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Sample records for acid dehydrogenase complex

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. Cyanobacterial NADPH dehydrogenase complexes

    SciTech Connect

    Ogawa, Teruo; Mi, Hualing

    2007-07-01

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

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

    PubMed Central

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

    2016-01-01

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

  4. 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.

  5. Which way does the citric acid cycle turn during hypoxia? The critical role of α-ketoglutarate dehydrogenase complex.

    PubMed

    Chinopoulos, Christos

    2013-08-01

    The citric acid cycle forms a major metabolic hub and as such it is involved in many disease states involving energetic imbalance. In spite of the fact that it is being branded as a "cycle", during hypoxia, when the electron transport chain does not oxidize reducing equivalents, segments of this metabolic pathway remain operational but exhibit opposing directionalities. This serves the purpose of harnessing high-energy phosphates through matrix substrate-level phosphorylation in the absence of oxidative phosphorylation. In this Mini-Review, these segments are appraised, pointing to the critical importance of the α-ketoglutarate dehydrogenase complex dictating their directionalities.

  6. Phosphorylation site on yeast pyruvate dehydrogenase complex

    SciTech Connect

    Uhlinger, D.J.

    1986-01-01

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

  7. Brain pyruvate and 2-oxoglutarate dehydrogenase complexes are mitochondrial targets of the CoA ester of the Refsum disease marker phytanic acid.

    PubMed

    Bunik, Victoria I; Raddatz, Günter; Wanders, Ronald J A; Reiser, Georg

    2006-06-12

    Pyruvate and 2-oxoglutarate dehydrogenase complexes are strongly inhibited by phytanoyl-CoA (IC(50) approximately 10(-6)-10(-7) M). Palmitoyl-CoA is 10-fold less potent. Phytanic or palmitic acids have no inhibitory effect up to 0.3 mM. At the substrate saturation, the acyl-CoA's affect the first and second enzymatic components of the 2-oxoglutarate dehydrogenase complex, while the third component is inhibited only at a low saturation with its substrate dihydrolipoamide. Thus, key regulatory branch points of mitochondrial metabolism are targets of a cellular derivative of phytanic acid. Decreased activity of the complexes might therefore contribute to neurological symptoms upon accumulation of phytanic acid in Refsum disease.

  8. Structural bases for the specific interactions between the E2 and E3 components of the Thermus thermophilus 2-oxo acid dehydrogenase complexes.

    PubMed

    Nakai, Tadashi; Kuramitsu, Seiki; Kamiya, Nobuo

    2008-06-01

    Pyruvate dehydrogenase (PDH), branched-chain 2-oxo acid dehydrogenase (BCDH) and 2-oxoglutarate dehydrogenase (OGDH) are multienzyme complexes that play crucial roles in several common metabolic pathways. These enzymes belong to a family of 2-oxo acid dehydrogenase complexes that contain multiple copies of three different components (E1, E2 and E3). For the Thermus thermophilus enzymes, depending on its substrate specificity (pyruvate, branched-chain 2-oxo acid or 2-oxoglutarate), each complex has distinctive E1 (E1p, E1b or E1o) and E2 (E2p, E2b or E2o) components and one of the two possible E3 components (E3b and E3o). (The suffixes, p, b and o identify their respective enzymes, PDH, BCDH and OGDH.) Our biochemical characterization demonstrates that only three specific E3*E2 complexes can form (E3b*E2p, E3b*E2b and E3o*E2o). X-ray analyses of complexes formed between the E3 components and the peripheral subunit-binding domains (PSBDs), derived from the corresponding E2-binding partners, reveal that E3b interacts with E2p and E2b in essentially the same manner as observed for Geobacillus stearothermophilus E3*E2p, whereas E3o interacts with E2o in a novel fashion. The buried intermolecular surfaces of the E3b*PSBDp/b and E3o*PSBDo complexes differ in size, shape and charge distribution and thus, these differences presumably confer the binding specificities for the complexes. PMID:18316329

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

    PubMed Central

    Williams, Michael; Randall, Douglas D.

    1979-01-01

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

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

    PubMed

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

    1977-05-01

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

  11. The Impact of the Branched-Chain Ketoacid Dehydrogenase Complex on Amino Acid Homeostasis in Arabidopsis1[OPEN

    PubMed Central

    Peng, Cheng; Uygun, Sahra; Shiu, Shin-Han; Last, Robert L.

    2015-01-01

    The branched-chain amino acids (BCAAs) Leu, Ile, and Val are among nine essential amino acids that must be obtained from the diet of humans and other animals, and can be nutritionally limiting in plant foods. Despite genetic evidence of its importance in regulating seed amino acid levels, the full BCAA catabolic network is not completely understood in plants, and limited information is available regarding its regulation. In this study, transcript coexpression analyses revealed positive correlations among BCAA catabolism genes in stress, development, diurnal/circadian, and light data sets. A core subset of BCAA catabolism genes, including those encoding putative branched-chain ketoacid dehydrogenase subunits, is highly expressed during the night in plants on a diel cycle and in prolonged darkness. Mutants defective in these subunits accumulate higher levels of BCAAs in mature seeds, providing genetic evidence for their function in BCAA catabolism. In addition, prolonged dark treatment caused the mutants to undergo senescence early and overaccumulate leaf BCAAs. These results extend the previous evidence that BCAAs can be catabolized and serve as respiratory substrates at multiple steps. Moreover, comparison of amino acid profiles between mature seeds and dark-treated leaves revealed differences in amino acid accumulation when BCAA catabolism is perturbed. Together, these results demonstrate the consequences of blocking BCAA catabolism during both normal growth conditions and under energy-limited conditions. PMID:25986129

  12. Limited proteolysis and sequence analysis of the 2-oxo acid dehydrogenase complexes from Escherichia coli. Cleavage sites and domains in the dihydrolipoamide acyltransferase components.

    PubMed Central

    Packman, L C; Perham, R N

    1987-01-01

    The structures of the dihydrolipoamide acyltransferase (E2) components of the 2-oxo acid dehydrogenase complexes from Escherichia coli were investigated by limited proteolysis. Trypsin and Staphylococcus aureus V8 proteinase were used to excise the three lipoyl domains from the E2p component of the pyruvate dehydrogenase complex and the single lipoyl domain from the E2o component of the 2-oxoglutarate dehydrogenase complex. The principal sites of action of these enzymes on each E2 chain were determined by sequence analysis of the isolated lipoyl fragments and of the truncated E2p and E2o chains. Each of the numerous cleavage sites (12 in E2p, six in E2o) fell within similar segments of the E2 chains, namely stretches of polypeptide rich in alanine, proline and/or charged amino acids. These regions are clearly accessible to proteinases of Mr 24,000-28,000 and, on the basis of n.m.r. spectroscopy, some of them have previously been implicated in facilitating domain movements by virtue of their conformational flexibility. The limited proteolysis data suggest that E2p and E2o possess closer architectural similarities than would be predicted from inspection of their amino acid sequences. As a result of this work, an error was detected in the sequence of E2o inferred from the previously published sequence of the encoding gene, sucB. The relevant peptides from E2o were purified and sequenced by direct means; an amended sequence is presented. Images Fig. 1. Fig. 2. PMID:3297046

  13. Effect of diet and starvation on the activity state of branched-chain 2-oxo-acid dehydrogenase complex in rat liver and heart.

    PubMed

    Solomon, M; Cook, K G; Yeaman, S J

    1987-12-10

    In rats fed a high-protein diet, the branched-chain 2-oxo-acid dehydrogenase complex in liver was essentially fully active and its activity state was unaffected by subsequent starvation for 48 h. Feeding with a low-protein diet led to a decrease in the activity state which was essentially reversed by 48 h of starvation. In heart, the enzyme was primarily inactive (activity state 18%) in rats fed a high-protein diet, with both low-protein diet and starvation leading to a further decrease in the activity state. PMID:3676350

  14. Pyruvate dehydrogenase complex from germinating castor bean endosperm.

    PubMed

    Rapp, B J; Randall, D D

    1980-02-01

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

  15. Leucine-induced activation of translational initiation is partly regulated by the branched-chain {alpha}-keto acid dehydrogenase complex in C2C12 cells

    SciTech Connect

    Nakai, Naoya . E-mail: nakai@hss.osaka-u.ac.jp; Shimomura, Yoshiharu; Tamura, Tomohiro; Tamura, Noriko; Hamada, Koichiro; Kawano, Fuminori; Ohira, Yoshinobu

    2006-05-19

    Branched-chain amino acid leucine has been shown to activate the translational regulators through the mammalian target of rapamycin. However, the leucine's effects are self-limiting because leucine promotes its own disposal by an oxidative pathway. The irreversible and rate-limiting step in the leucine oxidation pathway is catalyzed by the branched-chain {alpha}-keto acid dehydrogenase (BCKDH) complex. The complex contains E1 ({alpha}2{beta}2), E2, and E3 subunits, and its activity is abolished by phosphorylation of the E1{alpha} subunit by BCKDH kinase. The relationship between the activity of BCKDH complex and leucine-mediated activation of the protein translation was investigated using the technique of RNA interference. The activity of BCKDH complex in C2C12 cell was modulated by transfection of small interfering RNA (siRNA) for BCKDH E2 subunit or BCKDH kinase. Transfection of siRNAs decreased the mRNA expression and protein amount of corresponding gene. Suppression of either E2 subunit or kinase produced opposite effects on the cell proliferation and the activation of translational regulators by leucine. Suppression of BCKDH kinase for 48 h resulted in decreasing cell proliferation. In contrast, E2 suppression led to increased amount of total cellular protein. The phosphorylation of p70 S6 kinase by leucine was increased in E2-siRNA transfected C2C12 cells, whereas the leucine's effect was diminished in kinase-siRNA transfected cells. These results suggest that the activation of the translational regulators by leucine was partly regulated by the activity of BCKDH complex.

  16. Prenatal presentation of pyruvate dehydrogenase complex deficiency.

    PubMed

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

    2016-08-01

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

  17. Increased valinomycin production in mutants of Streptomyces sp. M10 defective in bafilomycin biosynthesis and branched-chain α-keto acid dehydrogenase complex expression.

    PubMed

    Lee, Dong Wan; Ng, Bee Gek; Kim, Beom Seok

    2015-11-01

    Streptomyces sp. M10 is a valinomycin-producing bacterial strain that shows potent bioactivity against Botrytis blight of cucumber plants. During studies to increase the yield of valinomycin (a cyclododecadepsipeptide) in strain M10, additional antifungal metabolites, including bafilomycin derivatives (macrolide antibiotics), were identified. To examine the effect of bafilomycin biosynthesis on valinomycin production, the bafilomycin biosynthetic gene cluster was cloned from the genome of strain M10, as were two branched-chain α-keto acid dehydrogenase (BCDH) gene clusters related to precursor supply for bafilomycin biosynthesis. A null mutant (M10bafm) of one bafilomycin biosynthetic gene (bafV) failed to produce bafilomycin, but resulted in a 1.2- to 1.5-fold increase in the amount of valinomycin produced. In another null mutant (M10bkdFm) of a gene encoding a subunit of the BCDH complex (bkdF), bafilomycin production was completely abolished and valinomycin production increased fourfold relative to that in the wild-type M10 strain. The higher valinomycin yield was likely the result of redistribution of the metabolic flux from bafilomycin to valinomycin biosynthesis, because the two antibiotics share a common precursor, 2-ketoisovaleric acid, a deamination product of valine. The results show that directing precursor flux toward active ingredient biosynthesis could be used as a prospective tool to increase the competence of biofungicides.

  18. R-lipoic acid inhibits mammalian pyruvate dehydrogenase kinase.

    PubMed

    Korotchkina, Lioubov G; Sidhu, Sukhdeep; Patel, Mulchand S

    2004-10-01

    The four pyruvate dehydrogenase kinase (PDK) and two pyruvate dehydrogenase phosphatase (PDP) isoenzymes that are present in mammalian tissues regulate activity of the pyruvate dehydrogenase complex (PDC) by phosphorylation/dephosphorylation of its pyruvate dehydrogenase (E1) component. The effect of lipoic acids on the activity of PDKs and PDPs was investigated in purified proteins system. R-lipoic acid, S-lipoic acid and R-dihydrolipoic acid did not significantly affect activities of PDPs and at the same time inhibited PDKs to different extents (PDK1>PDK4 approximately PDK2>PDK3 for R-LA). Since lipoic acids inhibited PDKs activity both when reconstituted in PDC and in the presence of E1 alone, dissociation of PDK from the lipoyl domains of dihydrolipoamide acetyltransferase in the presence of lipoic acids is not a likely explanation for inhibition. The activity of PDK1 towards phosphorylation sites 1, 2 and 3 of E1 was decreased to the same extent in the presence of R-lipoic acid, thus excluding protection of the E1 active site by lipoic acid from phosphorylation. R-lipoic acid inhibited autophosphorylation of PDK2 indicating that it exerted its effect on PDKs directly. Inhibition of PDK1 by R-lipoic acid was not altered by ADP but was decreased in the presence of pyruvate which itself inhibits PDKs. An inhibitory effect of lipoic acid on PDKs would result in less phosphorylation of E1 and hence increased PDC activity. This finding provides a possible mechanism for a glucose (and lactate) lowering effect of R-lipoic acid in diabetic subjects. PMID:15512796

  19. Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells

    PubMed Central

    Ohoka, Yoshiharu; Yokota-Nakatsuma, Aya; Maeda, Naoko; Takeuchi, Hajime; Iwata, Makoto

    2014-01-01

    Retinoic acid (RA)-producing dendritic cells (DCs) play critical roles in gut immunity. Retinal dehydrogenase 2 (RALDH2) encoded by Aldh1a2 is a key enzyme for generating RA in DCs. Granulocyte–macrophage colony-stimulating factor (GM-CSF) potently induces RALDH2 expression in DCs in an RA-dependent manner, and RA alone weakly induces the expression. However, how GM-CSF and RA induce RALDH2 expression remains unclear. Here, we show that GM-CSF-induced activation of the transcription factor Sp1 and RA-dependent signaling via the RA receptor (RAR)/retinoid X receptor (RXR) complex contribute to Aldh1a2 expression. The RAR antagonist LE540 and the Sp1 inhibitor mithramycin A inhibited GM-CSF-induced Aldh1a2 expression in fms-related tyrosine kinase 3 ligand-generated bone marrow-derived DCs (BM-DCs). ERK and p38 MAPK inhibitors suppressed GM-CSF-induced nuclear translocation of Sp1 and Aldh1a2 expression. Sp1 and the RARα/RXRα complex bound to GC-rich Sp1-binding sites and an RA response element (RARE) half-site, respectively, near the TATA box in the mouse Aldh1a2 promoter. The DNA sequences around these sites were highly conserved among different species. In the presence of RA, ectopic expression of RARα/RXRα and Sp1 synergistically enhanced Aldh1a2 promoter-reporter activity. GM-CSF did not significantly induce Aldh1a2 expression in plasmacytoid DCs, peritoneal macrophages, or T cells, and the Aldh1a2 promoter in these cells was mostly unmethylated. These results suggest that GM-CSF/RA-induced RALDH2 expression in DCs requires cooperative binding of Sp1 and the RAR/RXR complex to the Aldh1a2 promoter, and can be regulated by a DNA methylation-independent mechanism. PMID:24788806

  20. A mimic of the pyruvate dehydrogenase complex.

    PubMed

    Zhao, Huanyu; Breslow, Ronald

    2010-10-15

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

  1. The role of Pyruvate Dehydrogenase Complex in cardiovascular diseases.

    PubMed

    Sun, Wanqing; Liu, Quan; Leng, Jiyan; Zheng, Yang; Li, Ji

    2015-01-15

    The regulation of mammalian myocardial carbohydrate metabolism is complex; many factors such as arterial substrate and hormone levels, coronary flow, inotropic state and the nutritional status of the tissue play a role in regulating mammalian myocardial carbohydrate metabolism. The Pyruvate Dehydrogenase Complex (PDHc), a mitochondrial matrix multienzyme complex, plays an important role in energy homeostasis in the heart by providing the link between glycolysis and the tricarboxylic acid (TCA) cycle. In TCA cycle, PDHc catalyzes the conversion of pyruvate into acetyl-CoA. This review determines that there is altered cardiac glucose in various pathophysiological states consequently causing PDC to be altered. This review further summarizes evidence for the metabolism mechanism of the heart under normal and pathological conditions including ischemia, diabetes, hypertrophy and heart failure.

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

    SciTech Connect

    Park, Yun-Hee; Patel, Mulchand S.

    2010-05-07

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

  3. Making biochemistry count: life among the amino acid dehydrogenases.

    PubMed

    Engel, Paul C

    2011-04-01

    The guiding principle of the IAS Medal Lecture and of the research it covered was that searching mathematical analysis, depending on good measurements, must underpin sound biochemical conclusions. This was illustrated through various experiences with the amino acid dehydrogenases. Topics covered in the present article include: (i) the place of kinetic measurement in assessing the metabolic role of GDH (glutamate dehydrogenase); (ii) the discovery of complex regulatory behaviour in mammalian GDH, involving negative co-operativity in coenzyme binding; (iii) an X-ray structure solution for a bacterial GDH providing insight into catalysis; (iv) almost total positive co-operativity in glutamate binding to clostridial GDH; (v) unexpected outcomes with mutations at the catalytic aspartate site in GDH; (vi) reactive cysteine as a counting tool in the construction of hybrid oligomers to probe the basis of allosteric interaction; (vii) tryptophan-to-phenylalanine mutations in analysis of allosteric conformational change; (viii) site-directed mutagenesis to alter substrate specificity in GDH and PheDH (phenylalanine dehydrogenase); and (ix) varying strengths of binding of the 'wrong' enantiomer in engineered mutant enzymes and implications for resolution of racemates.

  4. Rv0132c of Mycobacterium tuberculosis Encodes a Coenzyme F420-Dependent Hydroxymycolic Acid Dehydrogenase

    PubMed Central

    Purwantini, Endang; Mukhopadhyay, Biswarup

    2013-01-01

    The ability of Mycobacterium tuberculosis to manipulate and evade human immune system is in part due to its extraordinarily complex cell wall. One of the key components of this cell wall is a family of lipids called mycolic acids. Oxygenation of mycolic acids generating methoxy- and ketomycolic acids enhances the pathogenic attributes of M. tuberculosis. Thus, the respective enzymes are of interest in the research on mycobacteria. The generation of methoxy- and ketomycolic acids proceeds through intermediary formation of hydroxymycolic acids. While the methyl transferase that generates methoxymycolic acids from hydroxymycolic acids is known, hydroxymycolic acids dehydrogenase that oxidizes hydroxymycolic acids to ketomycolic acids has been elusive. We found that hydroxymycolic acid dehydrogenase is encoded by the rv0132c gene and the enzyme utilizes F420, a deazaflavin coenzyme, as electron carrier, and accordingly we called it F420-dependent hydroxymycolic acid dehydrogenase. This is the first report on the involvement of F420 in the synthesis of a mycobacterial cell envelope. Also, F420-dependent hydroxymycolic acid dehydrogenase was inhibited by PA-824, and therefore, it is a previously unknown target for this new tuberculosis drug. PMID:24349169

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

    SciTech Connect

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

    1986-08-10

    Quinate dehydrogenase was isolated from young needles of the Siberian larch and partially purified by ammonium sulfate fractionation. It was found that in conifers, in contrast to other plants, quinate dehydrogenase is active both with NAD and with NADP. The values of K/sub m/ for quinate and NADP were 1.8 and 0.18 mM. The enzyme exhibits maximum activity at pH 9.0. It was assumed that NADP-dependent quinate dehydrogenase is responsible for quinic acid synthesis. The special features of the organization and regulation of the initial stages of the shikimate pathway in conifers are discussed.

  6. Often Ignored Facts about the Control of the 2-Oxoglutarate Dehydrogenase Complex

    ERIC Educational Resources Information Center

    Strumilo, Slawomir

    2005-01-01

    Information about the control of the activity of the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme in the citric acid cycle, is not well covered in the biochemical education literature, especially as it concerns the allosteric regulation of OGDHC by adenine nucleotide and ortophosphate. From experimental work published during the last…

  7. Phosphorylation of the pyruvate dehydrogenase complex isolated from Ascaris suum

    SciTech Connect

    Thissen, J.; Komuniecki, R.

    1987-05-01

    The pyruvate dehydrogenase complex (PDC) from body wall muscle of the porcine nematode, Ascaris suum, plays a pivotal role in anaerobic mitochondrial metabolism. As in mammalian mitochondria, PDC activity is inhibited by the phosphorylation of the ..cap alpha..PDH subunit, catalyzed by an associated PDH/sub a/ kinase. However, in contrast to PDC's isolated from all other eukaryotic sources, phosphorylation decreases the mobility of the ..cap alpha..PDH subunit on SDS-PAGE and permits the separation of the phosphorylated and nonphosphorylated ..cap alpha..PDH's. Phosphorylation and the inactivation of the Ascaris PDC correspond directly, and the additional phosphorylation that occurs after complete inactivation in mammalian PDC's is not observed. The purified ascarid PDC incorporates 10 nmoles /sup 32/P/mg P. Autoradiography of the radiolabeled PDC separated by SDS-PAGE yields a band which corresponds to the phosphorylated ..cap alpha..PDH and a second, faint band which is present only during the first three minutes of PDC inactivation, intermediate between the phosphorylated and nonphosphorylated ..cap alpha..PDH subunit. Tryptic digests of the /sup 32/P-PDC yields one major phosphopeptide, when separated by HPLC, and its amino acid sequence currently is being determined.

  8. Subunit structure of the dihydrolipoyl transacylase component of branched-chain. cap alpha. -keto acid dehydrogenase complex from bovine liver: mapping of the lipoyl-bearing domain by limited proteolysis

    SciTech Connect

    Not Available

    1986-01-05

    To characterize the lipoyl-bearing domain of the dihydrolipoyl transacylase (E/sub 2/) component, purified branched-chain ..cap alpha..-keto acid dehydrogenase complex from bovine liver was reductively acylated with (U-/sup 14/C)..cap alpha..-ketoisovalerate in the presence of thiamin pyrophosphate and N-ethylmaleimide. Digestion of the modified complex with increasing concentrations of trypsin sequentially cleaved the E/sub 2/ polypeptide chain (M/sub r/ = 52,000) into five radiolabeled lipoyl-containing fragments, L/sub 1/-L/sub 5/. In addition, a lipoate-free inner E/sub 2/ core consisting of fragment A and fragment B was produced. Fragment A contains the active site for transacylation reaction and fragment B is the subunit-binding domain. Fragment L/sub 5/ and fragment B were stable and resistant to further tryptic digestion. Mouse antiserium against E/sub 2/ reacted only with fragments L/sub 1/, L/sub 2/, and L/sub 3/, and did not bind fragments L/sub 4/, L/sub 5/, A, and B as judged by immunoblotting analysis. The anti-E/sub 2/ serum-strongly inhibited the overall reaction catalyzed by the complex, but was without effect on the transacylation activity of E/sub 2/. Measurement of incorporation of (1-/sup 14/C)isobutyryl groups into the E/sub 2/ subunit indicated the presence of 1 lipoyl residue/E/sub 2/ chain.

  9. A new role for α-ketoglutarate dehydrogenase complex: regulating metabolism through post-translational modification of other enzymes.

    PubMed

    McKenna, Mary C; Rae, Caroline D

    2015-07-01

    This Editorial highlights a study by Gibson et al. published in this issue of JNeurochem, in which the authors reveal a novel role for the α-ketoglutarate dehydrogenase complex (KGDHC) in post-translational modification of proteins. KGDHC may catalyze post-translational modification of itself as well as several other proteins by succinylation of lysine residues. The authors' report of an enzyme responsible for succinylation of key mitochondrial enzymes represents a major step toward our understanding of the complex functional metabolome. TCA, tricarboxylic acid; KG, α-ketoglutarate; KGDHC, α-ketoglutarate dehydrogenase complex; FUM, fumarase; MDH, malate dehydrogenase; ME, malic enzyme; GDH, glutamate dehydrogenase; AAT, aspartate aminotransferase; GS, glutamine synthetase; PAG, phosphate-activated glutaminase; SIRT3, silent information regulator 3; SIRT5, silent information regulator 5. PMID:26052752

  10. 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

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

    PubMed

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

    2016-05-01

    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'.

  12. Pyruvate dehydrogenase complex from higher plant mitochondria and proplastids: kinetics.

    PubMed

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

    1977-05-01

    A steady-state kinetic analysis has been performed on the pyruvate dehydrogenase complex from pea (Pisum sativum L.) mitochondria and castor bean (Ricinus communis L.) proplastids. Substrate interaction kinetics for all substrates gave parallel lines consistent with a multisite ping-pong mechanism. Product inhibition studies showed uncompetitive inhibition between acetyl-CoA and pyruvate and competitive inhibition between NADH and NAD(+), both of which are also consistent with this mechanism. In the mitochondrial complex, acetyl-CoA showed noncompetitive inhibition versus CoA which suggests that the intermediate complex is kinetically important in the lipoamide transacetylase component of this complex. In contrast, the proplastid complex showed competitive inhibition in this interaction. NADH is a noncompetitive inhibitor versus CoA in both complexes indicating that these complexes, like the mammalian complex, may have protein-protein interactions between the second and third enzymes of the complex. Since NADH also shows noncompetitive inhibition versus pyruvate, this interaction may extend to all components of the complex. Acetyl-CoA shows noncompetitive inhibition versus NAD(+) which may also be a result of interaction between the second and third enzymes of the complex. The limiting Michaelis constants for substrates and the inhibitor constants for both complexes were determined.

  13. The regulation of branched-chain 2-oxo acid dehydrogenase of liver, kidney and heart by phosphorylation.

    PubMed Central

    Hughes, W A; Halestrap, A P

    1981-01-01

    1. Incubation of mitochondria from heart, liver and kidney with [32P]phosphate allowed 32P incorporation into two intramitochondrial proteins, the decarboxylase alpha-subunit of the pyruvate dehydrogenase complex (mol.wt 42000) and a protein of mol.wt. 48000. 2. This latter protein incorporated 32P more slowly than did pyruvate dehydrogenase, was not precipitated by antibody to pyruvate dehydrogenase and showed behaviour distinct from that of pyruvate dehydrogenase towards high-speed centrifugation and pyruvate dehydrogenase phosphate phosphatase. 3. 32P incorporation into the protein was greatly diminished by the presence of 0.1 mM-4-methyl-2-oxopentanoate, but enhanced by pyruvate (1 mM), hypo-osmotic treatment of mitochondria and, under some conditions, by uncoupler. 4. The activity of branched-chain 2-oxo acid dehydrogenase was assayed in parallel experiments. Under appropriate conditions the enzyme was inhibited when 32P incorporation was increased and activated when incorporation was decreased. The data suggest that the 48000-mol.wt. phosphorylated protein is identical with the decarboxylase subunit of branched-chain 2-oxo acid dehydrogenase and that this enzyme may be controlled by a phosphorylation-dephosphorylation cycle akin to that for pyruvate dehydrogenase. 5. Strict correlation between activity and 32P incorporation was not observed, and a scheme for the regulation of the enzyme is proposed to account for these discrepancies. PMID:7316988

  14. Pyruvate dehydrogenase complex from higher plant mitochondria and proplastids: regulation.

    PubMed

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

    1977-05-01

    The activity of the pyruvate dehydrogenase complex from pea (Pisum sativum L.) mitochondria is inhibited when MgATP is added to the reaction mixture; 50% inhibition occurs at 4 mm ATP. The inhibition does not increase with time and is higher in the more highly purified preparations. Crude preparations of the complex show a time-dependent inactivation when incubated with 7.5 mm MgATP alone but this is not found with the more highly purified complex. This inactivation does not occur at 0 C. The complex could not be reactivated by high concentrations of Mg(2+). It is suggested that a phosphorylation-dephosphorylation mechanism may occur in plants, but the phosphatase and kinase are not tightly bound to the complex and are lost on isolation. The complex does not respond in a significant manner to energy charge. The NAD(+) to NADH ratio is the principal means of regulation of the complex, NADH being competitive with NAD(+) for the dihydrolipoamide component. The CoA to acetyl-CoA ratio is not important in regulation.The castor bean (Ricinus communis L.) proplastid complex is inhibited by the addition of 2 mm MgATP to the assay mixture. The inhibition is immediate, suggesting that phosphorylation of the enzyme is not involved or must be very rapid. Incubation of the complex with 20 mm MgCl(2) causes an activation of the complex. Maximum activity is not expressed in this case for 30 minutes. A similar activation can be achieved by preincubating the complex with 1 mm pyruvate. These data suggest that the complex is not fully activated on isolation.

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

    PubMed Central

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

    1982-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  17. Involvement of snapdragon benzaldehyde dehydrogenase in benzoic acid biosynthesis.

    PubMed

    Long, Michael C; Nagegowda, Dinesh A; Kaminaga, Yasuhisa; Ho, Kwok Ki; Kish, Christine M; Schnepp, Jennifer; Sherman, Debra; Weiner, Henry; Rhodes, David; Dudareva, Natalia

    2009-07-01

    Benzoic acid (BA) is an important building block in a wide spectrum of compounds varying from primary metabolites to secondary products. Benzoic acid biosynthesis from L-phenylalanine requires shortening of the propyl side chain by two carbons, which can occur via a beta-oxidative pathway or a non-beta-oxidative pathway, with benzaldehyde as a key intermediate. The non-beta-oxidative route requires benzaldehyde dehydrogenase (BALDH) to convert benzaldehyde to BA. Using a functional genomic approach, we identified an Antirrhinum majus (snapdragon) BALDH, which exhibits 40% identity to bacterial BALDH. Transcript profiling, biochemical characterization of the purified recombinant protein, molecular homology modeling, in vivo stable isotope labeling, and transient expression in petunia flowers reveal that BALDH is capable of oxidizing benzaldehyde to BA in vivo. GFP localization and immunogold labeling studies show that this biochemical step occurs in the mitochondria, raising a question about the role of subcellular compartmentalization in BA biosynthesis.

  18. Regulation of human class I alcohol dehydrogenases by bile acids

    PubMed Central

    Langhi, Cédric; Pedraz-Cuesta, Elena; Haro, Diego; Marrero, Pedro F.; Rodríguez, Joan C.

    2013-01-01

    Class I alcohol dehydrogenases (ADH1s) are the rate-limiting enzymes for ethanol and vitamin A (retinol) metabolism in the liver. Because previous studies have shown that human ADH1 enzymes may participate in bile acid metabolism, we investigated whether the bile acid-activated nuclear receptor farnesoid X receptor (FXR) regulates ADH1 genes. In human hepatocytes, both the endogenous FXR ligand chenodeoxycholic acid and synthetic FXR-specific agonist GW4064 increased ADH1 mRNA, protein, and activity. Moreover, overexpression of a constitutively active form of FXR induced ADH1A and ADH1B expression, whereas silencing of FXR abolished the effects of FXR agonists on ADH1 expression and activity. Transient transfection studies and electrophoretic mobility shift assays revealed functional FXR response elements in the ADH1A and ADH1B proximal promoters, thus indicating that both genes are direct targets of FXR. These findings provide the first evidence for direct connection of bile acid signaling and alcohol metabolism. PMID:23772048

  19. Regulation of human class I alcohol dehydrogenases by bile acids.

    PubMed

    Langhi, Cédric; Pedraz-Cuesta, Elena; Haro, Diego; Marrero, Pedro F; Rodríguez, Joan C

    2013-09-01

    Class I alcohol dehydrogenases (ADH1s) are the rate-limiting enzymes for ethanol and vitamin A (retinol) metabolism in the liver. Because previous studies have shown that human ADH1 enzymes may participate in bile acid metabolism, we investigated whether the bile acid-activated nuclear receptor farnesoid X receptor (FXR) regulates ADH1 genes. In human hepatocytes, both the endogenous FXR ligand chenodeoxycholic acid and synthetic FXR-specific agonist GW4064 increased ADH1 mRNA, protein, and activity. Moreover, overexpression of a constitutively active form of FXR induced ADH1A and ADH1B expression, whereas silencing of FXR abolished the effects of FXR agonists on ADH1 expression and activity. Transient transfection studies and electrophoretic mobility shift assays revealed functional FXR response elements in the ADH1A and ADH1B proximal promoters, thus indicating that both genes are direct targets of FXR. These findings provide the first evidence for direct connection of bile acid signaling and alcohol metabolism.

  20. Analysis of Loss-of-Function Mutants in Aspartate Kinase and Homoserine Dehydrogenase Genes Points to Complexity in the Regulation of Aspartate-Derived Amino Acid Contents1[OPEN

    PubMed Central

    Clark, Teresa J.; Lu, Yan

    2015-01-01

    Biosynthesis of aspartate (Asp)-derived amino acids lysine (Lys), methionine (Met), threonine (Thr), and isoleucine involves monofunctional Asp kinases (AKs) and dual-functional Asp kinase-homoserine dehydrogenases (AK-HSDHs). Four-week-old loss-of-function Arabidopsis (Arabidopsis thaliana) mutants in the AK-HSDH2 gene had increased amounts of Asp and Asp-derived amino acids, especially Thr, in leaves. To explore mechanisms behind this phenotype, we obtained single mutants for other AK and AK-HSDH genes, generated double mutants from ak-hsdh2 and ak mutants, and performed free and protein-bound amino acid profiling, transcript abundance, and activity assays. The increases of Asp, Lys, and Met in ak-hsdh2 were also observed in ak1-1, ak2-1, ak3-1, and ak-hsdh1-1. However, the Thr increase in ak-hsdh2 was observed in ak-hsdh1-1 but not in ak1-1, ak2-1, or ak3-1. Activity assays showed that AK2 and AK-HSDH1 are the major contributors to overall AK and HSDH activities, respectively. Pairwise correlation analysis revealed positive correlations between the amount of AK transcripts and Lys-sensitive AK activity and between the amount of AK-HSDH transcripts and both Thr-sensitive AK activity and total HSDH activity. In addition, the ratio of total AK activity to total HSDH activity negatively correlates with the ratio of Lys to the total amount of Met, Thr, and isoleucine. These data led to the hypothesis that the balance between Lys-sensitive AKs and Thr-sensitive AK-HSDHs is important for maintaining the amounts and ratios of Asp-derived amino acids. PMID:26063505

  1. Alpha-ketoglutarate dehydrogenase complex-dependent succinylation of proteins in neurons and neuronal cell lines

    PubMed Central

    Gibson, Gary E.; Xu, Hui; Chen, Huan-Lian; Chen, Wei; Denton, Travis; Zhang, Sheng

    2015-01-01

    Reversible post-translation modifications of proteins are common in all cells and appear to regulate many processes. Nevertheless, the enzyme(s) responsible for the alterations and the significance of the modification are largely unknown. Succinylation of proteins occurs and causes large changes in the structure of proteins; however, the source of the succinyl groups, the targets, and the consequences of these modifications on other proteins are unknown. These studies focused on succinylation of mitochondrial proteins. The results demonstrate that the α-ketoglutarate dehydrogenase complex (KGDHC) can serve as a trans-succinylase that mediates succinylation in an α-ketoglutarate-dependent manner. Inhibition of KGDHC reduced suc-cinylation of both cytosolic and mitochondrial proteins in cultured neurons and in a neuronal cell line. Purified KGDHC can succinylate multiple proteins including other enzymes of the tricarboxylic acid (TCA) cycle leading to modification of their activity. Inhibition of KGDHC also modifies acetylation by modifying the pyruvate dehydrogenase complex. The much greater effectiveness of KGDHC than succinyl CoA suggests that the catalysis due to the E2k suc-cinyltransferase is important. Succinylation appears to be a major signaling system and it can be mediated by KGDHC. PMID:25772995

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

    NASA Technical Reports Server (NTRS)

    2003-01-01

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

  3. A NEW LEVEL OF ARCHITECTURAL COMPLEXITY IN THE HUMAN PYRUVATE DEHYDROGENASE COMPLEX

    PubMed Central

    Smolle, Michaela; Prior, Alison Elizabeth; Brown, Audrey Elaine; Cooper, Alan; Byron, Olwyn; Lindsay, John Gordon

    2006-01-01

    SUMMARY Mammalian pyruvate dehydrogenase multi-enzyme complex (PDC) is a key metabolic assembly comprising a 60- meric pentagonal dodecahedral E2 core attached to which are 30 E1 heterotetramers and 6 E3 homodimers at maximal occupancy. Stable E3 integration is mediated by an accessory E3 binding protein (E3BP) located on each of the 12 E2 icosahedral faces. Here, we present evidence for a novel subunit organisation in which dihydrolipoamide dehydrogenase (E3) and E3BP form subcomplexes with a 1:2 stoichiometry implying the existence of a network of E3 ‘cross-bridges’ linking pairs of E3BPs across the surface of the E2 core assembly. We have also determined a low resolution structure for a truncated E3BP/E3 subcomplex using small angle xray scattering showing one of the E3BP lipoyl domains docked into the E3 active site. This new level of architectural complexity in mammalian PDC contrasts with the recently published crystal structure of human E3 complexed with its cognate subunit binding domain and provides important new insights into subunit organisation, its catalytic mechanism and regulation by the intrinsic PDC kinase. PMID:16679318

  4. In vivo regulation of alcohol dehydrogenase and lactate dehydrogenase in Rhizopus oryzae to improve L-lactic acid fermentation.

    PubMed

    Thitiprasert, Sitanan; Sooksai, Sarintip; Thongchul, Nuttha

    2011-08-01

    Rhizopus oryzae is becoming more important due to its ability to produce an optically pure L: -lactic acid. However, fermentation by Rhizopus usually suffers from low yield because of production of ethanol as a byproduct. Limiting ethanol production in living immobilized R. oryzae by inhibition of alcohol dehydrogenase (ADH) was observed in shake flask fermentation. The effects of ADH inhibitors added into the medium on the regulation of ADH and lactate dehydrogenase (LDH) as well as the production of cell biomass, lactic acid, and ethanol were elucidated. 1,2-diazole and 2,2,2-trifluroethanol were found to be the effective inhibitors used in this study. The highest lactic acid yield of 0.47 g/g glucose was obtained when 0.01 mM 2,2,2-trifluoroethanol was present during the production phase of the pregrown R. oryzae. This represents about 38% increase in yield as compared with that from the simple glucose fermentation. Fungal metabolism was suppressed when iodoacetic acid, N-ethylmaleimide, 4,4'-dithiodipyridine, or 4-hydroxymercury benzoic acid were present. Dramatic increase in ADH and LDH activities but slight change in product yields might be explained by the inhibitors controlling enzyme activities at the pyruvate branch point. This showed that in living R. oryzae, the inhibitors regulated the flux through the related pathways. PMID:21416338

  5. In vivo regulation of alcohol dehydrogenase and lactate dehydrogenase in Rhizopus oryzae to improve L-lactic acid fermentation.

    PubMed

    Thitiprasert, Sitanan; Sooksai, Sarintip; Thongchul, Nuttha

    2011-08-01

    Rhizopus oryzae is becoming more important due to its ability to produce an optically pure L: -lactic acid. However, fermentation by Rhizopus usually suffers from low yield because of production of ethanol as a byproduct. Limiting ethanol production in living immobilized R. oryzae by inhibition of alcohol dehydrogenase (ADH) was observed in shake flask fermentation. The effects of ADH inhibitors added into the medium on the regulation of ADH and lactate dehydrogenase (LDH) as well as the production of cell biomass, lactic acid, and ethanol were elucidated. 1,2-diazole and 2,2,2-trifluroethanol were found to be the effective inhibitors used in this study. The highest lactic acid yield of 0.47 g/g glucose was obtained when 0.01 mM 2,2,2-trifluoroethanol was present during the production phase of the pregrown R. oryzae. This represents about 38% increase in yield as compared with that from the simple glucose fermentation. Fungal metabolism was suppressed when iodoacetic acid, N-ethylmaleimide, 4,4'-dithiodipyridine, or 4-hydroxymercury benzoic acid were present. Dramatic increase in ADH and LDH activities but slight change in product yields might be explained by the inhibitors controlling enzyme activities at the pyruvate branch point. This showed that in living R. oryzae, the inhibitors regulated the flux through the related pathways.

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

    SciTech Connect

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

    2012-02-27

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

  7. Creation of a thermostable NADP⁺-dependent D-amino acid dehydrogenase from Ureibacillus thermosphaericus strain A1 meso-diaminopimelate dehydrogenase by site-directed mutagenesis.

    PubMed

    Akita, Hironaga; Doi, Katsumi; Kawarabayasi, Yutaka; Ohshima, Toshihisa

    2012-09-01

    A thermostable, NADP(+)-dependent D: -amino acid dehydrogenase (DAADH) was created from the meso-diaminopimelate dehydrogenase of Ureibacillus thermosphaericus strain A1 by introducing five point mutations into amino acid residues located in the active site. The recombinant protein, expressed in Escherichia coli, was purified to homogeneity using a two-step separation procedure and then characterized. In the presence of NADP(+), the protein catalyzed the oxidative deamination of several D: -amino acids, including D: -cyclohexylalanine, D: -isoleucine and D: -2-aminooctanoate, but not meso-diaminopimelate, confirming the creation of a NADP(+)-dependent DAADH. For the reverse reaction, the corresponding 2-oxo acids were aminated in the presence of NADPH and ammonia. In addition, the D: -amino acid dehydrogenase showed no loss of activity at 65 °C, indicating the mutant enzyme was more thermostable than its parental meso-diaminopimelate dehydrogenase.

  8. Inactivation of the pyruvate dehydrogenase complex of Escherichia coli by fluoropyruvate

    SciTech Connect

    Flournoy, D.S.; Frey, P.A. )

    1989-12-12

    The pyruvate dehydrogenase complex (PDH complex) of Escherichia coli and its pyruvate dehydrogenease component (E{sub 1}) are rapidly inactivated by low concentrations of fluoropyruvate in a thiamin pyrophosphate (TPP) dependent process. The inactivation rates for the PDH complex and for its E{sub 1} component are similar. Pyruvate protects the PDH complex and the E{sub 1} component against inactivation by fluoropyruvate. Dihydrolipoamide protects the E{sub 1} component from inactivation. TPP is not covalently bound to the PDH complex or to the E{sub 1} component by the inactivating reaction. When ({sup 14}C)fluoropyruvate is used to inactivate the PDH complex, {sup 14}C remains bound to the complex after gel filtration. This bound radioactivity is cleaved from the protein by NH{sub 2}OH, {sup {minus}}OH, and NaBH{sub 4} but not by dilute acid. When released by {sup {minus}}OH, greater than 90% of the {sup 14}C cochromatographs with acetate on DEAE-Sephadex. When released by NaBH{sub 4}, and {sup 14}C is recovered as ({sup 14}C)ethanol. Colorimetric analysis for sulfhydryl groups on the native E{sub 1} component and the inactivated E{sub 1} component, using 5,5{prime}-dithiobis(2-nitrobenzoate), reveals that complete inactivation results from covalent modification of 1.37 {plus minus} 0.03 sulfhydryl residues. Fluoropyruvate is known to generate acetyl-TPP at the active of E{sub 1}. The available evidence indicates that acetylation of a sulfhydryl group by acetyl-TPP at the active site of the E{sub 1} component inactivates the enzyme.

  9. Differential inhibition of PDKs by phenylbutyrate and enhancement of pyruvate dehydrogenase complex activity by combination with dichloroacetate.

    PubMed

    Ferriero, Rosa; Iannuzzi, Clara; Manco, Giuseppe; Brunetti-Pierri, Nicola

    2015-09-01

    Pyruvate dehydrogenase complex (PDHC) is a key enzyme in metabolism linking glycolysis to tricarboxylic acid cycle and its activity is tightly regulated by phosphorylation catalyzed by four pyruvate dehydrogenase kinase (PDK) isoforms. PDKs are pharmacological targets for several human diseases including cancer, diabetes, obesity, heart failure, and inherited PDHC deficiency. We investigated the inhibitory activity of phenylbutyrate toward PDKs and found that PDK isoforms 1-to-3 are inhibited whereas PDK4 is unaffected. Moreover, docking studies revealed putative binding sites of phenylbutyrate on PDK2 and 3 that are located on different sites compared to dichloroacetate (DCA), a previously known PDK inhibitor. Based on these findings, we showed both in cells and in mice that phenylbutyrate combined to DCA results in greater increase of PDHC activity compared to each drug alone. These results suggest that therapeutic efficacy can be enhanced by combination of drugs increasing PDHC enzyme activity. PMID:25601413

  10. Use of immobilized glutamate dehydrogenase to synthesize /sup 13/N-labeled L-amino acids

    SciTech Connect

    Cooper, A.J.L.; Gelbard, A.S.

    1981-02-01

    By utilizing glutamate dehydrogenase immobilized onto CNBr-activated Sepharose it is possible to synthesize six L-/sup 13/N-amino acids in high radiochemical yield (5-140 mCi) and in high (> 99%) radiochemical purity. These /sup 13/N-amino acid solutions are potentially suitable for whole body and organ imaging in large animals and man.

