent reactions but with lower sensitivity. Based on this substrate-decoupled selection scheme, we successfully identified the glycerol/1,2-propanediol dehydrogenase (Ec-GldA) as the major enzyme responsible for the acetoin reducing activity (k cat /K m ≈0.4mM -1 s -1 ) observed in E. coli. Significant shift of 2,3-BD configuration upon withdrawal of the heterologous acetolactate decarboxylase revealed that the endogenous synthesis of acetoin occurs via diacetyl. Among the predicted diacetyl reductase in E. coli, Ec-UcpA displayed the most significant activity towards diacetyl reduction into acetoin (V max ≈6U/mg). The final strain demonstrated a meso-2,3-BD production titer of 3g/L without introduction of foreign genes. The substrate-decoupled selection system allows redox balance regardless of the pathway stoichiometry thus enables segmented optimization of different reductive pathways through enzyme

  19. Investigation of tryptophan-NADH interactions in live human cells using three-photon fluorescence lifetime imaging and Förster resonance energy transfer microscopy

    NASA Astrophysics Data System (ADS)

    Jyothikumar, Vinod; Sun, Yuansheng; Periasamy, Ammasi

    2013-06-01

    A method to investigate the metabolic activity of intracellular tryptophan (TRP) and coenzyme-NADH using three-photon (3P) fluorescence lifetime imaging (FLIM) and Förster resonance energy transfer (FRET) is presented. Through systematic analysis of FLIM data from tumorigenic and nontumorigenic cells, a statistically significant decrease in the fluorescence lifetime of TRP was observed in response to the increase in protein-bound NADH as cells were treated with glucose. The results demonstrate the potential use of 3P-FLIM-FRET as a tool for label-free screening of the change in metabolic flux occurring in human diseases or other clinical conditions.

  20. Quantitative functional characterization of conserved molecular interactions in the active site of mannitol 2-dehydrogenase

    PubMed Central

    Lucas, James E; Siegel, Justin B

    2015-01-01

    Enzyme active site residues are often highly conserved, indicating a significant role in function. In this study we quantitate the functional contribution for all conserved molecular interactions occurring within a Michaelis complex for mannitol 2-dehydrogenase derived from Pseudomonas fluorescens (pfMDH). Through systematic mutagenesis of active site residues, we reveal that the molecular interactions in pfMDH mediated by highly conserved residues not directly involved in reaction chemistry can be as important to catalysis as those directly involved in the reaction chemistry. This quantitative analysis of the molecular interactions within the pfMDH active site provides direct insight into the functional role of each molecular interaction, several of which were unexpected based on canonical sequence conservation and structural analyses. PMID:25752240

  1. Purification and characterization of xylitol dehydrogenase with l-arabitol dehydrogenase activity from the newly isolated pentose-fermenting yeast Meyerozyma caribbica 5XY2.

    PubMed

    Sukpipat, Wiphat; Komeda, Hidenobu; Prasertsan, Poonsuk; Asano, Yasuhisa

    2017-01-01

    Meyerozyma caribbica strain 5XY2, which was isolated from an alcohol fermentation starter in Thailand, was found to catabolize l-arabinose as well as d-glucose and d-xylose. The highest production amounts of ethanol from d-glucose, xylitol from d-xylose, and l-arabitol from l-arabinose were 0.45 g/g d-glucose, 0.60 g/g d-xylose, and 0.61 g/g l-arabinose with 21.7 g/L ethanol, 20.2 g/L xylitol, and 30.3 g/l l-arabitol, respectively. The enzyme with l-arabitol dehydrogenase (LAD) activity was purified from the strain and found to exhibit broad specificity to polyols, such as xylitol, d-sorbitol, ribitol, and l-arabitol. Xylitol was the preferred substrate with K m =16.1 mM and k cat /K m =67.0 min -1 mM -1 , while l-arabitol was also a substrate for the enzyme with K m =31.1 mM and k cat /K m =6.5 min -1  mM -1 . Therefore, this enzyme from M. caribbica was named xylitol dehydrogenase (McXDH). McXDH had an optimum temperature and pH at 40°C and 9.5, respectively. The McXDH gene included a coding sequence of 1086 bp encoding a putative 362 amino acid protein of 39 kDa with an apparent homopentamer structure. Native McXDH and recombinant McXDH exhibited relative activities toward l-arabitol of approximately 20% that toward xylitol, suggesting the applicability of this enzyme with the functions of XDH and LAD to the development of pentose-fermenting Saccharomyces cerevisiae. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  2. Efficient Enzymatic Preparation of (13) N-Labelled Amino Acids: Towards Multipurpose Synthetic Systems.

    PubMed

    da Silva, Eunice S; Gómez-Vallejo, Vanessa; Baz, Zuriñe; Llop, Jordi; López-Gallego, Fernando

    2016-09-12

    Nitrogen-13 can be efficiently produced in biomedical cyclotrons in different chemical forms, and its stable isotopes are present in the majority of biologically active molecules. Hence, it may constitute a convenient alternative to Fluorine-18 and Carbon-11 for the preparation of positron-emitter-labelled radiotracers; however, its short half-life demands for the development of simple, fast, and efficient synthetic processes. Herein, we report the one-pot, enzymatic and non-carrier-added synthesis of the (13) N-labelled amino acids l-[(13) N]alanine, [(13) N]glycine, and l-[(13) N]serine by using l-alanine dehydrogenase from Bacillus subtilis, an enzyme that catalyses the reductive amination of α-keto acids by using nicotinamide adenine dinucleotide (NADH) as the redox cofactor and ammonia as the amine source. The integration of both l-alanine dehydrogenase and formate dehydrogenase from Candida boidinii in the same reaction vessel to facilitate the in situ regeneration of NADH during the radiochemical synthesis of the amino acids allowed a 50-fold decrease in the concentration of the cofactor without compromising reaction yields. After optimization of the experimental conditions, radiochemical yields were sufficient to carry out in vivo imaging studies in small rodents. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed Central

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

    1989-01-01

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

  4. Androgen-estrogen synergy in rat levator ani muscle Glucose-6-phosphate dehydrogenase

    NASA Technical Reports Server (NTRS)

    Max, S. R.

    1984-01-01

    The effects of castration and hormone administration on the activity of glucose-6-phosphate dehydrogenase in the rat levator ani muscle were studied. Castration caused a decrease in enzyme activity and in wet weight of the levator ani muscle. Chronic administration of testosterone propionate increased glucose-6-phosphate dehydrogenase activity in the levator ani muscle of castrated rats; the magnitude of the recovery of enzyme activity was related to the length of time of exposure to testosterone propionate after castration as well as to the length of time the animals were castrated. The longer the period of castration before exposure to testosterone propionate, the greater the effect. This result may be related to previously reported castration-mediated increases in androgen receptor binding in muscle. Dihydrotestosterone was less effective than testosterone propionate in enhancing glucose-6-phosphate dehydrogenase activity in the levator ani muscle from castrated rats; estradiol-17-beta alone was ineffective. Combined treatment with estradiol-17-beta and dihydrotestosterone, however, was as effective as testosterone alone. Thus, androgens and estrogens may exert synergistic effects on levator ani muscle.

  5. Pyruvate Formate-Lyase Is Essential for Fumarate-Independent Anaerobic Glycerol Utilization in the Enterococcus faecalis Strain W11

    PubMed Central

    Ikegami, Yuki

    2014-01-01

    Although anaerobic glycerol metabolism in Enterococcus faecalis requires exogenous fumarate for NADH oxidation, E. faecalis strain W11 can metabolize glycerol in the absence of oxygen without exogenous fumarate. In this study, metabolic end product analyses and reporter assays probing the expression of enzymes involved in pyruvate metabolism were performed to investigate this fumarate-independent anaerobic metabolism of glycerol in W11. Under aerobic conditions, the metabolic end products of W11 cultured with glycerol were similar to those of W11 cultured with glucose. However, when W11 was cultured anaerobically, most of the glucose was converted to l-lactate, but glycerol was converted to ethanol and formate. During anaerobic culture with glycerol, the expression of the l-lactate dehydrogenase and pyruvate dehydrogenase E1αβ genes in W11 was downregulated, whereas the expression of the pyruvate formate-lyase (Pfl) and aldehyde/alcohol dehydrogenase genes was upregulated. These changes in the expression levels caused the change in the composition of end products. A pflB gene disruptant (Δpfl mutant) of W11 could barely utilize glycerol under anaerobic conditions, but the growth of the Δpfl mutant cultured with either glucose or dihydroxyacetone (DHA) under anaerobic conditions was the same as that of W11. Glucose metabolism and DHA generates one NADH molecule per pyruvate molecule, whereas glycerol metabolism in the dehydrogenation pathway generates two NADH molecules per pyruvate molecule. These findings demonstrate that NADH generated from anaerobic glycerol metabolism in the absence of fumarate is oxidized through the Pfl-ethanol fermentation pathway. Thus, Pfl is essential to avoid the accumulation of excess NADH during fumarate-independent anaerobic glycerol metabolism. PMID:24769696