  11. Short-chain 3-hydroxyacyl-coenzyme A dehydrogenase associates with a protein super-complex integrating multiple metabolic pathways.

    PubMed

    Narayan, Srinivas B; Master, Stephen R; Sireci, Anthony N; Bierl, Charlene; Stanley, Paige E; Li, Changhong; Stanley, Charles A; Bennett, Michael J

    2012-01-01

    Proteins involved in mitochondrial metabolic pathways engage in functionally relevant multi-enzyme complexes. We previously described an interaction between short-chain 3-hydroxyacyl-coenzyme A dehydrogenase (SCHAD) and glutamate dehydrogenase (GDH) explaining the clinical phenotype of hyperinsulinism in SCHAD-deficient patients and adding SCHAD to the list of mitochondrial proteins capable of forming functional, multi-pathway complexes. In this work, we provide evidence of SCHAD's involvement in additional interactions forming tissue-specific metabolic super complexes involving both membrane-associated and matrix-dwelling enzymes and spanning multiple metabolic pathways. As an example, in murine liver, we find SCHAD interaction with aspartate transaminase (AST) and GDH from amino acid metabolic pathways, carbamoyl phosphate synthase I (CPS-1) from ureagenesis, other fatty acid oxidation and ketogenesis enzymes and fructose-bisphosphate aldolase, an extra-mitochondrial enzyme of the glycolytic pathway. Most of the interactions appear to be independent of SCHAD's role in the penultimate step of fatty acid oxidation suggesting an organizational, structural or non-enzymatic role for the SCHAD protein.

  12. Localization of the gene (OGDH) coding for the E1k component of the [alpha]-ketoglutarate dehydrogenase complex to chromosome 7p13-p11. 2

    SciTech Connect

    Szabo, P.; Cai, X.; Ali, G.; Blass, J.P. )

    1994-03-15

    [alpha]-Ketoglutarate dehydrogenase (E1k), also designated oxoglutarate dehydrogenase (OGDH; EC 1.2.4.2), is a component of the enzyme complex that catalyzes the conversion of [alpha]-ketogluterate to succinyl coenzyme A, a critical step in the Krebs tricarboxylic acid cycle. Deficiencies in the activity of this enzyme complex have been observed in brain and peripheral cells of patients with Alzheimer's disease. This finding led the authors to localize the genes for the polypeptides that compose the [alpha]-ketoglutarate dehydrogenase complex (KDGHC). The E1k locus was mapped to chromosome 7p13-p11.2 using a pair of human-rodent somatic cell hybrid panels. A second related sequence, possibly a pseudogene, was identified and mapped to chromosome 10. 16 refs., 1 fig.

  13. Mechanistic implications from structures of yeast alcohol dehydrogenase complexed with coenzyme and an alcohol.

    PubMed

    Plapp, Bryce V; Charlier, Henry A; Ramaswamy, S

    2016-02-01

    Yeast alcohol dehydrogenase I is a homotetramer of subunits with 347 amino acid residues, catalyzing the oxidation of alcohols using NAD(+) as coenzyme. A new X-ray structure was determined at 3.0 Å where both subunits of an asymmetric dimer bind coenzyme and trifluoroethanol. The tetramer is a pair of back-to-back dimers. Subunit A has a closed conformation and can represent a Michaelis complex with an appropriate geometry for hydride transfer between coenzyme and alcohol, with the oxygen of 2,2,2-trifluoroethanol ligated at 2.1 Å to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. Subunit B has an open conformation, and the coenzyme interacts with amino acid residues from the coenzyme binding domain, but not with residues from the catalytic domain. Coenzyme appears to bind to and dissociate from the open conformation. The catalytic zinc in subunit B has an alternative, inverted coordination with Cys-43, Cys-153, His-66 and the carboxylate of Glu-67, while the oxygen of trifluoroethanol is 3.5 Å from the zinc. Subunit B may represent an intermediate in the mechanism after coenzyme and alcohol bind and before the conformation changes to the closed form and the alcohol oxygen binds to the zinc and displaces Glu-67.

  14. Pyruvate dehydrogenase complex deficiency and its relationship with epilepsy frequency--An overview.

    PubMed

    Bhandary, Suman; Aguan, Kripamoy

    2015-10-01

    The pyruvate dehydrogenase complex (PDHc) is a member of a family of multienzyme complexes that provides the link between glycolysis and the tricarboxylic acid (TCA) cycle by catalyzing the physiologically irreversible decarboxylation of various 2-oxoacid substrates to their corresponding acyl-CoA derivatives, NADH and CO2. PDHc deficiency is a metabolic disorder commonly associated with lactic acidosis, progressive neurological and neuromuscular degeneration that vary with age and gender. In this review, we aim to discuss the relationship between occurrence of epilepsy and PDHc deficiency associated with the pyruvate dehydrogenase complex (E1α subunit (PDHA1) and E1β subunit (PDHB)) and PDH phosphatase (PDP) deficiency. PDHc plays a crucial role in the aerobic carbohydrate metabolism and regulates the use of carbohydrate as the source of oxidative energy. In severe PDHc deficiency, the energy deficit impairs brain development in utero resulting in physiological and structural changes in the brain that contributes to the subsequent onset of epileptogenesis. Epileptogenesis in PDHc deficiency is linked to energy failure and abnormal neurotransmitter metabolism that progressively alters neuronal excitability. This metabolic blockage might be restricted via inclusion of ketogenic diet that is broken up by β-oxidation and directly converting it to acetyl-CoA, and thereby improving the patient's health condition. Genetic counseling is essential as PDHA1 deficiency is X-linked. The demonstration of the X-chromosome localization of PDHA1 resolved a number of questions concerning the variable phenotype displayed by patients with E1 deficiency. Most patients show a broad range of neurological abnormalities, with the severity showing some dependence on the nature of the mutation in the Elα gene, while PDHB and PDH phosphatase (PDP) deficiencies are of autosomal recessive inheritance. However, in females, the disorder is further complicated by the pattern of X

  15. Expression of Aeromonas caviae ST pyruvate dehydrogenase complex components mediate tellurite resistance in Escherichia coli

    SciTech Connect

    Castro, Miguel E.; Molina, Roberto C.; Diaz, Waldo A.; Pradenas, Gonzalo A.; Vasquez, Claudio C.

    2009-02-27

    Potassium tellurite (K{sub 2}TeO{sub 3}) is harmful to most organisms and specific mechanisms explaining its toxicity are not well known to date. We previously reported that the lpdA gene product of the tellurite-resistant environmental isolate Aeromonas caviae ST is involved in the reduction of tellurite to elemental tellurium. In this work, we show that expression of A. caviae ST aceE, aceF, and lpdA genes, encoding pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, respectively, results in tellurite resistance and decreased levels of tellurite-induced superoxide in Escherichia coli. In addition to oxidative damage resulting from tellurite exposure, a metabolic disorder would be simultaneously established in which the pyruvate dehydrogenase complex would represent an intracellular tellurite target. These results allow us to widen our vision regarding the molecular mechanisms involved in bacterial tellurite resistance by correlating tellurite toxicity and key enzymes of aerobic metabolism.

  16. Retinoic acid response element in the human alcohol dehydrogenase gene ADH3: implications for regulation of retinoic acid synthesis.

    PubMed Central

    Duester, G; Shean, M L; McBride, M S; Stewart, M J

    1991-01-01

    Retinoic acid regulation of one member of the human class I alcohol dehydrogenase (ADH) gene family was demonstrated, suggesting that the retinol dehydrogenase function of ADH may play a regulatory role in the biosynthetic pathway for retinoic acid. Promoter activity of human ADH3, but not ADH1 or ADH2, was shown to be activated by retinoic acid in transient transfection assays of Hep3B human hepatoma cells. Deletion mapping experiments identified a region in the ADH3 promoter located between -328 and -272 bp which confers retinoic acid activation. This region was also demonstrated to confer retinoic acid responsiveness on the ADH1 and ADH2 genes in heterologous promoter fusions. Within a 34-bp stretch, the ADH3 retinoic acid response element (RARE) contains two TGACC motifs and one TGAAC motif, both of which exist in RAREs controlling other genes. A block mutation of the TGACC sequence located at -289 to -285 bp eliminated the retinoic acid response. As assayed by gel shift DNA binding studies, the RARE region (-328 to -272 bp) of ADH3 bound the human retinoic acid receptor beta (RAR beta) and was competed for by DNA containing a RARE present in the gene encoding RAR beta. Since ADH catalyzes the conversion of retinol to retinal, which can be further converted to retinoic acid by aldehyde dehydrogenase, these results suggest that retinoic acid activation of ADH3 constitutes a positive feedback loop regulating retinoic acid synthesis. Images PMID:1996113

  17. On dioxygen permeation through a dehydrogenase-pyrroloquinoline quinone complex. A molecular-dynamics investigation.

    PubMed

    Pietra, Francesco

    2014-02-01

    In this work, an all atom model of the quinoprotein dehydrogenase PqqC in complex with the PQQ (=4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid) cofactor and dioxygen (O2 ), solvated with TIP3 water in periodic boxes, was subjected to random-acceleration molecular dynamics (RAMD). It was found that O2 leaves the active binding pocket, in front of PQQ, to get to the solvent, as easily as with a variety of other O2 -activating enzymes, O2 carriers, and gas-sensing proteins. The shortest pathway, orthogonal to the center of the mean plane of PQQ, was largely preferred by O2 over pathways slightly deviating from this line. These observations challenge the interpretation of an impermeable active binding pocket of PqqC-PQQ, as drawn from both X-ray diffraction data of the crystal at low temperature and physiological experimentation.

  18. Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lignin is a significant recalcitrant in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired...

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

    PubMed

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

    2002-02-15

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

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

    PubMed

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

    2002-02-15

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

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

    PubMed

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

    2006-06-01

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

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

    PubMed Central

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

    2015-01-01

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

  3. Phosphorylation of the pyruvate dehydrogenase complex precedes HIF-1-mediated effects and pyruvate dehydrogenase kinase 1 upregulation during the first hours of hypoxic treatment in hepatocellular carcinoma cells

    PubMed Central

    Zimmer, Andreas David; Walbrecq, Geoffroy; Kozar, Ines; Behrmann, Iris; Haan, Claude

    2016-01-01

    The pyruvate dehydrogenase complex (PDC) is an important gatekeeper enzyme connecting glycolysis to the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Thereby, it has a strong impact on the glycolytic flux as well as the metabolic phenotype of a cell. PDC activity is regulated via reversible phosphorylation of three serine residues on the pyruvate dehydrogenase (PDH) E1α subunit. Phosphorylation of any of these residues by the PDH kinases (PDKs) leads to a strong decrease in PDC activity. Under hypoxia, the inactivation of the PDC has been described to be dependent on the hypoxia-inducible factor 1 (HIF-1)-induced PDK1 protein upregulation. In this study, we show in two hepatocellular carcinoma cell lines (HepG2 and JHH-4) that, during the adaptation to hypoxia, PDH is already phosphorylated at time points preceding HIF-1-mediated transcriptional events and PDK1 protein upregulation. Using siRNAs and small molecule inhibitor approaches, we show that this inactivation of PDC is independent of HIF-1α expression but that the PDKs need to be expressed and active. Furthermore, we show that reactive oxygen species might be important for the induction of this PDH phosphorylation since it correlates with the appearance of an altered redox state in the mitochondria and is also inducible by H2O2 treatment under normoxic conditions. Overall, these results show that neither HIF-1 expression nor PDK1 upregulation is necessary for the phosphorylation of PDH during the first hours of the adaptation to hypoxia. PMID:27800515

  4. Communication between Thiamin Cofactors in the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Active Centers

    PubMed Central

    Nemeria, Natalia S.; Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Mossad, Madouna; Tittmann, Kai; Furey, William; Jordan, Frank

    2010-01-01

    Kinetic, spectroscopic, and structural analysis tested the hypothesis that a chain of residues connecting the 4′-aminopyrimidine N1′ atoms of thiamin diphosphates (ThDPs) in the two active centers of the Escherichia coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (Glu571, Glu235, and Glu237) and Arg606 resulted in impaired binding of the second ThDP, once the first active center was filled, suggesting a pathway for communication between the two ThDPs. 1) Steady-state kinetic and fluorescence quenching studies revealed that upon E571A, E235A, E237A, and R606A substitutions, ThDP binding in the second active center was affected. 2) Analysis of the kinetics of thiazolium C2 hydrogen/deuterium exchange of enzyme-bound ThDP suggests half-of-the-sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for the slow exchanging site, indicating that only one of two active sites is filled with ThDP. 3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. 4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that Glu235 makes no direct contact with the cofactor. The role of the conserved Glu571 residue in both catalysis and cofactor orientation is revealed by the combined results for the first time. PMID:20106967

  5. Unusual NADPH conformation in the crystal structure of a cinnamyl alcohol dehydrogenase from Helicobacter pylori in complex with NADP(H) and substrate docking analysis.

    PubMed

    Seo, Kyung Hye; Zhuang, Ningning; Chen, Cong; Song, Jae-Young; Kang, Hyung-Lyun; Rhee, Kwang-Ho; Lee, Kon Ho

    2012-02-17

    Cinnamyl alcohol dehydrogenase is a zinc- and NADPH-dependent dehydrogenase catalyzing the reversible conversion of p-hydroxycinnamaldehydes to their corresponding hydroxycinnamyl alcohols. A CAD homolog from Helicobacter pylori (HpCAD) possesses broad substrate specificities like the plant CADs and additionally a dismutation activity converting benzaldehyde to benzyl alcohol and benzoic acid. We have determined the crystal structure of HpCAD complexed with NADP(H) at 2.18Å resolution to get a better understanding of this class of CAD outside of plants. The structure of HpCAD is highly homologous to the sinapyl alcohol dehydrogenase and the plant CAD with well-conserved residues involved in catalysis and zinc binding. However, the NADP(H) binding mode of the HpCAD has been found to be significantly different from those of plant CADs.

  6. Glutamate 190 is a general acid catalyst in the 6-phosphogluconate-dehydrogenase-catalyzed reaction.

    PubMed

    Karsten, W E; Chooback, L; Cook, P F

    1998-11-10

    Site-directed mutagenesis was used to change E190 of sheep liver 6-phosphogluconate dehydrogenase to A, D, H, K, Q, and R to probe its possible role as a general acid catalyst. Each of the mutant proteins was characterized with respect to the pH dependence of kinetic parameters. Mutations that eliminate a titrable group at position 190, result in pH-rate profiles with no observable pK on the basic side of the V/K6PG profile. Mutations that change the pK of the group at position 190 result in the expected pK perturbations in the V/K6PG profile. Kinetic parameters obtained at the pH optimum in the pH-rate profiles are consistent with a rate-limiting tautomerization of the 1,2-enediol of ribulose 5-phosphate consistent with the proposed role of E190. Data are also consistent with some participation of E190 in an isomerization required to form the active Michaelis complex.

  7. Effects of alpha-adrenergic stimulation on the regulation of the pyruvate dehydrogenase complex in the perfused rat liver

    SciTech Connect

    Fisher, R.A.; Tanabe, S.; Buxton, D.B.; Olson, M.S.

    1985-08-05

    The regulation of the pyruvate dehydrogenase multienzyme complex was investigated during alpha-adrenergic stimulation with phenylephrine in the isolated perfused rat liver. The metabolic flux through the pyruvate dehydrogenase reaction was monitored by measuring the production of UCO2 from infused (1- UC) pyruvate. In livers from fed animals perfused with a low concentration of pyruvate (0.05 mM), phenylephrine infusion significantly inhibited the rate of pyruvate decarboxylation without affecting the amount of pyruvate dehydrogenase in its active form. Results show that alpha-adrenergic agonists do not exert short term regulatory effects on pyruvate dehydrogenase in the liver. Furthermore, the results suggest either that the rat liver pyruvate dehydrogenase complex is insensitive to changes in mitochondrial calcium or that changes in intramitochondrial calcium levels as a result of alpha-adrenergic stimulation are considerably less than suggested by others.

  8. Molecular basis of maple syrup urine disease: Novel mutations at the E1[alpha] locus that impair E1([alpha][sub 2][beta][sub 2]) assembly or decrease steady-state E1[alpha] mRNA levels of branched-chain [alpha]-keto acid dehydrogenase complex

    SciTech Connect

    Chuang, J.L.; Fisher, C.R.; Chuang, D.T.; Cox, R.P. )

    1994-08-01

    The authors report the occurrence of three novel mutations in the E1[alpha] (BCKDHA) locus of the branched-chain [alpha]-keto acid dehydrogenase (BCKAD) complex that cause maple syrup urine disease (MSUD). An 8-bp deletion in exon 7 is present in one allele of a compound-heterozygous patient (GM-649). A single C nucleotide insertion in exon 2 occurs in one allele of an intermediate-MSUD patient (Lo). The second allele of patient Lo carries an A-to-G transition in exon 9 of the E1[alpha] gene. This missense mutation changes Tyr-368 to Cys (Y368C) in the E1[alpha] subunit. Both the 8-bp deletion and the single C insertion generate a downstream nonsense codon. Both mutations appear to be associated with a low abundance of the mutant E1[alpha] mRNA, as determined by allele-specific oligonucleotide probing. Transfection studies strongly suggest that the Y368C substitution in the E1[alpha] subunit impairs its proper assembly with the normal E1[beta]. Unassembled as well as misassembled E1[alpha] and E1[beta] subunits are degraded in the cell. 32 refs., 8 figs.

  9. Pyruvate dehydrogenase complex activity controls metabolic and malignant phenotype in cancer cells.

    PubMed

    McFate, Thomas; Mohyeldin, Ahmed; Lu, Huasheng; Thakar, Jay; Henriques, Jeremy; Halim, Nader D; Wu, Hong; Schell, Michael J; Tsang, Tsz Mon; Teahan, Orla; Zhou, Shaoyu; Califano, Joseph A; Jeoung, Nam Ho; Harris, Robert A; Verma, Ajay

    2008-08-15

    High lactate generation and low glucose oxidation, despite normal oxygen conditions, are commonly seen in cancer cells and tumors. Historically known as the Warburg effect, this altered metabolic phenotype has long been correlated with malignant progression and poor clinical outcome. However, the mechanistic relationship between altered glucose metabolism and malignancy remains poorly understood. Here we show that inhibition of pyruvate dehydrogenase complex (PDC) activity contributes to the Warburg metabolic and malignant phenotype in human head and neck squamous cell carcinoma. PDC inhibition occurs via enhanced expression of pyruvate dehydrogenase kinase-1 (PDK-1), which results in inhibitory phosphorylation of the pyruvate dehydrogenase alpha (PDHalpha) subunit. We also demonstrate that PDC inhibition in cancer cells is associated with normoxic stabilization of the malignancy-promoting transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha) by glycolytic metabolites. Knockdown of PDK-1 via short hairpin RNA lowers PDHalpha phosphorylation, restores PDC activity, reverts the Warburg metabolic phenotype, decreases normoxic HIF-1alpha expression, lowers hypoxic cell survival, decreases invasiveness, and inhibits tumor growth. PDK-1 is an HIF-1-regulated gene, and these data suggest that the buildup of glycolytic metabolites, resulting from high PDK-1 expression, may in turn promote HIF-1 activation, thus sustaining a feed-forward loop for malignant progression. In addition to providing anabolic support for cancer cells, altered fuel metabolism thus supports a malignant phenotype. Correction of metabolic abnormalities offers unique opportunities for cancer treatment and may potentially synergize with other cancer therapies. PMID:18541534

  10. New complexes containing the internal alternative NADH dehydrogenase (Ndi1) in mitochondria of Saccharomyces cerevisiae.

    PubMed

    Matus-Ortega, M G; Cárdenas-Monroy, C A; Flores-Herrera, O; Mendoza-Hernández, G; Miranda, M; González-Pedrajo, B; Vázquez-Meza, H; Pardo, J P

    2015-10-01

    Mitochondria of Saccharomyces cerevisiae lack the respiratory complex I, but contain three rotenone-insensitive NADH dehydrogenases distributed on both the external (Nde1 and Nde2) and internal (Ndi1) surfaces of the inner mitochondrial membrane. These enzymes catalyse the transfer of electrons from NADH to ubiquinone without the translocation of protons across the membrane. Due to the high resolution of the Blue Native PAGE (BN-PAGE) technique combined with digitonin solubilization, several bands with NADH dehydrogenase activity were observed on the gel. The use of specific S. cerevisiae single and double mutants of the external alternative elements (ΔNDE1, ΔNDE2, ΔNDE1/ΔNDE2) showed that the high and low molecular weight complexes contained the Ndi1. Some of the Ndi1 associations took place with complexes III and IV, suggesting the formation of respirasome-like structures. Complex II interacted with other proteins to form a high molecular weight supercomplex with a molecular mass around 600 kDa. We also found that the majority of the Ndi1 was in a dimeric form, which is in agreement with the recently reported three-dimensional structure of the protein. PMID:26173916

  11. New complexes containing the internal alternative NADH dehydrogenase (Ndi1) in mitochondria of Saccharomyces cerevisiae.

    PubMed

    Matus-Ortega, M G; Cárdenas-Monroy, C A; Flores-Herrera, O; Mendoza-Hernández, G; Miranda, M; González-Pedrajo, B; Vázquez-Meza, H; Pardo, J P

    2015-10-01

    Mitochondria of Saccharomyces cerevisiae lack the respiratory complex I, but contain three rotenone-insensitive NADH dehydrogenases distributed on both the external (Nde1 and Nde2) and internal (Ndi1) surfaces of the inner mitochondrial membrane. These enzymes catalyse the transfer of electrons from NADH to ubiquinone without the translocation of protons across the membrane. Due to the high resolution of the Blue Native PAGE (BN-PAGE) technique combined with digitonin solubilization, several bands with NADH dehydrogenase activity were observed on the gel. The use of specific S. cerevisiae single and double mutants of the external alternative elements (ΔNDE1, ΔNDE2, ΔNDE1/ΔNDE2) showed that the high and low molecular weight complexes contained the Ndi1. Some of the Ndi1 associations took place with complexes III and IV, suggesting the formation of respirasome-like structures. Complex II interacted with other proteins to form a high molecular weight supercomplex with a molecular mass around 600 kDa. We also found that the majority of the Ndi1 was in a dimeric form, which is in agreement with the recently reported three-dimensional structure of the protein.

  12. Comparative genomic analysis reveals 2-oxoacid dehydrogenase complex lipoylation correlation with aerobiosis in archaea.

    PubMed

    Borziak, Kirill; Posner, Mareike G; Upadhyay, Abhishek; Danson, Michael J; Bagby, Stefan; Dorus, Steve

    2014-01-01

    Metagenomic analyses have advanced our understanding of ecological microbial diversity, but to what extent can metagenomic data be used to predict the metabolic capacity of difficult-to-study organisms and their abiotic environmental interactions? We tackle this question, using a comparative genomic approach, by considering the molecular basis of aerobiosis within archaea. Lipoylation, the covalent attachment of lipoic acid to 2-oxoacid dehydrogenase multienzyme complexes (OADHCs), is essential for metabolism in aerobic bacteria and eukarya. Lipoylation is catalysed either by lipoate protein ligase (LplA), which in archaea is typically encoded by two genes (LplA-N and LplA-C), or by a lipoyl(octanoyl) transferase (LipB or LipM) plus a lipoic acid synthetase (LipA). Does the genomic presence of lipoylation and OADHC genes across archaea from diverse habitats correlate with aerobiosis? First, analyses of 11,826 biotin protein ligase (BPL)-LplA-LipB transferase family members and 147 archaeal genomes identified 85 species with lipoylation capabilities and provided support for multiple ancestral acquisitions of lipoylation pathways during archaeal evolution. Second, with the exception of the Sulfolobales order, the majority of species possessing lipoylation systems exclusively retain LplA, or either LipB or LipM, consistent with archaeal genome streamlining. Third, obligate anaerobic archaea display widespread loss of lipoylation and OADHC genes. Conversely, a high level of correspondence is observed between aerobiosis and the presence of LplA/LipB/LipM, LipA and OADHC E2, consistent with the role of lipoylation in aerobic metabolism. This correspondence between OADHC lipoylation capacity and aerobiosis indicates that genomic pathway profiling in archaea is informative and that well characterized pathways may be predictive in relation to abiotic conditions in difficult-to-study extremophiles. Given the highly variable retention of gene repertoires across the archaea

  13. Crystal structure of product-bound complex of UDP-N-acetyl-D-mannosamine dehydrogenase from Pyrococcus horikoshii OT3

    SciTech Connect

    Pampa, K.J.; Lokanath, N.K.; Girish, T.U.; Kunishima, N.; Rai, V.R.

    2014-10-24

    Highlights: • Determined the structure of UDP-D-ManNAcADH to a resolution of 1.55 Å. • First complex structure of PhUDP-D-ManNAcADH with UDP-D-ManMAcA. • The monomeric structure consists of three distinct domains. • Cys258 acting as catalytic nucleophilic and Lys204 acts as acid/base catalyst. • Oligomeric state plays an important role for the catalytic function. - Abstract: UDP-N-acetyl-D-mannosamine dehydrogenase (UDP-D-ManNAcDH) belongs to UDP-glucose/GDP-mannose dehydrogenase family and catalyzes Uridine-diphospho-N-acetyl-D-mannosamine (UDP-D-ManNAc) to Uridine-diphospho-N-acetyl-D-mannosaminuronic acid (UDP-D-ManNAcA) through twofold oxidation of NAD{sup +}. In order to reveal the structural features of the Pyrococcus horikoshii UDP-D-ManNAcADH, we have determined the crystal structure of the product-bound enzyme by X-ray diffraction to resolution of 1.55 Å. The protomer folds into three distinct domains; nucleotide binding domain (NBD), substrate binding domain (SBD) and oligomerization domain (OD, involved in the dimerization). The clear electron density of the UDP-D-ManNAcA is observed and the residues binding are identified for the first time. Crystal structures reveal a tight dimeric polymer chains with product-bound in all the structures. The catalytic residues Cys258 and Lys204 are conserved. The Cys258 acts as catalytic nucleophile and Lys204 as acid/base catalyst. The product is directly interacts with residues Arg211, Thr249, Arg244, Gly255, Arg289, Lys319 and Arg398. In addition, the structural parameters responsible for thermostability and oligomerization of the three dimensional structure are analyzed.

  14. Effect of Ganoderma lucidum on the activities of mitochondrial dehydrogenases and complex I and II of electron transport chain in the brain of aged rats.

    PubMed

    Ajith, T A; Sudheesh, N P; Roshny, D; Abishek, G; Janardhanan, K K

    2009-03-01

    Dysfunction of the mitochondrial respiratory chain, being direct intracellular source of reactive oxygen species (ROS), is important in the pathogenesis of number of ageing associated human disorders. Effect of ethanol extract of Ganoderma lucidum on the activities of mitochondrial dehydrogenases; complex I and II of electron transport chain have been evaluated in the aged rat brain. Aged male Wistar rats were administered with ethanol extract of G. lucidum (50 and 250mg/kg, p.o) once daily for 15 days. Similarly DL-alpha-lipoic acid (100mg/kg, p.o) administered group was kept as the reference standard. Young and aged rats administered with water were kept as young and aged control, respectively. The effect of treatment was assessed by estimating the activities of succinate dehydrogenase (SDH), malate dehydrogenase (MDH), alpha-ketoglutarate dehydrogenase (alpha-KGDH), pyruvate dehydrogenase (PDH), complex I and II in the mitochondria of rat brain. Results of the study demonstrated that the extract of G. lucidum (50 and 250mg/kg) significantly (p<0.01) enhanced the activities of PDH, alpha-KGDH, SDH, complex I and II when compared to that of the aged control animals. The level of the lipid peroxidation was significantly lowered (p<0.01) in the G. lucidum treated group with respect to that of aged control. However, we could not find any statistically significant difference between the activities of enzymes in groups treated with 50 and 250mg/kg of G. lucidum. The activity exhibited by the extract of G. lucidum in the present study can be partially correlated to its antioxidant activity. The results of the study concluded that the extract of G. lucidum may effective to improve the function of mitochondria in aged rat brain, suggest its possible therapeutic application against ageing associated neurodegenerative diseases. PMID:19041385

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

    PubMed Central

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

    2013-01-01

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

  16. [Effects of two UDP-glucose dehydrogenases on hyaluronic acid biotransformation].

    PubMed

    GuoI, Donghui; Han, Jian; Liu, Weifeng; Fu, Zhenzhou; Zhu, Qizhong; Tao, Yong

    2014-11-01

    We amplified genes encoding UDP-glucose dehydrogenase, ecohasB from Escherichia coli and spyhasB from Streptococcus pyogenes. Both ecohasB and spyhasB were inserted into T7 expression vector pRX2 to construct recombinant plasmids pRXEB and pRXSB, and to express in E. coli BL21(DE3). After nickel column purification of UDP-glucose dehydrogenases, the enzymes were characterized. The optimum reaction condition of spyHasB was at 30 °C and pH 10. The specific activity reached 12.2 U/mg under optimum condition. The optimum reaction condition of ecoHasB was at 30 °C and pH 9. Its specific activity reached 5.55 U/mg under optimum condition. The pmuhasA gene encoding hyaluronic acid synthase was amplified from Pasteurella multocida and ligated with ecohasB and spyhasB to construct the coexpression vectors pBPAEB and pBPASB, respectively. The co-expression vectors were transformed into E. coli BW25113. Hyaluronic acid (HA) was produced by biotransformation and the conditions were optimized. When recombinant strains were used to produce hyaluronic acid, the higher the activity of UDP-glucose dehydrogenase was, the better its stability was, and the higher the HA production could reach. Under the optimal conditions, the yields of HA produced by pBPAEB/BW25113 and pBPASB/BW25113 in shake flasks were 1.52 and 1.70 g/L, respectively, and the production increased more than 2-3 folds as previously reported. PMID:25985520

  17. Inhibition of Pyruvate Dehydrogenase Kinase 2 Protects Against Hepatic Steatosis Through Modulation of Tricarboxylic Acid Cycle Anaplerosis and Ketogenesis.

    PubMed

    Go, Younghoon; Jeong, Ji Yun; Jeoung, Nam Ho; Jeon, Jae-Han; Park, Bo-Yoon; Kang, Hyeon-Ji; Ha, Chae-Myeong; Choi, Young-Keun; Lee, Sun Joo; Ham, Hye Jin; Kim, Byung-Gyu; Park, Keun-Gyu; Park, So Young; Lee, Chul-Ho; Choi, Cheol Soo; Park, Tae-Sik; Lee, W N Paul; Harris, Robert A; Lee, In-Kyu

    2016-10-01

    Hepatic steatosis is associated with increased insulin resistance and tricarboxylic acid (TCA) cycle flux, but decreased ketogenesis and pyruvate dehydrogenase complex (PDC) flux. This study examined whether hepatic PDC activation by inhibition of pyruvate dehydrogenase kinase 2 (PDK2) ameliorates these metabolic abnormalities. Wild-type mice fed a high-fat diet exhibited hepatic steatosis, insulin resistance, and increased levels of pyruvate, TCA cycle intermediates, and malonyl-CoA but reduced ketogenesis and PDC activity due to PDK2 induction. Hepatic PDC activation by PDK2 inhibition attenuated hepatic steatosis, improved hepatic insulin sensitivity, reduced hepatic glucose production, increased capacity for β-oxidation and ketogenesis, and decreased the capacity for lipogenesis. These results were attributed to altered enzymatic capacities and a reduction in TCA anaplerosis that limited the availability of oxaloacetate for the TCA cycle, which promoted ketogenesis. The current study reports that increasing hepatic PDC activity by inhibition of PDK2 ameliorates hepatic steatosis and insulin sensitivity by regulating TCA cycle anaplerosis and ketogenesis. The findings suggest PDK2 is a potential therapeutic target for nonalcoholic fatty liver disease.

  18. In crystallo posttranslational modification within a MauG/pre-methylamine dehydrogenase complex.

    SciTech Connect

    Jensen, L. M. R.; Sanishvili, R.; Davidson, V. L.; Wilmot, C. M.; Biosciences Division; Univ. of Minnesota; Univ. of Mississippi

    2010-03-12

    MauG is a diheme enzyme responsible for the posttranslational modification of two tryptophan residues to form the tryptophan tryptophylquinone (TTQ) cofactor of methylamine dehydrogenase (MADH). MauG converts preMADH, containing monohydroxylated {beta}Trp{sup 57}, to fully functional MADH by catalyzing the insertion of a second oxygen atom into the indole ring and covalently linking {beta}Trp{sup 57} to {beta}Trp{sup 108}. We have solved the x-ray crystal structure of MauG complexed with preMADH to 2.1 angstroms. The c-type heme irons and the nascent TTQ site are separated by long distances over which electron transfer must occur to achieve catalysis. In addition, one of the hemes has an atypical His-Tyr axial ligation. The crystalline protein complex is catalytically competent; upon addition of hydrogen peroxide, MauG-dependent TTQ synthesis occurs.

  19. Crystal structures of apo-form and binary/ternary complexes of Podophyllum secoisolariciresinol dehydrogenase, an enzyme involved in formation of health-protecting and plant defense lignans.

    PubMed

    Youn, Buhyun; Moinuddin, Syed G A; Davin, Laurence B; Lewis, Norman G; Kang, Chulhee

    2005-04-01

    (-)-Matairesinol is a central biosynthetic intermediate to numerous 8-8'-lignans, including the antiviral agent podophyllotoxin in Podophyllum species and its semi-synthetic anticancer derivatives teniposide, etoposide, and Etopophos. It is formed by action of an enantiospecific secoisolariciresinol dehydrogenase, an NAD(H)-dependent oxidoreductase that catalyzes the conversion of (-)-secoisolariciresinol. Matairesinol is also a plant-derived precursor of the cancer-preventative "mammalian" lignan or "phytoestrogen" enterolactone, formed in the gut following ingestion of high fiber dietary foodstuffs, for example. Additionally, secoisolariciresinol dehydrogenase is involved in pathways to important plant defense molecules, such as plicatic acid in the western red cedar (Thuja plicata) heartwood. To understand the molecular and enantiospecific basis of Podophyllum secoisolariciresinol dehydrogenase, crystal structures of the apo-form and binary/ternary complexes were determined at 1.6, 2.8, and 2.0 angstrom resolution, respectively. The enzyme is a homotetramer, consisting of an alpha/beta single domain monomer containing seven parallel beta-strands flanked by eight alpha-helices on both sides. Its overall monomeric structure is similar to that of NAD(H)-dependent short-chain dehydrogenases/reductases, with a conserved Asp47 forming a hydrogen bond with both hydroxyl groups of the adenine ribose of NAD(H), and thus specificity toward NAD(H) instead of NADP(H). The highly conserved catalytic triad (Ser153, Tyr167, and Lys171) is adjacent to both NAD(+) and substrate molecules, where Tyr167 functions as a general base. Following analysis of high resolution structures of the apo-form and two complex forms, the molecular basis for both the enantio-specificity and the reaction mechanism of secoisolariciresinol dehydrogenase is discussed and compared with that of pinoresinol-lariciresinol reductase.

  20. Dye-linked D-amino acid dehydrogenases: biochemical characteristics and applications in biotechnology.

    PubMed

    Satomura, Takenori; Sakuraba, Haruhiko; Suye, Shin-Ichiro; Ohshima, Toshihisa

    2015-11-01

    Dye-linked D-amino acid dehydrogenases (Dye-DADHs) catalyze the dehydrogenation of free D-amino acids in the presence of an artificial electron acceptor. Although Dye-DADHs functioning in catabolism of L-alanine and as primary enzymes in electron transport chains are widely distributed in mesophilic Gram-negative bacteria, biochemical and biotechnological information on these enzymes remains scanty. This is in large part due to their instability after isolation. On the other hand, in the last decade, several novel types of Dye-DADH have been found in thermophilic bacteria and hyperthermophilic archaea, where they contribute not only to L-alanine catabolism but also to the catabolism of other amino acids, including D-arginine and L-hydroxyproline. In this minireview, we summarize recent developments in our understanding of the biochemical characteristics of Dye-DADHs and their specific application to electrochemical biosensors. PMID:26362681

  1. Dye-linked D-amino acid dehydrogenases: biochemical characteristics and applications in biotechnology.

    PubMed

    Satomura, Takenori; Sakuraba, Haruhiko; Suye, Shin-Ichiro; Ohshima, Toshihisa

    2015-11-01

    Dye-linked D-amino acid dehydrogenases (Dye-DADHs) catalyze the dehydrogenation of free D-amino acids in the presence of an artificial electron acceptor. Although Dye-DADHs functioning in catabolism of L-alanine and as primary enzymes in electron transport chains are widely distributed in mesophilic Gram-negative bacteria, biochemical and biotechnological information on these enzymes remains scanty. This is in large part due to their instability after isolation. On the other hand, in the last decade, several novel types of Dye-DADH have been found in thermophilic bacteria and hyperthermophilic archaea, where they contribute not only to L-alanine catabolism but also to the catabolism of other amino acids, including D-arginine and L-hydroxyproline. In this minireview, we summarize recent developments in our understanding of the biochemical characteristics of Dye-DADHs and their specific application to electrochemical biosensors.

  2. Acid-base catalysis in the chemical mechanism of inosine monophosphate dehydrogenase.

    PubMed

    Markham, G D; Bock, C L; Schalk-Hihi, C

    1999-04-01

    Inosine-5'-monophosphate dehydrogenase (IMPDH) catalyzes the K+-dependent reaction IMP + NAD + H2O --> XMP + NADH + H+ which is the rate-limiting step in guanine nucleotide biosynthesis. The catalytic mechanism of the human type-II IMPDH isozyme has been studied by measurement of the pH dependencies of the normal reaction, of the hydrolysis of 2-chloro-IMP (which yields XMP and Cl- in the absence of NAD), and of inactivation by the affinity label 6-chloro-purine-ribotide (6-Cl-PRT). The pH dependence of the IMPDH reaction shows bell-shaped profiles for kcat and the kcat/Km values for both IMP and NAD, illustrating the involvement of both acidic and basic groups in catalysis. Half-maximal kcat values occur at pH values of 7.2 and 9.8; similar pK values of 6.9 and 9.4 are seen in the kcat/Km profile for NAD. The kcat/Km profile for IMP, which binds first in the predominantly ordered kinetic mechanism, shows pK values of 8.1 and 7.3 for acidic and basic groups, respectively. None of the kinetic pK values correspond to ionizations of the free substrates and thus reflect ionization of the enzyme or enzyme-substrate complexes. The rate of inactivation by 6-Cl-PRT, which modifies the active site sulfhydryl of cysteine-331, increases with pH; the pK of 7.5 reflects the ionization of the sulfhydryl in the E.6-Cl-PRT complex. The pKs of the acids observed in the IMPDH reaction likely also reflect ionization of the cysteine-331 sulfhydryl which adds to C-2 of IMP prior to NAD reduction. The kcat and kcat/Km values for hydrolysis of 2-Cl-IMP show a pK value of 9.9 for a basic group, similar to that seen in the overall reaction, but do not exhibit the ionization of an acidic group. Surprisingly, the rates of 2-Cl-IMP hydrolysis and of inactivation by 6-Cl-PRT are not stimulated by K+, in contrast to the >100-fold K+ activation of the IMPDH reaction. Apparently the enigmatic role of K+ lies in the NAD(H)-dependent segment of the IMPDH reaction. To evaluate the importance of

  3. Random phage mimotopes recognized by monoclonal antibodies against the pyruvate dehydrogenase complex-E2 (PDC-E2).

    PubMed Central

    Cha, S; Leung, P S; Van de Water, J; Tsuneyama, K; Joplin, R E; Ansari, A A; Nakanuma, Y; Schatz, P J; Cwirla, S; Fabris, L E; Neuberger, J M; Gershwin, M E; Coppel, R L

    1996-01-01

    Dihydrolipoamide acetyltransferase, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), is the autoantigen most commonly recognized by autoantibodies in primary biliary cirrhosis (PBC). We identified a peptide mimotope(s) of PDC-E2 by screening a phage-epitope library expressing random dodecapeptides in the pIII coat protein of fd phage using C355.1, a murine monoclonal antibody (mAb) that recognizes a conformation-dependent epitope in the inner lipoyl domain of PDC-E2 and uniquely stains the apical region of bile duct epithelium (BDE) only in patients with PBC. Eight different sequences were identified in 36 phage clones. WMSYPDRTLRTS was present in 29 clones; WESYPFRVGTSL, APKTYVSVSGMV, LTYVSLQGRQGH, LDYVPLKHRHRH, AALWGVKVRHVS, KVLNRIMAGVRH and GNVALVSSRVNA were singly represented. Three common amino acid motifs (W-SYP, TYVS, and VRH) were shared among all peptide sequences. Competitive inhibition of the immunohistochemical staining of PBC BDE was performed by incubating the peptides WMSYPDRTLRTS, WESYPDRTLRTS, APKTYVSVSGMV, and AALWGVKVRHVS with either C355.1 or a second PDC-E2-specific mAb, C150.1. Both mAbs were originally generated to PDC-E2 but map to distinct regions of PDC-E2. Two of the peptides, although selected by reaction with C355.1, strongly inhibited the staining of BDE by C150.1, whereas the peptide APKTYVSVSGMV consistently inhibited the staining of C355.1 on biliary duct epithelium more strongly than the typical mitochondrial staining of hepatocytes. Rabbit sera raised against the peptide WMSYPDRTLRTS stained BDE of livers and isolated bile duct epithelial cells of PBC patients more intensively than controls. The rabbit sera stained all size ducts in normals, but only small/medium-sized ductules in PBC livers. These studies provide evidence that the antigen present in BDE is a molecular mimic of PDC-E2, and not PDC-E2 itself. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8855289

  4. Novel mutations in dihydrolipoamide dehydrogenase deficiency in two cousins with borderline-normal PDH complex activity.

    PubMed

    Cameron, Jessie M; Levandovskiy, Valeriy; Mackay, Neviana; Raiman, Julian; Renaud, Deborah L; Clarke, Joe T R; Feigenbaum, Annette; Elpeleg, Orly; Robinson, Brian H

    2006-07-15

    We have diagnosed dihydrolipoamide dehydrogenase (DLD) deficiency in two male second cousins, who presented with markedly different clinical phenotypes. Patient 1 had a recurrent encephalopathy, and patient 2 had microcephaly and lactic acidosis. Their presentation is unusual, in that the DLD subunit deficiency had little effect on pyruvate dehydrogenase complex activity, but caused a severe reduction in the activities of other enzymes that utilize this subunit. We have identified two mutations in the DLD gene in each patient. The second cousins have one novel mutation in common resulting in a substitution of isoleucine for threonine (I47T), which has not been previously reported in the literature. Patient 1 has a second mutation that has been reported to be common in the Ashkenazi Jewish population, G229C. Patient 2 has a second mutation, E375K, which has also been previously reported in the literature. Enzyme kinetic measurements on patient fibroblasts show that under certain conditions, one heteroallelic mutation may have a higher K(m). This may account for the differing clinical phenotypes. These findings have important repercussions for other patients with similar clinical phenotypes, as DLD activity is not normally measured in cases with normal PDHc activity.

  5. Structures of Michaelis and product complexes of plant cytokinin dehydrogenase: implications for flavoenzyme catalysis.

    PubMed

    Malito, Enrico; Coda, Alessandro; Bilyeu, Kristin D; Fraaije, Marco W; Mattevi, Andrea

    2004-08-27

    Cytokinins form a diverse class of compounds that are essential for plant growth. Cytokinin dehydrogenase has a major role in the control of the levels of these plant hormones by catalysing their irreversible oxidation. The crystal structure of Zea mays cytokinin dehydrogenase displays the same two-domain topology of the flavoenzymes of the vanillyl-alcohol oxidase family but its active site cannot be related to that of any other family member. The X-ray analysis reveals a bipartite architecture of the catalytic centre, which consists of a funnel-shaped region on the protein surface and an internal cavity lined by the flavin ring. A pore with diameter of about 4A connects the two active-site regions. Snapshots of two critical steps along the reaction cycle were obtained through the structural analysis of the complexes with a slowly reacting substrate and the reaction product, which correspond to the states immediately before (Michaelis complex) and after (product complex) oxidation has taken place. The substrate displays a "plug-into-socket" binding mode that seals the catalytic site and precisely positions the carbon atom undergoing oxidation in close contact with the reactive locus of the flavin. A polarising H-bond between the substrate amine group and an Asp-Glu pair may facilitate oxidation. Substrate to product conversion results in small atomic movements, which lead to a planar conformation of the reaction product allowing double-bond conjugation. These features in the mechanism of amine recognition and oxidation differ from those observed in other flavin-dependent amine oxidases.

  6. 15-oxoeicosatetraenoic acid is a 15-hydroxyprostaglandin dehydrogenase-derived electrophilic mediator of inflammatory signaling pathways

    PubMed Central

    Snyder, Nathaniel W.; Golin-Bisello, Franca; Gao, Yang; Blair, Ian A.; Freeman, Bruce A.; Wendell, Stacy Gelhaus

    2014-01-01

    Bioactive lipids govern cellular homeostasis and pathogenic inflammatory processes. Current dogma holds that bioactive lipids, such as prostaglandins and lipoxins, are inactivated by 15-hydroxyprostaglandin dehydrogenase (15PGDH). In contrast, the present results reveal that catabolic “inactivation” of hydroxylated polyunsaturated fatty acids (PUFAs) yields electrophilic α,β-unsaturated ketone derivatives. These endogenously produced species are chemically reactive signaling mediators that induce tissue protective events. Electrophilic fatty acids diversify the proteome through post-translational alkylation of nucleophilic cysteines in key transcriptional regulatory proteins and enzymes that govern cellular metabolic and inflammatory homeostasis. 15PGDH regulates these processes as it is responsible for the formation of numerous electrophilic fatty acids including the arachidonic acid metabolite, 15-oxoeicosatetraenoic acid (15-oxoETE). Herein, the role of 15-oxoETE in regulating signaling responses is reported. In cell cultures, 15-oxoETE activates Nrf2-regulated antioxidant responses (AR) and inhibits NF-κB-mediated pro-inflammatory responses via IKKβ inhibition. Inhibition of glutathione S-transferases using ethacrynic acid incrementally increased the signaling capacity of 15-oxoETE by decreasing 15-oxoETE-GSH adduct formation. This work demonstrates that 15PGDH plays a role in the regulation of cell and tissue homeostasis via the production of electrophilic fatty acid signaling mediators. PMID:25450232

  7. Surface Induced Dissociation Yields Quaternary Substructure of Refractory Noncovalent Phosphorylase B and Glutamate Dehydrogenase Complexes

    NASA Astrophysics Data System (ADS)

    Ma, Xin; Zhou, Mowei; Wysocki, Vicki H.

    2014-03-01

    Ion mobility (IM) and tandem mass spectrometry (MS/MS) coupled with native MS are useful for studying noncovalent protein complexes. Collision induced dissociation (CID) is the most common MS/MS dissociation method. However, some protein complexes, including glycogen phosphorylase B kinase (PHB) and L-glutamate dehydrogenase (GDH) examined in this study, are resistant to dissociation by CID at the maximum collision energy available in the instrument. Surface induced dissociation (SID) was applied to dissociate the two refractory protein complexes. Different charge state precursor ions of the two complexes were examined by CID and SID. The PHB dimer was successfully dissociated to monomers and the GDH hexamer formed trimeric subcomplexes that are informative of its quaternary structure. The unfolding of the precursor and the percentages of the distinct products suggest that the dissociation pathways vary for different charge states. The precursors at lower charge states (+21 for PHB dimer and +27 for GDH hexamer) produce a higher percentage of folded fragments and dissociate more symmetrically than the precusors at higher charge states (+29 for PHB dimer and +39 for GDH hexamer). The precursors at lower charge state may be more native-like than the higher charge state because a higher percentage of folded fragments and a lower percentage of highly charged unfolded fragments are detected. The combination of SID and charge reduction is shown to be a powerful tool for quaternary structure analysis of refractory noncovalent protein complexes, as illustrated by the data for PHB dimer and GDH hexamer.

  8. Mitochondrial complex I, aconitase, and succinate dehydrogenase during hypoxia-reoxygenation: modulation of enzyme activities by MnSOD.

    PubMed

    Powell, Charles S; Jackson, Robert M

    2003-07-01

    Both NADH dehydrogenase (complex I) and aconitase are inactivated partially in vitro by superoxide (O2-.) and other oxidants that cause loss of iron from enzyme cubane (4Fe-4S) centers. We tested whether hypoxia-reoxygenation (H-R) by itself would decrease lung epithelial cell NADH dehydrogenase, aconitase, and succinate dehydrogenase (SDH) activities and whether transfection with adenoviral vectors expressing MnSOD (Ad.MnSOD) would inhibit oxidative enzyme inactivation and thus confirm a mechanism involving O2-. Human lung carcinoma cells with alveolar epithelial cell characteristics (A549 cells) were exposed to <1% O2-5% CO2 (hypoxia) for 24 h followed by air-5% CO2 for 24 h (reoxygenation). NADH dehydrogenase activity was assayed in submitochondrial particles; aconitase and SDH activities were measured in cell lysates. H-R significantly decreased NADH dehydrogenase, aconitase, and SDH activities. Ad.MnSOD increased mitochondrial MnSOD substantially and prevented the inhibitory effects of H-R on enzyme activities. Addition of alpha-ketoglutarate plus aspartate, but not succinate, to medium prevented cytotoxicity due to 2,3-dimethoxy-1,4-naphthoquinone. After hypoxia, cells displayed significantly increased dihydrorhodamine fluorescence, indicating increased mitochondrial oxidant production. Inhibition of NADH dehydrogenase, aconitase, and SDH activities during reoxygenation are due to excess O2-. produced in mitochondria, because enzyme inactivation can be prevented by overexpression of MnSOD. PMID:12665464

  9. Dichloroacetate, the Pyruvate Dehydrogenase Complex and the Modulation of mESC Pluripotency

    PubMed Central

    Rodrigues, Ana Sofia; Correia, Marcelo; Gomes, Andreia; Pereira, Sandro L.; Perestrelo, Tânia; Sousa, Maria Inês; Ramalho-Santos, João

    2015-01-01

    Introduction The pyruvate dehydrogenase (PDH) complex is localized in the mitochondrial matrix catalyzing the irreversible decarboxylation of pyruvate to acetyl-CoA and NADH. For proper complex regulation the E1-α subunit functions as an on/off switch regulated by phosphorylation/dephosphorylation. In different cell types one of the four-pyruvate dehydrogenase kinase isoforms (PDHK1-4) can phosphorylate this subunit leading to PDH inactivation. Our previous results with human Embryonic Stem Cells (hESC), suggested that PDHK could be a key regulator in the metabolic profile of pluripotent cells, as it is upregulated in pluripotent stem cells. Therefore, we wondered if metabolic modulation, via inexpensive pharmacological inhibition of PDHK, could impact metabolism and pluripotency. Methods/Results In order to assess the importance of the PDH cycle in mouse Embryonic Stem Cells (mESC), we incubated cells with the PDHK inhibitor dichloroacetate (DCA) and observed that in its presence ESC started to differentiate. Changes in mitochondrial function and proliferation potential were also found and protein levels for PDH (both phosphorylated and non-phosphorylated) and PDHK1 were monitored. Interestingly, we were also able to describe a possible pathway that involves Hif-1α and p53 during DCA-induced loss of pluripotency. Results with ESCs treated with DCA were comparable to those obtained for cells grown without Leukemia Inhibitor Factor (LIF), used in this case as a positive control for differentiation. Conclusions DCA negatively affects ESC pluripotency by changing cell metabolism and elements related to the PDH cycle, suggesting that PDHK could function as a possible metabolic gatekeeper in ESC, and may be a good target to modulate metabolism and differentiation. Although further molecular biology-based experiments are required, our data suggests that inactive PDH favors pluripotency and that ESC have similar strategies as cancer cells to maintain a glycolytic

  10. 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.

  11. Alternative splicing isoform in succinate dehydrogenase complex, subunit C causes downregulation of succinate-coenzyme Q oxidoreductase activity in mitochondria.

    PubMed

    Satoh, Nana; Yokoyama, Chikako; Itamura, Noriaki; Miyajima-Nakano, Yoshiharu; Hisatomi, Hisashi

    2015-01-01

    Mitochondrial succinate dehydrogenase (SDH) is localized to the inner mitochondrial membrane and is responsible for the redox of succinic acid. SDH is a tetrameric iron-sulfur flavoprotein of the tricarboxylic acid cycle and respiratory chain. The SDH complex, subunit C (SDHC) transcript has deletion-type alternative splicing sites. Generally, alternative splicing produces variant proteins and expression patterns, as products of different genes. In certain cases, specific alternative splicing variants (ASVs) have been associated with human disease. Due to a frameshift mutation causing loss of the heme binding region, the SDHC Δ5 isoform (lacking exon 5) exhibits no SDHC activity. To investigate whether the SDHC splicing variants can function as dominant-negative inhibitors, SDHC ASVs were overexpressed in HCT-15 human colorectal cancer cells. Using real-time reverse transcription-polymerase chain reaction, a dominant-negative effect of the Δ5 isoform on SDHC mRNA was shown. In addition, Δ5 overexpression increased the levels of reactive oxygen species. Furthermore, in the Δ5 isoform-overexpressing cells, SDH activity was reduced. SDHC activation is a significant event during the electron transport chain, and the function of the SDHC Δ5 variant may be significant for the differentiation of tumor cells. PMID:25435987

  12. Mutations in PDX1, the human lipoyl-containing component X of the pyruvate dehydrogenase-complex gene on chromosome 11p1, in congenital lactic acidosis.

    PubMed Central

    Aral, B; Benelli, C; Ait-Ghezala, G; Amessou, M; Fouque, F; Maunoury, C; Créau, N; Kamoun, P; Marsac, C

    1997-01-01

    We have identified and sequenced a cDNA that encodes an apparent human orthologue of a yeast protein-X component (ScPDX1) of pyruvate dehydrogenase multienzyme complexes. The new human cDNA that has been referred to as "HsPDX1" cDNA was cloned by use of the "database cloning" strategy and had a 1,506-bp open reading frame. The amino acid sequence of the protein encoded by the cDNA was 20% identical with that encoded by the yeast PDX1 gene and 40% identical with that encoded by the lipoate acetyltransferase component of the pyruvate dehydrogenase and included a lipoyl-bearing domain that is conserved in some dehydrogenase enzyme complexes. Northern blot analysis demonstrated that the major HsPDX1 mRNA was 2.5 kb in length and was expressed mainly in human skeletal and cardiac muscles but was also present, at low levels, in other tissues. FISH analysis performed with a P1-derived artificial chromosome (PAC)-containing HsPDX1 gene sublocalized the gene to 11p1.3. Molecular investigation of PDX1 deficiency in four patients with neonatal lactic acidemias revealed mutations 78del85 and 965del59 in a homozygous state, and one other patient had no PDX1 mRNA expression. Images Figure 2 Figure 3 Figure 4 PMID:9399911

  13. Comparative studies of Acyl-CoA dehydrogenases for monomethyl branched chain substrates in amino acid metabolism.

    PubMed

    Liu, Xiaojun; Wu, Long; Deng, Guisheng; Chen, Gong; Li, Nan; Chu, Xiusheng; Li, Ding

    2013-04-01

    Short/branched chain acyl-CoA dehydrogenase (SBCAD), isovaleryl-CoA dehydrogenase (IVD), and isobutyryl-CoA dehydrogenase (IBD) are involved in metabolism of isoleucine, leucine, and valine, respectively. These three enzymes all belong to acyl-CoA dehydrogenase (ACD) family, and catalyze the dehydrogenation of monomethyl branched-chain fatty acid (mmBCFA) thioester derivatives. In the present work, the catalytic properties of rat SBCAD, IVD, and IBD, including their substrate specificity, isomerase activity, and enzyme inhibition, were comparatively studied. Our results indicated that SBCAD has its catalytic properties relatively similar to those of straight-chain acyl-CoA dehydrogenases in terms of their isomerase activity and enzyme inhibition, while IVD and IBD are different. IVD has relatively broader substrate specificity than those of the other two enzymes in accommodating various substrate analogs. The present study increased our understanding for the metabolism of monomethyl branched-chain fatty acids (mmBCFAs) and branched-chain amino acids (BCAAs), which should also be useful for selective control of a particular reaction through the design of specific inhibitors. PMID:23474214

  14. Acyl-CoA dehydrogenase drives heat adaptation by sequestering fatty acids

    PubMed Central

    Ma, Dengke K.; Li, Zhijie; Lu, Alice Y.; Sun, Fang; Chen, Sidi; Rothe, Michael; Menzel, Ralph; Sun, Fei; Horvitz, H. Robert

    2015-01-01

    Summary Cells adapt to temperature shifts by adjusting levels of lipid desaturation and membrane fluidity. This fundamental process occurs in nearly all forms of life, but its mechanism in eukaryotes is unknown. We discovered that the evolutionarily conserved C. elegans gene acdh-11 (acyl-CoA-dehydrogenase, ACDH) facilitates heat adaptation by regulating the lipid desaturase FAT-7. Human ACDH deficiency causes the most common inherited disorders of fatty acid oxidation, with syndromes that are exacerbated by hyperthermia. Heat up-regulates acdh-11 expression to decrease fat-7 expression. We solved the high-resolution crystal structure of ACDH-11 and established the molecular basis of its selective and high-affinity binding to C11/C12-chain fatty acids. ACDH-11 sequesters C11/C12-chain fatty acids and prevents these fatty acids from activating nuclear hormone receptors and driving fat-7 expression. Thus, the ACDH-11 pathway drives heat adaptation by linking temperature shifts to regulation of lipid desaturase levels and membrane fluidity via an unprecedented mode of fatty acid signaling. PMID:25981666

  15. Expression of Lactate Dehydrogenase in Aspergillus niger for L-Lactic Acid Production

    PubMed Central

    Dave, Khyati K.; Punekar, Narayan S.

    2015-01-01

    Different engineered organisms have been used to produce L-lactate. Poor yields of lactate at low pH and expensive downstream processing remain as bottlenecks. Aspergillus niger is a prolific citrate producer and a remarkably acid tolerant fungus. Neither a functional lactate dehydrogenase (LDH) from nor lactate production by A. niger is reported. Its genome was also investigated for the presence of a functional ldh. The endogenous A. niger citrate synthase promoter relevant to A. niger acidogenic metabolism was employed to drive constitutive expression of mouse lactate dehydrogenase (mldhA). An appraisal of different branches of the A. niger pyruvate node guided the choice of mldhA for heterologous expression. A high copy number transformant C12 strain, displaying highest LDH specific activity, was analyzed under different growth conditions. The C12 strain produced 7.7 g/l of extracellular L-lactate from 60 g/l of glucose, in non-neutralizing minimal media. Significantly, lactate and citrate accumulated under two different growth conditions. Already an established acidogenic platform, A. niger now promises to be a valuable host for lactate production. PMID:26683313

  16. Expression of Lactate Dehydrogenase in Aspergillus niger for L-Lactic Acid Production.

    PubMed

    Dave, Khyati K; Punekar, Narayan S

    2015-01-01

    Different engineered organisms have been used to produce L-lactate. Poor yields of lactate at low pH and expensive downstream processing remain as bottlenecks. Aspergillus niger is a prolific citrate producer and a remarkably acid tolerant fungus. Neither a functional lactate dehydrogenase (LDH) from nor lactate production by A. niger is reported. Its genome was also investigated for the presence of a functional ldh. The endogenous A. niger citrate synthase promoter relevant to A. niger acidogenic metabolism was employed to drive constitutive expression of mouse lactate dehydrogenase (mldhA). An appraisal of different branches of the A. niger pyruvate node guided the choice of mldhA for heterologous expression. A high copy number transformant C12 strain, displaying highest LDH specific activity, was analyzed under different growth conditions. The C12 strain produced 7.7 g/l of extracellular L-lactate from 60 g/l of glucose, in non-neutralizing minimal media. Significantly, lactate and citrate accumulated under two different growth conditions. Already an established acidogenic platform, A. niger now promises to be a valuable host for lactate production.

  17. Complex changes in the liver mitochondrial proteome of short chain acyl-CoA dehydrogenase deficient mice.

    PubMed

    Wang, Wei; Mohsen, Al-Walid; Uechi, Guy; Schreiber, Emanuel; Balasubramani, Manimalha; Day, Billy; Michael Barmada, M; Vockley, Jerry

    2014-05-01

    Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is an autosomal recessive inborn error of metabolism that leads to the impaired mitochondrial fatty acid β-oxidation of short chain fatty acids. It is heterogeneous in clinical presentation including asymptomatic in most patients identified by newborn screening. Multiple mutations have been identified in patients; however, neither clear genotype-phenotype relationships nor a good correlation between genotype and current biochemical markers for diagnosis has been identified. The definition and pathophysiology of this deficiency remain unclear. To better understand this disorder at a global level, quantitative alterations in the mitochondrial proteome in SCAD deficient mice were examined using a combined proteomics approach: two-dimensional gel difference electrophoresis (2DIGE) followed by protein identification with MALDI-TOF/TOF and iTRAQ labeling followed by nano-LC/MALDI-TOF/TOF. We found broad mitochondrial dysfunction in SCAD deficiency. Changes in the levels of multiple energy metabolism related proteins were identified indicating that a more complex mechanism for development of symptoms may exist. Affected pathways converge on disorders with neurologic symptoms, suggesting that even asymptomatic individuals with SCAD deficiency may be at risk to develop more severe disease. Our results also identified a pattern associated with hepatotoxicity implicated in mitochondrial dysfunction, fatty acid metabolism, decrease of depolarization of mitochondria and mitochondrial membranes, and swelling of mitochondria, demonstrating that SCAD deficiency relates more directly to mitochondrial dysfunction and alteration of fatty acid metabolism. We propose several candidate molecules that may serve as markers for recognition of clinical risk associated with this disorder.

  18. Effects of low molecular-weight organic acids and dehydrogenase activity in rhizosphere sediments of mangrove plants on phytoremediation of polycyclic aromatic hydrocarbons.

    PubMed

    Wang, Yuanyuan; Fang, Ling; Lin, Li; Luan, Tiangang; Tam, Nora F Y

    2014-03-01

    This work evaluated the roles of the low-molecular-weight organic acids (LMWOAs) from root exudates and the dehydrogenase activity in the rhizosphere sediments of three mangrove plant species on the removal of mixed PAHs. The results showed that the concentrations of LMWOAs and dehydrogenase activity changed species-specifically with the levels of PAH contamination. In all plant species, the concentration of citric acid was the highest, followed by succinic acid. For these acids, succinic acid was positively related to the removal of all the PAHs except Chr. Positive correlations were also found between the removal percentages of 4-and 5-ring PAHs and all LMWOAs, except citric acid. LMWOAs enhanced dehydrogenase activity, which positively related to PAH removal percentages. These findings suggested that LMWOAs and dehydrogenase activity promoted the removal of PAHs. Among three mangrove plants, Bruguiera gymnorrhiza, the plant with the highest root biomass, dehydrogenase activity and concentrations of LMWOAs, was most efficient in removing PAHs. PMID:24287262

  19. Effects of low molecular-weight organic acids and dehydrogenase activity in rhizosphere sediments of mangrove plants on phytoremediation of polycyclic aromatic hydrocarbons.

    PubMed

    Wang, Yuanyuan; Fang, Ling; Lin, Li; Luan, Tiangang; Tam, Nora F Y

    2014-03-01

    This work evaluated the roles of the low-molecular-weight organic acids (LMWOAs) from root exudates and the dehydrogenase activity in the rhizosphere sediments of three mangrove plant species on the removal of mixed PAHs. The results showed that the concentrations of LMWOAs and dehydrogenase activity changed species-specifically with the levels of PAH contamination. In all plant species, the concentration of citric acid was the highest, followed by succinic acid. For these acids, succinic acid was positively related to the removal of all the PAHs except Chr. Positive correlations were also found between the removal percentages of 4-and 5-ring PAHs and all LMWOAs, except citric acid. LMWOAs enhanced dehydrogenase activity, which positively related to PAH removal percentages. These findings suggested that LMWOAs and dehydrogenase activity promoted the removal of PAHs. Among three mangrove plants, Bruguiera gymnorrhiza, the plant with the highest root biomass, dehydrogenase activity and concentrations of LMWOAs, was most efficient in removing PAHs.

  20. An amino acid substitution in the pyruvate dehydrogenase E1{alpha} gene, affecting mitochondrial import of the precursor protein

    SciTech Connect

    Takakubo, F.; Thorburn, D.R.; Dahl, H.H.M.

    1995-10-01

    A mutation in the mitochondrial targeting sequence was characterized in a male patient with X chromosome-linked pyruvate dehydrogenase E1{alpha} deficiency. The mutation was a base substitution of G by C at nucleotide 134 in the mitochondrial targeting sequence of the PDHA1 gene, resulting in an arginine-to-proline substitution at codon 10 (R10P). Pyruvate dehydrogenase activity in cultured skin fibroblasts was 28% of the control value, and immunoblot analysis revealed a decreased level of pyruvate dehydrogenase E1{alpha}immunoreactivity. Chimeric constructs in which the normal and mutant pyruvate dehydrogenase E1{alpha} targeting sequences were attached to the mitochondrial matrix protein ornithine transcarbamylase were synthesized in a cell free translation system, and mitochondrial import of normal and mutant proteins was compared in vitro. The results show that ornithine transcarbamylase targeted by the mutant pyruvate dehydrogenase E1{alpha} sequence was translocated into the mitochondrial matrix at a reduced rate, suggesting that defective import is responsible for the reduced pyruvate dehydrogenase level in mitochondria. The mutation was also present in an affected brother and the mildly affected mother. The clinical presentations of this X chromosome-linked disorder in affected family members are discussed. To our knowledge, this is the first report of an amino acid substitution in a mitochondrial targeting sequence resulting in a human genetic disease. 58 refs., 5 figs., 1 tab.

  1. Amplification of the IMP dehydrogenase gene in Chinese hamster cells resistant to mycophenolic acid.

    PubMed Central

    Collart, F R; Huberman, E

    1987-01-01

    The regulation of IMP dehydrogenase (IMPDH) was analyzed in Chinese hamster V79 cell variants that exhibit different degrees of resistance to the cytotoxic effect of mycophenolic acid, a specific inhibitor of IMPDH. Western blot (immunoblot) analysis with an IMPDH antiserum revealed a 14- to 27-fold increase in the amount of enzyme in the mycophenolic acid-resistant cells. The antiserum was also used to screen for a phage containing the IMPDH cDNA sequence from a lambda gt11 expression library. Northern blot (RNA blot) analyses of total cellular and poly(A)+ RNA showed that an IMPDH cDNA probe hybridized to a 2.2-kilobase transcript, the amount of which was associated with increased resistance. Southern blotting with the probe indicated an amplification of the IMPDH gene in the mycophenolic acid-resistant cells. Our findings suggest that the acquired mycophenolic acid resistance of the V79 cell variants is associated with increases in the amount and activity of IMPDH and the number of IMPDH gene copies. Images PMID:2890098

  2. The fate of fluoropyruvate as an alternative substrate and inactivator of the pyruvate dehydrogenase complex of Escherichia coli

    SciTech Connect

    Flournoy, D.S.

    1988-01-01

    The pyruvate dehydrogenase component (E{sub 1}) of the pyruvate dehydrogenase complex of Escherichia coli catalyzes the decomposition of fluoropyruvate to CO{sub 2}, fluoride anion, and acetate. Acetyl thiamin pyrophosphate (acetyl-TPP) is an intermediate in this reaction. Incubation of the pyruvate dehydrogenase complex with ({sup 14}C)fluoropyruvate, TPP, CoASH, and either NADH or pyruvate as reducing agent leads to the formation of ({sup 14}C) acetylCoA. In this reaction the acetyl group of acetyl-TPP is partitioned by transfer to both CoASH and water. When the E{sub 1} component is incubated with ({sup 14}C)fluoropyruvate, TPP, and dihydrolipoamide, ({sup 14}C)acetyldihydrolipoamide is produced. The formation of ({sup 14}C)acetyldihydrolipoamide was examined as a function of dihydrolipoamide concentration. A plot of the extent of acetyl group-partitioning to dihydrolipoamide as a function of 1/(dihydrolipoamide) shows 95 {plus minus} 2% acetyl group transfer to dihydrolipoamide when dihydrolipoamide concentration is extrapolated to infinity. It is concluded that acetyl-TPP is chemically competent as an intermediate for the pyruvate dehydrogenase complex catalyzed oxidative decarboxylation of pyruvate.

  3. 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.

  4. Comparative 13C metabolic flux analysis of pyruvate dehydrogenase complex-deficient, L-valine-producing Corynebacterium glutamicum.

    PubMed

    Bartek, Tobias; Blombach, Bastian; Lang, Siegmund; Eikmanns, Bernhard J; Wiechert, Wolfgang; Oldiges, Marco; Nöh, Katharina; Noack, Stephan

    2011-09-01

    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 (13)C 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.

  5. Synthesis of acetyl coenzyme A by carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila.

    PubMed Central

    Abbanat, D R; Ferry, J G

    1990-01-01

    The carbon monoxide dehydrogenase (CODH) complex from Methanosarcina thermophila catalyzed the synthesis of acetyl coenzyme A (acetyl-CoA) from CH3I, CO, and coenzyme A (CoA) at a rate of 65 nmol/min/mg at 55 degrees C. The reaction ended after 5 min with the synthesis of 52 nmol of acetyl-CoA per nmol of CODH complex. The optimum temperature for acetyl-CoA synthesis in the assay was between 55 and 60 degrees C; the rate of synthesis at 55 degrees C was not significantly different between pHs 5.5 and 8.0. The rate of acetyl-CoA synthesis was independent of CoA concentrations between 20 microM and 1 mM; however, activity was inhibited 50% with 5 mM CoA. Methylcobalamin did not substitute for CH3I in acetyl-CoA synthesis; no acetyl-CoA or propionyl coenzyme A was detected when sodium acetate or CH3CH2I replaced CH3I in the assay mixture. CO could be replaced with CO2 and titanium(III) citrate. When CO2 and 14CO were present in the assay, the specific activity of the acetyl-CoA synthesized was 87% of the specific activity of 14CO, indicating that CO was preferentially incorporated into acetyl-CoA without prior oxidation to free CO2. Greater than 100 microM potassium cyanide was required to significantly inhibit acetyl-CoA synthesis, and 500 microM was required for 50% inhibition; in contrast, oxidation of CO by the CODH complex was inhibited 50% by approximately 10 microM potassium cyanide. PMID:2123865

  6. Heterologous Production of an Energy-Conserving Carbon Monoxide Dehydrogenase Complex in the Hyperthermophile Pyrococcus furiosus

    PubMed Central

    Schut, Gerrit J.; Lipscomb, Gina L.; Nguyen, Diep M. N.; Kelly, Robert M.; Adams, Michael W. W.

    2016-01-01

    Carbon monoxide (CO) is an important intermediate in anaerobic carbon fixation pathways in acetogenesis and methanogenesis. In addition, some anaerobes can utilize CO as an energy source. In the hyperthermophilic archaeon Thermococcus onnurineus, which grows optimally at 80°C, CO oxidation and energy conservation is accomplished by a respiratory complex encoded by a 16-gene cluster containing a CO dehydrogenase, a membrane-bound [NiFe]-hydrogenase and a Na+/H+ antiporter module. This complex oxidizes CO, evolves CO2 and H2, and generates a Na+ motive force that is used to conserve energy by a Na+-dependent ATP synthase. Herein we used a bacterial artificial chromosome to insert the 13.2 kb gene cluster encoding the CO-oxidizing respiratory complex of T. onnurineus into the genome of the heterotrophic archaeon, Pyrococcus furiosus, which grows optimally at 100°C. P. furiosus is normally unable to utilize CO, however, the recombinant strain readily oxidized CO and generated H2 at 80°C. Moreover, CO also served as an energy source and allowed the P. furiosus strain to grow with a limiting concentration of sugar or with peptides as the carbon source. Moreover, CO oxidation by P. furiosus was also coupled to the re-utilization, presumably for biosynthesis, of acetate generated by fermentation. The functional transfer of CO utilization between Thermococcus and Pyrococcus species demonstrated herein is representative of the horizontal gene transfer of an environmentally relevant metabolic capability. The transfer of CO utilizing, hydrogen-producing genetic modules also has applications for biohydrogen production and a CO-based industrial platform for various thermophilic organisms. PMID:26858706

  7. Heterologous Production of an Energy-Conserving Carbon Monoxide Dehydrogenase Complex in the Hyperthermophile Pyrococcus furiosus.

    PubMed

    Schut, Gerrit J; Lipscomb, Gina L; Nguyen, Diep M N; Kelly, Robert M; Adams, Michael W W

    2016-01-01

    Carbon monoxide (CO) is an important intermediate in anaerobic carbon fixation pathways in acetogenesis and methanogenesis. In addition, some anaerobes can utilize CO as an energy source. In the hyperthermophilic archaeon Thermococcus onnurineus, which grows optimally at 80°C, CO oxidation and energy conservation is accomplished by a respiratory complex encoded by a 16-gene cluster containing a CO dehydrogenase, a membrane-bound [NiFe]-hydrogenase and a Na(+)/H(+) antiporter module. This complex oxidizes CO, evolves CO2 and H2, and generates a Na(+) motive force that is used to conserve energy by a Na(+)-dependent ATP synthase. Herein we used a bacterial artificial chromosome to insert the 13.2 kb gene cluster encoding the CO-oxidizing respiratory complex of T. onnurineus into the genome of the heterotrophic archaeon, Pyrococcus furiosus, which grows optimally at 100°C. P. furiosus is normally unable to utilize CO, however, the recombinant strain readily oxidized CO and generated H2 at 80°C. Moreover, CO also served as an energy source and allowed the P. furiosus strain to grow with a limiting concentration of sugar or with peptides as the carbon source. Moreover, CO oxidation by P. furiosus was also coupled to the re-utilization, presumably for biosynthesis, of acetate generated by fermentation. The functional transfer of CO utilization between Thermococcus and Pyrococcus species demonstrated herein is representative of the horizontal gene transfer of an environmentally relevant metabolic capability. The transfer of CO utilizing, hydrogen-producing genetic modules also has applications for biohydrogen production and a CO-based industrial platform for various thermophilic organisms. PMID:26858706

  8. Heterologous production of an energy-conserving carbon monoxide dehydrogenase complex in the hyperthermophile Pyrococcus furiosus

    DOE PAGES

    Schut, Gerrit J.; Lipscomb, Gina L.; Nguyen, Diep M. N.; Kelly, Robert M.; Adams, Michael W. W.

    2016-01-29

    In this study, carbon monoxide (CO) is an important intermediate in anaerobic carbon fixation pathways in acetogenesis and methanogenesis. In addition, some anaerobes can utilize CO as an energy source. In the hyperthermophilic archaeon Thermococcus onnurineus, which grows optimally at 80°C, CO oxidation and energy conservation is accomplished by a respiratory complex encoded by a 16-gene cluster containing a CO dehydrogenase, a membrane-bound [NiFe]-hydrogenase and a Na+/H+ antiporter module. This complex oxidizes CO, evolves CO2 and H2, and generates a Na+ motive force that is used to conserve energy by a Na+-dependent ATP synthase. Herein we used a bacterial artificialmore » chromosome to insert the 13.2 kb gene cluster encoding the CO-oxidizing respiratory complex of T. onnurineus into the genome of the heterotrophic archaeon, Pyrococcus furiosus, which grows optimally at 100° C. P. furiosus is normally unable to utilize CO, however, the recombinant strain readily oxidized CO and generated H2 at 80° C. Moreover, CO also served as an energy source and allowed the P. furiosus strain to grow with a limiting concentration of sugar or with peptides as the carbon source. Moreover, CO oxidation by P. furiosus was also coupled to the re-utilization, presumably for biosynthesis, of acetate generated by fermentation. The functional transfer of CO utilization between Thermococcus and Pyrococcus species demonstrated herein is representative of the horizontal gene transfer of an environmentally relevant metabolic capability. The transfer of CO utilizing, hydrogen-producing genetic modules also has applications for biohydrogen production and a CO-based industrial platform for various thermophilic organisms.« less

  9. Overproduction of bioactive retinoic acid in cells expressing disease-associated mutants of retinol dehydrogenase 12.

    PubMed

    Lee, Seung-Ah; Belyaeva, Olga V; Popov, Ivan K; Kedishvili, Natalia Y

    2007-12-01

    Retinol dehydrogenase 12 (RDH12) is an NADP(+)-dependent oxidoreductase that in vitro catalyzes the reduction of all-trans-retinaldehyde to all-trans-retinol or the oxidation of retinol to retinaldehyde depending on substrate and cofactor availability. Recent studies have linked the mutations in RDH12 to severe early-onset autosomal recessive retinal dystrophy. The biochemical basis of photoreceptor cell death caused by mutations in RDH12 is not clear because the physiological role of RDH12 is not yet fully understood. Here we demonstrate that, although bi-directional in vitro, in living cells, RDH12 acts exclusively as a retinaldehyde reductase, shifting the retinoid homeostasis toward the increased levels of retinol and decreased levels of bioactive retinoic acid. The retinaldehyde reductase activity of RDH12 protects the cells from retinaldehyde-induced cell death, especially at high retinaldehyde concentrations, and this protective effect correlates with the lower levels of retinoic acid in RDH12-expressing cells. Disease-associated mutants of RDH12, T49M and I51N, exhibit significant residual activity in vitro, but are unable to control retinoic acid levels in the cells because of their dramatically reduced affinity for NADPH and much lower protein expression levels. These results suggest that RDH12 acts as a regulator of retinoic acid biosynthesis and protects photoreceptors against overproduction of retinoic acid from all-trans-retinaldehyde, which diffuses into the inner segments of photoreceptors from illuminated rhodopsin. These results provide a novel insight into the mechanism of retinal degeneration associated with mutations in RDH12 and are consistent with the observation that RDH12-null mice are highly susceptible to light-induced retinal apoptosis in cone and rod photoreceptors.

  10. Succinate-dependent energy generation and pyruvate dehydrogenase complex activity in isolated Ascaris suum mitochondria

    SciTech Connect

    Campbell, T.A.

    1988-01-01

    Body wall muscle from the parasitic nematode, Ascaris suum, contain unique anaerobic mitochondria that preferentially utilize fumarate and branched-chain enoyl CoA's as terminal electron acceptors instead of oxygen. While electron transport in these organelles is well characterized, the role of oxygen in succinate-dependent phosphorylation is still not clearly defined. Therefore, the present study was designed to more fully characterize succinate metabolism in these organelles as well as the in vitro regulation of a key mitochondrial enzyme, the pyruvate dehydrogenase complex (PDC). In the absence of added adenine nucleotides, incubations in succinate resulted in substantial elevations in intramitochrondrial ATP levels, but ATP/ADP ratios were considerably higher in incubations with malate. The stimulation of phosphorylation in aerobic incubations with succinate was rotenone sensitive and appears to be Site I dependent. Increase substrate level phosphorylation, coupled to propionate formation, or additional sites of electron-transport associated ATP synthesis were not significant. Under aerobic conditions, {sup 14}CO{sub 2} evolution from 1,4-({sup 14}C)succinate was stimulated and NADH/NAD{sup +} ratios were elevated, but the formation of {sup 14}C propionate was unchanged.

  11. Novel Inhibitors Complexed with Glutamate Dehydrogenase: ALLOSTERIC REGULATION BY CONTROL OF PROTEIN DYNAMICS

    SciTech Connect

    Li, Ming; Smith, Christopher J.; Walker, Matthew T.; Smith, Thomas J.

    2009-12-01

    Mammalian glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate using NAD(P){sup +} as coenzyme. Unlike its counterparts from other animal kingdoms, mammalian GDH is regulated by a host of ligands. The recently discovered hyperinsulinism/hyperammonemia disorder showed that the loss of allosteric inhibition of GDH by GTP causes excessive secretion of insulin. Subsequent studies demonstrated that wild-type and hyperinsulinemia/hyperammonemia forms of GDH are inhibited by the green tea polyphenols, epigallocatechin gallate and epicatechin gallate. This was followed by high throughput studies that identified more stable inhibitors, including hexachlorophene, GW5074, and bithionol. Shown here are the structures of GDH complexed with these three compounds. Hexachlorophene forms a ring around the internal cavity in GDH through aromatic stacking interactions between the drug and GDH as well as between the drug molecules themselves. In contrast, GW5074 and bithionol both bind as pairs of stacked compounds at hexameric 2-fold axes between the dimers of subunits. The internal core of GDH contracts when the catalytic cleft closes during enzymatic turnover. None of the drugs cause conformational changes in the contact residues, but all bind to key interfaces involved in this contraction process. Therefore, it seems likely that the drugs inhibit enzymatic turnover by inhibiting this transition. Indeed, this expansion/contraction process may play a major role in the inter-subunit communication and allosteric regulation observed in GDH.

  12. Atomic-Resolution Structures of Horse Liver Alcohol Dehydrogenase with NAD[superscript +] and Fluoroalcohols Define Strained Michaelis Complexes

    SciTech Connect

    Plapp, Bryce V.; Ramaswamy, S.

    2013-01-16

    Structures of horse liver alcohol dehydrogenase complexed with NAD{sup +} and unreactive substrate analogues, 2,2,2-trifluoroethanol or 2,3,4,5,6-pentafluorobenzyl alcohol, were determined at 100 K at 1.12 or 1.14 {angstrom} resolution, providing estimates of atomic positions with overall errors of 0.02 {angstrom}, the geometry of ligand binding, descriptions of alternative conformations of amino acid residues and waters, and evidence of a strained nicotinamide ring. The four independent subunits from the two homodimeric structures differ only slightly in the peptide backbone conformation. Alternative conformations for amino acid side chains were identified for 50 of the 748 residues in each complex, and Leu-57 and Leu-116 adopt different conformations to accommodate the different alcohols at the active site. Each fluoroalcohol occupies one position, and the fluorines of the alcohols are well-resolved. These structures closely resemble the expected Michaelis complexes with the pro-R hydrogens of the methylene carbons of the alcohols directed toward the re face of C4N of the nicotinamide rings with a C-C distance of 3.40 {angstrom}. The oxygens of the alcohols are ligated to the catalytic zinc at a distance expected for a zinc alkoxide (1.96 {angstrom}) and participate in a low-barrier hydrogen bond (2.52 {angstrom}) with the hydroxyl group of Ser-48 in a proton relay system. As determined by X-ray refinement with no restraints on bond distances and planarity, the nicotinamide rings in the two complexes are slightly puckered (quasi-boat conformation, with torsion angles of 5.9{sup o} for C4N and 4.8{sup o} for N1N relative to the plane of the other atoms) and have bond distances that are somewhat different compared to those found for NAD(P){sup +}. It appears that the nicotinamide ring is strained toward the transition state on the path to alcohol oxidation.

  13. Energy Landscape of the Michaelis Complex of Lactate Dehydrogenase: Relationship to Catalytic Mechanism

    PubMed Central

    2015-01-01

    Lactate dehydrogenase (LDH) catalyzes the interconversion between pyruvate and lactate with nicotinamide adenine dinucleotide (NAD) as a cofactor. Using isotope-edited difference Fourier transform infrared spectroscopy on the “live” reaction mixture (LDH·NADH·pyruvate ⇌ LDH·NAD+·lactate) for the wild-type protein and a mutant with an impaired catalytic efficiency, a set of interconverting conformational substates within the pyruvate side of the Michaelis complex tied to chemical activity is revealed. The important structural features of these substates include (1) electronic orbital overlap between pyruvate’s C2=O bond and the nicotinamide ring of NADH, as shown from the observation of a delocalized vibrational mode involving motions from both moieties, and (2) a characteristic hydrogen bond distance between the pyruvate C2=O group and active site residues, as shown by the observation of at least four C2=O stretch bands indicating varying degrees of C2=O bond polarization. These structural features form a critical part of the expected reaction coordinate along the reaction path, and the ability to quantitatively determine them as well as the substate population ratios in the Michaelis complex provides a unique opportunity to probe the structure–activity relationship in LDH catalysis. The various substates have a strong variance in their propensity toward on enzyme chemistry. Our results suggest a physical mechanism for understanding the LDH-catalyzed chemistry in which the bulk of the rate enhancement can be viewed as arising from a stochastic search through an available phase space that, in the enzyme system, involves a restricted ensemble of more reactive conformational substates as compared to the same chemistry in solution. PMID:24576110

  14. Energy landscape of the Michaelis complex of lactate dehydrogenase: relationship to catalytic mechanism.

    PubMed

    Peng, Huo-Lei; Deng, Hua; Dyer, R Brian; Callender, Robert

    2014-03-25

    Lactate dehydrogenase (LDH) catalyzes the interconversion between pyruvate and lactate with nicotinamide adenine dinucleotide (NAD) as a cofactor. Using isotope-edited difference Fourier transform infrared spectroscopy on the "live" reaction mixture (LDH·NADH·pyruvate ⇌ LDH·NAD(+)·lactate) for the wild-type protein and a mutant with an impaired catalytic efficiency, a set of interconverting conformational substates within the pyruvate side of the Michaelis complex tied to chemical activity is revealed. The important structural features of these substates include (1) electronic orbital overlap between pyruvate's C2═O bond and the nicotinamide ring of NADH, as shown from the observation of a delocalized vibrational mode involving motions from both moieties, and (2) a characteristic hydrogen bond distance between the pyruvate C2═O group and active site residues, as shown by the observation of at least four C2═O stretch bands indicating varying degrees of C2═O bond polarization. These structural features form a critical part of the expected reaction coordinate along the reaction path, and the ability to quantitatively determine them as well as the substate population ratios in the Michaelis complex provides a unique opportunity to probe the structure-activity relationship in LDH catalysis. The various substates have a strong variance in their propensity toward on enzyme chemistry. Our results suggest a physical mechanism for understanding the LDH-catalyzed chemistry in which the bulk of the rate enhancement can be viewed as arising from a stochastic search through an available phase space that, in the enzyme system, involves a restricted ensemble of more reactive conformational substates as compared to the same chemistry in solution.

  15. Combining Laue diffraction and molecular dynamics to study enzyme intermediates: Formation of the Michaelis complex in isocitrate dehydrogenase

    SciTech Connect

    Bash, P.A.; Stoddard, B.L.; Dean, A.

    1996-12-31

    Two separate techniques, Lane diffraction and computational molecular dynamics (MD) simulations, have been developed to allow the visualization and assessment of transient structural states and conformations motions in macromolecules. Recent studies on isocitrate dehydrogenase show that computational MD simulations of an enzymatic Michaelis complex are consistent with difference Fourier election-density maps of the same structure from a Lane experiment. The use of MD facilitates modeling of atoms into regions of experimental density that are poorly defined, due to missing and inaccurate low-resolution diffraction terms that degrade the X-ray scattering contribution from mobile atoms in the active site. The combination of these techniques is used to generate a model for the Michaelis complex of the enzyme isocitrate dehydrogenase with substrate isocitrate and coenzyme nicotinamide-adenine dinucleotide phosphate (NADP). This concerted use of time-resolved X-ray crystallography and of MD simulations has clear importance for the study of enzyme dynamics and function.

  16. Site-directed mutagenesis and /sup 1/H NMR spectroscopy of an interdomain segment in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

    SciTech Connect

    Texter, F.L.; Radford, S.E.; Laue, E.D.; Perham, R.N.; Miles, J.S.; Guest, J.R.

    1988-01-12

    Deletion of two of the three homologous lipoyl domains that form the N-terminal half of each dihydrolipoamide acetyltransferase (E2p) polypeptide chain of the Escherichia coli pyruvate dehydrogenase complex can be achieved by in vitro deletion in the structural gene aceF. A site-directed mutagenesis of this shortened aceF gene was carried out to replace the glutamine residue at position 291 (wild-type numbering) with a histidine residue. Residue 291 is near the middle of a long segment (about 30 amino acid residues) of polypeptide chain, rich in alanine, proline, and charged amino acids, that links the remaining lipoyl domain to the dihydrolipoamide dehydrogenase (E3) binding domain in the E2p chain. A fully active enzyme complex was still assembled, and despite the enormous size of the particle, sharp resonances attributable to the single new histidine residue per E2p chain could be detected in the 400-MHz /sup 1/H NMR spectrum of the complex. The sharpness of these resonances, their chemical shifts (7.94 and 7.05 ppm), and the apparent pK/sub a/ (6.4) of the side chain were all consistent with this histidine residue being exposed to solvent in a conformationally flexible region of the E2p polypeptide chain. These experiments provide direct proof for the conformational flexibility of this region of polypeptide chain, which is though to play an important part in the movement of the lipoyl domain required for active site coupling in the enzyme complex. The major sharp resonance (at l.39 ppm) in the 400-MHz /sup 1/H NMR spectrum of the mutated complex, was somewhat smaller than expected, suggesting that the insertion of the histidine residue at position 291 had diminished the flexibility of some at least of the alanine residues in this segment of polypeptide chain.

  17. Inhibition of snowshoe hare succinate dehydrogenase activity as a mechanism of deterrence for papyriferic acid in birch.

    PubMed

    Forbey, Jennifer Sorensen; Pu, Xinzhu; Xu, Dong; Kielland, Knut; Bryant, John

    2011-12-01

    The plant secondary metabolite papyriferic acid (PA) deters browsing by snowshoe hares (Lepus americanus) on the juvenile developmental stage of the Alaska paper birch (Betula neoalaskana). However, the physiological mechanism that reduces browsing remains unknown. We used pharmacological assays and molecular modeling to test the hypothesis that inhibition of succinate dehydrogenase (SDH) is a mode of action (MOA) of toxicity of PA in snowshoe hares. We tested this hypothesis by measuring the effect of PA on the activity of SDH in liver mitochondria isolated from wild hares. In addition, we used molecular modeling to determine the specific binding site of PA on SDH. We found that PA inhibits SDH from hares by an uncompetitive mechanism in a dose-dependent manner. Molecular modeling suggests that inhibition of SDH is a result of binding of PA at the ubiquinone binding sites in complex II. Our results provide a MOA for toxicity that may be responsible for the concentration-dependent anti-feedant effects of PA. We propose that snowshoe hares reduce the dose-dependent toxic consequences of PA by relying on efflux transporters and metabolizing enzymes that lower systemic exposure to dietary PA.