  6. Decrease in the cytosolic NADP+-dependent isocitrate dehydrogenase activity through porcine sperm capacitation.

    PubMed

    Katoh, Yuki; Tamba, Michiko; Matsuda, Manabu; Kikuchi, Kazuhiro; Okamura, Naomichi

    2018-02-26

    In order to understand the molecular mechanisms involved in the sperm capacitation, we have identified the proteins tyrosine-phosphorylated during the capacitation especially in conjunction with the regulation of the levels of reactive oxygen species (ROS) in sperm. In the present study, the effects of the tyrosine phosphorylation of cytosolic NADP + -dependent isocitrate dehydrogenase (IDPc) on its catalytic activity and on the levels of ROS in sperm have been studied. The tyrosine phosphorylated IDPc showed a significantly lowered enzymatic activity. The immunocytochemical analyses using the highly specific antisera against IDPc revealed that IDPc was mainly localized to the principal piece of the porcine sperm flagellum. As IDPc is one of the major NADPH regenerating enzymes in porcine sperm, it is strongly suggested that the decrease in IDPc activity is involved in the increased levels of ROS, which results in the induction of hyperactivated flagellar movement and capacitation. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. Real-time evaluation of tissue vitality by monitoring of microcirculatory blood flow, HbO2, and mitochondrial NADH redox state

    NASA Astrophysics Data System (ADS)

    Deutsch, Assaf; Pevzner, Eliyahu; Jaronkin, Alex; Mayevsky, Avraham

    2004-06-01

    Monitoring of tissue vitality (oxygen supply/demand) in real time is very rare in clinical practice although its use as an early warning alarming system, for clinical care medicine, is very practical. In our previous communication (SPIE 2003) we described the Tissue Spectroscope - TiSpec02, by which tissue microcirculatory blood flow (TBF) and mitochondrial NADH fluorescence were measured using a single light source (390nm). In order to improve the measurement capabilities as well as to decrease dramatically the size and cost of this clinical device, we have changed the TiSpec02 into a multi-wavelength illumination system in the new TiSpec03. In order to measure microcirculatory blood flow by laser Doppler flowmetry we used a 785nm laser diode. For mitochondrial NADH fluorescence measurement we adopted the 370nm LED. For the determination of the oxygenation level of hemoglobin (HbO2) we used the 2-wavelength reflectance technique. This new monitored parameter that was added to the TiSpec03 increases the accuracy of the diagnosis of tissue vitality. The bundle of optical fibers used to connect the tissue to the TiSpec03, was integrated into a special anchoring methodology depending on the monitored tissue or organ. In order to test the performance of the improved TiSpec we have used it in experimental animals brain models exposed to various pathophysiological conditions. Rats and gerbils were anesthetized and the fiber optic probe was located epidurally used dental acrylic cement. During anoxia and ischemia the lack of O2 led to a clear decrease in TBF and HbO2 while NADH shows a large elevation. When brain activation was induced by cortical spreading depression (SD), the elevated O2 consumption was recorded as a large oxidation (decrease) of mitochondrial NADH while TBF increase dramatically. Blood HbO2 was not affected significantly by the SD wave.

  8. Studies on the mechanism of electron bifurcation catalyzed by electron transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (Bcd) of Acidaminococcus fermentans.

    PubMed

    Chowdhury, Nilanjan Pal; Mowafy, Amr M; Demmer, Julius K; Upadhyay, Vikrant; Koelzer, Sebastian; Jayamani, Elamparithi; Kahnt, Joerg; Hornung, Marco; Demmer, Ulrike; Ermler, Ulrich; Buckel, Wolfgang

    2014-02-21

    Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (EtfAf) and butyryl-CoA dehydrogenase (BcdAf) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. EtfAf contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The EtfAf-NAD(+) complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH(-) is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH(-) by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD, which donates this electron further to Dh-FAD of BcdAf after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH(•), immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH(-) that converts crotonyl-CoA to butyryl-CoA.