  18. Genetics Home Reference: pyruvate dehydrogenase deficiency

    MedlinePlus

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

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

    PubMed

    Posch, K C; Napoli, J L

    1992-05-28

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

  20. Escherichia coli Pyruvate Dehydrogenase Complex Is an Important Component of CXCL10-Mediated Antimicrobial Activity

    PubMed Central

    Schutte, Kirsten M.; Fisher, Debra J.; Burdick, Marie D.; Mehrad, Borna; Mathers, Amy J.; Mann, Barbara J.; Nakamoto, Robert K.

    2015-01-01

    Chemokines are best recognized for their role within the innate immune system as chemotactic cytokines, signaling and recruiting host immune cells to sites of infection. Certain chemokines, such as CXCL10, have been found to play an additional role in innate immunity, mediating CXCR3-independent killing of a diverse array of pathogenic microorganisms. While this is still not clearly understood, elucidating the mechanisms underlying chemokine-mediated antimicrobial activity may facilitate the development of novel therapeutic strategies effective against antibiotic-resistant Gram-negative pathogens. Here, we show that CXCL10 exerts antibacterial effects on clinical and laboratory strains of Escherichia coli and report that disruption of pyruvate dehydrogenase complex (PDHc), which converts pyruvate to acetyl coenzyme A, enables E. coli to resist these antimicrobial effects. Through generation and screening of a transposon mutant library, we identified two mutants with increased resistance to CXCL10, both with unique disruptions of the gene encoding the E1 subunit of PDHc, aceE. Resistance to CXCL10 also occurred following deletion of either aceF or lpdA, genes that encode the remaining two subunits of PDHc. Although PDHc resides within the bacterial cytosol, electron microscopy revealed localization of immunogold-labeled CXCL10 to the bacterial cell surface in both the E. coli parent and aceE deletion mutant strains. Taken together, our findings suggest that while CXCL10 interacts with an as-yet-unidentified component on the cell surface, PDHc is an important mediator of killing by CXCL10. To our knowledge, this is the first description of PDHc as a key bacterial component involved in the antibacterial effect of a chemokine. PMID:26553462

  1. Escherichia coli Pyruvate Dehydrogenase Complex Is an Important Component of CXCL10-Mediated Antimicrobial Activity.

    PubMed

    Schutte, Kirsten M; Fisher, Debra J; Burdick, Marie D; Mehrad, Borna; Mathers, Amy J; Mann, Barbara J; Nakamoto, Robert K; Hughes, Molly A

    2016-01-01

    Chemokines are best recognized for their role within the innate immune system as chemotactic cytokines, signaling and recruiting host immune cells to sites of infection. Certain chemokines, such as CXCL10, have been found to play an additional role in innate immunity, mediating CXCR3-independent killing of a diverse array of pathogenic microorganisms. While this is still not clearly understood, elucidating the mechanisms underlying chemokine-mediated antimicrobial activity may facilitate the development of novel therapeutic strategies effective against antibiotic-resistant Gram-negative pathogens. Here, we show that CXCL10 exerts antibacterial effects on clinical and laboratory strains of Escherichia coli and report that disruption of pyruvate dehydrogenase complex (PDHc), which converts pyruvate to acetyl coenzyme A, enables E. coli to resist these antimicrobial effects. Through generation and screening of a transposon mutant library, we identified two mutants with increased resistance to CXCL10, both with unique disruptions of the gene encoding the E1 subunit of PDHc, aceE. Resistance to CXCL10 also occurred following deletion of either aceF or lpdA, genes that encode the remaining two subunits of PDHc. Although PDHc resides within the bacterial cytosol, electron microscopy revealed localization of immunogold-labeled CXCL10 to the bacterial cell surface in both the E. coli parent and aceE deletion mutant strains. Taken together, our findings suggest that while CXCL10 interacts with an as-yet-unidentified component on the cell surface, PDHc is an important mediator of killing by CXCL10. To our knowledge, this is the first description of PDHc as a key bacterial component involved in the antibacterial effect of a chemokine.

  2. Abscisic acid effects on activity and expression of barley (Hordeum vulgare) plastidial glucose-6-phosphate dehydrogenase

    PubMed Central

    Cardi, Manuela; Chibani, Kamel; Cafasso, Donata; Rouhier, Nicolas; Jacquot, Jean-Pierre; Esposito, Sergio

    2011-01-01

    Total glucose-6-phosphate dehydrogenase (G6PDH) activity, protein abundance, and transcript levels of G6PDH isoforms were measured in response to exogenous abscisic acid (ABA) supply to barley (Hordeum vulgare cv Nure) hydroponic culture. Total G6PDH activity increased by 50% in roots treated for 12 h with exogenous 0.1 mM ABA. In roots, a considerable increase (35%) in plastidial P2-G6PDH transcript levels was observed during the first 3 h of ABA treatment. Similar protein variations were observed in immunoblotting analyses. In leaves, a 2-fold increase in total G6PDH activity was observed after ABA treatment, probably related to an increase in the mRNA level (increased by 50%) and amount of protein (increased by 85%) of P2-G6PDH. Together these results suggest that the plastidial P2-isoform plays an important role in ABA-treated barley plants. PMID:21464159

  3. Three-dimensional solution structure of the E3-binding domain of the dihydrolipoamide succinyltransferase core from the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli.

    PubMed

    Robien, M A; Clore, G M; Omichinski, J G; Perham, R N; Appella, E; Sakaguchi, K; Gronenborn, A M

    1992-04-01

    The three-dimensional solution structure of a 51-residue synthetic peptide comprising the dihydrolipoamide dehydrogenase (E3)-binding domain of the dihydrolipoamide succinyltransferase (E2) core of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli has been determined by nuclear magnetic resonance spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. The structure is based on 630 approximate interproton distance and 101 torsion angle (phi, psi, chi 1) restraints. A total of 56 simulated annealing structures were calculated, and the atomic rms distribution about the mean coordinate positions for residues 12-48 of the synthetic peptide is 1.24 A for the backbone atoms, 1.68 A for all atoms, and 1.33 A for all atoms excluding the six side chains which are disordered at chi 1 and the seven which are disordered at chi 2; when the irregular partially disordered loop from residues 31 to 39 is excluded, the rms distribution drops to 0.77 A for the backbone atoms, 1.55 A for all atoms, and 0.89 A for ordered side chains. Although proton resonance assignments for the N-terminal 11 residues and the C-terminal 3 residues were obtained, these two segments of the polypeptide are disordered in solution as evidenced by the absence of nonsequential nuclear Overhauser effects. The solution structure of the E3-binding domain consists of two parallel helices (residues 14-23 and 40-48), a short extended strand (24-26), a five-residue helical-like turn, and an irregular (and more disordered) loop (residues 31-39). This report presents the first structure of an E3-binding domain from a 2-oxo acid dehydrogenase complex.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1554728

  4. Novel binding motif and new flexibility revealed by structural analyses of a pyruvate dehydrogenase-dihydrolipoyl acetyltransferase subcomplex from the Escherichia coli pyruvate dehydrogenase multienzyme complex.

    PubMed

    Arjunan, Palaniappa; Wang, Junjie; Nemeria, Natalia S; Reynolds, Shelley; Brown, Ian; Chandrasekhar, Krishnamoorthy; Calero, Guillermo; Jordan, Frank; Furey, William

    2014-10-24

    The Escherichia coli pyruvate dehydrogenase multienzyme complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subcomplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subcomplex, both of which probably have consequences in the overall multienzyme complex assembly. This represents the first structure of an E1p-E2p didomain subcomplex involving a homodimeric E1p, and the results may be applicable to a large range of complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subcomplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly. PMID:25210042

  5. Dehydrogenases, Acid and Alkaline Phosphatases, and Esterases for Chemotaxonomy of Selected Meloidogyne, Ditylenchus, Heterodera and Aphelenchus spp.

    PubMed Central

    Dickson, D. W.; Huisingh, D.; Sasser, J. N.

    1971-01-01

    Various taxonomically useful profiles of four dehydrogenases (lactate, malate, glucose-6-phosphate, and a-glycerophosphate) and three hydrolases (acid and alkaline phosphatase and esterase) were detected in whole nematode homogenates of Meloidogyne javanica, M. hapla, M. incognita, M. arenaria, Ditylenchus dipsaci, D. triformis, Heterodera glycines, and Aphelenchus avenae. The enzyme profiles were stable in populations cultured on several different hosts. A tentative enzymically-determined phylogeny of Meloidogyne is given. PMID:19322334

  6. Isolation of an enzyme complex with carbon monoxide dehydrogenase activity containing corrinoid and nickel from acetate-grown Methanosarcina thermophila.

    PubMed Central

    Terlesky, K C; Nelson, M J; Ferry, J G

    1986-01-01

    Fast protein liquid chromatography of cell extract from methanol- or acetate-grown Methanosarcina thermophila resolved two peaks of CO dehydrogenase activity. The activity of one of the CO dehydrogenases was sixfold greater in acetate-grown compared with methanol-grown cells. This CO dehydrogenase was purified to apparent homogeneity (70 mumol of methyl viologen reduced per min per mg of protein) and made up greater than 10% of the cellular protein of acetate-grown cells. The native enzyme (Mr 250,000) formed aggregates with an Mr of approximately 1,000,000. The enzyme contained five subunits (Mrs 89,000, 71,000, 60,000, 58,000, and 19,000), suggesting a multifunctional enzyme complex. Nickel, iron, cobalt, zinc, inorganic sulfide, and a corrinoid were present in the complex. The UV-visible spectrum suggested the presence of iron-sulfur centers. The electron paramagnetic resonance spectrum contained g values of 2.073, 2.049, and 2.028; these features were broadened in enzyme that was purified from cells grown in the presence of medium enriched with 61Ni, indicating the involvement of this metal in the spectrum. The pattern of potassium cyanide inhibition indicated that cyanide binds at or near the CO binding site. The properties of the enzyme imply an involvement in the dissimilation of acetate to methane, possibly by cleavage of acetate or activated acetate. Images PMID:3023296

  7. Solution Structure and Characterisation of the Human Pyruvate Dehydrogenase Complex Core Assembly

    PubMed Central

    Vijayakrishnan, S.; Kelly, S.M.; Gilbert, R.J.C.; Callow, P.; Bhella, D.; Forsyth, T.; Lindsay, J.G.; Byron, O.

    2010-01-01

    Mammalian pyruvate dehydrogenase complex (PDC) is a key multi-enzyme assembly that is responsible for glucose homeostasis maintenance and conversion of pyruvate into acetyl-CoA. It comprises a central pentagonal dodecahedral core consisting of two subunit types (E2 and E3BP) to which peripheral enzymes (E1 and E3) bind tightly but non-covalently. Currently, there are two conflicting models of PDC (E2 + E3BP) core organisation: the ‘addition’ model (60 + 12) and the ‘substitution’ model (48 + 12). Here we present the first ever low-resolution structures of human recombinant full-length PDC core (rE2/E3BP), truncated PDC core (tE2/E3BP) and native bovine heart PDC core (bE2/E3BP) obtained by small-angle X-ray scattering and small-angle neutron scattering. These structures, corroborated by negative-stain and cryo electron microscopy data, clearly reveal open pentagonal core faces, favouring the ‘substitution’ model of core organisation. The native and recombinant core structures are all similar to the truncated bacterial E2 core crystal structure obtained previously. Cryo-electron microscopy reconstructions of rE2/E3BP and rE2/E3BP:E3 directly confirm that the core has open pentagonal faces, agree with scattering-derived models and show density extending outwards from their surfaces, which is much more structurally ordered in the presence of E3. Additionally, analytical ultracentrifugation characterisation of rE2/E3BP, rE2 (full-length recombinant E2-only) and tE2/E3BP supports the substitution model. Superimposition of the small-angle neutron scattering tE2/E3BP and truncated bacterial E2 crystal structures demonstrates conservation of the overall pentagonal dodecahedral morphology, despite evolutionary diversity. In addition, unfolding studies using circular dichroism and tryptophan fluorescence spectroscopy show that the rE2/E3BP is less stable than its rE2 counterpart, indicative of a role for E3BP in core destabilisation. The architectural

  8. Stimulation of rat liver branched-chain alpha-keto acid dehydrogenase activity by low doses of bezafibrate.

    PubMed

    Knapik-Czajka, Malgorzata

    2013-04-01

    Multienzyme branched-chain alpha-ketoacid dehydrogenase complex (BCKDH) catalyzes the regulatory step of oxidative catabolism of indispensable branched-chain amino acids (BCAA). The activity of the BCKDH complex is regulated by a reversible phosphorylation, end-product inhibition and by changes in the gene expression of BCKDH component enzymes. It has been shown previously that a high dose of bezafibrate (an agent added to rat chow at final concentration of 0.5%) changes mRNA levels of BCKDH-related enzymes and increases dephosphorylation of the complex leading to stimulation of liver BCKDH activity and the enhanced BCAA catabolism. The aim of the present study was to determine an in vivo effect of low, clinically relevant doses of bezafibrate on BCKDH activity in rat liver. Bezafibrate was administrated for 14 days by gastric gavage to Wistar male rats (fed low-protein chow; 8% protein) at one of the following daily doses of 5, 10 and 20mg/kgb.wt. The control group was given the vehicle (0.3% methylcellulose) only. The actual BCKDH and total BCKDH activities were assayed spectrophotometrically before and after incubation with a broad-specificity phosphatase, respectively. The mRNA levels of the selected genes (BCKDH catalytic subunits and regulatory enzymes) were quantified by means of semi-quantitative RT-PCR. Current catalytic activity of BCKDH (described as BCKDH activity state - the proportion of the BCKDH complex in its active dephosphorylated form) increased by 2.1 ± 0.2, 2.3 ± 0.2 and 2.7 ± 0.2 fold (p<0.01). Changes in BCKDH activity did not correspond with changes in mRNA levels of the complex catalytic subunits. Moreover, mRNA levels of regulatory enzymes remained unaltered. Initially bezafibrate caused a transient insignificant reduction in body weight, but it had no effect on the final body weight. The highest dose of bezafibrate induced hepatomegaly. In conclusion, these data indicate that under conditions of dietary protein restriction low

  9. Nicotinamide adenine dinucleotide-dependent and nicotinamide adenine dinucleotide-independent lactate dehydrogenases in homofermentative and heterofermentative lactic acid bacteria.

    PubMed

    Doelle, H W

    1971-12-01

    Three homofermentative (Lactobacillus plantarum B38, L. plantarum B33, Pediococcus pentosaceus B30) and three heterofermentative (Leuconostoc mesenteroides 39, L. oenos B70, Lactobacillus brevis) lactic acid bacteria were examined for the presence or absence of nicotinamide adenine dinucleotide (NAD)-dependent and NAD-independent d- and l-lactate dehydrogenases. Two of the six strains investigated, P. pentosaceus and L. oenos, did not exhibit an NAD-independent enzyme activity capable of reducing dichlorophenol indophenol. The pH optima of the lactic dehydrogenases were determined. The NAD-dependent enzymes from homofermentative strains exhibited optima at pH 7.8 to 8.8, whereas values from 9.0 to 10.0 were noted for these enzymes from heterofermentative organisms. The optima for the NAD-independent enzymes were between 5.8 and 6.6. The apparent Michaelis-Menten constants determined for both NAD and the substrates demonstrated the existence of a greater affinity for d- than l-lactic acid. A comparison of the specific NAD-dependent and NAD-independent lactate dehydrogenase activities revealed a direct correlation of the d/l ratios of these activities with the type of lactic acid produced during the growth of the organism.

  10. The two active sites in human branched-chain alpha-keto acid dehydrogenase operate independently without an obligatory alternating-site mechanism.

    PubMed

    Li, Jun; Machius, Mischa; Chuang, Jacinta L; Wynn, R Max; Chuang, David T

    2007-04-20

    A long standing controversy is whether an alternating activesite mechanism occurs during catalysis in thiamine diphosphate (ThDP)-dependent enzymes. We address this question by investigating the ThDP-dependent decarboxylase/dehydrogenase (E1b) component of the mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC). Our crystal structure reveals that conformations of the two active sites in the human E1b heterotetramer harboring the reaction intermediate are identical. Acidic residues in the core of the E1b heterotetramer, which align with the proton-wire residues proposed to participate in active-site communication in the related pyruvate dehydrogenase from Bacillus stearothermophilus, are mutated. Enzyme kinetic data show that, except in a few cases because of protein misfolding, these alterations are largely without effect on overall activity of BCKDC, ruling out the requirement of a proton-relay mechanism in E1b. BCKDC overall activity is nullified at 50% phosphorylation of E1b, but it is restored to nearly half of the pre-phosphorylation level after dissociation and reconstitution of BCKDC with the same phosphorylated E1b. The results suggest that the abolition of overall activity likely results from the specific geometry of the half-phosphorylated E1b in the BCKDC assembly and not due to a disruption of the alternating active-site mechanism. Finally, we show that a mutant E1b containing only one functional active site exhibits half of the wild-type BCKDC activity, which directly argues against the obligatory communication between active sites. The above results provide evidence that the two active sites in the E1b heterotetramer operate independently during the ThDP-dependent decarboxylation reaction. PMID:17329260

  11. Activity of soil dehydrogenases, urease, and acid and alkaline phosphatases in soil polluted with petroleum.

    PubMed

    Wyszkowska, Jadwiga; Wyszkowski, Mirosław

    2010-01-01

    This study was undertaken to (1) determine the effects of petroleum pollution on changes in the biochemical properties of soil and (2) demonstrate whether the application of compost, bentonite, and calcium oxide is likely to restore biological balance. Petroleum soil pollution at a dose ranging from 2.5 to 10 cm(3)/kg disturbed the biochemical balance as evidenced by inhibition of the activities of soil dehydrogenases (SDH), urease (URE), and acid phosphatase (ACP). The greatest change was noted in the activity of SDH, whereas the least change occurred in URE. Petroleum significantly increased the activity of soil alkaline phosphatase (ALP) in soil used for spring rape, whereas in soil used for oat harvest there was decreased ALP activity. The application of compost, bentonite, and calcium oxide to soil proved effective in mitigating the adverse effects of petroleum on the activities of soil enzymes. Soil enrichment with compost, bentonite, and calcium oxide was found to stimulate the activities of URE and ALP and inhibit the activity of ACP. The influence of bentonite and calcium oxide was greater than that of compost. Calcium oxide and, to a lesser extent, compost were found to increase the activity of SDH, whereas bentonite exerted the opposite effect, especially in the case of the main crop, spring rape. The activities of SDH, URE, and ACP were higher in soil used for rape than that for oats. In contrast the activity of ALP was higher in soil used for oats. Data thus indicate that compost and especially bentonite and calcium oxide exerted a positive effect on activities of some enzymes in soil polluted with petroleum. Application of neutralizing additives to soil restored soil biological balance by counteracting the negative influence of petroleum on activities of URE and ALP. PMID:20706945

  12. Regulation of the activity of lactate dehydrogenases from four lactic acid bacteria.

    PubMed

    Feldman-Salit, Anna; Hering, Silvio; Messiha, Hanan L; Veith, Nadine; Cojocaru, Vlad; Sieg, Antje; Westerhoff, Hans V; Kreikemeyer, Bernd; Wade, Rebecca C; Fiedler, Tomas

    2013-07-19

    Despite high similarity in sequence and catalytic properties, the l-lactate dehydrogenases (LDHs) in lactic acid bacteria (LAB) display differences in their regulation that may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the effects of fructose 1,6-bisphosphate (FBP), phosphate (Pi), and ionic strength (NaCl concentration) on six LDHs from four LABs studied at pH 6 and pH 7. We found that 1) the extent of activation by FBP (Kact) differs. Lactobacillus plantarum LDH is not regulated by FBP, but the other LDHs are activated with increasing sensitivity in the following order: Enterococcus faecalis LDH2 ≤ Lactococcus lactis LDH2 < E. faecalis LDH1 < L. lactis LDH1 ≤ Streptococcus pyogenes LDH. This trend reflects the electrostatic properties in the allosteric binding site of the LDH enzymes. 2) For L. plantarum, S. pyogenes, and E. faecalis, the effects of Pi are distinguishable from the effect of changing ionic strength by adding NaCl. 3) Addition of Pi inhibits E. faecalis LDH2, whereas in the absence of FBP, Pi is an activator of S. pyogenes LDH, E. faecalis LDH1, and L. lactis LDH1 and LDH2 at pH 6. These effects can be interpreted by considering the computed binding affinities of Pi to the catalytic and allosteric binding sites of the enzymes modeled in protonation states corresponding to pH 6 and pH 7. Overall, the results show a subtle interplay among the effects of Pi, FBP, and pH that results in different regulatory effects on the LDHs of different LABs.

  13. Methemoglobin reduction mediated by D-amino acid dehydrogenase in Propsilocerus akamusi (Tokunaga) larvae.

    PubMed

    Kobori, Hiroki; Tanigawa, Minoru; Maeda, Shintaro; Hori, Hiroshi; Yubisui, Toshitsugu; Nagata, Yoko

    2015-06-01

    A methemoglobin (metHb) reduction system is required for aerobic respiration. In humans, Fe(III)-heme-bearing metHb (the oxidized form of hemoglobin), which cannot bind oxygen, is converted to Fe(II)-heme-bearing oxyhemoglobin (oxyHb, the reduced form), which can bind oxygen, in a system comprising NADH, NADH-cytochrome b5 reductase, and cytochrome b5. However, the mechanism of metHb reduction in organisms that inhabit oxygen-deficient environments is unknown. In the coelomic fluid of the larvae of Propsilocerus akamusi, which inhabit a microaerobic environment, we found that metHb was reduced by D-alanine. We purified an FAD-containing enzyme, D-amino acid dehydrogenase (DAD), and component V hemoglobin from the larvae. Using the purified components and spectrophotometric analyses, we showed a novel function of DAD: DAD-mediation of P. akamusi component V metHb reduction with using D-alanine as an electron donor. P. akamusi larvae possess this D-alanine-DAD metHb reduction system in addition to a previously discovered NADH-NADH-cytochrome b5 reductase system. This is the first report of the presence of DAD in a multicellular organism. The molecular mass of DAD was estimated to be 45 kDa. The optimal pH and temperature of the enzyme were 7.4 and 20 °C, respectively, and the optimal substrate was D-alanine. The enzyme activity was inhibited by benzoate and sulfhydryl-binding reagents. PMID:25896287

  14. Structural Basis for Inactivation of the Human Pyruvate Dehydrogenase Complex by Phosphorylation: Role of Disordered Phosphorylation Loops

    SciTech Connect

    Kato, Masato; Wynn, R. Max; Chuang, Jacinta L.; Tso, Shih-Chia; Machius, Mischa; Li, Jun; Chuang, David T.

    2009-09-11

    We report the crystal structures of the phosporylated pyruvate dehydrogenase (E1p) component of the human pyruvate dehydrogenase complex (PDC). The complete phosphorylation at Ser264-{alpha} (site 1) of a variant E1p protein was achieved using robust pyruvate dehydrogenase kinase 4 free of the PDC core. We show that unlike its unmodified counterpart, the presence of a phosphoryl group at Ser264-{alpha} prevents the cofactor thiamine diphosphate-induced ordering of the two loops carrying the three phosphorylation sites. The disordering of these phosphorylation loops is caused by a previously unrecognized steric clash between the phosphoryl group at site 1 and a nearby Ser266-{alpha}, which nullifies a hydrogen-bonding network essential for maintaining the loop conformations. The disordered phosphorylation loops impede the binding of lipoyl domains of the PDC core to E1p, negating the reductive acetylation step. This results in the disruption of the substrate channeling in the PDC, leading to the inactivation of this catalytic machine.

  15. Structure of the pyruvate dehydrogenase multienzyme complex E1 component from Escherichia coli at 1.85 A resolution.

    PubMed

    Arjunan, Palaniappa; Nemeria, Natalia; Brunskill, Andrew; Chandrasekhar, Krishnamoorthy; Sax, Martin; Yan, Yan; Jordan, Frank; Guest, John R; Furey, William

    2002-04-23

    The crystal structure of the recombinant thiamin diphosphate-dependent E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined at a resolution of 1.85 A. The E. coli PDHc E1 component E1p is a homodimeric enzyme and crystallizes with an intact dimer in an asymmetric unit. Each E1p subunit consists of three domains: N-terminal, middle, and C-terminal, with all having alpha/beta folds. The functional dimer contains two catalytic centers located at the interface between subunits. The ThDP cofactors are bound in the "V" conformation in clefts between the two subunits (binding involves the N-terminal and middle domains), and there is a common ThDP binding fold. The cofactors are completely buried, as only the C2 atoms are accessible from solution through the active site clefts. Significant structural differences are observed between individual domains of E1p relative to heterotetrameric multienzyme complex E1 components operating on branched chain substrates. These differences may be responsible for reported alternative E1p binding modes to E2 components within the respective complexes. This paper represents the first structural example of a functional pyruvate dehydrogenase E1p component from any species. It also provides the first representative example for the entire family of homodimeric (alpha2) E1 multienzyme complex components, and should serve as a model for this class of enzymes. PMID:11955070

  16. Crystal structures of complexes of NAD+-dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101 with formate

    NASA Astrophysics Data System (ADS)

    Filippova, E. V.; Polyakov, K. M.; Tikhonova, T. V.; Stekhanova, T. N.; Boiko, K. M.; Sadykhov, I. G.; Tishkov, V. I.; Popov, V. O.; Labru, N.

    2006-07-01

    Formate dehydrogenase (FDH) from the methylotrophic bacterium Pseudomonas sp. 101 catalyzes oxidation of formate to NI2 with the coupled reduction of nicotinamide adenine dinucleotide (NAD+). The three-dimensional structures of the apo form (the free enzyme) and the holo form (the ternary FDH-NAD+-azide complex) of FDH have been established earlier. In the present study, the structures of FDH complexes with formate are solved at 2.19 and 2.28 Å resolution by the molecular replacement method and refined to the R factors of 22.3 and 20.5%, respectively. Both crystal structures contain four protein molecules per asymmetric unit. These molecules form two dimers identical to the dimer of the apo form of FDH. Two possible formatebinding sites are found in the active site of the FDH structure. In the complexes the sulfur atom of residue Cys354 exists in the oxidized state.

  17. Crystal structures of complexes of NAD{sup +}-dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101 with formate

    SciTech Connect

    Filippova, E. V. Polyakov, K. M.; Tikhonova, T. V.; Stekhanova, T. N.; Boiko, K. M.; Sadykhov, I. G.; Tishkov, V. I.; Popov, V. O.; Labru, N.

    2006-07-15

    Formate dehydrogenase (FDH) from the methylotrophic bacterium Pseudomonas sp. 101 catalyzes oxidation of formate to NI{sub 2} with the coupled reduction of nicotinamide adenine dinucleotide (NAD{sup +}). The three-dimensional structures of the apo form (the free enzyme) and the holo form (the ternary FDH-NAD{sup +}-azide complex) of FDH have been established earlier. In the present study, the structures of FDH complexes with formate are solved at 2.19 and 2.28 A resolution by the molecular replacement method and refined to the R factors of 22.3 and 20.5%, respectively. Both crystal structures contain four protein molecules per asymmetric unit. These molecules form two dimers identical to the dimer of the apo form of FDH. Two possible formatebinding sites are found in the active site of the FDH structure. In the complexes the sulfur atom of residue Cys354 exists in the oxidized state.

  18. Characterization of the major dehydrogenase related to d-lactic acid synthesis in Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293.

    PubMed

    Li, Ling; Eom, Hyun-Ju; Park, Jung-Mi; Seo, Eunyoung; Ahn, Ji Eun; Kim, Tae-Jip; Kim, Jeong Hwan; Han, Nam Soo

    2012-10-10

    Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 is a lactic acid bacterium that converts pyruvate mainly to d-(-)-lactic acid by using d-(-)-lactate dehydrogenase (ldhD). The aim of this study was to identify the gene responsible for d-lactic acid formation in this organism and to characterize the enzyme to facilitate the production of optically pure d-lactic acid. A genomic analysis of L. mesenteroides ATCC 8293 revealed that 7 genes encode lactate-related dehydrogenase. According to transcriptomic, proteomic, and phylogenetic analyses, LEUM_1756 was the major gene responsible for the production of d-lactic acid. The LEUM_1756 gene, of 996bp and encoding 332 amino acids (36.5kDa), was cloned and overexpressed in Escherichia coli BL21(DE3) Star from an inducible pET-21a(+) vector. The enzyme was purified by Ni-NTA column chromatography and showed a specific activity of 4450U/mg, significantly higher than those of other previously reported ldhDs. The gel permeation chromatography analysis showed that the purified enzyme exists as tetramers in solution and this was the first report among lactic acid bacteria. The pH and temperature optima were pH 8.0 and 30°C, respectively, for the pyruvate reduction reaction, and pH 11.0 and 20°C, respectively, for the lactate oxidation reaction. The K(m) kinetic parameters for pyruvate and lactate were 0.58mM and 260mM, respectively. In addition, the k(cat) values for pyruvate and lactate were 2900s(-1) and 2280s(-1), respectively. The enzyme was not inhibited by Ca(2+), Co(2+), Cu(2+), Mg(2+), Mn(2+), Na(+), or urea, but was inhibited by 1mM Zn(2+) and 1mM SDS.

  19. Isolation of a multi-enzyme complex of fatty acid oxidation from Escherichia coli.

    PubMed Central

    Binstock, J F; Pramanik, A; Schulz, H

    1977-01-01

    A multi-enzyme complex of fatty acid oxidation has been isolated from E. coli B cells and has been purified to near homogeneity by a simple two-step procedure. The complex exhibits thiolase (EC 2.3.1.9), enoyl-CoA hydratase (EC 4.2.1.17), and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activities towards short-, medium-, and long-chain substrates. The complex has been estimated to have a molecular weight of approximately 300,000 and is apparently composed of two types of subunits with molecular weights of 78,000 and 42,000. Images PMID:322129

  20. Pistacia lentiscus Oleoresin: Virtual Screening and Identification of Masticadienonic and Isomasticadienonic Acids as Inhibitors of 11β-Hydroxysteroid Dehydrogenase 1.

    PubMed

    Vuorinen, Anna; Seibert, Julia; Papageorgiou, Vassilios P; Rollinger, Judith M; Odermatt, Alex; Schuster, Daniela; Assimopoulou, Andreana N

    2015-04-01

    In traditional medicine, the oleoresinous gum of Pistacia lentiscus var. chia, so-called mastic gum, has been used to treat multiple conditions such as coughs, sore throats, eczema, dyslipidemia, and diabetes. Mastic gum is rich in triterpenes, which have been postulated to exert antidiabetic effects and improve lipid metabolism. In fact, there is evidence of oleanonic acid, a constituent of mastic gum, acting as a peroxisome proliferator-activated receptor γ agonist, and mastic gum being antidiabetic in mice in vivo. Despite these findings, the exact antidiabetic mechanism of mastic gum remains unknown. Glucocorticoids play a key role in regulating glucose and fatty acid metabolism, and inhibition of 11β-hydroxysteroid dehydrogenase 1 that converts inactive cortisone to active cortisol has been proposed as a promising approach to combat metabolic disturbances including diabetes. In this study, a pharmacophore-based virtual screening was applied to filter a natural product database for possible 11β-hydroxysteroid dehydrogenase 1 inhibitors. The hit list analysis was especially focused on the triterpenoids present in Pistacia species. Multiple triterpenoids, such as masticadienonic acid and isomasticadienonic acid, main constituents of mastic gum, were identified. Indeed, masticadienonic acid and isomasticadienonic acid selectively inhibited 11β-hydroxysteroid dehydrogenase 1 over 11β-hydroxysteroid dehydrogenase 2 at low micromolar concentrations. These findings suggest that inhibition of 11β-hydroxysteroid dehydrogenase 1 contributes to the antidiabetic activity of mastic gum.

  1. Metabolic fate of unsaturated glucuronic/iduronic acids from glycosaminoglycans: molecular identification and structure determination of streptococcal isomerase and dehydrogenase.

    PubMed

    Maruyama, Yukie; Oiki, Sayoko; Takase, Ryuichi; Mikami, Bunzo; Murata, Kousaku; Hashimoto, Wataru

    2015-03-01

    Glycosaminoglycans in mammalian extracellular matrices are degraded to their constituents, unsaturated uronic (glucuronic/iduronic) acids and amino sugars, through successive reactions of bacterial polysaccharide lyase and unsaturated glucuronyl hydrolase. Genes coding for glycosaminoglycan-acting lyase, unsaturated glucuronyl hydrolase, and the phosphotransferase system are assembled into a cluster in the genome of pathogenic bacteria, such as streptococci and clostridia. Here, we studied the streptococcal metabolic pathway of unsaturated uronic acids and the structure/function relationship of its relevant isomerase and dehydrogenase. Two proteins (gbs1892 and gbs1891) of Streptococcus agalactiae strain NEM316 were overexpressed in Escherichia coli, purified, and characterized. 4-Deoxy-l-threo-5-hexosulose-uronate (Dhu) nonenzymatically generated from unsaturated uronic acids was converted to 2-keto-3-deoxy-d-gluconate via 3-deoxy-d-glycero-2,5-hexodiulosonate through successive reactions of gbs1892 isomerase (DhuI) and gbs1891 NADH-dependent reductase/dehydrogenase (DhuD). DhuI and DhuD enzymatically corresponded to 4-deoxy-l-threo-5-hexosulose-uronate ketol-isomerase (KduI) and 2-keto-3-deoxy-d-gluconate dehydrogenase (KduD), respectively, involved in pectin metabolism, although no or low sequence identity was observed between DhuI and KduI or between DhuD and KduD, respectively. Genes for DhuI and DhuD were found to be included in the streptococcal genetic cluster, whereas KduI and KduD are encoded in clostridia. Tertiary and quaternary structures of DhuI and DhuD were determined by x-ray crystallography. Distinct from KduI β-barrels, DhuI adopts an α/β/α-barrel structure as a basic scaffold similar to that of ribose 5-phosphate isomerase. The structure of DhuD is unable to accommodate the substrate/cofactor, suggesting that conformational changes are essential to trigger enzyme catalysis. This is the first report on the bacterial metabolism of

  2. Creation of a Broad-Range and Highly Stereoselective d-Amino Acid Dehydrogenase for the One-Step Synthesis of d-Amino Acids

    PubMed Central

    Vedha-Peters, Kavitha; Gunawardana, Manjula; Rozzell, J. David; Novick, Scott J.

    2008-01-01

    Using both rational and random mutagenesis, we have created the first known broad substrate range, nicotinamide cofactor dependent, and highly stereoselective d-amino acid dehydrogenase. This new enzyme is capable of producing d-amino acids via the reductive amination of the corresponding 2-keto acid with ammonia. This biocatalyst was the result of three rounds of mutagenesis and screening performed on the enzyme meso-diaminopimelate d-dehydrogenase. The first round targeted the active site of the wild-type enzyme and produced mutants that were no longer strictly dependent on the native substrate. The second and third rounds produced mutants that had an increased substrate range including straight- and branched-aliphatic amino acids and aromatic amino acids. The very high selectivity towards the d-enantiomer (95 to > 99% e.e) was shown to be preserved even after the addition of the five mutations found in the three rounds of mutagenesis and screening. This new enzyme could complement and improve upon current methods for d-amino acid synthesis. PMID:16910688

  3. Human 2-oxoglutarate dehydrogenase complex E1 component forms a thiamin-derived radical by aerobic oxidation of the enamine intermediate.

    PubMed

    Nemeria, Natalia S; Ambrus, Attila; Patel, Hetalben; Gerfen, Gary; Adam-Vizi, Vera; Tretter, Laszlo; Zhou, Jieyu; Wang, Junjie; Jordan, Frank

    2014-10-24

    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.

  4. Two shikimate dehydrogenases, VvSDH3 and VvSDH4, are involved in gallic acid biosynthesis in grapevine.

    PubMed

    Bontpart, Thibaut; Marlin, Thérèse; Vialet, Sandrine; Guiraud, Jean-Luc; Pinasseau, Lucie; Meudec, Emmanuelle; Sommerer, Nicolas; Cheynier, Véronique; Terrier, Nancy

    2016-05-01

    In plants, the shikimate pathway provides aromatic amino acids that are used to generate numerous secondary metabolites, including phenolic compounds. In this pathway, shikimate dehydrogenases (SDH) 'classically' catalyse the reversible dehydrogenation of 3-dehydroshikimate to shikimate. The capacity of SDH to produce gallic acid from shikimate pathway metabolites has not been studied in depth. In grapevine berries, gallic acid mainly accumulates as galloylated flavan-3-ols. The four grapevine SDH proteins have been produced in Escherichia coli In vitro, VvSDH1 exhibited the highest 'classical' SDH activity. Two genes, VvSDH3 and VvSDH4, mainly expressed in immature berry tissues in which galloylated flavan-3-ols are accumulated, encoded enzymes with lower 'classical' activity but were able to produce gallic acid in vitro The over-expression of VvSDH3 in hairy-roots increased the content of aromatic amino acids and hydroxycinnamates, but had little or no effect on molecules more distant from the shikimate pathway (stilbenoids and flavan-3-ols). In parallel, the contents of gallic acid, β-glucogallin, and galloylated flavan-3-ols were increased, attesting to the influence of this gene on gallic acid metabolism. Phylogenetic analysis from dicotyledon SDHs opens the way for the examination of genes from other plants which accumulate gallic acid-based metabolites. PMID:27241494

  5. Two shikimate dehydrogenases, VvSDH3 and VvSDH4, are involved in gallic acid biosynthesis in grapevine.

    PubMed

    Bontpart, Thibaut; Marlin, Thérèse; Vialet, Sandrine; Guiraud, Jean-Luc; Pinasseau, Lucie; Meudec, Emmanuelle; Sommerer, Nicolas; Cheynier, Véronique; Terrier, Nancy

    2016-05-01

    In plants, the shikimate pathway provides aromatic amino acids that are used to generate numerous secondary metabolites, including phenolic compounds. In this pathway, shikimate dehydrogenases (SDH) 'classically' catalyse the reversible dehydrogenation of 3-dehydroshikimate to shikimate. The capacity of SDH to produce gallic acid from shikimate pathway metabolites has not been studied in depth. In grapevine berries, gallic acid mainly accumulates as galloylated flavan-3-ols. The four grapevine SDH proteins have been produced in Escherichia coli In vitro, VvSDH1 exhibited the highest 'classical' SDH activity. Two genes, VvSDH3 and VvSDH4, mainly expressed in immature berry tissues in which galloylated flavan-3-ols are accumulated, encoded enzymes with lower 'classical' activity but were able to produce gallic acid in vitro The over-expression of VvSDH3 in hairy-roots increased the content of aromatic amino acids and hydroxycinnamates, but had little or no effect on molecules more distant from the shikimate pathway (stilbenoids and flavan-3-ols). In parallel, the contents of gallic acid, β-glucogallin, and galloylated flavan-3-ols were increased, attesting to the influence of this gene on gallic acid metabolism. Phylogenetic analysis from dicotyledon SDHs opens the way for the examination of genes from other plants which accumulate gallic acid-based metabolites.

  6. Two shikimate dehydrogenases, VvSDH3 and VvSDH4, are involved in gallic acid biosynthesis in grapevine

    PubMed Central

    Bontpart, Thibaut; Marlin, Thérèse; Vialet, Sandrine; Guiraud, Jean-Luc; Pinasseau, Lucie; Meudec, Emmanuelle; Sommerer, Nicolas; Cheynier, Véronique; Terrier, Nancy

    2016-01-01

    In plants, the shikimate pathway provides aromatic amino acids that are used to generate numerous secondary metabolites, including phenolic compounds. In this pathway, shikimate dehydrogenases (SDH) ‘classically’ catalyse the reversible dehydrogenation of 3-dehydroshikimate to shikimate. The capacity of SDH to produce gallic acid from shikimate pathway metabolites has not been studied in depth. In grapevine berries, gallic acid mainly accumulates as galloylated flavan-3-ols. The four grapevine SDH proteins have been produced in Escherichia coli. In vitro, VvSDH1 exhibited the highest ‘classical’ SDH activity. Two genes, VvSDH3 and VvSDH4, mainly expressed in immature berry tissues in which galloylated flavan-3-ols are accumulated, encoded enzymes with lower ‘classical’ activity but were able to produce gallic acid in vitro. The over-expression of VvSDH3 in hairy-roots increased the content of aromatic amino acids and hydroxycinnamates, but had little or no effect on molecules more distant from the shikimate pathway (stilbenoids and flavan-3-ols). In parallel, the contents of gallic acid, β-glucogallin, and galloylated flavan-3-ols were increased, attesting to the influence of this gene on gallic acid metabolism. Phylogenetic analysis from dicotyledon SDHs opens the way for the examination of genes from other plants which accumulate gallic acid-based metabolites. PMID:27241494

  7. Antisense Inhibition of the 2-Oxoglutarate Dehydrogenase Complex in Tomato Demonstrates Its Importance for Plant Respiration and during Leaf Senescence and Fruit Maturation[W][OA

    PubMed Central

    Araújo, Wagner L.; Tohge, Takayuki; Osorio, Sonia; Lohse, Marc; Balbo, Ilse; Krahnert, Ina; Sienkiewicz-Porzucek, Agata; Usadel, Björn; Nunes-Nesi, Adriano; Fernie, Alisdair R.

    2012-01-01

    Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the gene encoding the E1 subunit of the 2-oxoglutarate dehydrogenase complex in the antisense orientation and exhibiting substantial reductions in the activity of this enzyme exhibit a considerably reduced rate of respiration. They were, however, characterized by largely unaltered photosynthetic rates and fruit yields but restricted leaf, stem, and root growth. These lines displayed markedly altered metabolic profiles, including changes in tricarboxylic acid cycle intermediates and in the majority of the amino acids but unaltered pyridine nucleotide content both in leaves and during the progression of fruit ripening. Moreover, they displayed a generally accelerated development exhibiting early flowering, accelerated fruit ripening, and a markedly earlier onset of leaf senescence. In addition, transcript and selective hormone profiling of gibberellins and abscisic acid revealed changes only in the former coupled to changes in transcripts encoding enzymes of gibberellin biosynthesis. The data obtained are discussed in the context of the importance of this enzyme in both photosynthetic and respiratory metabolism as well as in programs of plant development connected to carbon–nitrogen interactions. PMID:22751214

  8. 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.

  9. Uric acid substantially enhances the free radical-induced inactivation of alcohol dehydrogenase.

    PubMed

    Kittridge, K J; Willson, R L

    1984-05-01

    Lactate dehydrogenase (LDH) and yeast alcohol dehydrogenase ( YADH ) are inactivated when attacked by hydroxy free radicals (OH). Organic molecules with a high rate constant of reaction with OH such as ascorbate or urate can compete with the enzymes for these strongly oxidising radicals. However, although 10(-3)M ascorbate can substantially protect both LDH and YADH from OH attack, in the presence of 10(-3)M urate only LDH is protected. In the case of YADH an even greater degree of inactivation than with OH occurs. The extent of inactivation is considerably reduced when oxygen is absent, in agreement with a urate peroxy radical perhaps being partly responsible for the increased inactivation of the enzyme.

  10. 7alpha- and 12alpha-Hydroxysteroid dehydrogenases from Acinetobacter calcoaceticus lwoffii: a new integrated chemo-enzymatic route to ursodeoxycholic acid.

    PubMed

    Giovannini, Pier Paolo; Grandini, Alessandro; Perrone, Daniela; Pedrini, Paola; Fantin, Giancarlo; Fogagnolo, Marco

    2008-12-22

    We report the very efficient biotransformation of cholic acid to 7-keto- and 7,12-diketocholic acids with Acinetobacter calcoaceticus lwoffii. The enzymes responsible of the biotransformation (i.e. 7alpha- and 12alpha-hydroxysteroid dehydrogenases) are partially purified and employed in a new chemo-enzymatic synthesis of ursodeoxycholic acid starting from cholic acid. The first step is the 12alpha-HSDH-mediated total oxidation of sodium cholate followed by the Wolf-Kishner reduction of the carbonyl group to chenodeoxycholic acid. This acid is then quantitatively oxidized with 7alpha-HSDH to 7-ketochenodeoxycholic acid, that was chemically reduced to ursodeoxycholic acid (70% overall yield).

  11. Nuclear magnetic resonance and molecular modeling study on mycophenolic acid: implications for binding to inosine monophosphate dehydrogenase.