  9. A conjugated fatty acid present at high levels in bitter melon seed favorably affects lipid metabolism in hepatocytes by increasing NAD(+)/NADH ratio and activating PPARα, AMPK and SIRT1 signaling pathway.

    PubMed

    Chen, Gou-Chun; Su, Hui-Min; Lin, Yu-Shun; Tsou, Po-Yen; Chyuan, Jong-Ho; Chao, Pei-Min

    2016-07-01

    α-Eleostearic acid (α-ESA), or the cis-9, trans-11, trans-13 isomer of conjugated linolenic acid, is a special fatty acid present at high levels in bitter melon seed oil. The aim of this study was to examine the effect of α-ESA on hepatic lipid metabolism. Using H4IIEC3 hepatoma cell line, we showed that α-ESA significantly lowered intracellular triglyceride accumulation compared to α-linolenic acid (LN), used as a fatty acid control, in a dose- and time-dependent manner. The effects of α-ESA on enzyme activities and mRNA profiles in H4IIEC3 cells suggested that enhanced fatty acid oxidation and lowered lipogenesis were involved in α-ESA-mediated triglyceride lowering effects. In addition, α-ESA triggered AMP-activated protein kinase (AMPK) activation without altering sirtuin 1 (SIRT1) protein levels. When cells were treated with vehicle control (VC), LN alone (LN; 100μmol/L) or in combination with α-ESA (LN+α-ESA; 75+25μmol/L) for 24h, acetylation of forkhead box protein O1 was decreased, while the NAD(+)/NADH ratio, mRNA levels of NAMPT and PTGR1 and enzyme activity of nicotinamide phosphoribosyltransferase were increased by LN+α-ESA treatment compared to treatment with LN alone, suggesting that α-ESA activates SIRT1 by increasing NAD(+) synthesis and NAD(P)H consumption. The antisteatosis effect of α-ESA was confirmed in mice treated with a high-sucrose diet supplemented with 1% α-ESA for 5weeks. We conclude that α-ESA favorably affects hepatic lipid metabolism by increasing cellular NAD(+)/NADH ratio and activating PPARα, AMPK and SIRT1 signaling pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Contributory roles of two l-lactate dehydrogenases for l-lactic acid production in thermotolerant Bacillus coagulans.

    PubMed

    Sun, Lifan; Zhang, Caili; Lyu, Pengcheng; Wang, Yanping; Wang, Limin; Yu, Bo

    2016-11-25

    Thermotolerant Bacillus coagulans is considered to be a more promising producer for bio-chemicals, due to its capacity to withstand harsh conditions. Two L-lactate dehydrogenase (LDH) encoding genes (ldhL1 and ldhL2) and one D-LDH encoding gene (ldhD) were annotated from the B. coagulans DSM1 genome. Transcriptional analysis revealed that the expression of ldhL2 was undetectable while the ldhL1 transcription level was much higher than that of ldhD at all growth phases. Deletion of the ldhL2 gene revealed no difference in fermentation profile compared to the wild-type strain, while ldhL1 single deletion or ldhL1ldhL2 double deletion completely blocked L-lactic acid production. Complementation of ldhL1 in the above knockout strains restored fermentation profiles to those observed in the wild-type strain. This study demonstrates ldhL1 is crucial for L-lactic acid production and NADH balance in B. coagulans DSM1 and lays the fundamental for engineering the thermotolerant B. coagulans strain as a platform chemicals producer.

  11. Free energy landscape of the Michaelis complex of lactate dehydrogenase: A network analysis of atomistic simulations

    NASA Astrophysics Data System (ADS)

    Pan, Xiaoliang; Schwartz, Steven

    2015-03-01

    It has long been recognized that the structure of a protein is a hierarchy of conformations interconverting on multiple time scales. However, the conformational heterogeneity is rarely considered in the context of enzymatic catalysis in which the reactant is usually represented by a single conformation of the enzyme/substrate complex. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of two forms of the cofactor nicotinamide adenine dinucleotide (NADH and NAD+). Recent experimental results suggest that multiple substates exist within the Michaelis complex of LDH, and they are catalytic competent at different reaction rates. In this study, millisecond-scale all-atom molecular dynamics simulations were performed on LDH to explore the free energy landscape of the Michaelis complex, and network analysis was used to characterize the distribution of the conformations. Our results provide a detailed view of the kinetic network the Michaelis complex and the structures of the substates at atomistic scale. It also shed some light on understanding the complete picture of the catalytic mechanism of LDH.