    PubMed

    Makara, G M; Keserû, G M; Kajtár-Peredy, M; Anderson, W K

    1996-03-15

    The conformation of the sodium salt of mycophenolic acid (MPA), a potent inhibitor of inosine monophosphate dehydrogenase (IMPD), derived from 1D DIFNOE and 2D ROESY experiments in water and molecular dynamics (MD) is described. The hexenoic acid side chain conformation consistent with the NMR data was similar to that seen in the X-ray structure of MPA. The solution conformation was applied in a molecular modeling study in order to explore the potential features of enzyme binding. Our results, based on striking similarities in molecular volume and electrostatic isopotential between MPA and cofactor NAD+, lead to the suggestion that MPA is capable of binding to the nicotinamide site of IMPD and mimicking the NAD+ inverse regulation of the enzyme. In addition, our proposed model is in good agreement with the observed high affinity of the dinucleotide analogues thiazole- and selenazole-4-carboxamide adenine dinucleotide to IMPD.

  12. Hydroperoxidic inhibitor of horse liver alcohol dehydrogenase activity, tightly bound to the enzyme-NAD+ complex, characteristically degrades the coenzyme.

    PubMed

    Skurský, L; Rezác, M; Khan, A N; Zídek, L; Rocek, J

    1992-01-01

    The strong inhibition of horse liver alcohol dehydrogenase (HLAD) by p-methylbenzyl hydroperoxide (XyHP) is only transient, XyHP behaves also as a pseudo-substrate of the enzyme and in the presence of NAD+, is degraded by HLAD to (as yet unidentified) non-inhibiting products while the NAD+ is converted to a derivative similar to the "NADX", originally observed in an analogous reaction of HLAD with hydrogen peroxide. The apparent KM for XyHP is approximately 10(4) times smaller than that for H2O2. The catalytic constant kcat for HLAD degradation of XyHP is two orders of magnitude less than that for ethanol dehydrogenation. XyHP inhibits both directions of the alcohol-aldehyde interconversion with equal potency. The first step of the inhibition mechanism is a tight binding of XyHP to the binary HLAD-NAD+ complex. PMID:1284958

  13. Dye-linked D-amino acid dehydrogenase from the thermophilic bacterium Rhodothermus marinus JCM9785: characteristics and role in trans-4-hydroxy-L-proline catabolism.

    PubMed

    Satomura, Takenori; Ishikura, Masaru; Koyanagi, Takashi; Sakuraba, Haruhiko; Ohshima, Toshihisa; Suye, Shin-ichiro

    2015-05-01

    A gene from the thermophilic Gram-negative bacterium Rhodothermus marinus JCM9785, encoding a dye-linked D-amino acid dehydrogenase homologue, was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme was a highly thermostable dye-linked D-amino acid dehydrogenase that retained more than 80% of its activity after incubation for 10 min at up to 70 °C. When enzyme-catalyzed dehydrogenation of several D-amino acids was carried out using 2,6-dichloroindophenol as the electron acceptor, D-phenylalanine was the most preferable substrate among the D-amino acids tested. Immediately upstream of the dye-linked D-amino acid dehydrogenase gene (dadh) was a gene encoding a 4-hydroxyproline 2-epimerase homologue (hypE). That gene was successfully expressed in E. coli, and the gene product exhibited strong 4-hydroxyproline 2-epimerase activity. Reverse transcription PCR and quantitative real-time PCR showed that the six genes containing the dadh and hypE genes were arranged in an operon and were required for catabolism of trans-4-hydroxy-L-proline in R. marinus. This is the first description of a dye-linked D-amino acid dehydrogenase (Dye-DADH) with broad substrate specificity involved in trans-4-hydroxy-L-proline catabolism. PMID:25472442

  14. [Stability of glucose 6-phosphate dehydrogenase complexed with its substrate and/or cofactor in aqueous and micellar environment].

    PubMed

    Puchkaev, A V; Vlasov, A P; Metelitsa, D I

    2002-01-01

    Inactivation of glucose 6-phosphate dehydrogenase (G6PDH) complexed with its substrate, glucose 6-phosphate (GP), and/or cofactor, NADP+, has been studied within the range 20-40 degrees C in three media: (a) 0.04 M NaOH-glycine buffer (pH 9.1); (b) Aerosol OT (AOT) reversed micelles in octane; and (c) Triton X-100 micelles in octane supplemented with 10% hexanol. The enzyme inactivation was characterized quantitatively by first order rate constants, kin (s-1). In the case of G6PDH-NADP+ complexes, the values of kin were independent of the initial concentrations of G6PDH, either in aqueous medium or AOT micelles. The values of kin for the complex G6PDH-GP were inversely related to the initial concentration of the enzyme, in both aqueous and micellar media. When inactivation of both complexes were studied in AOT micelles, minimum values of kin corresponded to the degree of hydration W0 = 16.7; at W0 > 16.7 and W0 < 16.7, kin increased. Within the range 20-40 degrees C, the values of kin measured for both complexes in aqueous medium were significantly lower than those measured in AOT micelles. Temperature dependences of kin were characterized by inflections in Arrhenius plots, which corresponded, depending on the medium, to certain temperatures from 33.6 degrees C to 40 degrees C. In all media studied, NADP+ complexes of the enzyme exhibited higher stability than their GP counterparts. The parameters of G6PDH and G6PDH-NADP+ melting, measured by differential scanning microcalorimetry (maximum temperature and half-width of the transition, enthalpy of denaturation, and van't Hoff enthalpy), provided unequivocal evidence of the higher stability of the complex as compared to that of the enzyme. In addition, this approach demonstrated that G6PDH undergoes destabilization in AOT micelles.

  15. Crystal Structures of CO and NO Adducts of MauG in Complex with Pre-Methylamine Dehydrogenase: Implications for the Mechanism of Dioxygen Activation

    SciTech Connect

    Yukl, Erik T.; Goblirsch, Brandon R.; Davidson, Victor L.; Wilmot, Carrie M.

    2011-09-28

    MauG is a diheme enzyme responsible for the post-translational formation of the catalytic tryptophan tryptophylquinone (TTQ) cofactor in methylamine dehydrogenase (MADH). MauG can utilize hydrogen peroxide, or molecular oxygen and reducing equivalents, to complete this reaction via a catalytic bis-Fe(IV) intermediate. Crystal structures of diferrous, Fe(II)-CO, and Fe(II)-NO forms of MauG in complex with its preMADH substrate have been determined and compared to one another as well as to the structure of the resting diferric MauG-preMADH complex. CO and NO each bind exclusively to the 5-coordinate high-spin heme with no change in ligation of the 6-coordinate low-spin heme. These structures reveal likely roles for amino acid residues in the distal pocket of the high-spin heme in oxygen binding and activation. Glu113 is implicated in the protonation of heme-bound diatomic oxygen intermediates in promoting cleavage of the O-O bond. Pro107 is shown to change conformation on the binding of each ligand and may play a steric role in oxygen activation by positioning the distal oxygen near Glu113. Gln103 is in a position to provide a hydrogen bond to the Fe(IV){double_bond}O moiety that may account for the unusual stability of this species in MauG.

  16. HLA DRB4 0101-restricted immunodominant T cell autoepitope of pyruvate dehydrogenase complex in primary biliary cirrhosis: evidence of molecular mimicry in human autoimmune diseases

    PubMed Central

    1995-01-01

    We established six T cell clones specific for pyruvate dehydrogenase complex (PDC)-E2 peptides from four different patients with primary biliary cirrhosis using 33 different peptides of 17-20 amino acid residues corresponding to human PDC-E2 as stimulating antigens. The minimal T cell epitopes of these six T cell clones were all mapped to the same region of the PDC-E2 peptide 163-176 (GDLLAEIETDKATI), which corresponds to the inner lipoyl domain of PDC-E2. The HLA restriction molecules for this epitope were all identified as HLA DRB4 0101. The common essential amino acids of this epitope for these T cell clones were E, D, and K at positions 170, 172, and 173, respectively; other crucial amino acids for this epitope differed in each T cell clone. In addition, the alanine-substituted peptides at positions 170 and 173, but not 172, inhibited the proliferation of all T cell clones induced by the original peptide of human PDC-E2 163-176, indicating that amino acid D at position 172 is a critical MHC-binding site for all T cell clones tested. Interestingly, all T cell clones reacted to PDC-E2 peptide 36-49 (GDLIAEVETDKATV), which corresponds to the outer lipoyl domain of human PDC-E2. Furthermore, one T cell clone cross-reacted with exogenous antigens such as Escherichia coli PDC-E2 peptide 31- 44/134-147/235-248 (EQSLITVEGDKASM), which has an EXDK sequence. This is a definite demonstration of the presence of molecular mimicry at the T cell clonal level in human autoimmune diseases. It is also considered possible to design peptide-specific immunotherapy based on the findings of T cell autoepitopes in primary biliary cirrhosis. PMID:7536796

  17. Complexation and molecular modeling studies of europium(III)-gallic acid-amino acid complexes.

    PubMed

    Taha, Mohamed; Khan, Imran; Coutinho, João A P

    2016-04-01

    With many metal-based drugs extensively used today in the treatment of cancer, attention has focused on the development of new coordination compounds with antitumor activity with europium(III) complexes recently introduced as novel anticancer drugs. The aim of this work is to design new Eu(III) complexes with gallic acid, an antioxida'nt phenolic compound. Gallic acid was chosen because it shows anticancer activity without harming health cells. As antioxidant, it helps to protect human cells against oxidative damage that implicated in DNA damage, cancer, and accelerated cell aging. In this work, the formation of binary and ternary complexes of Eu(III) with gallic acid, primary ligand, and amino acids alanine, leucine, isoleucine, and tryptophan was studied by glass electrode potentiometry in aqueous solution containing 0.1M NaNO3 at (298.2 ± 0.1) K. Their overall stability constants were evaluated and the concentration distributions of the complex species in solution were calculated. The protonation constants of gallic acid and amino acids were also determined at our experimental conditions and compared with those predicted by using conductor-like screening model for realistic solvation (COSMO-RS) model. The geometries of Eu(III)-gallic acid complexes were characterized by the density functional theory (DFT). The spectroscopic UV-visible and photoluminescence measurements are carried out to confirm the formation of Eu(III)-gallic acid complexes in aqueous solutions.

  18. Teneligliptin Decreases Uric Acid Levels by Reducing Xanthine Dehydrogenase Expression in White Adipose Tissue of Male Wistar Rats

    PubMed Central

    2016-01-01

    We investigated the effects of teneligliptin on uric acid metabolism in male Wistar rats and 3T3-L1 adipocytes. The rats were fed with a normal chow diet (NCD) or a 60% high-fat diet (HFD) with or without teneligliptin for 4 weeks. The plasma uric acid level was not significantly different between the control and teneligliptin groups under the NCD condition. However, the plasma uric acid level was significantly decreased in the HFD-fed teneligliptin treated rats compared to the HFD-fed control rats. The expression levels of xanthine dehydrogenase (Xdh) mRNA in liver and epididymal adipose tissue of NCD-fed rats were not altered by teneligliptin treatment. On the other hand, Xdh expression was reduced significantly in the epididymal adipose tissue of the HFD-fed teneligliptin treated rats compared with that of HFD-fed control rats, whereas Xdh expression in liver did not change significantly in either group. Furthermore, teneligliptin significantly decreased Xdh expression in 3T3-L1 adipocytes. DPP-4 treatment significantly increased Xdh expression in 3T3-L1 adipocytes. With DPP-4 pretreatment, teneligliptin significantly decreased Xdh mRNA expression compared to the DPP-4-treated 3T3-L1 adipocytes. In conclusion, our studies suggest that teneligliptin reduces uric acid levels by suppressing Xdh expression in epididymal adipose tissue of obese subjects.

  19. Teneligliptin Decreases Uric Acid Levels by Reducing Xanthine Dehydrogenase Expression in White Adipose Tissue of Male Wistar Rats

    PubMed Central

    2016-01-01

    We investigated the effects of teneligliptin on uric acid metabolism in male Wistar rats and 3T3-L1 adipocytes. The rats were fed with a normal chow diet (NCD) or a 60% high-fat diet (HFD) with or without teneligliptin for 4 weeks. The plasma uric acid level was not significantly different between the control and teneligliptin groups under the NCD condition. However, the plasma uric acid level was significantly decreased in the HFD-fed teneligliptin treated rats compared to the HFD-fed control rats. The expression levels of xanthine dehydrogenase (Xdh) mRNA in liver and epididymal adipose tissue of NCD-fed rats were not altered by teneligliptin treatment. On the other hand, Xdh expression was reduced significantly in the epididymal adipose tissue of the HFD-fed teneligliptin treated rats compared with that of HFD-fed control rats, whereas Xdh expression in liver did not change significantly in either group. Furthermore, teneligliptin significantly decreased Xdh expression in 3T3-L1 adipocytes. DPP-4 treatment significantly increased Xdh expression in 3T3-L1 adipocytes. With DPP-4 pretreatment, teneligliptin significantly decreased Xdh mRNA expression compared to the DPP-4-treated 3T3-L1 adipocytes. In conclusion, our studies suggest that teneligliptin reduces uric acid levels by suppressing Xdh expression in epididymal adipose tissue of obese subjects. PMID:27652270

  20. Semi-Rational Design of Geobacillus stearothermophilus L-Lactate Dehydrogenase to Access Various Chiral α-Hydroxy Acids.

    PubMed

    Aslan, Aşkın Sevinç; Birmingham, William R; Karagüler, Nevin Gül; Turner, Nicholas J; Binay, Barış

    2016-06-01

    Chiral α-hydroxy acids (AHAs) are rapidly becoming important synthetic building blocks, in particular for the production of pharmaceuticals and other fine chemicals. Chiral compounds of a variety of functionalities are now often derived using enzymes, and L-lactate dehydrogenase from the thermophilic organism Geobacillus stearothermophilus (bsLDH) has the potential to be employed for the industrial synthesis of chiral α-hydroxy acids. Despite the thorough characterization of this enzyme, generation of variants with high activity on non-natural substrates has remained difficult and therefore limits the use of bsLDH in industry. Here, we present the engineering of bsLDH using semi-rational design as a method of focusing screening in a small and smart library for novel biocatalysts. In this study, six mutant libraries were designed in an effort to expand the substrate range of bsLDH. The eight variants identified as having enhanced activity toward the selected α-keto acids belonged to the same library, which targeted two positions simultaneously. These new variants now may be useful biocatalysts for chiral synthesis of α-hydroxy acids.

  1. Teneligliptin Decreases Uric Acid Levels by Reducing Xanthine Dehydrogenase Expression in White Adipose Tissue of Male Wistar Rats.

    PubMed

    Moriya, Chihiro; Satoh, Hiroaki

    2016-01-01

    We investigated the effects of teneligliptin on uric acid metabolism in male Wistar rats and 3T3-L1 adipocytes. The rats were fed with a normal chow diet (NCD) or a 60% high-fat diet (HFD) with or without teneligliptin for 4 weeks. The plasma uric acid level was not significantly different between the control and teneligliptin groups under the NCD condition. However, the plasma uric acid level was significantly decreased in the HFD-fed teneligliptin treated rats compared to the HFD-fed control rats. The expression levels of xanthine dehydrogenase (Xdh) mRNA in liver and epididymal adipose tissue of NCD-fed rats were not altered by teneligliptin treatment. On the other hand, Xdh expression was reduced significantly in the epididymal adipose tissue of the HFD-fed teneligliptin treated rats compared with that of HFD-fed control rats, whereas Xdh expression in liver did not change significantly in either group. Furthermore, teneligliptin significantly decreased Xdh expression in 3T3-L1 adipocytes. DPP-4 treatment significantly increased Xdh expression in 3T3-L1 adipocytes. With DPP-4 pretreatment, teneligliptin significantly decreased Xdh mRNA expression compared to the DPP-4-treated 3T3-L1 adipocytes. In conclusion, our studies suggest that teneligliptin reduces uric acid levels by suppressing Xdh expression in epididymal adipose tissue of obese subjects. PMID:27652270

  2. Semi-Rational Design of Geobacillus stearothermophilus L-Lactate Dehydrogenase to Access Various Chiral α-Hydroxy Acids.

    PubMed

    Aslan, Aşkın Sevinç; Birmingham, William R; Karagüler, Nevin Gül; Turner, Nicholas J; Binay, Barış

    2016-06-01

    Chiral α-hydroxy acids (AHAs) are rapidly becoming important synthetic building blocks, in particular for the production of pharmaceuticals and other fine chemicals. Chiral compounds of a variety of functionalities are now often derived using enzymes, and L-lactate dehydrogenase from the thermophilic organism Geobacillus stearothermophilus (bsLDH) has the potential to be employed for the industrial synthesis of chiral α-hydroxy acids. Despite the thorough characterization of this enzyme, generation of variants with high activity on non-natural substrates has remained difficult and therefore limits the use of bsLDH in industry. Here, we present the engineering of bsLDH using semi-rational design as a method of focusing screening in a small and smart library for novel biocatalysts. In this study, six mutant libraries were designed in an effort to expand the substrate range of bsLDH. The eight variants identified as having enhanced activity toward the selected α-keto acids belonged to the same library, which targeted two positions simultaneously. These new variants now may be useful biocatalysts for chiral synthesis of α-hydroxy acids. PMID:26852025

  3. A new high phenyl lactic acid-yielding Lactobacillus plantarum IMAU10124 and a comparative analysis of lactate dehydrogenase gene.

    PubMed

    Zhang, Xiqing; Zhang, Shuli; Shi, Yan; Shen, Fadi; Wang, Haikuan

    2014-07-01

    Phenyl lactic acid (PLA) has been widely reported as a new natural antimicrobial compound. In this study, 120 Lactobacillus plantarum strains were demonstrated to produce PLA using high-performance liquid chromatography. Lactobacillus plantarum IMAU10124 was screened with a PLA yield of 0.229 g L(-1) . Compared with all previous reports, this is the highest PLA-producing lactic acid bacteria (LAB) when grown in MRS broth without any optimizing conditions. When 3.0 g L(-1) phenyl pyruvic acid (PPA) was added to the medium as substrate, PLA production reached 2.90 g L(-1) , with the highest 96.05% conversion rate. A lowest PLA-yielding L. plantarum IMAU40105 (0.043 g L(-1) ) was also screened. It was shown that the conversion from PPA to PLA by lactic dehydrogenase (LDH) is the key factor in the improvement of PLA production by LAB. Comparing the LDH gene of two strains, four amino acid mutation sites were found in this study in the LDH of L. plantarum IMAU10124.

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

    PubMed

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

    1989-06-01

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

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

    PubMed Central

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

    2003-01-01

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

  6. Increased expression of hepatic pyruvate dehydrogenase kinases 2 and 4 in young and middle-aged Otsuka Long-Evans Tokushima Fatty rats: induction by elevated levels of free fatty acids.

    PubMed

    Bajotto, Gustavo; Murakami, Taro; Nagasaki, Masaru; Qin, Bolin; Matsuo, Yoshiyuki; Maeda, Ken; Ohashi, Masayo; Oshida, Yoshiharu; Sato, Yuzo; Shimomura, Yoshiharu

    2006-03-01

    The activity of the pyruvate dehydrogenase complex (PDC) is regulated by covalent modification of its E1 component, which is catalyzed by specific pyruvate dehydrogenase kinases (PDKs) and phosphatases. In the liver, PDK2 and PDK4 are the most abundant PDK isoforms, which are responsible for inactivation of PDC when glucose availability is scarce in the body. In the present study, regulatory mechanisms of hepatic PDC were examined before and after the onset of type 2 diabetes mellitus in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, using Long-Evans Tokushima Otsuka (LETO) rats as controls. Plasma glucose and insulin concentrations were at normal levels in rats aged 8 weeks, but were significantly higher in OLETF than in LETO rats aged 25 weeks, indicating insulin resistance in OLETF rats. Plasma free fatty acids (FFAs) were 1.6-fold concentrated, and the liver PDC activity was significantly lower in OLETF than in LETO rats at both ages, suggesting suppression of pyruvate oxidative decarboxylation in OLETF rats before and after the onset of diabetes. Pyruvate dehydrogenase kinase activity and abundance of PDK2 and PDK4 proteins, as well as mRNAs, were greater in OLETF rats at both ages. These results suggest that persistently elevated levels of circulating free fatty acid in normal and diabetic OLETF rats play an important role in stimulating PDK2 and PDK4 expression in liver. PMID:16483874

  7. Phosphorescence maxima and triplet state lifetimes of NAD+ and epsilon-NAD+ in ternary complexes with horse liver alcohol dehydrogenase.

    PubMed

    Rousslang, K; Allen, L; Ross, J B

    1989-02-01

    This paper describes the phosphorescence emission and decay times of NAD+ and its fluorescent etheno derivative, epsilon-NAD+, in the pyrazole ternary complex with horse liver alcohol dehydrogenase (ADH). We show that the epsilon-NAD+ triplet state, as well as the tryptophan triplet state, can be utilized to monitor the coenzyme-enzyme interaction. The decays of NAD+ and AMP are single exponential, and the lifetimes are the same within experimental error. The phosphorescence lifetimes, evaluated as single exponentials, are slightly shorter in epsilon-NAD+ than they are in epsilon-AMP. Whereas the decay of epsilon-AMP was adequately fit by a single exponential with a time constant of very close to 0.5 s, it was necessary to fit the decay of epsilon-NAD+ to a double exponential. Ternary complexes with NAD+ excited at 297 nm exhibit decay kinetics nearly identical to those of ADH by itself. On the other hand, when excitation of the epsilon-NAD+ ternary complex is provided at 313 nm, where there is very little absorption by either tryptophan residue, the decay law of the ternary complex is similar to that of epsilon-NAD+ in solution. Our results demonstrate that NAD+ and epsilon-NAD+ quench tryptophan phosphorescence in ADH. Normalizing the phosphorescence intensity to the 0-0 vibronic band assigned to Trp-15 (blue-edge), we calculate a 21% decrease in the phosphorescence associated with Trp-314 at stoichiometric saturation of the coenzyme binding sites with NAD+ in the ternary complex. When the active sites are saturated with epsilon-NAD+, the relative phosphorescence due to Trp-314 decreases by 63%.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. SdhE-dependent formation of a functional Acetobacter pasteurianus succinate dehydrogenase in Gluconobacter oxydans--a first step toward a complete tricarboxylic acid cycle.

    PubMed

    Kiefler, Ines; Bringer, Stephanie; Bott, Michael

    2015-11-01

    The obligatory aerobic α-proteobacterium Gluconobacter oxydans 621H possesses an unusual metabolism in which the majority of the carbohydrate substrates are incompletely oxidized in the periplasm and only a small fraction is metabolized in the cytoplasm. The cytoplasmic oxidation capabilities are limited due to an incomplete tricarboxylic acid (TCA) cycle caused by the lack of succinate dehydrogenase (Sdh) and succinyl-CoA synthetase. As a first step to test the consequences of a functional TCA cycle for growth, metabolism, and bioenergetics of G. oxydans, we attempted to establish a heterologous Sdh in this species. Expression of Acetobacter pasteurianus sdhCDAB in G. oxydans did not yield an active succinate dehydrogenase. Co-expression of a putative sdhE gene from A. pasteurianus, which was assumed to encode an assembly factor for covalent attachment of flavin adenine dinucleotide (FAD) to SdhA, stimulated Sdh activity up to 400-fold to 4.0 ± 0.4 U (mg membrane protein)(‒1). The succinate/oxygen reductase activity of membranes was 0.68 ± 0.04 U (mg membrane protein)(‒1), indicating the formation of functional Sdh complex capable of transferring electrons from succinate to ubiquinone. A. pasteurianus SdhE could be functionally replaced by SdhE from the γ-proteobacterium Serratia sp. According to these results, the accessory protein SdhE was necessary and sufficient for heterologous synthesis of an active A. pasteurianus Sdh in G. oxydans. Studies with the Sdh-positive G. oxydans strain provided evidence for a limited functionality of the TCA cycle despite the absence of succinyl-CoA synthetase.

  9. A role for AMPK in the inhibition of glucose-6-phosphate dehydrogenase by polyunsaturated fatty acids

    SciTech Connect

    Kohan, Alison B.; Talukdar, Indrani; Walsh, Callee M.; Salati, Lisa M.

    2009-10-09

    Both polyunsaturated fatty acids and AMPK promote energy partitioning away from energy consuming processes, such as fatty acid synthesis, towards energy generating processes, such as {beta}-oxidation. In this report, we demonstrate that arachidonic acid activates AMPK in primary rat hepatocytes, and that this effect is p38 MAPK-dependent. Activation of AMPK mimics the inhibition by arachidonic acid of the insulin-mediated induction of G6PD. Similar to intracellular signaling by arachidonic acid, AMPK decreases insulin signal transduction, increasing Ser{sup 307} phosphorylation of IRS-1 and a subsequent decrease in AKT phosphorylation. Overexpression of dominant-negative AMPK abolishes the effect of arachidonic acid on G6PD expression. These data suggest a role for AMPK in the inhibition of G6PD by polyunsaturated fatty acids.

  10. Developmental Defects of Caenorhabditis elegans Lacking Branched-chain α-Ketoacid Dehydrogenase Are Mainly Caused by Monomethyl Branched-chain Fatty Acid Deficiency.

    PubMed

    Jia, Fan; Cui, Mingxue; Than, Minh T; Han, Min

    2016-02-01

    Branched-chain α-ketoacid dehydrogenase (BCKDH) catalyzes the critical step in the branched-chain amino acid (BCAA) catabolic pathway and has been the focus of extensive studies. Mutations in the complex disrupt many fundamental metabolic pathways and cause multiple human diseases including maple syrup urine disease (MSUD), autism, and other related neurological disorders. BCKDH may also be required for the synthesis of monomethyl branched-chain fatty acids (mmBCFAs) from BCAAs. The pathology of MSUD has been attributed mainly to BCAA accumulation, but the role of mmBCFA has not been evaluated. Here we show that disrupting BCKDH in Caenorhabditis elegans causes mmBCFA deficiency, in addition to BCAA accumulation. Worms with deficiency in BCKDH function manifest larval arrest and embryonic lethal phenotypes, and mmBCFA supplementation suppressed both without correcting BCAA levels. The majority of developmental defects caused by BCKDH deficiency may thus be attributed to lacking mmBCFAs in worms. Tissue-specific analysis shows that restoration of BCKDH function in multiple tissues can rescue the defects, but is especially effective in neurons. Taken together, we conclude that mmBCFA deficiency is largely responsible for the developmental defects in the worm and conceivably might also be a critical contributor to the pathology of human MSUD.

  11. Developmental Defects of Caenorhabditis elegans Lacking Branched-chain α-Ketoacid Dehydrogenase Are Mainly Caused by Monomethyl Branched-chain Fatty Acid Deficiency.

    PubMed

    Jia, Fan; Cui, Mingxue; Than, Minh T; Han, Min

    2016-02-01

    Branched-chain α-ketoacid dehydrogenase (BCKDH) catalyzes the critical step in the branched-chain amino acid (BCAA) catabolic pathway and has been the focus of extensive studies. Mutations in the complex disrupt many fundamental metabolic pathways and cause multiple human diseases including maple syrup urine disease (MSUD), autism, and other related neurological disorders. BCKDH may also be required for the synthesis of monomethyl branched-chain fatty acids (mmBCFAs) from BCAAs. The pathology of MSUD has been attributed mainly to BCAA accumulation, but the role of mmBCFA has not been evaluated. Here we show that disrupting BCKDH in Caenorhabditis elegans causes mmBCFA deficiency, in addition to BCAA accumulation. Worms with deficiency in BCKDH function manifest larval arrest and embryonic lethal phenotypes, and mmBCFA supplementation suppressed both without correcting BCAA levels. The majority of developmental defects caused by BCKDH deficiency may thus be attributed to lacking mmBCFAs in worms. Tissue-specific analysis shows that restoration of BCKDH function in multiple tissues can rescue the defects, but is especially effective in neurons. Taken together, we conclude that mmBCFA deficiency is largely responsible for the developmental defects in the worm and conceivably might also be a critical contributor to the pathology of human MSUD. PMID:26683372

  12. A unique enzyme of acetic acid bacteria, PQQ-dependent alcohol dehydrogenase, is also present in Frateuria aurantia.

    PubMed

    Trček, Janja; Matsushita, Kazunobu

    2013-08-01

    A membrane-bound, pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) was purified from Frateuria aurantia LMG 1558(T). Although F. aurantia belongs to a group of γ-Proteobacteria, the characteristics of its PQQ-ADH were similar to the enzyme characteristics of the typical high-acetic acid-resistant bacterium Gluconacetobacter europaeus from the group of α-Proteobacteria. The PQQ-dependent ADH was solubilized from the membranes and purified after anionic, cationic, and affinity chromatography with specific activity of 117 U/mg. The purified enzyme was estimated to be composed of two subunits of ca. 72 and 45 kDa, as judged by SDS-polyacrylamide gel electrophoresis. The purified enzyme had maximum activity at pH 4.5 and showed the highest substrate specificity to ethanol, isoamyl alcohol, 1-butanol, and 1-propanol. The deduced sequences of cloned genes adhA and adhB encoding subunits I and II of PQQ-ADH showed 80 % amino acid (AA) identity to AdhA and 68 % AA identity to AdhB of Ga. europaeus V3 (LMG 18494). Because of the high similarity between genes encoding subunits I and II of PQQ-ADH and its homologous genes found in a distantly related taxonomic group of acetic acid bacteria, the results suggest the possibility of horizontal gene transfer between these two groups of genera.

  13. Serum albumin complexation of acetylsalicylic acid metabolites.

    PubMed

    Jurkowski, Wiktor; Porebski, Grzegorz; Obtułowicz, Krystyna; Roterman, Irena

    2009-06-01

    One possible origin of the type I hypersensitivity reaction is reaction of drugs such as acetylsalicylic acid and its metabolites being complexed with human serum albumin. Albumin, being transporting molecule abundant in blood plasma is able to bind large array of ligands varying from small single carbon particles to long hydrophobic tailed lipidic acids (e.g. myristic acid). This non specificity is possible because of multi domain scaffold and large flexibility of inter-domain loops, which results in serious reorientation of domains. Hypothesis that acetylsalicylic acid metabolites may play indirect role in activation of allergic reaction has been tested. Binding of acetylsalicylic acid metabolites in intra-domain space causes significant increase of liability of domains IIIA and IIIB. One of metabolites, salicyluric acid, once is bound causes distortion and partial unfolding of helices in domains IA, IIB and IIIB. Changed are both directions and amplitude of relative motions as well as intra-domain distances. In result albumin is able to cross-link of adjacent IgE receptors which subsequently starts allergic reaction.

  14. Serum albumin complexation of acetylsalicylic acid metabolites.

    PubMed

    Jurkowski, Wiktor; Porebski, Grzegorz; Obtułowicz, Krystyna; Roterman, Irena

    2009-06-01

    One possible origin of the type I hypersensitivity reaction is reaction of drugs such as acetylsalicylic acid and its metabolites being complexed with human serum albumin. Albumin, being transporting molecule abundant in blood plasma is able to bind large array of ligands varying from small single carbon particles to long hydrophobic tailed lipidic acids (e.g. myristic acid). This non specificity is possible because of multi domain scaffold and large flexibility of inter-domain loops, which results in serious reorientation of domains. Hypothesis that acetylsalicylic acid metabolites may play indirect role in activation of allergic reaction has been tested. Binding of acetylsalicylic acid metabolites in intra-domain space causes significant increase of liability of domains IIIA and IIIB. One of metabolites, salicyluric acid, once is bound causes distortion and partial unfolding of helices in domains IA, IIB and IIIB. Changed are both directions and amplitude of relative motions as well as intra-domain distances. In result albumin is able to cross-link of adjacent IgE receptors which subsequently starts allergic reaction. PMID:19689242

  15. Succinate dehydrogenase (SDHx) mutations in pituitary tumors: could this be a new role for mitochondrial complex II and/or Krebs cycle defects?

    PubMed

    Xekouki, Paraskevi; Stratakis, Constantine A

    2012-12-01

    Succinate dehydrogenase (SDH) or mitochondrial complex II is a multimeric enzyme that is bound to the inner membrane of mitochondria and has a dual role as it serves both as a critical step of the tricarboxylic acid or Krebs cycle and as a member of the respiratory chain that transfers electrons directly to the ubiquinone pool. Mutations in SDH subunits have been implicated in the formation of familial paragangliomas (PGLs) and/or pheochromocytomas (PHEOs) and in Carney-Stratakis syndrome. More recently, SDH defects were associated with predisposition to a Cowden disease phenotype, renal, and thyroid cancer. We recently described a kindred with the coexistence of familial PGLs and an aggressive GH-secreting pituitary adenoma, harboring an SDHD mutation. The pituitary tumor showed loss of heterozygosity at the SDHD locus, indicating the possibility that SDHD's loss was causatively linked to the development of the neoplasm. In total, 29 cases of pituitary adenomas presenting in association with PHEOs and/or extra-adrenal PGLs have been reported in the literature since 1952. Although a number of other genetic defects are possible in these cases, we speculate that the association of PHEOs and/or PGLs with pituitary tumors is a new syndromic association and a novel phenotype for SDH defects.

  16. Crystal structures of Tritrichomonasfoetus inosine monophosphate dehydrogenase in complex with substrate, cofactor and analogs: a structural basis for the random-in ordered-out kinetic mechanism.

    PubMed

    Prosise, Glen L; Luecke, Hartmut

    2003-02-14

    The enzyme inosine monophosphate dehydrogenase (IMPDH) is responsible for the rate-limiting step in guanine nucleotide biosynthesis. Because it is up-regulated in rapidly proliferating cells, human type II IMPDH is actively targeted for immunosuppressive, anticancer, and antiviral chemotherapy. The enzyme employs a random-in ordered-out kinetic mechanism where substrate or cofactor can bind first but product is only released after the cofactor leaves. Due to structural and kinetic differences between mammalian and microbial enzymes, most drugs that are successful in the inhibition of mammalian IMPDH are far less effective against the microbial forms of the enzyme. It is possible that with greater knowledge of the structural mechanism of the microbial enzymes, an effective and selective inhibitor of microbial IMPDH will be developed for use as a drug against multi-drug resistant bacteria and protists. The high-resolution crystal structures of four different complexes of IMPDH from the protozoan parasite Tritrichomonas foetus have been solved: with its substrate IMP, IMP and the inhibitor mycophenolic acid (MPA), the product XMP with MPA, and XMP with the cofactor NAD(+). In addition, a potassium ion has been located at the dimer interface. A structural model for the kinetic mechanism is proposed.

  17. Engineering acetyl coenzyme A supply: functional expression of a bacterial pyruvate dehydrogenase complex in the cytosol of Saccharomyces cerevisiae.

    PubMed

    Kozak, Barbara U; van Rossum, Harmen M; Luttik, Marijke A H; Akeroyd, Michiel; Benjamin, Kirsten R; Wu, Liang; de Vries, Simon; Daran, Jean-Marc; Pronk, Jack T; van Maris, Antonius J A

    2014-10-21

    The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs(+) reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. Importance: Genetically engineered microorganisms are intensively investigated and applied for production of biofuels and chemicals from renewable sugars. To make such processes economically and environmentally sustainable, the energy

  18. Engineering acetyl coenzyme A supply: functional expression of a bacterial pyruvate dehydrogenase complex in the cytosol of Saccharomyces cerevisiae.

    PubMed

    Kozak, Barbara U; van Rossum, Harmen M; Luttik, Marijke A H; Akeroyd, Michiel; Benjamin, Kirsten R; Wu, Liang; de Vries, Simon; Daran, Jean-Marc; Pronk, Jack T; van Maris, Antonius J A

    2014-01-01

    The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs(+) reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. Importance: Genetically engineered microorganisms are intensively investigated and applied for production of biofuels and chemicals from renewable sugars. To make such processes economically and environmentally sustainable, the energy

  19. 21 CFR 172.315 - Nicotinamide-ascorbic acid complex.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Nicotinamide-ascorbic acid complex. 172.315... Nutritional Additives § 172.315 Nicotinamide-ascorbic acid complex. Nicotinamide-ascorbic acid complex may be... the controlled reaction between ascorbic acid and nicotinamide, melting in the range 141 °C to 145...

  20. Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate.

    PubMed

    Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Sax, Martin; Brunskill, Andrew; Nemeria, Natalia; Jordan, Frank; Furey, William

    2004-03-01

    Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP). To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg(2+) has been determined at 2.1 A and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg(2+) complex. When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others. These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP. The observed hydrogen bonding pattern is not an invariant feature of ThDP-dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues. Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP. The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor. Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release. This study also provides additional insight into the role of water in enzyme inhibition and catalysis. PMID:14992577

  1. Crystal Structure of Binary and Ternary Complexes of Archaeal UDP-galactose 4-Epimerase-like l-Threonine Dehydrogenase from Thermoplasma volcanium*

    PubMed Central

    Yoneda, Kazunari; Sakuraba, Haruhiko; Araki, Tomohiro; Ohshima, Toshihisa

    2012-01-01

    A gene from the thermophilic archaeon Thermoplasma volcanium encoding an l-threonine dehydrogenase (l-ThrDH) with a predicted amino acid sequence that was remarkably similar to the sequence of UDP-galactose 4-epimerase (GalE) was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme was moderately thermostable, retaining more than 90% of its activity after incubation for 10 min at up to 70 °C. The catalytic residue was assessed using site-directed mutagenesis, and Tyr137 was found to be essential for catalysis. To clarify the structural basis of the catalytic mechanism, four different crystal structures were determined using the molecular replacement method: l-ThrDH-NAD+, l-ThrDH in complex with NAD+ and pyruvate, Y137F mutant in complex with NAD+ and l-threonine, and Y137F in complex with NAD+ and l-3-hydroxynorvaline. Each monomer consisted of a Rossmann-fold domain and a C-terminal catalytic domain, and the fold of the catalytic domain showed notable similarity to that of the GalE-like l-ThrDH from the psychrophilic bacterium Flavobacterium frigidimaris KUC-1. The substrate binding model suggests that the reaction proceeds through abstraction of the β-hydroxyl hydrogen of l-threonine via direct proton transfer driven by Tyr137. The factors contributing to the thermostability of T. volcanium l-ThrDH were analyzed by comparing its structure to that of F. frigidimaris l-ThrDH. This comparison showed that the presence of extensive inter- and intrasubunit ion pair networks are likely responsible for the thermostability of T. volcanium l-ThrDH. This is the first description of the molecular basis for the substrate recognition and thermostability of a GalE-like l-ThrDH. PMID:22374996

  2. Function of Several Critical Amino Acids in Human Pyruvate Dehydrogenase Revealed by Its Structure

    NASA Technical Reports Server (NTRS)

    Korotchkina, Lioubov G.; Ciszak, E.; Patel, M.

    2004-01-01

    Pyruvate dehydrogenase (E1), an alpha 2 beta 2 tetramer, catalyzes the oxidative decarboxylation of pyruvate and reductive acetylation of lipoyl moieties of the dihydrolipoamide acetyltransferase. The roles of beta W135, alpha P188, alpha M181, alpha H15 and alpha R349 of E1 determined by kinetic analysis were reassessed by analyzing the three-dimensional structure of human E1. The residues identified above are found to play a structural role rather than being directly involved in catalysis: beta W135 is the center residue in the hydrophobic interaction between beta and beta' subunits; alpha P188 and alpha M181 are critical for the conformation of the TPP-binding motif and interaction between alpha and beta subunits; alpha H15, is necessary for the organization of the N-terminus of alpha and alpha'; subunits and alpha R349 supports the interaction of the C-terminus of the alpha subunits with the beta subunits. Analysis of several critical E1 residues confirms the importance of residues distant from the active site for subunit interactions and enzyme function.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-05-01

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

  5. Correlation of loss of activity of human aldehyde dehydrogenase with reaction of bromoacetophenone with glutamic acid-268 and cysteine-302 residues. Partial-sites reactivity of aldehyde dehydrogenase.

    PubMed Central

    Abriola, D P; MacKerell, A D; Pietruszko, R

    1990-01-01

    Bromoacetophenone (2-bromo-1-phenylethanone) has been characterized as an affinity reagent for human aldehyde dehydrogenase (EC 1.2.1.3) [MacKerell, MacWright & Pietruszko (1986) Biochemistry 25, 5182-5189], and has been shown to react specifically with the Glu-268 residue [Abriola, Fields, Stein, MacKerell & Pietruszko (1987) Biochemistry 26, 5679-5684] with an apparent inactivation stoichiometry of two molecules of bromoacetophenone per molecule of enzyme. The specificity of bromoacetophenone for reaction with Glu-268, however, is not absolute, owing to the extreme reactivity of this reagent. When bromo[14C]acetophenone was used to label the human cytoplasmic E1 isoenzyme radioactively and tryptic fragmentation was carried out, peptides besides that containing Glu-268 were found to have reacted with reagent. These peptides were purified by h.p.l.c. and analysed by sequencing and scintillation counting to quantify radioactive label in the material from each cycle of sequencing. Reaction of bromoacetophenone with the aldehyde dehydrogenase molecule during enzyme activity loss occurs with two residues, Glu-268 and Cys-302. The activity loss, however, appears to be proportional to incorporation of label at Glu-268. The large part of incorporation of label at Cys-302 occurs after the activity loss is essentially complete. With both Glu-268 and Cys-302, however, the incorporation of label stops after one molecule of bromoacetophenone has reacted with each residue. Reaction with other residues continues after activity loss is complete. PMID:1968743

  6. Isolation, characterization, and mapping of gene encoding dihydrolipoyl succinyltransferase (E2k) of human [alpha]-ketoglutarate dehydrogenase complex

    SciTech Connect

    Ali, G.; Cai, Xingang; Sheu, Kwan-Fu R.; Blass, J.P. ); Wasco, W.; Gaston, S.M.; Tanzi, R.E.; Cooper, A.J.L.; Gusella, J.F. ); Szabo, P. )

    1994-03-01

    The authors have isolated and sequenced cDNAs representing the full-length (2987-bp) gene for dihydrolipoyl succinyltransferase (E2k component) of the human [alpha]-ketoglutarate dehydrogenase complex (KHDHC) from a human fetal brain cDNA library. The E2k cDNA was mapped to human chromosome 14 using a somatic cell hybrid panel, and more precisely to band 14q24.3 by in situ hybridization. This cDNA also cross-hybridized to an apparent E2k pseudogene on chromosome 1p31. Northern analysis revealed the E2k gene to be ubiquitously expressed in peripheral tissues and brain. Interestingly, chromosome 14q24.3 has recently been reported to contain gene defects for an early-onset form of familial Alzheimer's disease and for Machado-Joseph disease. Future studies will be necessary to determine whether the E2K gene plays a role in either of these two disorders.

  7. DLAT subunit of the pyruvate dehydrogenase complex is upregulated in gastric cancer-implications in cancer therapy

    PubMed Central

    Goh, Wen Quan Jonathan; Ow, Ghim Siong; Kuznetsov, Vladimir A; Chong, Shirly; Lim, Yoon Pin

    2015-01-01

    An iTRAQ-based tandem mass spectrometry approach was employed to relatively quantify proteins in the membrane proteome of eleven gastric cancer cell lines relative to a denominator non-cancer gastric epithelial cell line HFE145. Of the 882 proteins detected, 57 proteins were found to be upregulated with > 1.3-fold change in at least 6 of the 11 cell lines. Bioinformatics analysis revealed that these proteins are significantly associated with cancer, cell growth and proliferation, death, survival and cell movement. The catalogue of membrane proteins presented that are potential regulators/effectors of gastric cancer progression has implications in cancer therapy. DLAT, a subunit of the pyruvate dehydrogenase complex, was selected as a candidate protein for further studies as its function in gastric cancer has yet to be established. SiRNA studies supported a role of DLAT in gastric cancer cell proliferation and carbohydrate metabolism, reprogramming of which is a hallmark of cancer. Our study contributes to recent interest and discussion in cancer energetics and related phenomena such as the Warburg and Reverse Warburg effects. Future mechanistic studies should lead to the elucidation of the mode of action of DLAT in human gastric cancer and establish DLAT as a viable drug target. PMID:26279757

  8. Importance of serum sialic acid and lactate dehydrogenase in diagnosis and treatment monitoring of cervical cancer patients.

    PubMed

    Patel, P S; Rawal, G N; Balar, D B

    1993-09-01

    In an attempt to establish a blood-based biochemical index for diagnosis of cervical cancer and treatment monitoring of patients suffering from the disease, serum levels of total sialic acid (TSA), lipid-bound sialic acid (LSA), and lactate dehydrogenase were estimated by highly specific spectrophotometric methods. Serum concentrations of the markers in 108 untreated cervical cancer patients were compared with the levels of the biomarkers in 125 healthy, age-matched female individuals (controls). The alterations in serum levels of the markers after radiotherapy in cervical cancer patients were also observed. The levels of all markers were significantly higher (P < 0.001) in untreated cervical cancer patients compared to the controls. TSA was found to be the most sensitive (90.74%) marker for diagnosis of cervical cancer. Combined use of the markers revealed maximum (100%) sensitivity. In comparison between early (stage I+II) and advanced (stage III+IV) malignant disease, the markers showed insignificant changes. TSA and LSA values in patients who did not respond to radiotherapy were significantly higher (P < 0.05 and P < 0.001, respectively) than those of the responders. The results suggest that combined evaluation of the markers is helpful for diagnosis as well as for treatment monitoring of cervical carcinoma patients.

  9. Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels.

    PubMed

    Parkhomenko, Yulia M; Kudryavtsev, Pavel A; Pylypchuk, Svetlana Yu; Chekhivska, Lilia I; Stepanenko, Svetlana P; Sergiichuk, Andrej A; Bunik, Victoria I

    2011-06-01

    Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability.

  10. A Spontaneous Missense Mutation in Branched Chain Keto Acid Dehydrogenase Kinase in the Rat Affects Both the Central and Peripheral Nervous Systems.

    PubMed

    Zigler, J Samuel; Hodgkinson, Colin A; Wright, Megan; Klise, Andrew; Sundin, Olof; Broman, Karl W; Hejtmancik, Fielding; Huang, Hao; Patek, Bonnie; Sergeev, Yuri; Hose, Stacey; Brayton, Cory; Xaiodong, Jiao; Vasquez, David; Maragakis, Nicholas; Mori, Susumu; Goldman, David; Hoke, Ahmet; Sinha, Debasish

    2016-01-01

    A novel mutation, causing a phenotype we named frogleg because its most obvious characteristic is a severe splaying of the hind limbs, arose spontaneously in a colony of Sprague-Dawley rats. Frogleg is a complex phenotype that includes abnormalities in hind limb function, reduced brain weight with dilated ventricles and infertility. Using micro-satellite markers spanning the entire rat genome, the mutation was mapped to a region of rat chromosome 1 between D1Rat131 and D1Rat287. Analysis of whole genome sequencing data within the linkage interval, identified a missense mutation in the branched-chain alpha-keto dehydrogenase kinase (Bckdk) gene. The protein encoded by Bckdk is an integral part of an enzyme complex located in the mitochondrial matrix of many tissues which regulates the levels of the branched-chain amino acids (BCAAs), leucine, isoleucine and valine. BCAAs are essential amino acids (not synthesized by the body), and circulating levels must be tightly regulated; levels that are too high or too low are both deleterious. BCKDK phosphorylates Ser293 of the E1α subunit of the BCKDH protein, which catalyzes the rate-limiting step in the catabolism of the BCAAs, inhibiting BCKDH and thereby, limiting breakdown of the BCAAs. In contrast, when Ser293 is not phosphorylated, BCKDH activity is unchecked and the levels of the BCAAs will decrease dramatically. The mutation is located within the kinase domain of Bckdk and is predicted to be damaging. Consistent with this, we show that in rats homozygous for the mutation, phosphorylation of BCKDH in the brain is markedly decreased relative to wild type or heterozygous littermates. Further, circulating levels of the BCAAs are reduced by 70-80% in animals homozygous for the mutation. The frogleg phenotype shares important characteristics with a previously described Bckdk knockout mouse and with human subjects with Bckdk mutations. In addition, we report novel data regarding peripheral neuropathy of the hind limbs

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

    SciTech Connect

    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 use {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.

  12. A Spontaneous Missense Mutation in Branched Chain Keto Acid Dehydrogenase Kinase in the Rat Affects Both the Central and Peripheral Nervous Systems

    PubMed Central

    Zigler, J. Samuel; Hodgkinson, Colin A.; Wright, Megan; Klise, Andrew; Broman, Karl W.; Huang, Hao; Patek, Bonnie; Sergeev, Yuri; Hose, Stacey; Xaiodong, Jiao; Vasquez, David; Maragakis, Nicholas; Mori, Susumu; Goldman, David; Sinha, Debasish

    2016-01-01

    A novel mutation, causing a phenotype we named frogleg because its most obvious characteristic is a severe splaying of the hind limbs, arose spontaneously in a colony of Sprague-Dawley rats. Frogleg is a complex phenotype that includes abnormalities in hind limb function, reduced brain weight with dilated ventricles and infertility. Using micro-satellite markers spanning the entire rat genome, the mutation was mapped to a region of rat chromosome 1 between D1Rat131 and D1Rat287. Analysis of whole genome sequencing data within the linkage interval, identified a missense mutation in the branched-chain alpha-keto dehydrogenase kinase (Bckdk) gene. The protein encoded by Bckdk is an integral part of an enzyme complex located in the mitochondrial matrix of many tissues which regulates the levels of the branched-chain amino acids (BCAAs), leucine, isoleucine and valine. BCAAs are essential amino acids (not synthesized by the body), and circulating levels must be tightly regulated; levels that are too high or too low are both deleterious. BCKDK phosphorylates Ser293 of the E1α subunit of the BCKDH protein, which catalyzes the rate-limiting step in the catabolism of the BCAAs, inhibiting BCKDH and thereby, limiting breakdown of the BCAAs. In contrast, when Ser293 is not phosphorylated, BCKDH activity is unchecked and the levels of the BCAAs will decrease dramatically. The mutation is located within the kinase domain of Bckdk and is predicted to be damaging. Consistent with this, we show that in rats homozygous for the mutation, phosphorylation of BCKDH in the brain is markedly decreased relative to wild type or heterozygous littermates. Further, circulating levels of the BCAAs are reduced by 70–80% in animals homozygous for the mutation. The frogleg phenotype shares important characteristics with a previously described Bckdk knockout mouse and with human subjects with Bckdk mutations. In addition, we report novel data regarding peripheral neuropathy of the hind limbs

  13. A Spontaneous Missense Mutation in Branched Chain Keto Acid Dehydrogenase Kinase in the Rat Affects Both the Central and Peripheral Nervous Systems.

    PubMed

    Zigler, J Samuel; Hodgkinson, Colin A; Wright, Megan; Klise, Andrew; Sundin, Olof; Broman, Karl W; Hejtmancik, Fielding; Huang, Hao; Patek, Bonnie; Sergeev, Yuri; Hose, Stacey; Brayton, Cory; Xaiodong, Jiao; Vasquez, David; Maragakis, Nicholas; Mori, Susumu; Goldman, David; Hoke, Ahmet; Sinha, Debasish

    2016-01-01

    A novel mutation, causing a phenotype we named frogleg because its most obvious characteristic is a severe splaying of the hind limbs, arose spontaneously in a colony of Sprague-Dawley rats. Frogleg is a complex phenotype that includes abnormalities in hind limb function, reduced brain weight with dilated ventricles and infertility. Using micro-satellite markers spanning the entire rat genome, the mutation was mapped to a region of rat chromosome 1 between D1Rat131 and D1Rat287. Analysis of whole genome sequencing data within the linkage interval, identified a missense mutation in the branched-chain alpha-keto dehydrogenase kinase (Bckdk) gene. The protein encoded by Bckdk is an integral part of an enzyme complex located in the mitochondrial matrix of many tissues which regulates the levels of the branched-chain amino acids (BCAAs), leucine, isoleucine and valine. BCAAs are essential amino acids (not synthesized by the body), and circulating levels must be tightly regulated; levels that are too high or too low are both deleterious. BCKDK phosphorylates Ser293 of the E1α subunit of the BCKDH protein, which catalyzes the rate-limiting step in the catabolism of the BCAAs, inhibiting BCKDH and thereby, limiting breakdown of the BCAAs. In contrast, when Ser293 is not phosphorylated, BCKDH activity is unchecked and the levels of the BCAAs will decrease dramatically. The mutation is located within the kinase domain of Bckdk and is predicted to be damaging. Consistent with this, we show that in rats homozygous for the mutation, phosphorylation of BCKDH in the brain is markedly decreased relative to wild type or heterozygous littermates. Further, circulating levels of the BCAAs are reduced by 70-80% in animals homozygous for the mutation. The frogleg phenotype shares important characteristics with a previously described Bckdk knockout mouse and with human subjects with Bckdk mutations. In addition, we report novel data regarding peripheral neuropathy of the hind limbs.

  14. Microwave structure for the propiolic acid-formic acid complex.

    PubMed

    Kukolich, Stephen G; Mitchell, Erik G; Carey, Spencer J; Sun, Ming; Sargus, Bryan A

    2013-10-01

    New microwave spectra were measured to obtain rotational constants and centrifugal distortion constants for the DCCCOOH···HOOCH and HCCCOOD···DOOCH isotopologues. Rotational transitions were measured in the frequency range of 4.9-15.4 GHz, providing accurate rotational constants, which, combined with previous rotational constants, allowed an improved structural fit for the propiolic acid-formic acid complex. The new structural fit yields reasonably accurate orientations for both the propiolic and formic acid monomers in the complex and more accurate structural parameters describing the hydrogen bonding. The structure is planar, with a positive inertial defect of Δ = 1.33 amu Å(2). The experimental structure exhibits a greater asymmetry for the two hydrogen bond lengths than was obtained from the ab initio mp2 calculations. The best-fit hydrogen bond lengths have an r(O1-H1···O4) of 1.64 Å and an r(O3-H2···O2) of 1.87 Å. The average of the two hydrogen bond lengths is r(av)(exp) = 1.76 Å, in good agreement with r(av)(theory) = 1.72 Å. The center of mass separation of the monomers is R(CM) = 3.864 Å. Other structural parameters from the least-squares fit using the experimental rotational constants are compared with theoretical values. The spectra were obtained using two different pulsed beam Fourier transform microwave spectrometers.

  15. The complex structures of isocitrate dehydrogenase from Clostridium thermocellum and Desulfotalea psychrophila suggest a new active site locking mechanism

    PubMed Central

    Leiros, Hanna-Kirsti S.; Fedøy, Anita-Elin; Leiros, Ingar; Steen, Ida Helene

    2012-01-01

    Isocitrate dehydrogenase (IDH) catalyzes the oxidative NAD(P)+-dependent decarboxylation of isocitrate into α-ketoglutarate and CO2 and is present in organisms spanning the biological range of temperature. We have solved two crystal structures of the thermophilic Clostridium thermocellum IDH (CtIDH), a native open apo CtIDH to 2.35 Å and a quaternary complex of CtIDH with NADP+, isocitrate and Mg2+ to 2.5 Å. To compare to these a quaternary complex structure of the psychrophilic Desulfotalea psychrophila IDH (DpIDH) was also resolved to 1.93 Å. CtIDH and DpIDH showed similar global thermal stabilities with melting temperatures of 67.9 and 66.9 °C, respectively. CtIDH represents a typical thermophilic enzyme, with a large number of ionic interactions and hydrogen bonds per residue combined with stabilization of the N and C termini. CtIDH had a higher activity temperature optimum, and showed greater affinity for the substrates with an active site that was less thermolabile compared to DpIDH. The uncompensated negative surface charge and the enlarged methionine cluster in the hinge region both of which are important for cold activity in DpIDH, were absent in CtIDH. These structural comparisons revealed that prokaryotic IDHs in subfamily II have a unique locking mechanism involving Arg310, Asp251′ and Arg255 (CtIDH). These interactions lock the large domain to the small domain and direct NADP+ into the correct orientation, which together are important for NADP+ selectivity. PMID:23650595

  16. Nicotine Dehydrogenase Complexed with 6-Hydroxypseudooxynicotine Oxidase Involved in the Hybrid Nicotine-Degrading Pathway in Agrobacterium tumefaciens S33

    PubMed Central

    Li, Huili; Xie, Kebo; Yu, Wenjun; Hu, Liejie; Huang, Haiyan; Xie, Huijun

    2016-01-01

    Nicotine, a major toxic alkaloid in tobacco wastes, is degraded by bacteria, mainly via pyridine and pyrrolidine pathways. Previously, we discovered a new hybrid of the pyridine and pyrrolidine pathways in Agrobacterium tumefaciens S33 and characterized its key enzyme 6-hydroxy-3-succinoylpyridine (HSP) hydroxylase. Here, we purified the nicotine dehydrogenase initializing the nicotine degradation from the strain and found that it forms a complex with a novel 6-hydroxypseudooxynicotine oxidase. The purified complex is composed of three different subunits encoded by ndhAB and pno, where ndhA and ndhB overlap by 4 bp and are ∼26 kb away from pno. As predicted from the gene sequences and from chemical analyses, NdhA (82.4 kDa) and NdhB (17.1 kDa) harbor a molybdopterin cofactor and two [2Fe-2S] clusters, respectively, whereas Pno (73.3 kDa) harbors an flavin mononucleotide and a [4Fe-4S] cluster. Mutants with disrupted ndhA or ndhB genes did not grow on nicotine but grew well on 6-hydroxynicotine and HSP, whereas the pno mutant did not grow on nicotine or 6-hydroxynicotine but grew well on HSP, indicating that NdhA and NdhB are responsible for initialization of nicotine oxidation. We successfully expressed pno in Escherichia coli and found that the recombinant Pno presented 2,6-dichlorophenolindophenol reduction activity when it was coupled with 6-hydroxynicotine oxidation. The determination of reaction products catalyzed by the purified enzymes or mutants indicated that NdhAB catalyzed nicotine oxidation to 6-hydroxynicotine, whereas Pno oxidized 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde pyridine. These results provide new insights into this novel hybrid pathway of nicotine degradation in A. tumefaciens S33. PMID:26729714

  17. The complex structures of isocitrate dehydrogenase from Clostridium thermocellum and Desulfotalea psychrophila suggest a new active site locking mechanism.

    PubMed

    Leiros, Hanna-Kirsti S; Fedøy, Anita-Elin; Leiros, Ingar; Steen, Ida Helene

    2012-01-01

    Isocitrate dehydrogenase (IDH) catalyzes the oxidative NAD(P)(+)-dependent decarboxylation of isocitrate into α-ketoglutarate and CO2 and is present in organisms spanning the biological range of temperature. We have solved two crystal structures of the thermophilic Clostridium thermocellum IDH (CtIDH), a native open apo CtIDH to 2.35 Å and a quaternary complex of CtIDH with NADP(+), isocitrate and Mg(2+) to 2.5 Å. To compare to these a quaternary complex structure of the psychrophilic Desulfotalea psychrophila IDH (DpIDH) was also resolved to 1.93 Å. CtIDH and DpIDH showed similar global thermal stabilities with melting temperatures of 67.9 and 66.9 °C, respectively. CtIDH represents a typical thermophilic enzyme, with a large number of ionic interactions and hydrogen bonds per residue combined with stabilization of the N and C termini. CtIDH had a higher activity temperature optimum, and showed greater affinity for the substrates with an active site that was less thermolabile compared to DpIDH. The uncompensated negative surface charge and the enlarged methionine cluster in the hinge region both of which are important for cold activity in DpIDH, were absent in CtIDH. These structural comparisons revealed that prokaryotic IDHs in subfamily II have a unique locking mechanism involving Arg310, Asp251' and Arg255 (CtIDH). These interactions lock the large domain to the small domain and direct NADP(+) into the correct orientation, which together are important for NADP(+) selectivity.

  18. Biosynthesis of Germacrene A Carboxylic Acid in Chicory Roots. Demonstration of a Cytochrome P450 (+)-Germacrene A Hydroxylase and NADP+-Dependent Sesquiterpenoid Dehydrogenase(s) Involved in Sesquiterpene Lactone Biosynthesis

    PubMed Central

    de Kraker, Jan-Willem; Franssen, Maurice C. R.; Dalm, Marcella C. F.; de Groot, Aede; Bouwmeester, Harro J.

    2001-01-01

    Sprouts of chicory (Cichorium intybus), a vegetable grown in the dark, have a slightly bitter taste associated with the presence of guaianolides, eudesmanolides, and germacranolides. The committed step in the biosynthesis of these compounds is catalyzed by a (+)-germacrene A synthase. Formation of the lactone ring is the postulated next step in biosynthesis of the germacrene-derived sesquiterpene lactones. The present study confirms this hypothesis by isolation of enzyme activities from chicory roots that introduce a carboxylic acid function in the germacrene A isopropenyl side chain, which is necessary for lactone ring formation. (+)-Germacrene A is hydroxylated to germacra-1(10),4,11(13)-trien-12-ol by a cytochrome P450 enzyme, and is subsequently oxidized to germacra-1(10),4,11(13)-trien-12-oic acid by NADP+-dependent dehydrogenase(s). Both oxidized germacrenes were detected as their Cope-rearrangement products elema-1,3,11(13)-trien-12-ol and elema-1,3,11(13)-trien-12-oic acid, respectively. The cyclization products of germacra-1(10),4,11(13)-trien-12-ol, i.e. costol, were also observed. The (+)-germacrene A hydroxylase is inhibited by carbon monoxide (blue-light reversible), has an optimum pH at 8.0, and hydroxylates β-elemene with a modest degree of enantioselectivity. PMID:11299372

  19. Application of a genetically encoded biosensor for live cell imaging of L-valine production in pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum strains.

    PubMed

    Mustafi, Nurije; Grünberger, Alexander; Mahr, Regina; Helfrich, Stefan; Nöh, Katharina; Blombach, Bastian; Kohlheyer, Dietrich; Frunzke, Julia

    2014-01-01

    The majority of biotechnologically relevant metabolites do not impart a conspicuous phenotype to the producing cell. Consequently, the analysis of microbial metabolite production is still dominated by bulk techniques, which may obscure significant variation at the single-cell level. In this study, we have applied the recently developed Lrp-biosensor for monitoring of amino acid production in single cells of gradually engineered L-valine producing Corynebacterium glutamicum strains based on the pyruvate dehydrogenase complex-deficient (PDHC) strain C. glutamicum ΔaceE. Online monitoring of the sensor output (eYFP fluorescence) during batch cultivation proved the sensor's suitability for visualizing different production levels. In the following, we conducted live cell imaging studies on C. glutamicum sensor strains using microfluidic chip devices. As expected, the sensor output was higher in microcolonies of high-yield producers in comparison to the basic strain C. glutamicum ΔaceE. Microfluidic cultivation in minimal medium revealed a typical Gaussian distribution of single cell fluorescence during the production phase. Remarkably, low amounts of complex nutrients completely changed the observed phenotypic pattern of all strains, resulting in a phenotypic split of the population. Whereas some cells stopped growing and initiated L-valine production, others continued to grow or showed a delayed transition to production. Depending on the cultivation conditions, a considerable fraction of non-fluorescent cells was observed, suggesting a loss of metabolic activity. These studies demonstrate that genetically encoded biosensors are a valuable tool for monitoring single cell productivity and to study the phenotypic pattern of microbial production strains.

  20. Communication between thiamin cofactors in the Escherichia coli pyruvate dehydrogenase complex E1 component active centers: evidence for a "direct pathway" between the 4'-aminopyrimidine N1' atoms.

    PubMed

    Nemeria, Natalia S; Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Mossad, Madouna; Tittmann, Kai; Furey, William; Jordan, Frank

    2010-04-01

    Kinetic, spectroscopic, and structural analysis tested the hypothesis that a chain of residues connecting the 4'-aminopyrimidine N1' atoms of thiamin diphosphates (ThDPs) in the two active centers of the Escherichia coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (Glu(571), Glu(235), and Glu(237)) and Arg(606) resulted in impaired binding of the second ThDP, once the first active center was filled, suggesting a pathway for communication between the two ThDPs. 1) Steady-state kinetic and fluorescence quenching studies revealed that upon E571A, E235A, E237A, and R606A substitutions, ThDP binding in the second active center was affected. 2) Analysis of the kinetics of thiazolium C2 hydrogen/deuterium exchange of enzyme-bound ThDP suggests half-of-the-sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for the slow exchanging site, indicating that only one of two active sites is filled with ThDP. 3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. 4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that Glu(235) makes no direct contact with the cofactor. The role of the conserved Glu(571) residue in both catalysis and cofactor orientation is revealed by the combined results for the first time.

  1. Communication between thiamin cofactors in the Escherichia coli pyruvate dehydrogenase complex E1 component active centers: evidence for a "direct pathway" between the 4'-aminopyrimidine N1' atoms.

    PubMed

    Nemeria, Natalia S; Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Mossad, Madouna; Tittmann, Kai; Furey, William; Jordan, Frank

    2010-04-01

    Kinetic, spectroscopic, and structural analysis tested the hypothesis that a chain of residues connecting the 4'-aminopyrimidine N1' atoms of thiamin diphosphates (ThDPs) in the two active centers of the Escherichia coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (Glu(571), Glu(235), and Glu(237)) and Arg(606) resulted in impaired binding of the second ThDP, once the first active center was filled, suggesting a pathway for communication between the two ThDPs. 1) Steady-state kinetic and fluorescence quenching studies revealed that upon E571A, E235A, E237A, and R606A substitutions, ThDP binding in the second active center was affected. 2) Analysis of the kinetics of thiazolium C2 hydrogen/deuterium exchange of enzyme-bound ThDP suggests half-of-the-sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for the slow exchanging site, indicating that only one of two active sites is filled with ThDP. 3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. 4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that Glu(235) makes no direct contact with the cofactor. The role of the conserved Glu(571) residue in both catalysis and cofactor orientation is revealed by the combined results for the first time. PMID:20106967

  2. Amino acid changes within antenna helix are responsible for different regulatory preferences of human glutamate dehydrogenase isozymes.

    PubMed

    Choi, Myung-Min; Kim, Eun-A; Yang, Seung-Ju; Choi, Soo Young; Cho, Sung-Woo; Huh, Jae-Wan

    2007-07-01

    Human glutamate dehydrogenase (hGDH) exists in hGDH1 (housekeeping isozyme) and in hGDH2 (nerve-specific isozyme), which differ markedly in their allosteric regulation. Because they differ in only 16 of their 505 amino acids, the regulatory preferences must arise from amino acid residues that are not common between hGDH1 and hGDH2. To our knowledge none of the mutagenesis studies on the hGDH isozymes to date have identified the amino acid residues fully responsible for the different regulatory preferences between hGDH1 and hGDH2. In this study we constructed hGDH1(hGDH2(390-448))hGDH1 (amino acid segment 390-448 of hGDH1 replaced by the corresponding hGDH2 segment) and hGDH2(hGDH1(390-448))hGDH2 (amino acid segment 390-448 of hGDH2 replaced by the corresponding hGDH1 segment) by swapping the corresponding amino acid segments in hGDH1 and hGDH2. The chimeric enzymes by reciprocal swapping resulted in double mutations in amino acid sequences at 415 and 443 residues that are not common between hGDH1 and hGDH2 and are located in the C-terminal 48-residue "antenna" helix, which is thought to be part of the regulatory domain of mammalian GDHs. Functional analyses revealed that the doubly mutated chimeric enzymes almost completely acquired most of the different regulatory preferences between hGDH1 and hGDH2 for electrophoretic mobility, heat-stability, ADP activation, palmitoyl-CoA inhibition, and l-leucine activation, except for GTP inhibition. Our results indicate that substitutions of the residues in the antenna region may be important evolutionary changes that led to the adaptation of hGDH2 to the unique metabolic needs of the nerve tissue.

  3. Structure and Function of Plasmodium falciparum malate dehydrogenase: Role of Critical Amino Acids in C-substrate Binding Procket

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our lab have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal g...

  4. Comparative analysis of the catalytic components in the archaeal dye-linked L-proline dehydrogenase complexes.

    PubMed

    Kawakami, Ryushi; Noguchi, Chiaki; Higashi, Marie; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2013-04-01

    Two types of hetero-oligomeric dye-linked L-proline dehydrogenases (α4β4 and αβγδ types) are expressed in the hyperthermophilic archaea belonging to Thermococcales. In both enzymes, the β subunit (PDHβ) is responsible for catalyzing L-proline dehydrogenation. The genes encoding the two enzyme types form respective clusters that are completely conserved among Pyrococcus and Thermococcus strains. To compare the enzymatic properties of PDHβs from α4β4- and αβγδ-type enzyme complexes, eight PDHβs (four of each type) from Pyrococcus furiosus DSM3638, Pyrococcus horikoshii OT-3, Thermococcus kodakaraensis KOD1 JCM12380 and Thermococcus profundus DSM9503 were expressed in Escherichia coli cells and purified to homogeneity using one-step Ni-chelating chromatography. The α4β4-type PDHβs showed greater thermostability than most of the αβγδ-type PDHβs: the former retained more than 80 % of their activity after heating at 70 °C for 20 min, while the latter showed different thermostabilities under the same conditions. In addition, the α4β4-type PDHβs utilized ferricyanide as the most preferable electron acceptor, whereas αβγδ-type PDHβs preferred 2, 6-dichloroindophenol, with one exception. These results indicate that the differences in the enzymatic properties of the PDHβs likely reflect whether they were from an αβγδ- or α4β4-type complex, though the wider divergence observed within αβγδ-type PDHβs based on the phylogenetic analysis may also be responsible for their inconsistent enzymatic properties. By contrast, differences in the kinetic parameters among the PDHβs did not reflect the complex type. Interestingly, the k cat value for free α4β4-type PDHβ from P. horikoshii was much larger than the value for the same subunit within the α4β4-complex. This indicates that the isolated PDHβ could be a useful element for an electrochemical system for detection of L-proline. PMID:22752365

  5. Use of transmitochondrial cybrids to assign a complex I defect to the mitochondrial DNA-encoded NADH dehydrogenase subunit 6 gene mutation at nucleotide pair 14459 that causes Leber hereditary optic neuropathy and dystonia.

    PubMed Central

    Jun, A S; Trounce, I A; Brown, M D; Shoffner, J M; Wallace, D C

    1996-01-01

    A heteroplasmic G-to-A transition at nucleotide pair (np) 14459 within the mitochondrial DNA (mtDNA)-encoded NADH dehydrogenase subunit 6 (ND6) gene has been identified as the cause of Leber hereditary optic neuropathy (LHON) and/or pediatric-onset dystonia in three unrelated families. This ND6 np 14459 mutation changes a moderately conserved alanine to a valine at amino acid position 72 of the ND6 protein. Enzymologic analysis of mitochondrial NADH dehydrogenase (complex I) with submitochondrial particles isolated from Epstein-Barr virus-transformed lymphoblasts revealed a 60% reduction (P < 0.005) of complex I-specific activity in patient cell lines compared with controls, with no differences in enzymatic activity for complexes II plus III, III and IV. This biochemical defect was assigned to the ND6 np 14459 mutation by using transmitochondrial cybrids in which patient Epstein-Barr virus-transformed lymphoblast cell lines were enucleated and the cytoplasts were fused to a mtDNA-deficient (p 0) lymphoblastoid recipient cell line. Cybrids harboring the np 14459 mutation exhibited a 39% reduction (p < 0.02) in complex I-specific activity relative to wild-type cybrid lines but normal activity for the other complexes. Kinetic analysis of the np 14459 mutant complex I revealed that the Vmax of the enzyme was reduced while the Km remained the same as that of wild type. Furthermore, specific activity was inhibited by increasing concentrations of the reduced coenzyme Q analog decylubiquinol. These observations suggest that the np 14459 mutation may alter the coenzyme Q-binding site of complex I. PMID:8622678

  6. 21 CFR 172.315 - Nicotinamide-ascorbic acid complex.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Nicotinamide-ascorbic acid complex. 172.315 Section... HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.315 Nicotinamide-ascorbic acid complex. Nicotinamide-ascorbic acid complex may be safely used in accordance with the following prescribed...

  7. 21 CFR 172.315 - Nicotinamide-ascorbic acid complex.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Nicotinamide-ascorbic acid complex. 172.315... HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.315 Nicotinamide-ascorbic acid complex. Nicotinamide-ascorbic acid complex may be safely used in accordance with the following prescribed...

  8. 21 CFR 172.315 - Nicotinamide-ascorbic acid complex.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Nicotinamide-ascorbic acid complex. 172.315... HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.315 Nicotinamide-ascorbic acid complex. Nicotinamide-ascorbic acid complex may be safely used in accordance with the following prescribed...

  9. 21 CFR 172.315 - Nicotinamide-ascorbic acid complex.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Nicotinamide-ascorbic acid complex. 172.315... HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.315 Nicotinamide-ascorbic acid complex. Nicotinamide-ascorbic acid complex may be safely used in accordance with the following prescribed...

  10. Protein engineering of alcohol dehydrogenase--1. Effects of two amino acid changes in the active site of yeast ADH-1.

    PubMed

    Murali, C; Creaser, E H

    1986-01-01

    One of the promises held out by protein engineering is the ability to alter predictably the properties of an enzyme to enable it to find new substrates or catalyse existing substrates more efficiently, such manipulations being of interest both enzymologically and, potentially, industrially. It has been postulated that in yeast alcohol dehydrogenase (YADH-1) certain amino acids such as Trp 93 and Thr 48 constrict the active site due to their bulky side chains and thus impede catalysis of molecules larger than ethanol. To study effects of enlarging the active site we have made two changes into YADH-1, replacing Trp 93 with Phe and Thr 48 with Ser. Kinetic experiments showed that this enzyme had marked increases in reaction velocity for the n-alcohols propanol, butanol, pentanol, hexanol, heptanol, octanol and cinnamyl alcohol compared to the parent, agreeing with the prediction that expanding the active site should facilitate the oxidation of larger alcohols. The substrate affinities were slightly reduced in the altered enzyme, possibly due to its having reduced hydrophobicity at Phe 93.

  11. Detection of glyceraldehyde 3-phosphate dehydrogenase messenger RNA using a peptide nucleic acid probe in paraffin-embedded archival specimens.

    PubMed

    Hiroyasu, Makoto; Akatsuka, Shinya; Shirase, Tomoyuki; Toda, Yoshinobu; Hiai, Hiroshi; Toyokuni, Shinya

    2004-04-01

    Although the human genome project has been completed, the functions of many genes remain undetermined. In situ hybridization (ISH) is a key method for identifying cells in which a given messenger RNA is transcribed. Paraffin-embedded specimens remain precious materials for research, but preservation of high-quality RNA in these specimens is not expected unless ample caution was taken during fixation. Peptide nucleic acid (PNA) is a recently developed hybrid molecule with genetic information that has high stability and high affinity to the complementary DNA or RNA. We applied a PNA probe to mRNA ISH of liver specimens obtained by autopsy and embedded in paraffin 28-48 years ago. An 18-mer PNA probe for glyceraldehyde 3-phosphate dehydrogenase was used. Staining was then analyzed in association with morphology by hematoxylin and eosin staining, and with the time between death of the patient and tissue fixation. Notably, specimens fixed with formalin and embedded in paraffin 48 years ago yielded excellent results if the time before fixation was short enough (<8 h). There was a significant inverse correlation between the intensity of ISH staining and the time before fixation. Oligonucleotide PNA probe, albeit at high cost, would increase the value of paraffin-embedded specimens in storage for use in human medical research.

  12. Isocitrate dehydrogenase 1 mutations prime the all-trans retinoic acid myeloid differentiation pathway in acute myeloid leukemia

    PubMed Central

    Boutzen, Héléna; Saland, Estelle; Larrue, Clément; de Toni, Fabienne; Gales, Lara; Castelli, Florence A.; Cathebas, Mathilde; Zaghdoudi, Sonia; Stuani, Lucille; Kaoma, Tony; Riscal, Romain; Yang, Guangli; Hirsch, Pierre; David, Marion; De Mas-Mansat, Véronique; Delabesse, Eric; Vallar, Laurent; Delhommeau, François; Jouanin, Isabelle; Ouerfelli, Ouathek; Le Cam, Laurent; Linares, Laetitia K.; Junot, Christophe; Portais, Jean-Charles; Vergez, François; Récher, Christian

    2016-01-01

    Acute myeloid leukemia (AML) is characterized by the accumulation of malignant blasts with impaired differentiation programs caused by recurrent mutations, such as the isocitrate dehydrogenase (IDH) mutations found in 15% of AML patients. These mutations result in the production of the oncometabolite (R)-2-hydroxyglutarate (2-HG), leading to a hypermethylation phenotype that dysregulates hematopoietic differentiation. In this study, we identified mutant R132H IDH1-specific gene signatures regulated by key transcription factors, particularly CEBPα, involved in myeloid differentiation and retinoid responsiveness. We show that treatment with all-trans retinoic acid (ATRA) at clinically achievable doses markedly enhanced terminal granulocytic differentiation in AML cell lines, primary patient samples, and a xenograft mouse model carrying mutant IDH1. Moreover, treatment with a cell-permeable form of 2-HG sensitized wild-type IDH1 AML cells to ATRA-induced myeloid differentiation, whereas inhibition of 2-HG production significantly reduced ATRA effects in mutant IDH1 cells. ATRA treatment specifically decreased cell viability and induced apoptosis of mutant IDH1 blasts in vitro. ATRA also reduced tumor burden of mutant IDH1 AML cells xenografted in NOD–Scid–IL2rγnull mice and markedly increased overall survival, revealing a potent antileukemic effect of ATRA in the presence of IDH1 mutation. This therapeutic strategy holds promise for this AML patient subgroup in future clinical studies. PMID:26951332

  13. Microsomal aldehyde dehydrogenase is localized to the endoplasmic reticulum via its carboxyl-terminal 35 amino acids

    PubMed Central

    1994-01-01

    Rat microsomal aldehyde dehydrogenase (msALDH) has no amino-terminal signal sequence, but instead it has a characteristic hydrophobic domain at the carboxyl terminus (Miyauchi, K., R. Masaki, S. Taketani, A. Yamamoto, A. Akayama, and Y. Tashiro. 1991. J. Biol. Chem. 266:19536- 19542). This membrane-bound enzyme is a useful model protein for studying posttranslational localization to its final destination. When expressed from cDNA in COS-1 cells, wild-type msALDH is localized exclusively in the well-developed ER. The removal of the hydrophobic domain results in the cytosolic localization of truncated proteins, thus suggesting that the portion is responsible for membrane anchoring. The last 35 amino acids of msALDH, including the hydrophobic domain, are sufficient for targeting of E. coli beta-galactosidase to the ER membrane. Further studies using chloramphenicol acetyltransferase fusion proteins suggest that two hydrophilic sequences on either side of the hydrophobic domain play an important role in ER targeting. PMID:8089174

  14. Engineering Acetyl Coenzyme A Supply: Functional Expression of a Bacterial Pyruvate Dehydrogenase Complex in the Cytosol of Saccharomyces cerevisiae

    PubMed Central

    Kozak, Barbara U.; van Rossum, Harmen M.; Luttik, Marijke A. H.; Akeroyd, Michiel; Benjamin, Kirsten R.; Wu, Liang; de Vries, Simon; Daran, Jean-Marc; Pronk, Jack T.

    2014-01-01

    ABSTRACT The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs+ reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. PMID:25336454

  15. Aldehyde dehydrogenase enzyme ALDH3H1 from Arabidopsis thaliana: Identification of amino acid residues critical for cofactor specificity.

    PubMed

    Stiti, Naim; Podgórska, Karolina; Bartels, Dorothea

    2014-03-01

    The cofactor-binding site of the NAD(+)-dependent Arabidopsis thaliana aldehyde dehydrogenase ALDH3H1 was analyzed to understand structural features determining cofactor-specificity. Homology modeling and mutant analysis elucidated important amino acid residues. Glu149 occupies a central position in the cofactor-binding cleft, and its carboxylate group coordinates the 2'- and 3'-hydroxyl groups of the adenosyl ribose ring of NAD(+) and repels the 2'-phosphate moiety of NADP(+). If Glu149 is mutated to Gln, Asp, Asn or Thr the binding of NAD(+) is altered and rendered the enzyme capable of using NADP(+). This change is attributed to a weaker steric hindrance and elimination of the electrostatic repulsion force of the 2'-phosphate of NADP(+). Simultaneous mutations of Glu149 and Ile200, which is situated opposite of the cofactor binding cleft, improved the enzyme efficiency with NADP(+). The double mutant ALDH3H1Glu149Thr/Ile200Val showed a good catalysis with NADP(+). Subsequently a triple mutation was generated by replacing Val178 by Arg in order to create a "closed" cofactor binding site. The cofactor specificity was shifted even further in favor of NADP(+), as the mutant ALDH3H1E149T/V178R/I200V uses NADP(+) with almost 7-fold higher catalytic efficiency compared to NAD(+). Our experiments suggest that residues occupying positions equivalent to 149, 178 and 200 constitute a group of amino acids in the ALDH3H1 protein determining cofactor affinity.

  16. Mechanism for enhanced 5-aminolevulinic acid fluorescence in isocitrate dehydrogenase 1 mutant malignant gliomas

    PubMed Central

    Kim, Ji Young; Kim, Sung Kwon; Kim, Seung-Ki; Park, Sung-Hye; Kim, Hyeonjin; Lee, Se-Hoon; Choi, Seung Hong; Park, Sunghyouk; Park, Chul-Kee

    2015-01-01

    Fluorescence-guided surgery using 5-aminolevulinic acid (5-ALA) has become the main treatment modality in malignant gliomas. However unlike glioblastomas, there are inconsistent result about fluorescence status in WHO grade III gliomas. Here, we show that mutational status of IDH1 is linked to 5-ALA fluorescence. Using genetically engineered malignant glioma cells harboring wild type (U87MG-IDH1WT) or mutant (U87MG-IDH1R132H) IDH1, we demonstrated a lag in 5-ALA metabolism and accumulation of protoporphyrin IX (PpIX) in U87MG-IDH1R132H cells. Next, we used liquid chromatography–mass spectrometry (LC-MS) to screen for tricarboxylic acid (TCA) cycle-related metabolite changes caused by 5-ALA exposure. We observed low baseline levels of NADPH, an essential cofactor for the rate-limiting step of heme degradation, in U87MG-IDH1R132H cells. High levels of NADPH are required to metabolize excessive 5-ALA, giving a plausible reason for the temporarily enhanced 5-ALA fluorescence in mutant IDH1 cells. This hypothesis was supported by the results of metabolic screening in human malignant glioma samples. In conclusion, we have discovered a relationship between enhanced 5-ALA fluorescence and IDH1 mutations in WHO grade III gliomas. Low levels of NADPH in tumors with mutated IDH1 is responsible for the enhanced fluorescence. PMID:26008980

  17. The effect of central chemical sympathectomy on the oxygen uptake; anaerobic glycolysis and lactic acid dehydrogenase activity in the retina of white rats.

    PubMed

    Pojda, S M; Brus, R

    1976-01-01

    Male Wistar rats were injected intraventricularly with two doses of 250 mcg of 6-hydroxydopamine (6-OHDA) in two consecutive days. Two weeks later the oxygen uptake, anaerobic glycolysis and lactic acid dehydrogenase (LDH) activity in the retina were determined. The decrease of oxygen uptake (-28%), anaerobic glycolysis (-31%) and LDH activity (-12%) in rats treated with 6-OHDA in comparison to control animals was found. The possible role of the adrenergic system in regulation of the metabolism of the retina is discussed.

  18. The catabolic function of the alpha-aminoadipic acid pathway in plants is associated with unidirectional activity of lysine-oxoglutarate reductase, but not saccharopine dehydrogenase.

    PubMed Central

    Zhu, X; Tang, G; Galili, G

    2000-01-01

    Whereas plants and animals use the alpha-aminoadipic acid pathway to catabolize lysine, yeast and fungi use the very same pathway to synthesize lysine. These two groups of organisms also possess structurally distinct forms of two enzymes in this pathway, namely lysine-oxoglutarate reductase (lysine-ketoglutarate reductase; LKR) and saccharopine dehydrogenase (SDH): in plants and animals these enzymes are linked on to a single bifunctional polypeptide, while in yeast and fungi they exist as separate entities. In addition, yeast LKR and SDH possess bi-directional activities, and their anabolic function is regulated by complex transcriptional and post-transcriptional controls, which apparently ascertain differential accumulation of intermediate metabolites; in plants, the regulation of the catabolic function of these two enzymes is not known. To elucidate the regulation of the catabolic function of plant bifunctional LKR/SDH enzymes, we have used yeast as an expression system to test whether a plant LKR/SDH also possesses bi-directional LKR and SDH activities, similar to the yeast enzymes. The Arabidopsis enzyme complemented a yeast SDH, but not LKR, null mutant. Identical results were obtained when deletion mutants encoding only the LKR or SDH domains of this bifunctional polypeptide were expressed individually in the yeast cells. Moreover, activity assays showed that the Arabidopsis LKR possessed catabolic, but not anabolic, activity, and its uni-directional activity stems from its structure rather than its linkage to SDH. Our results suggest that the uni-directional activity of LKR plays an important role in regulating the catabolic function of the alpha-amino adipic acid pathway in plants. PMID:10998364

  19. Metabolic bypass of the tricarboxylic acid cycle during lipid mobilization in germinating oilseeds. Regulation Of nad+-dependent isocitrate dehydrogenase versus fumarase

    PubMed

    Falk; Behal; Xiang; Oliver

    1998-06-01

    Biosynthesis of sucrose from triacylglycerol requires the bypass of the CO2-evolving reactions of the tricarboxylic acid (TCA) cycle. The regulation of the TCA cycle bypass during lipid mobilization was examined. Lipid mobilization in Brassica napus was initiated shortly after imbibition of the seed and proceeded until 2 d postimbibition, as measured by in vivo [1-14C]acetate feeding to whole seedlings. The activity of NAD+-isocitrate dehydrogenase (a decarboxylative enzyme) was not detected until 2 d postimbibition. RNA-blot analysis of B. napus seedlings demonstrated that the mRNA for NAD+-isocitrate dehydrogenase was present in dry seeds and that its level increased through the 4 d of the experiment. This suggested that NAD+-isocitrate dehydrogenase activity was regulated by posttranscriptional mechanisms during early seedling development but was controlled by mRNA level after the 2nd or 3rd d. The activity of fumarase (a component of the nonbypassed section of the TCA cycle) was low but detectable in B. napus seedlings at 12 h postimbibition, coincident with germination, and increased for the next 4 d. RNA-blot analysis suggested that fumarase activity was regulated primarily by the level of its mRNA during germination and early seedling development. It is concluded that posttranscriptional regulation of NAD+-isocitrate dehydrogenase activity is one mechanism of restricting carbon flux through the decarboxylative section of the TCA cycle during lipid mobilization in germinating oilseeds.

  20. Investigation of potential mechanisms regulating protein expression of hepatic pyruvate dehydrogenase kinase isoforms 2 and 4 by fatty acids and thyroid hormone.

    PubMed Central

    Holness, Mark J; Bulmer, Karen; Smith, Nicholas D; Sugden, Mary C

    2003-01-01

    Liver contains two pyruvate dehydrogenase kinases (PDKs), namely PDK2 and PDK4, which regulate glucose oxidation through inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Starvation increases hepatic PDK2 and PDK4 protein expression, the latter occurring, in part, via a mechanism involving peroxisome proliferator-activated receptor-alpha (PPARalpha). High-fat feeding and hyperthyroidism, which increase circulating lipid supply, enhance hepatic PDK2 protein expression, but these increases are insufficient to account for observed increases in hepatic PDK activity. Enhanced expression of PDK4, but not PDK2, occurs in part via a mechanism involving PPAR-alpha. Heterodimerization partners for retinoid X receptors (RXRs) include PPARalpha and thyroid-hormone receptors (TRs). We therefore investigated the responses of hepatic PDK protein expression to high-fat feeding and hyperthyroidism in relation to hepatic lipid delivery and disposal. High-fat feeding increased hepatic PDK2, but not PDK4, protein expression whereas hyperthyroidism increased both hepatic PDK2 and PDK4 protein expression. Both manipulations decreased the sensitivity of hepatic carnitine palmitoyltransferase I (CPT I) to suppression by malonyl-CoA, but only hyperthyrodism elevated plasma fatty acid and ketone-body concentrations and CPT I maximal activity. Administration of the selective PPAR-alpha activator WY14,643 significantly increased PDK4 protein to a similar extent in both control and high-fat-fed rats, but WY14,643 treatment and hyperthyroidism did not have additive effects on hepatic PDK4 protein expression. PPARalpha activation did not influence hepatic PDK2 protein expression in euthyroid rats, suggesting that up-regulation of PDK2 by hyperthyroidism does not involve PPARalpha, but attenuated the effect of hyperthyroidism to increase hepatic PDK2 expression. The results indicate that hepatic PDK4 up-regulation can be achieved by heterodimerization of either PPARalpha or

  1. Investigation of potential mechanisms regulating protein expression of hepatic pyruvate dehydrogenase kinase isoforms 2 and 4 by fatty acids and thyroid hormone.