  12. Free energy surface of the Michaelis complex of lactate dehydrogenase: a network analysis of microsecond simulations.

    PubMed

    Pan, Xiaoliang; Schwartz, Steven D

    2015-04-30

    It has long been recognized that the structure of a protein creates a hierarchy of conformations interconverting on multiple time scales. The conformational heterogeneity of the Michaelis complex is of particular interest in the context of enzymatic catalysis in which the reactant is usually represented by a single conformation of the enzyme/substrate complex. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of two forms of the cofactor nicotinamide adenine dinucleotide (NADH and NAD(+)). Recent experimental results suggest that multiple substates exist within the Michaelis complex of LDH, and they show a strong variance in their propensity toward the on-enzyme chemical step. In this study, microsecond-scale all-atom molecular dynamics simulations were performed on LDH to explore the free energy landscape of the Michaelis complex, and network analysis was used to characterize the distribution of the conformations. Our results provide a detailed view of the kinetic network of the Michaelis complex and the structures of the substates at atomistic scales. They also shed light on the complete picture of the catalytic mechanism of LDH.

  13. The plasma membrane-associated NADH oxidase (ECTO-NOX) of mouse skin responds to blue light

    NASA Technical Reports Server (NTRS)

    Morre, D. James; Morre, Dorothy M.

    2003-01-01

    NADH oxidases of the external plasma membrane surface (ECTO-NOX proteins) are characterized by oscillations in activity with a regular period length of 24 min. Explants of mouse skin exhibit the oscillatory activity as estimated from the decrease in A(340) suggesting that individual ECTO-NOX molecules must somehow be induced to function synchronously. Transfer of explants of mouse skin from darkness to blue light (495 nm, 2 min, 50 micromol m(-1) s(-1)) resulted in initiation of a new activity maximum (entrainment) with a midpoint 36 min after light exposure followed by maxima every 24 min thereafter. Addition of melatonin resulted in a new maximum 24 min after melatonin addition. The findings suggest that the ECTO-NOX proteins play a central role in the entrainment of the biological clock both by light and by melatonin.

  14. Intracellular Redox State Revealed by In Vivo 31P MRS Measurement of NAD+ and NADH Contents in Brains

    PubMed Central

    Lu, Ming; Zhu, Xiao-Hong; Zhang, Yi; Chen, Wei

    2015-01-01

    Purpose Nicotinamide adenine dinucleotide (NAD), in oxidized (NAD+) or reduced (NADH) form, plays key roles in cellular metabolism. Intracellular NAD+/NADH ratio represents the cellular redox state; however, it is difficult to measure in vivo. We report here a novel in vivo 31P MRS method for noninvasive measurement of intracellular NAD concentrations and NAD+/NADH ratio in the brain. Methods It uses a theoretical model to describe the NAD spectral patterns at a given field for quantification. Standard NAD solutions and independent cat brain measurements at 9.4 T and 16.4 T were used to evaluate this method. We also measured T1 values of brain NAD. Results Model simulation and studies of solutions and brains indicate that the proposed method can quantify submillimolar NAD concentrations with reasonable accuracy if adequate 31P MRS signal-to-noise ratio and linewidth were obtained. The NAD concentrations and NAD+/NADH ratio of cat brains measured at 16.4 T and 9.4 T were consistent despite the significantly different T1 values and NAD spectra patterns at two fields. Conclusion This newly established 31P MRS method makes it possible for the first time to noninvasively study the intracellular redox state and its roles in brain functions and diseases, and it can potentially be applied to other organs. PMID:23843330