    PubMed

    Holness, Mark J; Bulmer, Karen; Smith, Nicholas D; Sugden, Mary C

    2003-02-01

    Liver contains two pyruvate dehydrogenase kinases (PDKs), namely PDK2 and PDK4, which regulate glucose oxidation through inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Starvation increases hepatic PDK2 and PDK4 protein expression, the latter occurring, in part, via a mechanism involving peroxisome proliferator-activated receptor-alpha (PPARalpha). High-fat feeding and hyperthyroidism, which increase circulating lipid supply, enhance hepatic PDK2 protein expression, but these increases are insufficient to account for observed increases in hepatic PDK activity. Enhanced expression of PDK4, but not PDK2, occurs in part via a mechanism involving PPAR-alpha. Heterodimerization partners for retinoid X receptors (RXRs) include PPARalpha and thyroid-hormone receptors (TRs). We therefore investigated the responses of hepatic PDK protein expression to high-fat feeding and hyperthyroidism in relation to hepatic lipid delivery and disposal. High-fat feeding increased hepatic PDK2, but not PDK4, protein expression whereas hyperthyroidism increased both hepatic PDK2 and PDK4 protein expression. Both manipulations decreased the sensitivity of hepatic carnitine palmitoyltransferase I (CPT I) to suppression by malonyl-CoA, but only hyperthyrodism elevated plasma fatty acid and ketone-body concentrations and CPT I maximal activity. Administration of the selective PPAR-alpha activator WY14,643 significantly increased PDK4 protein to a similar extent in both control and high-fat-fed rats, but WY14,643 treatment and hyperthyroidism did not have additive effects on hepatic PDK4 protein expression. PPARalpha activation did not influence hepatic PDK2 protein expression in euthyroid rats, suggesting that up-regulation of PDK2 by hyperthyroidism does not involve PPARalpha, but attenuated the effect of hyperthyroidism to increase hepatic PDK2 expression. The results indicate that hepatic PDK4 up-regulation can be achieved by heterodimerization of either PPARalpha or

  2. Behavior of carboxylic acids upon complexation with beryllium compounds.

    PubMed

    Mykolayivna-Lemishko, Kateryna; Montero-Campillo, M Merced; Mó, Otilia; Yáñez, Manuel

    2014-07-31

    A significant acidity enhancement and changes on aromaticity were previously observed in squaric acid and its derivatives when beryllium bonds are present in those systems. In order to know if these changes on the chemical properties could be considered a general behavior of carboxylic acids upon complexation with beryllium compounds, complexes between a set of representative carboxylic acids RCOOH (formic acid, acetic acid, propanoic acid, benzoic acid, and oxalic acid) and beryllium compounds BeX2 (X = H, F, Cl) were studied by means of density functional theory calculations. Complexes that contain a dihydrogen bond or a OH···X interaction are the most stable in comparison with other possible BeX2 complexation patterns in which no other weak interactions are involved apart from the beryllium bond. Formic, acetic, propanoic, benzoic, and oxalic acid complexes with BeX2 are much stronger acids than their related free forms. The analysis of the topology of the electron density helps to clarify the reasons behind this acidity enhancement. Importantly, when the halogen atom is replaced by hydrogen in the beryllium compound, the dihydrogen bond complex spontaneously generates a new neutral complex [RCOO:BeH] in which a hydrogen molecule is lost. This seems to be a trend for carboxylic acids on complexing BeX2 compounds.

  3. Acid-induced folding of yeast alcohol dehydrogenase under low pH conditions.

    PubMed

    Le, W P; Yan, S X; Zhang, Y X; Zhou, H M

    1996-04-01

    Under conditions of low pH, the conformational states of holo-YADH and apo-YADH were examined by protein intrinsic fluorescence, ANS fluorescence, and far-UV CD measurements. The results obtained show that a low ionic strength, with the addition of HCl, the holo- and apo- YADH denatured gradually to reach the ultimate unfolded conformation in the vicinity of pH 2.0 and 2.5, respectively. With the decrease of pH from 7.0 to 2.0, the fluorescence emission decreased markedly, with its emission maximum red-shifting from 335 to 355 nm, indicating complete exposure of the buried tryptophan residues to the solvent. The far-UV CD spectra show the loss of the arrayed secondary structure, though the acid-denatured enzyme still maintained a partially arrayed secondary structure. A further decrease in pH by increasing the concentration of HClO4 induced a cooperative folding of the denatured enzyme to a compact conformation with the properties of a molten globule, described previously by Goto et al. [Proc. Natl. Acad. Sci. USA 87, 573-577 (1990)]. More extensive studies showed that although apo-YADH and holo-YADH exhibited similar behavior, the folding cooperative ability of apo-YADH was lower than that of the holo-enzyme. From the above results, it is suggested that the zinc ion plays an important role in the proper folding of YADH and in stabilizing its native conformation.

  4. Role of Lipoylation of the Immunodominant Epitope of Pyruvate Dehydrogenase Complex: Toward a Peptide-Based Diagnostic Assay for Primary Biliary Cirrhosis.

    PubMed

    Pacini, Giulia; Carotenuto, Alfonso; Rentier, Cedric; Nuti, Francesca; Real-Fernandez, Feliciana; Brancaccio, Diego; Sabatino, Giuseppina; Larregola, Maud; Peroni, Elisa; Migliorini, Paola; Novellino, Ettore; Battezzati, Pier Maria; Selmi, Carlo; Papini, Anna Maria; Rovero, Paolo

    2015-08-27

    Primary biliary cirrhosis is an immune-mediated chronic liver disease whose diagnosis relies on the detection of serum antimitochondrial antibodies directed against a complex set of proteins, among which pyruvate dehydrogenase complex is considered the main autoantigen. We studied the immunological role of the lipoyl domain of this protein using synthetic lipoylated peptides, showing that the lipoyl chain chirality does not affect autoantibody recognition and, most importantly, confirming that both lipoylated and unlipoylated peptides are able to recognize specific autoantibodies in patients sera. In fact, 74% of patients sera recognize at least one of the tested peptides but very few positive sera recognized exclusively the lipoylated peptide, suggesting that the lipoamide moiety plays a marginal role within the autoreactive epitope. These results are supported by a conformational analysis showing that the lipoyl moiety of pyruvate dehydrogenase complex appears to be involved in hydrophobic interactions, which may limit its exposition and thus its contribution to the complex antigenic epitope. A preliminary analysis of the specificity of the two most active peptides indicates that they could be part of a panel of synthetic antigens collectively able to mimic in a simple immunoenzymatic assay the complex positivity pattern detected in immunofluorescence. PMID:26214254

  5. 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). PMID:24742683

  6. Supplementation of medium with diammonium hydrogen phosphate enhanced the D-lactate dehydrogenase levels leading to increased D-lactic acid productivity.

    PubMed

    Singhvi, Mamata; Jadhav, Akanksha; Gokhale, Digambar

    2013-10-01

    The production of D-lactic acid by Lactobacillus lactis RM2-24 was investigated using modified media to increase the efficiency of the fermentation process. The results indicated that the addition of 5 g/l peptone and 1 g/l (NH4)2HPO4 enhanced D-lactic acid production by 32%, as compared to that obtained from non supplemented media, with a productivity of 3.0 g/l/h. Lactate dehydrogenase (LDH) expression profile in these different media was studied which resulted in appearance of additional LDH isoform produced by cells when they were grown in HSYE supplemented with (NH4)2HPO4. The additional LDH appears to be L-LDH contributing to production of L-lactic acid in the fermented broth. This is totally new information in the lactic acid fermentation and could be very useful to industries engaged in D-lactic acid production. PMID:23932744

  7. Supplementation of medium with diammonium hydrogen phosphate enhanced the D-lactate dehydrogenase levels leading to increased D-lactic acid productivity.

    PubMed

    Singhvi, Mamata; Jadhav, Akanksha; Gokhale, Digambar

    2013-10-01

    The production of D-lactic acid by Lactobacillus lactis RM2-24 was investigated using modified media to increase the efficiency of the fermentation process. The results indicated that the addition of 5 g/l peptone and 1 g/l (NH4)2HPO4 enhanced D-lactic acid production by 32%, as compared to that obtained from non supplemented media, with a productivity of 3.0 g/l/h. Lactate dehydrogenase (LDH) expression profile in these different media was studied which resulted in appearance of additional LDH isoform produced by cells when they were grown in HSYE supplemented with (NH4)2HPO4. The additional LDH appears to be L-LDH contributing to production of L-lactic acid in the fermented broth. This is totally new information in the lactic acid fermentation and could be very useful to industries engaged in D-lactic acid production.

  8. Demonstration of carbon-carbon bond cleavage of acetyl coenzyme A by using isotopic exchange catalyzed by the CO dehydrogenase complex from acetate-grown Methanosarcina thermophila.

    PubMed Central

    Raybuck, S A; Ramer, S E; Abbanat, D R; Peters, J W; Orme-Johnson, W H; Ferry, J G; Walsh, C T

    1991-01-01

    The purified nickel-containing CO dehydrogenase complex isolated from methanogenic Methanosarcina thermophila grown on acetate is able to catalyze the exchange of [1-14C] acetyl-coenzyme A (CoA) (carbonyl group) with 12CO as well as the exchange of [3'-32P]CoA with acetyl-CoA. Kinetic parameters for the carbonyl exchange have been determined: Km (acetyl-CoA) = 200 microM, Vmax = 15 min-1. CoA is a potent inhibitor of this exchange (Ki = 25 microM) and is formed under the assay conditions because of a slow but detectable acetyl-CoA hydrolase activity of the enzyme. Kinetic parameters for both exchanges are compared with those previously determined for the acetyl-CoA synthase/CO dehydrogenase from the acetogenic Clostridium thermoaceticum. Collectively, these results provide evidence for the postulated role of CO dehydrogenase as the key enzyme for acetyl-CoA degradation in acetotrophic bacteria. PMID:1987173

  9. Mycophenolic acid inhibits inosine 5'-monophosphate dehydrogenase and suppresses production of pro-inflammatory cytokines, nitric oxide, and LDH in macrophages.

    PubMed

    Jonsson, Charlotte A; Carlsten, Hans

    2002-01-01

    Mycophenolic acid (MPA) inhibits reversibly inosine 5(')-monophosphate dehydrogenase, an enzyme involved in the de novo synthesis of guanine nucleotides. Previously, mycophenolate mofetil (MMF), the pro-drug of MPA, was shown to exert beneficial effects on the systemic lupus erythematosus (SLE)-like disease in MRLlpr/lpr mice. In this study MPA's immunomodulating effects in vitro on the murine macrophage cell line IC-21 were investigated. The cells were exposed to MPA together with lipopolysaccharide and IFN-gamma. Cytokine, NO(2)(-), and lactate dehydrogenase levels in supernatants and cell lysates were analysed as well as the proliferation of IC-21 cells. MPA exposure reduced the total levels of all molecules investigated and suppressed the proliferation. All MPA-induced effects were reversed by the addition of guanosine to the cultures. Since macrophages play a role in lupus nephritis, our results indicate that modulation of macrophages may be involved in the ameliorating effects of MMF in SLE. PMID:12381354

  10. Characterization of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate and malate dehydrogenases.

    PubMed

    Goullet, P; Picard, B

    1988-02-01

    Acid phosphatase, esterases, and glutamate and malate dehydrogenases of 192 strains of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gels. The six species were clearly separated from each other by their distinct enzyme electrophoretic polymorphism. For Y. enterocolitica, the strains of biotype 5 were differentiated from the other biotypes by the mobility of glutamate dehydrogenase. For Y. frederiksenii, six zymotypes were delineated by pI and by the mobility of the enzymes. Variation in number or mobility of esterases within each species could represent a marker for epidemiological and ecological analyses. A linear relationship was obtained between the mean genetic diversity coefficient of enzymes and the mean percentage DNA-DNA relatedness of Y. intermedia, Y. aldovae, Y. enterocolitica and Y. frederiksenii.

  11. Sulfur-rich zinc chemistry: new tris(thioimidazolyl)hydroborate ligands and their zinc complex chemistry related to the structure and function of alcohol dehydrogenase.

    PubMed

    Tesmer, M; Shu, M; Vahrenkamp, H

    2001-07-30

    The 1-substituted tris(2-thioimidazolyl)hydroborate ligands Tt(R) were prepared as the potassium salts from KBH(4) and the corresponding 1-R-2-thioimidazole for R = t-Bu and C(6)H(4)-p-CH(CH(3))(2) (Cum). Their reactions with zinc salts yielded the tetrahedral complexes Tt(R)Zn-X with X = F, Cl, ONO(2) and (Tt(t)()(-)(Bu))(2)Zn. With zinc perchlorate the labile perchlorate complexes Tt(R)Zn-OClO(3) were obtained. They served as starting materials for the incorporation of substrates which are relevant for the chemistry of horse liver alcohol dehydrogenase: Ethanol led to [Tt(t)()(-Bu)Zn.EtOH] ClO(4).EtOH, p-nitrophenol (NitOH) yielded Tt(Cum)Zn-ONit. Pyridine-2-carbaldehyde and salicylic aldehyde were incorporated as N(pyridine) and O(phenolate) coligands with possible additional O(aldehyde) coordination. Substituted pyridyl methanols (R-PyCH(2)OH) yielded the trinuclear complexes [(Tt(t)()(-Bu))(2)Zn(3)(R-PyCH(2)O)(2)] (ClO(4))(2) with bridging Tt and pyridylmethoxide ligands. Preliminary experiments on the functional modeling of alcohol dehydrogenase have shown that TtZn complexes promote both the dehydrogenation of 2-propanol and the hydrogenation of pentafluorobenzaldehyde. PMID:11466063

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

    PubMed

    Sazanov, L A; Burrows, P A; Nixon, P J

    1998-02-01

    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.

  13. L-Galactono-1,4-lactone dehydrogenase is an assembly factor of the membrane arm of mitochondrial complex I in Arabidopsis.

    PubMed

    Schimmeyer, Joram; Bock, Ralph; Meyer, Etienne H

    2016-01-01

    L-Galactono-1,4-lactone dehydrogenase (GLDH) catalyses the last enzymatic step of the ascorbate biosynthetic pathway in plants. GLDH is localised to mitochondria and several reports have shown that GLDH is associated with complex I of the respiratory chain. In a gldh knock-out mutant, complex I is not detectable, suggesting that GLDH is essential for complex I assembly or stability. GLDH has not been identified as a genuine complex I subunit, instead, it is present in a smaller, lowly abundant version of complex I called complex I*. In addition, GLDH activity has also been detected in smaller protein complexes within mitochondria membranes. Here, we investigated the role of GLDH during complex I assembly. We identified GLDH in complexes co-localising with some complex I assembly intermediates. Using a mutant that accumulates complex I assembly intermediates, we confirmed that GLDH is associated with the complex I assembly intermediates of 400 and 450 kDa. In addition, we detected accumulation of the 200 kDa complex I assembly intermediate in the gldh mutant. Taken together, our data suggest that GLDH is an assembly factor of the membrane arm of complex I. This function appears to be independent of the role of GLDH in ascorbate synthesis, as evidenced by the ascorbate-deficient mutant vtc2-1 accumulating wild-type levels of complex I. Therefore, we propose that GLDH is a dual-function protein that has a second, non-enzymatic function in complex I assembly as a plant-specific assembly factor. We propose an updated model for complex I assembly that includes complex I* as an assembly intermediate.

  14. Additive effects of clofibric acid and pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high-saturated fat diet

    PubMed Central

    Hwang, Byounghoon; Wu, Pengfei; Harris, Robert A.

    2012-01-01

    SUMMARY Although improving glucose metabolism by inhibition of pyruvate dehydrogenase kinase 4 (PDK4) might prove beneficial in the treatment of type 2 diabetes or diet-induced obesity, it might induce detrimental effects by inhibiting fatty acid oxidation. PPARα agonists are often used to treat dyslipidemia in patients, especially in type 2 diabetes. Combinational treatment with a PDK4 inhibitor and PPARα agonists may prove beneficial. However, PPARα agonists may be less effective in the presence of a PDK4 inhibitor because PPARα agonists induce PDK4 expression. In the present study, the effects of clofibric acid, a PPARα agonist, on blood and liver lipids were determined in wild type and PDK4 knockout mice fed a high fat diet. As expected, treatment of wild type mice with clofibric acid resulted in less body weight gain, smaller epididymal fat pads, greater insulin sensitivity, and lower levels of serum and liver triacylglycerol. Surprisingly, rather than decreasing the effectiveness of clofibric acid, PDK4 deficiency enhanced the beneficial effects of clofibric acid on hepatic steatosis, lowered blood glucose levels, and did not prevent the positive effects of clofibric acid on serum triacylglycerols and free fatty acids. The metabolic effects of clofibric acid are therefore independent of the induction of PDK4 expression. The additive beneficial effects on hepatic steatosis may be due to induction of increased capacity for fatty acid oxidation and partial uncoupling of oxidative phosphorylation by clofibric acid and a reduction in the capacity for fatty acid synthesis by PDK4 deficiency. PMID:22429297

  15. Major Role of NAD-Dependent Lactate Dehydrogenases in the Production of l-Lactic Acid with High Optical Purity by the Thermophile Bacillus coagulans

    PubMed Central

    Wang, Limin; Cai, Yumeng; Zhu, Lingfeng; Guo, Honglian

    2014-01-01

    Bacillus coagulans 2-6 is an excellent producer of optically pure l-lactic acid. However, little is known about the mechanism of synthesis of the highly optically pure l-lactic acid produced by this strain. Three enzymes responsible for lactic acid production—NAD-dependent l-lactate dehydrogenase (l-nLDH; encoded by ldhL), NAD-dependent d-lactate dehydrogenase (d-nLDH; encoded by ldhD), and glycolate oxidase (GOX)—were systematically investigated in order to study the relationship between these enzymes and the optical purity of lactic acid. Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (a d-lactic acid producer) and Lactobacillus plantarum subsp. plantarum DSM 20174 (a dl-lactic acid producer) were also examined in this study as comparative strains, in addition to B. coagulans. The specific activities of key enzymes for lactic acid production in the three strains were characterized in vivo and in vitro, and the levels of transcription of the ldhL, ldhD, and GOX genes during fermentation were also analyzed. The catalytic activities of l-nLDH and d-nLDH were different in l-, d-, and dl-lactic acid producers. Only l-nLDH activity was detected in B. coagulans 2-6 under native conditions, and the level of transcription of ldhL in B. coagulans 2-6 was much higher than that of ldhD or the GOX gene at all growth phases. However, for the two Lactobacillus strains used in this study, ldhD transcription levels were higher than those of ldhL. The high catalytic efficiency of l-nLDH toward pyruvate and the high transcription ratios of ldhL to ldhD and ldhL to the GOX gene provide the key explanations for the high optical purity of l-lactic acid produced by B. coagulans 2-6. PMID:25217009

  16. Major Role of NAD-Dependent Lactate Dehydrogenases in the Production of l-Lactic Acid with High Optical Purity by the Thermophile Bacillus coagulans.

    PubMed

    Wang, Limin; Cai, Yumeng; Zhu, Lingfeng; Guo, Honglian; Yu, Bo

    2014-12-01

    Bacillus coagulans 2-6 is an excellent producer of optically pure l-lactic acid. However, little is known about the mechanism of synthesis of the highly optically pure l-lactic acid produced by this strain. Three enzymes responsible for lactic acid production-NAD-dependent l-lactate dehydrogenase (l-nLDH; encoded by ldhL), NAD-dependent d-lactate dehydrogenase (d-nLDH; encoded by ldhD), and glycolate oxidase (GOX)-were systematically investigated in order to study the relationship between these enzymes and the optical purity of lactic acid. Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (a d-lactic acid producer) and Lactobacillus plantarum subsp. plantarum DSM 20174 (a dl-lactic acid producer) were also examined in this study as comparative strains, in addition to B. coagulans. The specific activities of key enzymes for lactic acid production in the three strains were characterized in vivo and in vitro, and the levels of transcription of the ldhL, ldhD, and GOX genes during fermentation were also analyzed. The catalytic activities of l-nLDH and d-nLDH were different in l-, d-, and dl-lactic acid producers. Only l-nLDH activity was detected in B. coagulans 2-6 under native conditions, and the level of transcription of ldhL in B. coagulans 2-6 was much higher than that of ldhD or the GOX gene at all growth phases. However, for the two Lactobacillus strains used in this study, ldhD transcription levels were higher than those of ldhL. The high catalytic efficiency of l-nLDH toward pyruvate and the high transcription ratios of ldhL to ldhD and ldhL to the GOX gene provide the key explanations for the high optical purity of l-lactic acid produced by B. coagulans 2-6.

  17. Photochemical properties of copper(II)-amino acid complexes

    SciTech Connect

    Natarajan, P.; Ferraudi, G.

    1981-11-01

    The photochemistry of copper(II)-amino acid complexes (amino acid = glutamic acid, ..beta..-alanine, or glycine) has been investigated by continuous and flash photolysis. The charge-transfer irradiations induce the oxidation of the ligand and the reduction of copper(II) to copper(I). Transients observed in flash photolysis have been assigned as copper-alkyl complexes. Moreover, other copper-alkyl species are produced when carbon-centered radicals react with excess of copper(II) complexes. The photochemical properties of the copper(II)-amino acid complexes are discussed in terms of population of charge transfer to copper excited states.

  18. Species-specific differences in tissue-specific expression of alcohol dehydrogenase are under the control of complex cis-acting loci: Evidence from Drosophila hybrids

    SciTech Connect

    Ranganayakulu, G.; Reddy, A.R. ); Kirkpatrick, R.B.; Martin, P.F. )

    1991-12-01

    Differences in the expression of alcohol dehydrogenase in the hindgut and testis of adult Drosophila virilis, D. texana, D. novamexicana and D. borealis flies were observed. These heritable differences do not arise due to chromosomal rearrangements, since the polytene chromosome banding patterns did not reveal any such gross chromosomal rearrangements near the Adh locus in any of the tested species. Analysis of the interspecific hybrids revealed that these differences are controlled by complex cis-acting genetic loci. Further, the cis-acting locus controlling the expression of ADH in testis was found to be separable by crossing-over.

  19. Expression, crystallization and preliminary X-ray crystallographic analysis of glucose-6-phosphate dehydrogenase from the human pathogen Trypanosoma cruzi in complex with substrate

    PubMed Central

    Ortíz, Cecilia; Larrieux, Nicole; Medeiros, Andrea; Botti, Horacio; Comini, Marcelo; Buschiazzo, Alejandro

    2011-01-01

    An N-terminally truncated version of the enzyme glucose-6-phosphate dehydrogenase from Trypanosoma cruzi lacking the first 37 residues was crystallized both in its apo form and in a binary complex with glucose 6-­phosphate. The crystals both belonged to space group P21 and diffracted to 2.85 and 3.35 Å resolution, respectively. Self-rotation function maps were consistent with point group 222. The structure was solved by molecular replacement, confirming a tetrameric quaternary structure. PMID:22102256

  20. Overexpression of the NADP+-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L.

    PubMed

    Kobayashi, Keiichi; Hattori, Takasumi; Hayashi, Rie; Kirimura, Kohtaro

    2014-01-01

    In the tricarboxylic acid (TCA) cycle, NADP(+)-specific isocitrate dehydrogenase (NADP(+)-ICDH) catalyzes oxidative decarboxylation of isocitric acid to form α-ketoglutaric acid with NADP(+) as a cofactor. We constructed an NADP(+)-ICDH gene (icdA)-overexpressing strain (OPI-1) using Aspergillus niger WU-2223L as a host and examined the effects of increase in NADP(+)-ICDH activity on citric acid production. Under citric acid-producing conditions with glucose as the carbon source, the amounts of citric acid produced and glucose consumed by OPI-1 for the 12-d cultivation period decreased by 18.7 and 10.5%, respectively, compared with those by WU-2223L. These results indicate that the amount of citric acid produced by A. niger can be altered with the NADP(+)-ICDH activity. Therefore, NADP(+)-ICDH is an important regulator of citric acid production in the TCA cycle of A. niger. Thus, we propose that the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering.

  1. JWH-018 ω-OH, a shared hydroxy metabolite of the two synthetic cannabinoids JWH-018 and AM-2201, undergoes oxidation by alcohol dehydrogenase and aldehyde dehydrogenase enzymes in vitro forming the carboxylic acid metabolite.

    PubMed

    Holm, Niels Bjerre; Noble, Carolina; Linnet, Kristian

    2016-09-30

    Synthetic cannabinoids are new psychoactive substances (NPS) acting as agonists at the cannabinoid receptors. The aminoalkylindole-type synthetic cannabinoid naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) was among the first to appear on the illicit drug market and its metabolism has been extensively investigated. The N-pentyl side chain is a major site of human cytochrome P450 (CYP)-mediated oxidative metabolism, and the ω-carboxylic acid metabolite appears to be a major in vivo human urinary metabolite. This metabolite is, however, not formed to any significant extent in human liver microsomal (HLM) incubations raising the possibility that the discrepancy is due to involvement of cytosolic enzymes. Here we demonstrate in incubations with human liver cytosol (HLC), that JWH-018 ω-OH, but not the JWH-018 parent compound, is a substrate for nicotinamide adenine dinucleotide (NAD(+))-dependent alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. The sole end-product identified in HLC was the JWH-018 ω-COOH metabolite, while trapping tests with methoxyamine proved the presence of the aldehyde intermediate. ADH/ALDH and UDP-glucuronosyl-transferases (UGT) enzymes may therefore both act on the JWH-018 ω-OH substrate. Finally, we note that for [1-(5-fluoropentyl)indol-3-yl]-naphthalen-1-yl-methanone (AM-2201), the ω-fluorinated analog of JWH-018, a high amount of JWH-018 ω-OH was formed in HLM incubated without NADPH, suggesting that the oxidative defluorination is efficiently catalyzed by non-CYP enzyme(s). The pathway presented here may therefore be especially important for N-(5-fluoropentyl) substituted synthetic cannabinoids, because the oxidative defluorination can occur even if the CYP-mediated metabolism preferentially takes place on other parts of the molecule than the N-alkyl side chain. Controlled clinical studies in humans are ultimately required to demonstrate the in vivo importance of the oxidation pathway presented here

  2. JWH-018 ω-OH, a shared hydroxy metabolite of the two synthetic cannabinoids JWH-018 and AM-2201, undergoes oxidation by alcohol dehydrogenase and aldehyde dehydrogenase enzymes in vitro forming the carboxylic acid metabolite.

    PubMed

    Holm, Niels Bjerre; Noble, Carolina; Linnet, Kristian

    2016-09-30

    Synthetic cannabinoids are new psychoactive substances (NPS) acting as agonists at the cannabinoid receptors. The aminoalkylindole-type synthetic cannabinoid naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) was among the first to appear on the illicit drug market and its metabolism has been extensively investigated. The N-pentyl side chain is a major site of human cytochrome P450 (CYP)-mediated oxidative metabolism, and the ω-carboxylic acid metabolite appears to be a major in vivo human urinary metabolite. This metabolite is, however, not formed to any significant extent in human liver microsomal (HLM) incubations raising the possibility that the discrepancy is due to involvement of cytosolic enzymes. Here we demonstrate in incubations with human liver cytosol (HLC), that JWH-018 ω-OH, but not the JWH-018 parent compound, is a substrate for nicotinamide adenine dinucleotide (NAD(+))-dependent alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. The sole end-product identified in HLC was the JWH-018 ω-COOH metabolite, while trapping tests with methoxyamine proved the presence of the aldehyde intermediate. ADH/ALDH and UDP-glucuronosyl-transferases (UGT) enzymes may therefore both act on the JWH-018 ω-OH substrate. Finally, we note that for [1-(5-fluoropentyl)indol-3-yl]-naphthalen-1-yl-methanone (AM-2201), the ω-fluorinated analog of JWH-018, a high amount of JWH-018 ω-OH was formed in HLM incubated without NADPH, suggesting that the oxidative defluorination is efficiently catalyzed by non-CYP enzyme(s). The pathway presented here may therefore be especially important for N-(5-fluoropentyl) substituted synthetic cannabinoids, because the oxidative defluorination can occur even if the CYP-mediated metabolism preferentially takes place on other parts of the molecule than the N-alkyl side chain. Controlled clinical studies in humans are ultimately required to demonstrate the in vivo importance of the oxidation pathway presented here.

  3. Identification of D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in Corynebacterium glutamicum.

    PubMed

    Lee, Jung-Hoon; Kim, Yong-Jae; Shin, Hee-Sung; Lee, Heung-Shick; Jin, Shouguang; Ha, Un-Hwan

    2016-06-01

    Expression of a putative acyltransferase encoded by NCgl- 0350 of Corynebacterium glutamicum is induced by cell-free culture fluids obtained from stationary-phase growth of both C. glutamicum and Pseudomonas aeruginosa, providing evidence for interspecies communication. Here, we further confirmed that such communication occurs by showing that acyltransferase expression is induced by culture fluid obtained from diverse Gram-negative and -positive bacterial strains, including Escherichia coli, Salmonella Typhimurium, Bacillus subtilis, Staphylococcus aureus, Mycobacterium sp. strain JC1, and Mycobacterium smegmatis. A homologous acyltransferase encoded by PA5238 of P. aeruginosa was also induced by fluids obtained from P. aeruginosa as well as other bacterial strains, as observed for NCgl0350 of C. glutamicum. Because C. glutamicum is difficult to study using molecular approaches, the homologous gene PA5238 of P. aeruginosa was used to identify PA5309 as an upstream regulator of expression. A homologous D-amino acid dehydrogenase encoded by NCgl- 2909 of C. glutamicum was cloned based on amino acid similarity to PA5309, and its role in the regulation of NCgl0350 expression was confirmed. Moreover, NCgl2909 played positive roles in growth of C. glutamicum. Thus, we identified a D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in C. glutamicum. PMID:27225460

  4. Pyruvate dehydrogenase alpha 1 as a target of omega-3 polyunsaturated fatty acids in human prostate cancer through a global phosphoproteomic analysis.

    PubMed

    Zhao, Heng; Pflug, Beth R; Lai, Xianyin; Wang, Mu

    2016-09-01

    Prostate cancer is one of the leading cancers in men. Taking dietary supplements, such as fish oil (FO), which is rich in n-3 polyunsaturated fatty acids (PUFAs), has been employed as a strategy to lower prostate cancer risk and control disease progression. In this study, we investigated the global phosphoproteomic changes induced by FO using a combination of phosphoprotein-enrichment strategy and high-resolution tandem mass spectrometry. We found that FO induces many more phosphorylation changes than oleic acid when they both are compared to control group. Quantitative comparison between untreated group and FO- or oleic acid-treated groups uncovered a number of important protein phosphorylation changes induced by n-3PUFAs. This phosphoproteomic discovery study and the follow-up Western Blot validation study elucidate that phosphorylation levels of the two regulatory serine residues in pyruvate dehydrogenase alpha 1 (PDHA1), serine-232 and serine-300, are significantly decreased upon FO treatment. As expected, increased pyruvate dehydrogenase activity was also observed. This study suggests that FO-induced phosphorylation changes in PDHA1 is more likely related to the glucose metabolism pathway, and n-3 PUFAs may have a role in controlling the balance between lipid and glucose oxidation. PMID:27357730

  5. A dynamic loop at the active center of the Escherichia coli pyruvate dehydrogenase complex E1 component modulates substrate utilization and chemical communication with the E2 component.

    PubMed

    Kale, Sachin; Arjunan, Palaniappa; Furey, William; Jordan, Frank

    2007-09-21

    Our crystallographic studies have shown that two active center loops (an inner loop formed by residues 401-413 and outer loop formed by residues 541-557) of the E1 component of the Escherichia coli pyruvate dehydrogenase complex become organized only on binding a substrate analog that is capable of forming a stable thiamin diphosphate-bound covalent intermediate. We showed that residue His-407 on the inner loop has a key role in the mechanism, especially in the reductive acetylation of the E. coli dihydrolipoamide transacetylase component, whereas crystallographic results showed a role of this residue in a disorder-order transformation of these two loops, and the ordered conformation gives rise to numerous new contacts between the inner loop and the active center. We present mapping of the conserved residues on the inner loop. Kinetic, spectroscopic, and crystallographic studies on some inner loop variants led us to conclude that charged residues flanking His-407 are important for stabilization/ordering of the inner loop thereby facilitating completion of the active site. The results further suggest that a disorder to order transition of the dynamic inner loop is essential for substrate entry to the active site, for sequestering active site chemistry from undesirable side reactions, as well as for communication between the E1 and E2 components of the E. coli pyruvate dehydrogenase multienzyme complex.

  6. [Polymorphism of the gene for subunit 6 of the NADh dehydrogenase complex (ND6) in ethnic russian population in Russia].

    PubMed

    Kornienko, I V; Vodolazhskiĭ, D I; Mikhalkovich, L S; Pavlichenko, G N; Ivanov, P L

    2003-01-01

    A sample of ethnic Russians of Russia was tested for polymorphism of the NADH dehydrogenase subunit 6 (ND6) gene mapping to the mtDNA region 14,170-14,569. Genetic diversity of ND6 haplotypes was estimated at 0.406, and probability of haplotype random match, at 0.598. Combined with typing the mtDNA control region, analysis of the ND6 gene polymorphism was assumed to improve the reliability of forensic identification. Several point substitutions in the ND6 gene region proved to be associated with particular transitions in the mtDNA control region; the association was characterized with the phi coefficient.

  7. Identification and molecular characterization of the aco genes encoding the Pelobacter carbinolicus acetoin dehydrogenase enzyme system.

    PubMed Central

    Oppermann, F B; Steinbüchel, A

    1994-01-01

    Use of oligonucleotide probes, which were deduced from the N-terminal sequences of the purified enzyme components, identified the structural genes for the alpha and beta subunits of E1 (acetoin:2,6-dichlorophenolindophenol oxidoreductase), E2 (dihydrolipoamide acetyltransferase), and E3 (dihydrolipoamide dehydrogenase) of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system, which were designated acoA, acoB, acoC, and acoL, respectively. The nucleotide sequences of acoA (979 bp), acoB (1,014 bp), acoC (1,353 bp), and acoL (1,413 bp) as well as of acoS (933 bp), which encodes a protein with an M(r) of 34,421 exhibiting 64.7% amino acid identity to the Escherichia coli lipA gene product, were determined. These genes are clustered on a 6.1-kbp region. Heterologous expression of acoA, acoB, acoC, acoL, and acoS in E. coli was demonstrated. The amino acid sequences deduced from acoA, acoB, acoC, and acoL for E1 alpha (M(r), 34,854), E1 beta (M(r), 36,184), E2 (M(r), 47,281), and E3 (M(r), 49,394) exhibited striking similarities to the amino acid sequences of the components of the Alcaligenes eutrophus acetoin-cleaving system. Homologies of up to 48.7% amino acid identity to the primary structures of the enzyme components of various 2-oxo acid dehydrogenase complexes also were found. In addition, the respective genes of the 2-oxo acid dehydrogenase complexes and of the acetoin dehydrogenase enzyme system were organized very similarly, indicating a close relationship of the P. carbinolicus acetoin dehydrogenase enzyme system to 2-oxo acid dehydrogenase complexes. Images PMID:8110297

  8. Ellagic acid protects against arsenic toxicity in isolated rat mitochondria possibly through the maintaining of complex II.

    PubMed

    Keshtzar, E; Khodayar, M J; Javadipour, M; Ghaffari, M A; Bolduc, D L; Rezaei, M

    2016-10-01

    Chronic arsenic exposure has been linked to many health problems including diabetes and cancer. In the present study, we assessed the protective effect of ellagic acid (EA) against toxicity induced by arsenic in isolated rat liver mitochondria. Reactive oxygen species (ROS) and mitochondrial membrane potential decline were assayed using dichlorofluorescein diacetate and rhodamine 123, respectively, and dehydrogenase activity obtained by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide conversion assay. Arsenic increased ROS levels and mitochondrial dysfunction, which led to a reduction in mitochondrial total dehydrogenase activity. Mitochondria pretreated with EA exposed to arsenic at various concentrations led to a reversal of ROS production and mitochondrial damage. Our results showed that mitochondria were significantly affected when exposed to arsenic, which resulted in excessive ROS production and mitochondrial membrane disruption. Pretreatment with EA, reduced ROS amounts, mitochondrial damage, and restored total dehydrogenase activity specifically associated with mitochondrial complex II. EA protective characteristics may be accomplished particularly throughout the mitochondrial maintenance either directly by its antioxidant property or indirectly through its maintaining of complex II. These findings also suggest a potential role for EA in treating or preventing mitochondria associated disorders.

  9. Crystal structure of a ternary complex of D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei, NAD+ and 2-oxoisocaproate at 1.9 A resolution.

    PubMed

    Dengler, U; Niefind, K; Kiess, M; Schomburg, D

    1997-04-01

    D-2-hydroxyisocaproate dehydrogenase (D-HicDH) from Lactobacillus casei is a homodimer with 333 amino acids and a molecular mass of 37 kDa per subunit. The enzyme belongs to the protein family of NAD+-dependent D-2-hydroxycarboxylate dehydrogenases and within this family to the subgroup of D-lactate dehydrogenases (D-LDHs). Compared with other D-LDHs D-HicDH is characterized by a very low specificity regarding size and chemical constitution of the accepted D-2-hydroxycarboxylates. Hexagonal crystals of recombinant D-HicDH in the presence of NAD+ and 2-oxoisocaproate (4-methyl-2-oxopentanoate) were grown with ammonium sulphate as precipitating agent. The structure of these crystals was solved by molecular replacement and refined to a final R-factor of 19.6% for all measured X-ray reflections in the resolution range (infinity to 1.86 A). Both NAD+ and 2-oxoisocaproate were identified in the electron density map; binding of the latter in the active site, however, competes with a sulphate ion, which is also defined by electron density. Additionally the final model contains 182 water molecules and a second sulphate ion. The binding of both an in vitro substrate and the natural cosubstrate in the active site provides substantial insight into the catalytic mechanism and allows us to assess previously published active site models for this enzyme family, in particular the two most controversial points, the role of the conserved Arg234 and substrate binding. Furthermore the overall topology and details of the D-HicDH structure are described, discussed against the background of homologous structures and compared with one closely and one distantly related protein.

  10. Rationally re-designed mutation of NAD-independent l-lactate dehydrogenase: high optical resolution of racemic mandelic acid by the engineered Escherichia coli

    PubMed Central

    2012-01-01

    Background NAD-independent l-lactate dehydrogenase (l-iLDH) from Pseudomonas stutzeri SDM can potentially be used for the kinetic resolution of small aliphatic 2-hydroxycarboxylic acids. However, this enzyme showed rather low activity towards aromatic 2-hydroxycarboxylic acids. Results Val-108 of l-iLDH was changed to Ala by rationally site-directed mutagenesis. The l-iLDH mutant exhibited much higher activity than wide-type l-iLDH towards l-mandelate, an aromatic 2-hydroxycarboxylic acid. Using the engineered Escherichia coli expressing the mutant l-iLDH as a biocatalyst, 40 g·L-1 of dl-mandelic acid was converted to 20.1 g·L-1 of d-mandelic acid (enantiomeric purity higher than 99.5%) and 19.3 g·L-1 of benzoylformic acid. Conclusions A new biocatalyst with high catalytic efficiency toward an unnatural substrate was constructed by rationally re-design mutagenesis. Two building block intermediates (optically pure d-mandelic acid and benzoylformic acid) were efficiently produced by the one-pot biotransformation system. PMID:23176608

  11. Succinic semialdehyde dehydrogenase deficiency (SSADHD): Pathophysiological complexity and multifactorial trait associations in a rare monogenic disorder of GABA metabolism.

    PubMed

    Malaspina, P; Roullet, J-B; Pearl, P L; Ainslie, G R; Vogel, K R; Gibson, K M

    2016-10-01

    Discovered some 35 years ago, succinic semialdehyde dehydrogenase deficiency (SSADHD) represents a rare, autosomal recessively-inherited defect in the second step of the GABA degradative pathway. Some 200 patients have been reported, with broad phenotypic and genotypic heterogeneity. SSADHD represents an unusual neurometabolic disorder in which two neuromodulatory agents, GABA (and the GABA analogue, 4-hydroxybutyrate), accumulate to supraphysiological levels. The unexpected occurrence of epilepsy in several patients is counterintuitive in view of the hyperGABAergic state, in which sedation might be expected. However, the epileptic status of some patients is most likely represented by broader imbalances of GABAergic and glutamatergic neurotransmission. Cumulative research encompassing decades of basic and clinical study of SSADHD reveal a monogenic disease with broad pathophysiological and clinical phenotypes. Numerous metabolic perturbations unmasked in SSADHD include alterations in oxidative stress parameters, dysregulation of autophagy and mitophagy, dysregulation of both inhibitory and excitatory neurotransmitters and gene expression, and unique subsets of SNP alterations of the SSADH gene (so-called ALDH5A1, or aldehyde dehydrogenase 5A1 gene) on the 6p22 chromosomal arm. While seemingly difficult to collate and interpret, these anomalies have continued to open novel pathways for pharmacotherapeutic considerations. Here, we present an update on selected aspects of SSADHD, the ALDH5A1 gene, and future avenues for research on this rare disorder of GABA metabolism. PMID:27311541

  12. Formate dehydrogenase from Pseudomonas oxalaticus.

    PubMed

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

    1978-02-01

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

  13. (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (FadB’) from fatty acid degradation operon of Ralstonia eutropha H16

    PubMed Central

    2014-01-01

    In this study (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (H16_A0461/FadB’, gene ID: 4247876) from one of two active fatty acid degradation operons of Ralstonia eutropha H16 has been heterologously expressed in Escherichia coli, purified as protein possessing a His-Tag and initially characterized. FadB’ is an enzyme with two catalytic domains exhibiting a single monomeric structure and possessing a molecular weight of 86 kDa. The C-terminal part of the enzyme harbors enoyl-CoA hydratase activity and is able to convert trans-crotonyl-CoA to 3-hydroxybutyryl-CoA. The N-terminal part of FadB’ comprises an NAD+ binding site and is responsible for 3-hydroxyacyl-CoA dehydrogenase activity converting (S)-3-hydroxybutyryl-CoA to acetoacetyl-CoA. Enoyl-CoA hydratase activity was detected spectrophotometrically with trans-crotonyl-CoA. (S)-3-Hydroxyacyl-CoA dehydrogenase activity was measured in both directions with acetoacetyl-CoA and 3-hydroxybutyryl-CoA. FadB’ was found to be strictly stereospecific to (S)-3-hydroxybutyryl-CoA and to prefer NAD+. The Km value for acetoacetyl-CoA was 48 μM and Vmax 149 μmol mg−1 min−1. NADP(H) was utilized at a rate of less than 10% in comparison to activity with NAD(H). FadB’ exhibited optimal activity at pH 6–7 and the activity decreased at alkaline and acidic pH values. Acetyl-CoA, propionyl-CoA and CoA were found to have an inhibitory effect on FadB’. This study is a first report on biochemical properties of purified (S)-stereospecific 3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase with the inverted domain order from R. eutropha H16. In addition to fundamental information about FadB’ and fatty acid metabolism, FadB’ might be also interesting for biotechnological applications. PMID:25401070

  14. Yeast peroxisomal multifunctional enzyme: (3R)-hydroxyacyl-CoA dehydrogenase domains A and B are required for optimal growth on oleic acid.

    PubMed

    Qin, Y M; Marttila, M S; Haapalainen, A M; Siivari, K M; Glumoff, T; Hiltunen, J K

    1999-10-01

    The yeast peroxisomal (3R)-hydroxyacyl-CoA dehydrogenase/2-enoyl-CoA hydratase 2 (multifunctional enzyme type 2; MFE-2) has two N-terminal domains belonging to the short chain alcohol dehydrogenase/reductase superfamily. To investigate the physiological roles of these domains, here called A and B, Saccharomyces cerevisiae fox-2 cells (devoid of Sc MFE-2) were taken as a model system. Gly(16) and Gly(329) of the S. cerevisiae A and B domains, corresponding to Gly(16), which is mutated in the human MFE-2 deficiency, were mutated to serine and cloned into the yeast expression plasmid pYE352. In oleic acid medium, fox-2 cells transformed with pYE352:: ScMFE-2(aDelta) and pYE352::ScMFE-2(bDelta) grew slower than cells transformed with pYE352::ScMFE-2, whereas cells transformed with pYE352::ScMFE-2(aDeltabDelta) failed to grow. Candida tropicalis MFE-2 with a deleted hydratase 2 domain (Ct MFE- 2(h2Delta)) and mutational variants of the A and B domains (Ct MFE- 2(h2DeltaaDelta), Ct MFE- 2(h2DeltabDelta), and Ct MFE- 2(h2DeltaaDeltabDelta)) were overexpressed and characterized. All proteins were dimers with similar secondary structure elements. Both wild type domains were enzymatically active, with the B domain showing the highest activity with short chain and the A domain with medium and long chain (3R)-hydroxyacyl-CoA substrates. The data show that the dehydrogenase domains of yeast MFE-2 have different substrate specificities required to allow the yeast to propagate optimally on fatty acids as the carbon source.

  15. 3, 4-dihydroxyl-phenyl lactic acid restores NADH dehydrogenase 1 α subunit 10 to ameliorate cardiac reperfusion injury.