  15. Autodisplay of active sorbitol dehydrogenase (SDH) yields a whole cell biocatalyst for the synthesis of rare sugars.

    PubMed

    Jose, Joachim; von Schwichow, Steffen

    2004-04-02

    Whole cell biocatalysts are attractive technological tools for the regio- and enantioselective synthesis of products, especially from substrates with several identical reactive groups. In the present study, a whole cell biocatalyst for the synthesis of rare sugars from polyalcohols was constructed. For this purpose, sorbitol dehydrogenase (SDH) from Rhodobacter sphaeroides, a member of the short-chain dehydrogenase/reductase (SDR) family, was expressed on the surface of Escherichia coli using Autodisplay. Autodisplay is an efficient surface display system for Gram-negative bacteria and is based on the autotransporter secretion pathway. Transport of SDH to the outer membrane was monitored by SDS-PAGE and Western blotting of different cell fractions. The surface exposure of the enzyme could be verified by immunofluorescence microscopy and fluorescence activated cell sorting (FACS). The activity of whole cells displaying SDH at the surface was determined in an optical test. Specific activities were found to be 12 mU per 3.3 x 10(8) cells for the conversion of D-glucitol (sorbitol) to D-fructose, 7 mU for the conversion D-galactitol to D-tagatose, and 17 mU for the conversion of L-arabitol to L-ribulose. The whole cell biocatalyst obtained by surface display of SDH could also produce D-glucitol from D-fructose (29 mU per 3.3 x 10(8) cells).

  16. Engineering of Corynebacterium glutamicum for high-yield L-valine production under oxygen deprivation conditions.

    PubMed

    Hasegawa, Satoshi; Suda, Masako; Uematsu, Kimio; Natsuma, Yumi; Hiraga, Kazumi; Jojima, Toru; Inui, Masayuki; Yukawa, Hideaki

    2013-02-01

    We previously demonstrated efficient L-valine production by metabolically engineered Corynebacterium glutamicum under oxygen deprivation. To achieve the high productivity, a NADH/NADPH cofactor imbalance during the synthesis of l-valine was overcome by engineering NAD-preferring mutant acetohydroxy acid isomeroreductase (AHAIR) and using NAD-specific leucine dehydrogenase from Lysinibacillus sphaericus. Lactate as a by-product was largely eliminated by disrupting the lactate dehydrogenase gene ldhA. Nonetheless, a few other by-products, particularly succinate, were still produced and acted to suppress the L-valine yield. Eliminating these by-products therefore was deemed key to improving theL-valine yield. By additionally disrupting the phosphoenolpyruvate carboxylase gene ppc, succinate production was effectively suppressed, but both glucose consumption and L-valine production dropped considerably due to the severely elevated intracellular NADH/NAD(+) ratio. In contrast, this perturbed intracellular redox state was more than compensated for by deletion of three genes associated with NADH-producing acetate synthesis and overexpression of five glycolytic genes, including gapA, encoding NADH-inhibited glyceraldehyde-3-phosphate dehydrogenase. Inserting feedback-resistant mutant acetohydroxy acid synthase and NAD-preferring mutant AHAIR in the chromosome resulted in higher L-valine yield and productivity. Deleting the alanine transaminase gene avtA suppressed alanine production. The resultant strain produced 1,280 mM L-valine at a yield of 88% mol mol of glucose(-1) after 24 h under oxygen deprivation, a vastly improved yield over our previous best.

  17. Engineering of Corynebacterium glutamicum for High-Yield l-Valine Production under Oxygen Deprivation Conditions

    PubMed Central

    Hasegawa, Satoshi; Suda, Masako; Uematsu, Kimio; Natsuma, Yumi; Hiraga, Kazumi; Jojima, Toru; Inui, Masayuki

    2013-01-01

    We previously demonstrated efficient l-valine production by metabolically engineered Corynebacterium glutamicum under oxygen deprivation. To achieve the high productivity, a NADH/NADPH cofactor imbalance during the synthesis of l-valine was overcome by engineering NAD-preferring mutant acetohydroxy acid isomeroreductase (AHAIR) and using NAD-specific leucine dehydrogenase from Lysinibacillus sphaericus. Lactate as a by-product was largely eliminated by disrupting the lactate dehydrogenase gene ldhA. Nonetheless, a few other by-products, particularly succinate, were still produced and acted to suppress the l-valine yield. Eliminating these by-products therefore was deemed key to improving the l-valine yield. By additionally disrupting the phosphoenolpyruvate carboxylase gene ppc, succinate production was effectively suppressed, but both glucose consumption and l-valine production dropped considerably due to the severely elevated intracellular NADH/NAD+ ratio. In contrast, this perturbed intracellular redox state was more than compensated for by deletion of three genes associated with NADH-producing acetate synthesis and overexpression of five glycolytic genes, including gapA, encoding NADH-inhibited glyceraldehyde-3-phosphate dehydrogenase. Inserting feedback-resistant mutant acetohydroxy acid synthase and NAD-preferring mutant AHAIR in the chromosome resulted in higher l-valine yield and productivity. Deleting the alanine transaminase gene avtA suppressed alanine production. The resultant strain produced 1,280 mM l-valine at a yield of 88% mol mol of glucose−1 after 24 h under oxygen deprivation, a vastly improved yield over our previous best. PMID:23241971

  18. Crystal structure of homoisocitrate dehydrogenase from Schizosaccharomyces pombe

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

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

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

  19. Studies on associations of glycolytic and glutaminolytic enzymes in MCF-7 cells: role of P36.

    PubMed

    Mazurek, S; Hugo, F; Failing, K; Eigenbrodt, E

    1996-05-01

    Isoelectric focusing of MCF-7 cell extracts revealed an association of the glycolytic enzymes glyceraldehyde 3-phosphate-dehydrogenase, phosphoglycerate kinase, enolase, and pyruvate kinase. This complex between the glycolytic enzymes is sensitive to RNase. p36 could not be detected within this association of glycolytic enzymes; however an association of p36 with a specific form of malate dehydrogenase was found. In MCF-7 cells three forms of malate dehydrogenase can be detected by isoelectric focusing: the mitochondrial form with an isoelectric point between 8.9 and 9.5, the cytosolic form with pl 5.0, and a p36-associated form with pl 7.8. The mitochondrial form comprises the mature mitochondrial isoenzyme (pl 9.5) and its precursor form (pl 8.9). Refocusing of the pl 7.8 form of malate dehydrogenase also gave rise to the mitochondrial isoenzyme. Thus, the pl 7.8 form of malate dehydrogenase is actually the mitochondrial isoenzyme retained in the cytosol by the association with p36. Addition of fructose 1,6-bisphosphate to the initial focusing column induced a quantitative shift of the pl 7.8 form of malate dehydrogenase to the mitochondrial forms (pl 8.9 and 9.5). In MCF-7 cells p36 is not phosphorylated in tyrosine. Kinetic measurements revealed that the pl 7.8 form of malate dehydrogenase has the lowest affinity for NADH. Compared to both mitochondrial forms the cytosolic isoenzyme has a high capacity when measured in the NAD --> NADH direction (malate --> oxaloacetate direction). The association of p36 with the mitochondrial isoenzyme may favor the flow of hydrogen from the cytosol into the mitochondria. Inhibition of cell proliferation by AMP which leads to an inhibition of glycolysis has no effect on complex formation by glycolytic and glutaminolytic enzymes in MCF-7 cells. AMP treatment leads to an activation of malate dehydrogenase, which correlates with the increase of pyruvate and the decrease of lactate levels, but has no effect on the distribution of

  20. Symmetrical refolding of protein domains and subunits: example of the dimeric two-domain 3-isopropylmalate dehydrogenases.

    PubMed

    Gráczer, Eva; Varga, Andrea; Melnik, Bogdan; Semisotnov, Gennady; Závodszky, Péter; Vas, Mária

    2009-02-10

    The refolding mechanism of the homodimeric two-domain 3-isopropylmalate dehydrogenase (IPMDH) from the organisms adapted to different temperatures, Thermus thermophilus (Tt), Escherichia coli (Ec), and Vibrio sp. I5 (Vib), is described. In all three cases, instead of a self-template mechanism, the high extent of symmetry and cooperativity in folding of subunits and domains have been concluded from the following experimental findings: The complex time course of refolding, monitored by Trp fluorescence, consists of a fast (the rate constant varies as 16.5, 25.0, and 11.7 min-1 in the order of Tt, Ec, and Vib IPMDHs) and a slow (the rate constants are 0.11, 0.80, and 0.23 min-1 for the three different species) first-order process. However, a burst increase of Trp fluorescence anisotropy to the value of the native states indicates that in all three cases the association of the two polypeptide chains occurs at the beginning of refolding. This dimeric species binds the substrate IPM, but the native-like interactions of the tertiary and quaternary structures are only formed during the slow phase of refolding, accompanied by further increase of protein fluorescence and appearance of FRET between Trp side chain(s) and the bound NADH. Joining the contacting arms of each subunit also takes place exclusively during this slow phase. To monitor refolding of each domain within the intact molecule of T. thermophilus IPMDH, Trp's (located in separate domains) were systematically replaced with Phe's. The refolding processes of the mutants were followed by measuring changes in Trp fluorescence and in FRET between the particular Trp and NADH. The high similarity of time courses (both in biphasicity and in their rates) strongly suggests cooperative folding of the domains during formation of the native three-dimensional structure of IPMDH.