    PubMed

    Yang, Xiao-Yuan; He, Ke; Pan, Chun-Shui; Li, Quan; Liu, Yu-Ying; Yan, Li; Wei, Xiao-Hong; Hu, Bai-He; Chang, Xin; Mao, Xiao-Wei; Huang, Dan-Dan; Wang, Li-Jun; Hu, Shui-Wang; Jiang, Yong; Wang, Guo-Cheng; Fan, Jing-Yu; Fan, Tai-Ping; Han, Jing-Yan

    2015-01-01

    The present study aimed to detect the role of 3, 4-dihydroxyl-phenyl lactic acid (DLA) during ischemia/reperfusion (I/R) induced myocardial injury with emphasis on the underlying mechanism of DLA antioxidant. Male Spragu-Dawley (SD) rats were subjected to left descending artery occlusion followed by reperfusion. Treatment with DLA ameliorated myocardial structure and function disorder, blunted the impairment of Complex I activity and mitochondrial function after I/R. The results of 2-D fluorescence difference gel electrophoresis revealed that DLA prevented the decrease in NDUFA10 expression, one of the subunits of Complex I. To find the target of DLA, the binding affinity of Sirtuin 1 (SIRT1) to DLA and DLA derivatives with replaced two phenolic hydroxyls was detected using surface plasmon resonance and bilayer interferometry. The results showed that DLA could activate SIRT1 after I/R probably by binding to this protein, depending on phenolic hydroxyl. Moreover, the importance of SIRT1 to DLA effectiveness was confirmed through siRNA transfection in vitro. These results demonstrated that DLA was able to prevent I/R induced decrease in NDUFA10 expression, improve Complex I activity and mitochondrial function, eventually attenuate cardiac structure and function injury after I/R, which was possibly related to its ability of binding to and activating SIRT1. PMID:26030156

  16. Homo-D-lactic acid fermentation from arabinose by redirection of the phosphoketolase pathway to the pentose phosphate pathway in L-lactate dehydrogenase gene-deficient Lactobacillus plantarum.

    PubMed

    Okano, Kenji; Yoshida, Shogo; Tanaka, Tsutomu; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

    2009-08-01

    Optically pure d-lactic acid fermentation from arabinose was achieved by using the Lactobacillus plantarum NCIMB 8826 strain whose l-lactate dehydrogenase gene was deficient and whose phosphoketolase gene was substituted with a heterologous transketolase gene. After 27 h of fermentation, 38.6 g/liter of d-lactic acid was produced from 50 g/liter of arabinose.

  17. Decomposition of peracetic acid catalyzed by vanadium complexes

    SciTech Connect

    Makarov, A.P.; Gekhman, A.E.; Moiseev, I.I.; Polotryuk, O.Y.

    1986-02-01

    This paper studies the decomposition of peracetic acid (AcOOH) in acetic acid (AcOH) catalyzed by vanadium complexes. It is shown that peractic acid in acetic acid solutions of ammonium anadate decomposes with the predominant formation of 0/sub 2/ and small amounts of CO/sub 2/, the yield of which increases with increasing temperature and peracetic acid concentration. Both reactions proceed without the formation of free radicals in amounts detectable by ESR spectroscopy. The rate of oxygen release under conditions in which the formation of CO/sub 2/ is insignificant obeys a kinetic equation indicating the intermediate formation of a complex between V/sup 5 +/ ions and peracetic acid and the slow conversion of this complex into the observed products.

  18. Additivity of the Stabilization Effect of Single Amino Acid Substitutions in Triple Mutants of Recombinant Formate Dehydrogenase from the Soybean Glycine max.

    PubMed

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

    2015-01-01

    Recently, we demonstrated that the amino acid substitutions Ala267Met and Ala267Met/Ile272Val (Alekseeva et al., Biochemistry, 2012), Phe290Asp, Phe290Asn and Phe290Ser (Alekseeva et al., Prot. Eng. Des. Select, 2012) in recombinant formate dehydrogenase from soya Glycine max (SoyFDH) lead to a significant (up to 30-100 times) increase in the thermal stability of the enzyme. The substitutions Phe290Asp, Phe290Asn and Phe290Ser were introduced into double mutant SoyFDH Ala267Met/Ile272Val by site-directed mutagenesis. Combinations of three substitutions did not lead to a noticeable change in the catalytic properties of the mutant enzymes. The stability of the resultant triple mutants was studied through thermal inactivation kinetics and differential scanning calorimetry. The thermal stability of the new mutant SoyFDHs was shown to be much higher than that of their precursors. The stability of the best mutant SoyFDH Ala267Met/Ile272Val/Phe290Asp turned out to be comparable to that of the most stable wild-type formate dehydrogenases from other sources. The results obtained with both methods indicate a great synergistic contribution of individual amino acid substitutions to the common stabilization effect.

  19. Determinants of dual substrate specificity revealed by the crystal structure of homoisocitrate dehydrogenase from Thermus thermophilus in complex with homoisocitrate·Mg(2+)·NADH.

    PubMed

    Takahashi, Kento; Tomita, Takeo; Kuzuyama, Tomohisa; Nishiyama, Makoto

    2016-09-30

    HICDH (Homoisocitrate dehydrogenase) is a member of the β-decarboxylating dehydrogenase family that catalyzes the conversion of homoisocitrate to α-ketoadipate using NAD(+) as a coenzyme, which is the fourth reaction involved in lysine biosynthesis through the α-aminoadipate pathway. Although typical HICDHs from fungi and yeast exhibit strict substrate specificities toward homoisocitrate (HIC), HICDH from a thermophilic bacterium Thermus thermophilus (TtHICDH) catalyzes the reactions using both HIC and isocitrate (IC) as substrates at similar efficiencies. We herein determined the crystal structure of the quaternary complex of TtHICDH with HIC, NADH, and Mg(2+) ion at a resolution of 2.5 Å. The structure revealed that the distal carboxyl group of HIC was recognized by the side chains of Ser72 and Arg85 from one subunit, and Asn173 from another subunit of a dimer unit. Model structures were constructed for TtHICDH in complex with IC and also for HICDH from Saccharomyces cerevisiae (ScHICDH) in complex with HIC. TtHICDH recognized the distal carboxyl group of IC by Arg85 in the model. In ScHICDH, the distal carboxyl group of HIC was recognized by the side chains of Ser98 and Ser108 from one subunit and Asn208 from another subunit of a dimer unit. By contrast, in ScHICDH, which lacks an Arg residue at the position corresponding to Arg85 in TtHICDH, these residues may not interact with the distal carboxyl group of shorter IC. These results provide a molecular basis for the differences in substrate specificities between TtHICDH and ScHICDH. PMID:27601325

  20. Determination of pre-steady-state rate constants on the Escherichia coli pyruvate dehydrogenase complex reveals that loop movement controls the rate-limiting step.

    PubMed

    Balakrishnan, Anand; Nemeria, Natalia S; Chakraborty, Sumit; Kakalis, Lazaros; Jordan, Frank

    2012-11-14

    Spectroscopic identification and characterization of covalent and noncovalent intermediates on large enzyme complexes is an exciting and challenging area of modern enzymology. The Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), consisting of multiple copies of enzymic components and coenzymes, performs the oxidative decarboxylation of pyruvate to acetyl-CoA and is central to carbon metabolism linking glycolysis to the Krebs cycle. On the basis of earlier studies, we hypothesized that the dynamic regions of the E1p component, which undergo a disorder-order transition upon substrate binding to thiamin diphosphate (ThDP), play a critical role in modulation of the catalytic cycle of PDHc. To test our hypothesis, we kinetically characterized ThDP-bound covalent intermediates on the E1p component, and the lipoamide-bound covalent intermediate on the E2p component in PDHc and in its variants with disrupted active-site loops. Our results suggest that formation of the first covalent predecarboxylation intermediate, C2α-lactylthiamin diphosphate (LThDP), is rate limiting for the series of steps culminating in acetyl-CoA formation. Substitutions in the active center loops produced variants with up to 900-fold lower rates of formation of the LThDP, demonstrating that these perturbations directly affected covalent catalysis. This rate was rescued by up to 5-fold upon assembly to PDHc of the E401K variant. The E1p loop dynamics control covalent catalysis with ThDP and are modulated by PDHc assembly, presumably by selection of catalytically competent loop conformations. This mechanism could be a general feature of 2-oxoacid dehydrogenase complexes because such interfacial dynamic regions are highly conserved.

  1. Conversion of L-sorbosone to L-ascorbic acid by a NADP-dependent dehydrogenase in bean and spinach leaf. [Phaseolus vulgaris L. ; Spinacia oleracea L

    SciTech Connect

    Loewus, M.W.; Bedgar, D.L.; Saito, Kazumi; Loewus, F.A. )

    1990-11-01

    An NADP-dependent dehydrogenase catalyzing the conversion of L-sorbosone to L-ascorbic acid has been isolated from Phaseolus vulgaris L. and Spinacia oleracea L. and partially purified. It is stable at {minus}20{degree}C for up to 8 months. Molecular masses, as determined by gel filtration, were 21 and 29 kilodaltons for bean and spinach enzymes, respectively. K{sub m} for sorbosone were 12 {plus minus} 2 and 18 {plus minus} 2 millimolar and for NADP{sup +}, 0.14 {plus minus} 0.05 and 1.2 {plus minus} 0.5 millimolar, for bean and spinach, respectively. Lycorine, a purported inhibitor of L-ascorbic acid biosynthesis, had no effect on the reaction.

  2. NADP+ and NAD+ binding to the dual coenzyme specific enzyme Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase: different interdomain hinge angles are seen in different binary and ternary complexes.

    PubMed

    Naylor, C E; Gover, S; Basak, A K; Cosgrove, M S; Levy, H R; Adams, M J

    2001-05-01

    The reduced coenzymes NADH and NADPH only differ by one phosphate, but in the cell NADH provides reducing power for catabolism while NADPH is utilized in biosynthetic pathways. Enzymes almost invariably discriminate between the coenzymes, but glucose 6-phosphate dehydrogenase (G6PD) from Leuconostoc mesenteroides is rare in being functionally dual specific. In order to elucidate the coenzyme selectivity, the structures of NADP(+)- and NAD(+)-complexed L. mesenteroides G6PD have been determined including data to 2.2 and 2.5 A resolution, respectively, and compared with unliganded G6PD crystallized in the same space groups. Coenzyme binding is also compared with that in a ternary complex of a mutant in which Asp177 in the active site has been mutated to asparagine. There are no gross structural differences between the complexes. In both binary complexes, the enzyme interdomain hinge angle has opened. NADP(+) binds to the furthest open form; of the residues within the coenzyme domain, only Arg46 moves, interacting with the 2'-phosphate and adenine. NAD(+) is less well defined in the binding site; smaller hinge opening is seen but larger local changes: Arg46 is displaced, Thr14 bonds the 3'-hydroxyl and Gln47 bonds the 2'-hydroxyl. In the ternary complex, the hinge angle has closed; only the adenine nucleotide is ordered in the binding site. Arg46 again provides most binding interactions.

  3. Complex formation between benzene carboxylic acids and β-cyclodextrin

    NASA Astrophysics Data System (ADS)

    Belyakova, L. A.; Lyashenko, D. Yu.

    2008-05-01

    Complex formation between benzene carboxylic acids and β-cyclodextrin in aqueous solutions at 290 300 K was studied using UV spectroscopy. The formation of 1:1 supramolecular inclusion compounds β-cyclodextrin-benzene and β-cyclodextrin-salicylic acid was found. Stability constants (Ks) of the complexes and thermodynamic parameters for formation of the inclusion compounds (ΔG, ΔH, and ΔS) were calculated.

  4. Caenorhabditis elegans expressing the Saccharomyces cerevisiae NADH alternative dehydrogenase Ndi1p, as a tool to identify new genes involved in complex I related diseases

    PubMed Central

    Cossard, Raynald; Esposito, Michela; Sellem, Carole H.; Pitayu, Laras; Vasnier, Christelle; Delahodde, Agnès; Dassa, Emmanuel P.

    2015-01-01

    Isolated complex I deficiencies are one of the most commonly observed biochemical features in patients suffering from mitochondrial disorders. In the majority of these clinical cases the molecular bases of the diseases remain unknown suggesting the involvement of unidentified factors that are critical for complex I function. The Saccharomyces cerevisiae NDI1 gene, encoding the mitochondrial internal NADH dehydrogenase was previously shown to complement a complex I deficient strain in Caenorhabditis elegans with notable improvements in reproduction and whole organism respiration. These features indicate that Ndi1p can functionally integrate the respiratory chain, allowing complex I deficiency complementation. Taking into account the Ndi1p ability to bypass complex I, we evaluate the possibility to extend the range of defects/mutations causing complex I deficiencies that can be alleviated by NDI1 expression. We report here that NDI1 expressing animals unexpectedly exhibit a slightly shortened lifespan, a reduction in the progeny, and a depletion of the mitochondrial genome. However, Ndi1p is expressed and targeted to the mitochondria as a functional protein that confers rotenone resistance to those animals without affecting their respiration rate and ATP content. We show that the severe embryonic lethality level caused by the RNAi knockdowns of complex I structural subunit encoding genes (e.g., NDUFV1, NDUFS1, NDUFS6, NDUFS8, or GRIM-19 human orthologs) in wild type animals is significantly reduced in the Ndi1p expressing worm. All together these results open up the perspective to identify new genes involved in complex I function, assembly, or regulation by screening an RNAi library of genes leading to embryonic lethality that should be rescued by NDI1 expression. PMID:26124772

  5. β-Cyclodextrin- para-aminosalicylic acid inclusion complexes

    NASA Astrophysics Data System (ADS)

    Roik, N. V.; Belyakova, L. A.; Oranskaya, E. I.

    2010-11-01

    Complex formation of β-cyclodextrin with para-aminosalicylic acid in buffer solutions is studied by UV spectroscopy. It is found that the stoichiometric proportion of the components in the β-cyclodextrin-para-aminosalicylic acid inclusion complex is 1:1. The Ketelar equation is used to calculate the stability constants of the inclusion complexes at different temperatures. The thermodynamic parameters of the complex formation process (ΔG, ΔH, ΔS) are calculated using the van't Hoff equation. The 1:1 β-cyclodextrin-para-aminosalicylic acid inclusion complex is prepared in solid form and its characteristics are determined by IR spectroscopic and x-ray diffraction techniques.

  6. The lysine-ketoglutarate reductase-saccharopine dehydrogenase is involved in the osmo-induced synthesis of pipecolic acid in rapeseed leaf tissues.

    PubMed

    Moulin, M; Deleu, C; Larher, F; Bouchereau, A

    2006-01-01

    Higher plant responses to abiotic stresses are associated with physiological and biochemical changes triggering a number of metabolic adjustments. We focused on L-lysine catabolism, and have previously demonstrated that degradation of this amino acid is osmo-regulated at the level of lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9) in Brassica napus. LKR and SDH activities are enhanced by decreasing osmotic potential and decrease when the upshock osmotic treatment is followed by a downshock osmotic one. Moreover we have shown that the B. napus LKR/SDH gene is up-regulated in osmotically-stressed tissues. The LKR/SDH activity produces alpha-aminoadipate semialdehyde which could be further converted into alpha-aminoadipate and acetyl CoA. Alternatively alpha-aminoadipate could behave as a precursor for pipecolic acid. Pipecolic acid is described as an osmoprotectant in bacteria and is co-accumulated with proline in halophytic plants. We suggest that osmo-induction of the LKR/SDH activity could be partly responsible for pipecolic acid accumulation. This proposal has been assessed in this study through pipecolic acid amounts determination in rape leaf discs subjected to various upshift and downshift osmotic treatments. Changes in pipecolic acid level actually behave as those observed for LKR and SDH activities, since it increases or decreases in rape leaf discs treated under hyper- or hypo-osmotic conditions, respectively. In addition we show that pipecolic acid level is positively correlated with the external osmotic potential as well as with the duration of the applied treatment. On the other hand pipecolic acid level is related to the availability of L-lysine and not to that of D-lysine. Collectively the results obtained demonstrate that lysine catabolism through LKR/SDH activity is involved in osmo-induced synthesis of pipecolic acid.

  7. NADP-specific electron-bifurcating [FeFe]-hydrogenase in a functional complex with formate dehydrogenase in Clostridium autoethanogenum grown on CO.

    PubMed

    Wang, Shuning; Huang, Haiyan; Kahnt, Jörg; Mueller, Alexander P; Köpke, Michael; Thauer, Rudolf K

    2013-10-01

    Flavin-based electron bifurcation is a recently discovered mechanism of coupling endergonic to exergonic redox reactions in the cytoplasm of anaerobic bacteria and archaea. Among the five electron-bifurcating enzyme complexes characterized to date, one is a heteromeric ferredoxin- and NAD-dependent [FeFe]-hydrogenase. We report here a novel electron-bifurcating [FeFe]-hydrogenase that is NADP rather than NAD specific and forms a complex with a formate dehydrogenase. The complex was found in high concentrations (6% of the cytoplasmic proteins) in the acetogenic Clostridium autoethanogenum autotrophically grown on CO, which was fermented to acetate, ethanol, and 2,3-butanediol. The purified complex was composed of seven different subunits. As predicted from the sequence of the encoding clustered genes (fdhA/hytA-E) and from chemical analyses, the 78.8-kDa subunit (FdhA) is a selenocysteine- and tungsten-containing formate dehydrogenase, the 65.5-kDa subunit (HytB) is an iron-sulfur flavin mononucleotide protein harboring the NADP binding site, the 51.4-kDa subunit (HytA) is the [FeFe]-hydrogenase proper, and the 18.1-kDa (HytC), 28.6-kDa (HytD), 19.9-kDa (HytE1), and 20.1-kDa (HytE2) subunits are iron-sulfur proteins. The complex catalyzed both the reversible coupled reduction of ferredoxin and NADP(+) with H2 or formate and the reversible formation of H2 and CO2 from formate. We propose the complex to have two functions in vivo, namely, to normally catalyze CO2 reduction to formate with NADPH and reduced ferredoxin in the Wood-Ljungdahl pathway and to catalyze H2 formation from NADPH and reduced ferredoxin when these redox mediators get too reduced during unbalanced growth of C. autoethanogenum on CO (E0' = -520 mV).

  8. NADP-Specific Electron-Bifurcating [FeFe]-Hydrogenase in a Functional Complex with Formate Dehydrogenase in Clostridium autoethanogenum Grown on CO

    PubMed Central

    Wang, Shuning; Huang, Haiyan; Kahnt, Jörg; Mueller, Alexander P.; Köpke, Michael

    2013-01-01

    Flavin-based electron bifurcation is a recently discovered mechanism of coupling endergonic to exergonic redox reactions in the cytoplasm of anaerobic bacteria and archaea. Among the five electron-bifurcating enzyme complexes characterized to date, one is a heteromeric ferredoxin- and NAD-dependent [FeFe]-hydrogenase. We report here a novel electron-bifurcating [FeFe]-hydrogenase that is NADP rather than NAD specific and forms a complex with a formate dehydrogenase. The complex was found in high concentrations (6% of the cytoplasmic proteins) in the acetogenic Clostridium autoethanogenum autotrophically grown on CO, which was fermented to acetate, ethanol, and 2,3-butanediol. The purified complex was composed of seven different subunits. As predicted from the sequence of the encoding clustered genes (fdhA/hytA-E) and from chemical analyses, the 78.8-kDa subunit (FdhA) is a selenocysteine- and tungsten-containing formate dehydrogenase, the 65.5-kDa subunit (HytB) is an iron-sulfur flavin mononucleotide protein harboring the NADP binding site, the 51.4-kDa subunit (HytA) is the [FeFe]-hydrogenase proper, and the 18.1-kDa (HytC), 28.6-kDa (HytD), 19.9-kDa (HytE1), and 20.1-kDa (HytE2) subunits are iron-sulfur proteins. The complex catalyzed both the reversible coupled reduction of ferredoxin and NADP+ with H2 or formate and the reversible formation of H2 and CO2 from formate. We propose the complex to have two functions in vivo, namely, to normally catalyze CO2 reduction to formate with NADPH and reduced ferredoxin in the Wood-Ljungdahl pathway and to catalyze H2 formation from NADPH and reduced ferredoxin when these redox mediators get too reduced during unbalanced growth of C. autoethanogenum on CO (E0′ = −520 mV). PMID:23893107

  9. Evidence for messenger ribonucleic acid of an ammonium-inducible glutamate dehydrogenase and synthesis, covalent modification, and degradation of enzyme subunits in uninduced Chlorella sorokiniana cells.

    PubMed Central

    Turner, K J; Bascomb, N F; Lynch, J J; Molin, W T; Thurston, C F; Schmidt, R R

    1981-01-01

    The cells of Chlorella sorokiniana cultured in nitrate medium contain no detectable catalytic activity of an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH). However, several lines of experimental evidence indicated that the NADP-GDH messenger ribonucleic acid was present at high levels and was being translated in uninduced cells. First, binding studies with 125I-labeled anti-NADP-GDH immunoglobulin G and total polysomes isolated from uninduced and induced cells showed that NADP-GDH subunits were being synthesized on polysomes from both types of cells. Second, when polyadenylic acid-containing ribonucleic acid was extracted from polysomes from uninduced and induced cells and placed into a messenger ribonucleic acid-dependent in vitro translation system, NADP-GDH subunits were synthesized from the ribonucleic acid from both sources. Third, when ammonia was added to uninduced cells, NADP-GDH antigen accumulated without an apparent induction lag. Fourth, by use of a specific immunoprecipitation procedure coupled to pulse-chase studies with [35S]sulfate, it was shown that the NADP-GDH subunits are rapidly synthesized, covalently modified, and then degraded in uninduced cells. PMID:7217012

  10. Elucidation of the interaction loci of the human pyruvate dehydrogenase complex E2·E3BP core with pyruvate dehydrogenase kinase 1 and kinase 2 by H/D exchange mass spectrometry and nuclear magnetic resonance.

    PubMed

    Wang, Junjie; Kumaran, Sowmini; Zhou, Jieyu; Nemeria, Natalia S; Tao, Hu; Kakalis, Lazaros; Park, Yun-Hee; Birkaya, Barbara; Patel, Mulchand S; Jordan, Frank

    2015-01-13

    The human pyruvate dehydrogenase complex (PDC) comprises three principal catalytic components for its mission: E1, E2, and E3. The core of the complex is a strong subcomplex between E2 and an E3-binding protein (E3BP). The PDC is subject to regulation at E1 by serine phosphorylation by four kinases (PDK1-4), an inactivation reversed by the action of two phosphatases (PDP1 and -2). We report H/D exchange mass spectrometric (HDX-MS) and nuclear magnetic resonance (NMR) studies in the first attempt to define the interaction loci between PDK1 and PDK2 with the intact E2·E3BP core and their C-terminally truncated proteins. While the three lipoyl domains (L1 and L2 on E2 and L3 on E3BP) lend themselves to NMR studies and determination of interaction maps with PDK1 and PDK2 at the individual residue level, HDX-MS allowed studies of interaction loci on both partners in the complexes, PDKs, and other regions of the E2·E3BP core, as well, at the peptide level. HDX-MS suggested that the intact E2·E3BP core enhances the binding specificity of L2 for PDK2 over PDK1, while NMR studies detected lipoyl domain residues unique to interaction with PDK1 and PDK2. The E2·E3BP core induced more changes on PDKs than any C-terminally truncated protein, with clear evidence of greater plasticity of PDK1 than of PDK2. The effect of L1L2S paralleled HDX-MS results obtained with the intact E2·E3BP core; hence, L1L2S is an excellent candidate with which to define interaction loci with these two PDKs. Surprisingly, L3S' induced moderate interaction with both PDKs according to both methods. PMID:25436986

  11. Glutaric acid and its metabolites cause apoptosis in immature oligodendrocytes: a novel mechanism of white matter degeneration in glutaryl-CoA dehydrogenase deficiency.

    PubMed

    Gerstner, Bettina; Gratopp, Alexander; Marcinkowski, Monika; Sifringer, Marco; Obladen, Michael; Bührer, Christoph

    2005-06-01

    Glutaryl-CoA dehydrogenase deficiency is an inherited metabolic disease characterized by elevated concentrations of glutaric acid (GA) and its metabolites glutaconic acid (GC) and 3-hydroxy-glutaric acid (3-OH-GA). Its hallmarks are striatal and cortical degeneration, which have been linked to excitotoxic neuronal cell death. However, magnetic resonance imaging studies have also revealed widespread white matter disease. Correspondingly, we decided to investigate the effects of GA, GC, and 3-OH-GA on the rat immature oligodendroglia cell line, OLN-93. For comparison, we also exposed the neuroblastoma line SH-SY5Y and the microglia line BV-2 to GA, GC, and 3-OH-GA. Cell viability was measured by metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium. Flow cytometry was used to assess apoptosis via annexin-V, anti-active caspase-3 antibody, and propidium iodide staining. GA, GC, and 3-OH-GA reduced OLN-93 oligodendroglia cell viability in a dose-dependent manner. Toxicity of GA, GC, and 3-OH-GA was abrogated by preincubation with the pan-caspase inhibitor z-VAD-fmk. Apoptosis but not necrosis was detected at various stages (early: annexin-V; effector: caspase-3) after 24-48 h of incubation with GA, GC, or 3-OH-GA in OLN-93 but not in neuroblastoma or microglia cells. OLN-93 lacked expression of N-methyl-d-aspartate receptors, making classical glutamatergic excitotoxicity an unlikely explanation for the selective toxicity of GA, GC, and 3-OH-GA for OLN-93 cells. GA, GC, and 3-OH-GA directly initiate the apoptotic cascade in oligodendroglia cells. This mechanism may contribute to the white matter damage observed in glutaryl-CoA dehydrogenase deficiency.

  12. Bioinorganic modeling chemistry of carbon monoxide dehydrogenases: description of model complexes, current status and possible future scopes.

    PubMed

    Majumdar, Amit

    2014-08-28

    Carbon monoxide dehydrogenases (CODHs) use CO as their sole source of carbon and energy and are found in both aerobic and anaerobic carboxidotrophic bacteria. Reversible transformation of CO to CO2 is catalyzed by a bimetallic [Mo-(μ2-S)-Cu] system in aerobic and by a highly asymmetric [Ni-Fe-S] cluster in anaerobic CODH active sites. The CODH activity in the microorganisms effects the removal of almost 10(8) tons of CO annually from the lower atmosphere and earth and thus help to maintain a sub-toxic concentration of CO. Despite an appreciable amount of work, the mechanism of CODH activity is not clearly understood yet. Moreover, biomimetic chemistry directed towards the active sites of CODHs faces several synthetic challenges. The synthetic problems associated with the modeling chemistry and strategies adopted to overcome those problems are discussed along with their limitations. A critical analysis of the exciting results delineating the present status of CODH modeling chemistry and its future prospects are presented.

  13. Complex compound polyvinyl alcohol-titanic acid/titanium dioxide

    NASA Astrophysics Data System (ADS)

    Prosanov, I. Yu.

    2013-02-01

    A complex compound polyvinyl alcohol-titanic acid has been produced and investigated by means of IR and Raman spectroscopy, X-ray diffraction, and synchronous thermal analysis. It is claimed that it represents an interpolymeric complex of polyvinyl alcohol and hydrated titanium oxide.

  14. Characterization of a group of pyrroloquinoline quinone-dependent dehydrogenases that are involved in the conversion of L-sorbose to 2-Keto-L-gulonic acid in Ketogulonicigenium vulgare WSH-001.

    PubMed

    Gao, Lili; Du, Guocheng; Zhou, Jingwen; Chen, Jian; Liu, Jie

    2013-01-01

    Ketogulonicigenium vulgare WSH-001 is an industrial strain used for vitamin C production. Based on genome sequencing and pathway analysis of the bacterium, some of its potential pyrroloquinoline quinone (PQQ)-dependent dehydrogenases were predicted, including KVU_pmdA_0245, KVU_2142, KVU_2159, KVU_1366, KVU_0203, KVU_0095, and KVU_pmdB_0115. BLAST and function domain searches showed that enzymes encoded by these genes may act as putative PQQ-dependent L-sorbose dehydrogenases (SDH) or L-sorbosone dehydrogenases (SNDH). To validate whether these dehydrogenases are PQQ-dependent or not, these seven putative dehyrogenases were overexpressed in Escherichia coli BL21 (DE3) and purified for characterization. Biochemical and kinetic characterization of the purified proteins have led to the identification of seven enzymes that possess the ability to oxidize L-sorbose or L-sorbosone to varying degrees. In addition, the dehydrogenation of sorbose in K. vulgare is validated to be PQQ dependent, identification of these PQQ-dependent dehydrogenases expanded the PQQ-dependent dehydrogenase family. Besides, the optimal combination of enzymes that could more efficiently catalyze the conversion of sorbose to gulonic acid was proposed. These are important in supporting the development of metabolic engineering strategies and engineering of efficient strains for one-step production of vitamin C in the future.

  15. Characterization of a group of pyrroloquinoline quinone-dependent dehydrogenases that are involved in the conversion of L-sorbose to 2-Keto-L-gulonic acid in Ketogulonicigenium vulgare WSH-001.

    PubMed

    Gao, Lili; Du, Guocheng; Zhou, Jingwen; Chen, Jian; Liu, Jie

    2013-01-01

    Ketogulonicigenium vulgare WSH-001 is an industrial strain used for vitamin C production. Based on genome sequencing and pathway analysis of the bacterium, some of its potential pyrroloquinoline quinone (PQQ)-dependent dehydrogenases were predicted, including KVU_pmdA_0245, KVU_2142, KVU_2159, KVU_1366, KVU_0203, KVU_0095, and KVU_pmdB_0115. BLAST and function domain searches showed that enzymes encoded by these genes may act as putative PQQ-dependent L-sorbose dehydrogenases (SDH) or L-sorbosone dehydrogenases (SNDH). To validate whether these dehydrogenases are PQQ-dependent or not, these seven putative dehyrogenases were overexpressed in Escherichia coli BL21 (DE3) and purified for characterization. Biochemical and kinetic characterization of the purified proteins have led to the identification of seven enzymes that possess the ability to oxidize L-sorbose or L-sorbosone to varying degrees. In addition, the dehydrogenation of sorbose in K. vulgare is validated to be PQQ dependent, identification of these PQQ-dependent dehydrogenases expanded the PQQ-dependent dehydrogenase family. Besides, the optimal combination of enzymes that could more efficiently catalyze the conversion of sorbose to gulonic acid was proposed. These are important in supporting the development of metabolic engineering strategies and engineering of efficient strains for one-step production of vitamin C in the future. PMID:23970495

  16. Evolution of cytochrome bc complexes: from membrane-anchored dehydrogenases of ancient bacteria to triggers of apoptosis in vertebrates

    PubMed Central

    Dibrova, Daria V.; Cherepanov, Dmitry A.; Galperin, Michael Y.; Skulachev, Vladimir P.; Mulkidjanian, Armen Y.

    2013-01-01

    This review traces the evolution of the cytochrome bc complexes from their early spread among prokaryotic lineages and up to the mitochondrial cytochrome bc1 complex (complex III) and its role in apoptosis. The results of phylogenomic analysis suggest that the bacterial cytochrome b6f-type complexes with short cytochromes b were the ancient form that preceded in evolution the cytochrome bc1-type complexes with long cytochromes b. The common ancestor of the b6f-type and the bc1-type complexes probably resembled the b6f-type complexes found in Heliobacteriaceae and in some Planctomycetes. Lateral transfers of cytochrome bc operons could account for the several instances of acquisition of different types of bacterial cytochrome bc complexes by archaea. The gradual oxygenation of the atmosphere could be the key evolutionary factor that has driven further divergence and spread of the cytochrome bc complexes. On one hand, oxygen could be used as a very efficient terminal electron acceptor. On the other hand, auto-oxidation of the components of the bc complex results in the generation of reactive oxygen species (ROS), which necessitated diverse adaptations of the b6f-type and bc1-type complexes, as well as other, functionally coupled proteins. A detailed scenario of the gradual involvement of the cardiolipin-containing mitochondrial cytochrome bc1 complex into the intrinsic apoptotic pathway is proposed, where the functioning of the complex as an apoptotic trigger is viewed as a way to accelerate the elimination of the cells with irreparably damaged, ROS-producing mitochondria. PMID:23871937

  17. Fermentation and alternative respiration compensate for NADH dehydrogenase deficiency in a prokaryotic model of DJ-1-associated Parkinsonism.

    PubMed

    Messaoudi, Nadia; Gautier, Valérie; Dairou, Julien; Mihoub, Mouhad; Lelandais, Gaëlle; Bouloc, Philippe; Landoulsi, Ahmed; Richarme, Gilbert

    2015-11-01

    YajL is the closest prokaryotic homologue of Parkinson's disease-associated DJ-1, a protein of undefined function involved in the oxidative stress response. We reported recently that YajL and DJ-1 protect cells against oxidative stress-induced protein aggregation by acting as covalent chaperones for the thiol proteome, including the NuoG subunit of NADH dehydrogenase 1, and that NADH dehydrogenase 1 activity is negligible in the yajL mutant. We report here that this mutant compensates for low NADH dehydrogenase activity by utilizing NADH-independent alternative dehydrogenases, including pyruvate oxidase PoxB and d-amino acid dehydrogenase DadA, and mixed acid aerobic fermentations characterized by acetate, lactate, succinate and ethanol excretion. The yajL mutant has a low adenylate energy charge favouring glycolytic flux, and a high NADH/NAD ratio favouring fermentations over pyruvate dehydrogenase and the Krebs cycle. DNA array analysis showed upregulation of genes involved in glycolytic and pentose phosphate pathways and alternative respiratory pathways. Moreover, the yajL mutant preferentially catabolized pyruvate-forming amino acids over Krebs cycle-related amino acids, and thus the yajL mutant utilizes pyruvate-centred respiro-fermentative metabolism to compensate for the NADH dehydrogenase 1 defect and constitutes an interesting model for studying eukaryotic respiratory complex I deficiencies, especially those associated with Alzheimer's and Parkinson's diseases.

  18. Geometry and cooperativity effects in adenosine-carboxylic acid complexes.

    PubMed

    Schlund, Sebastian; Mladenovic, Milena; Basílio Janke, Eline M; Engels, Bernd; Weisz, Klaus

    2005-11-23

    NMR experiments and theoretical investigations were performed on hydrogen bonded complexes of specifically 1- and 7-15N-labeled adenine nucleosides with carboxylic acids. By employing a freonic solvent of CDClF2 and CDF3, NMR spectra were acquired at temperatures as low as 123 K, where the regime of slow hydrogen bond exchange is reached and several higher-order complexes were found to coexist in solution. Unlike acetic acid, chloroacetic acid forms Watson-Crick complexes with the proton largely displaced from oxygen to the nitrogen acceptor in an ion pairing structure. Calculated geometries and chemical shifts of the proton in the hydrogen bridge favorably agree with experimentally determined values if vibrational averaging and solvent effects are taken into account. The results indicate that binding a second acidic ligand at the adenine Hoogsteen site in a ternary complex weakens the hydrogen bond to the Watson-Crick bound carboxylic acid. However, substituting a second adenine nucleobase for a carboxylic acid in the trimolecular complex leads to cooperative binding at Watson-Crick and Hoogsteen faces of adenosine.

  19. Relationships within the aldehyde dehydrogenase extended family.

    PubMed

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

    1999-01-01

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

  20. A salicylic acid-based analogue discovered from virtual screening as a potent inhibitor of human 20alpha-hydroxysteroid dehydrogenase.

    PubMed

    Dhagat, Urmi; Carbone, Vincenzo; Chung, Roland P-T; Matsunaga, Toshihiro; Endo, Satoshi; Hara, Akira; El-Kabbani, Ossama

    2007-11-01

    20alpha-hydroxysteroid dehydrogenase (AKR1C1) plays a key role in the metabolism of progesterone and other steroid hormones, thereby regulating their action at the pre-receptor level. AKR1C1 is implicated in neurological and psychiatric conditions such as catamenial epilepsy and depressive disorders. Increased activity of AKR1C1 is associated with termination of pregnancy and the development of breast cancer, endometriosis and endometrial cancer. Inhibition of the undesired activity of AKR1C1 will help reduce risks of premature birth, neurological disorders and the development of cancer. In order to identify potential leads for new inhibitors of AKR1C1 we adopted a virtual screening-based approach using the automated DOCK program. Approximately 250,000 compounds from the NCI database were screened for potential ligands based on their chemical complementarity and steric fit within the active site of AKR1C1. Kinetic analysis revealed 3,5-diiodosalicylic acid, an analogue of salicylic acid, as a potent competitive inhibitor with respect to the substrate 5beta-pregnane-3alpha,20alpha-diol with a K(i) of 9 nM. Aspirin, which is a well known salicylic acid-based drug, was also found to inhibit AKR1C1 activity. This is the first report to show aspirin (IC(50)=21 microM) and its metabolite salicylic acid (IC(50)=7.8 microM) as inhibitors of AKR1C1.

  1. Identification of a novel operon in Lactococcus lactis encoding three enzymes for lactic acid synthesis: phosphofructokinase, pyruvate kinase, and lactate dehydrogenase.

    PubMed Central

    Llanos, R M; Harris, C J; Hillier, A J; Davidson, B E

    1993-01-01

    The discovery of a novel multicistronic operon that encodes phosphofructokinase, pyruvate kinase, and lactate dehydrogenase in the lactic acid bacterium Lactococcus lactis is reported. The three genes in the operon, designated pfk, pyk, and ldh, contain 340, 502, and 325 codons, respectively. The intergenic distances are 87 bp between pfk and pyk and 117 bp between pyk and ldh. Plasmids containing pfk and pyk conferred phosphofructokinase and pyruvate kinase activity, respectively, on their host. The identity of ldh was established previously by the same approach (R. M. Llanos, A. J. Hillier, and B. E. Davidson, J. Bacteriol. 174:6956-6964, 1992). Each of the genes is preceded by a potential ribosome binding site. The operon is expressed in a 4.1-kb transcript. The 5' end of the transcript was determined to be a G nucleotide positioned 81 bp upstream from the pfk start codon. The pattern of codon usage within the operon is highly biased, with 11 unused amino acid codons. This degree of bias suggests that the operon is highly expressed. The three proteins encoded on the operon are key enzymes in the Embden-Meyerhoff pathway, the central pathway of energy production and lactic acid synthesis in L. lactis. For this reason, we have called the operon the las (lactic acid synthesis) operon. Images PMID:8478320

  2. Anaerobic degradation of nitrilotriacetate (NTA) in a denitrifying bacterium: purification and characterization of the NTA dehydrogenase-nitrate reductase enzyme complex.

    PubMed Central

    Jenal-Wanner, U; Egli, T

    1993-01-01

    The initial step in the anoxic metabolism of nitrilotriacetate (NTA) was investigated in a denitrifying member of the gamma subgroup of the Proteobacteria. In membrane-free cell extracts, the first step of NTA oxidation was catalyzed by a protein complex consisting of two enzymes, NTA dehydrogenase (NTADH) and nitrate reductase (NtR). The products formed were iminodiacetate and glyoxylate. Electrons derived from the oxidation of NTA were transferred to nitrate only via the artificial dye phenazine methosulfate, and nitrate was stoichiometrically reduced to nitrite. NTADH activity could be measured only in the presence of NtrR and vice versa. The NTADH-NtrR enzyme complex was purified and characterized. NTADH and NtrR were both alpha 2 dimers and had molecular weights of 170,000 and 105,000, respectively. NTADH contained covalently bound flavin cofactor, and NtrR contained a type b cytochrome. Optimum NTA-oxidizing activity was achieved at a molar ratio of NTADH to NtrR of approximately 1:1. So far, NTA is the only known substrate for NTADH. This is the first report of a redox enzyme complex catalyzing the oxidation of a substrate and concomitantly reducing nitrate. Images PMID:8250558

  3. Biochemical and Structural Studies of Uncharacterized Protein PA0743 from Pseudomonas aeruginosa Revealed NAD+-dependent l-Serine Dehydrogenase*

    PubMed Central

    Tchigvintsev, Anatoli; Singer, Alexander; Brown, Greg; Flick, Robert; Evdokimova, Elena; Tan, Kemin; Gonzalez, Claudio F.; Savchenko, Alexei; Yakunin, Alexander F.

    2012-01-01

    The β-hydroxyacid dehydrogenases form a large family of ubiquitous enzymes that catalyze oxidation of various β-hydroxy acid substrates to corresponding semialdehydes. Several known enzymes include β-hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, 2-(hydroxymethyl)glutarate dehydrogenase, and phenylserine dehydrogenase, but the vast majority of β-hydroxyacid dehydrogenases remain uncharacterized. Here, we demonstrate that the predicted β-hydroxyisobutyrate dehydrogenase PA0743 from Pseudomonas aeruginosa catalyzes an NAD+-dependent oxidation of l-serine and methyl-l-serine but exhibits low activity against β-hydroxyisobutyrate. Two crystal structures of PA0743 were solved at 2.2–2.3-Å resolution and revealed an N-terminal Rossmann fold domain connected by a long α-helix to the C-terminal all-α domain. The PA0743 apostructure showed the presence of additional density modeled as HEPES bound in the interdomain cleft close to the predicted catalytic Lys-171, revealing the molecular details of the PA0743 substrate-binding site. The structure of the PA0743-NAD+ complex demonstrated that the opposite side of the enzyme active site accommodates the cofactor, which is also bound near Lys-171. Site-directed mutagenesis of PA0743 emphasized the critical role of four amino acid residues in catalysis including the primary catalytic residue Lys-171. Our results provide further insight into the molecular mechanisms of substrate selectivity and activity of β-hydroxyacid dehydrogenases. PMID:22128181

  4. Pressure adaptation of 3-isopropylmalate dehydrogenase from an extremely piezophilic bacterium is attributed to a single amino acid substitution.

    PubMed

    Hamajima, Yuki; Nagae, Takayuki; Watanabe, Nobuhisa; Ohmae, Eiji; Kato-Yamada, Yasuyuki; Kato, Chiaki

    2016-03-01

    3-Isopropylmalate dehydrogenase (IPMDH) from the extreme piezophile Shewanella benthica (SbIPMDH) is more pressure-tolerant than that from the atmospheric pressure-adapted Shewanella oneidensis (SoIPMDH). To understand the molecular mechanisms of this pressure tolerance, we analyzed mutated enzymes. The results indicate that only a single mutation at position 266, corresponding to Ala (SbIPMDH) and Ser (SoIPMDH), essentially affects activity under higher-pressure conditions. Structural analyses of SoIPMDH suggests that penetration of three water molecules into the cleft around Ser266 under high-pressure conditions could reduce the activity of the wild-type enzyme; however, no water molecule is observed in the Ala266 mutant. PMID:26847201

  5. Production of gamma-aminobutyric acid from glucose by introduction of synthetic scaffolds between isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase in recombinant Escherichia coli.

    PubMed

    Pham, Van Dung; Lee, Seung Hwan; Park, Si Jae; Hong, Soon Ho

    2015-08-10

    Escherichia coli were engineered for the direct production of gamma-aminobutyric acid from glucose by introduction of synthetic protein scaffold. In this study, three enzymes consisting GABA pathway (isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase) were connected via synthetic protein scaffold. By introduction of scaffold, 0.92g/L of GABA was produced from 10g/L of glucose while no GABA was produced in wild type E. coli. The optimum pH and temperature for GABA production were 4.5 and 30°C, respectively. When competing metabolic network was inactivated by knockout mutation, maximum GABA concentration of 1.3g/L was obtained from 10g/L glucose. The recombinant E. coli strain which produces GABA directly from glucose was successfully constructed by introduction of protein scaffold.

  6. Biosynthesis of ascorbic acid in kidney bean. L-galactono-gamma-lactone dehydrogenase is an intrinsic protein located at the mitochondrial inner membrane

    PubMed

    Siendones; Gonzalez-Reyes; Santos-Ocana; Navas; C rdoba F

    1999-07-01

    Hypocotyls of kidney beans (Phaseolus vulgaris L.) accumulated ascorbate after preincubation with a number of possible precursors, mainly L-galactono-gamma-lactone (L-GL) and L-gulono-gamma-lactone. The increase in the intracellular ascorbate concentration was parallel to the high stimulation of the L-GL dehydrogenase (L-GLD) activity measured in vitro using L-GL as a substrate and cytochrome c as an electron acceptor. Cell fractionation using a continuous linear Percoll gradient demonstrated that L-GLD is associated with mitochondria; therefore, pure mitochondria were isolated and subjected to detergent treatment to separate soluble from membrane-linked proteins. L-GLD activity was mainly associated with the detergent phase, suggesting that a membrane-intrinsic protein is responsible for the ascorbic acid biosynthetic activity. Subfractionation of mitochondria demonstrated that L-GLD is located at the inner membrane. PMID:10398727

  7. Pharmacological activation of the pyruvate dehydrogenase complex reduces statin-mediated upregulation of FOXO gene targets and protects against statin myopathy in rodents.

    PubMed

    Mallinson, Joanne E; Constantin-Teodosiu, Dumitru; Glaves, Philip D; Martin, Elizabeth A; Davies, Wendy J; Westwood, F Russell; Sidaway, James E; Greenhaff, Paul L

    2012-12-15

    We previously reported that statin myopathy is associated with impaired carbohydrate (CHO) oxidation in fast-twitch rodent skeletal muscle, which we hypothesised occurred as a result of forkhead box protein O1 (FOXO1) mediated upregulation of pyruvate dehydrogenase kinase-4 (PDK4) gene transcription. Upregulation of FOXO gene targets known to regulate proteasomal and lysosomal muscle protein breakdown was also evident. We hypothesised that increasing CHO oxidation in vivo, using the pyruvate dehydrogenase complex (PDC) activator, dichloroacetate (DCA), would blunt activation of FOXO gene targets and reduce statin myopathy. Female Wistar Hanover rats were dosed daily for 12 days (oral gavage) with either vehicle (control, 0.5% w/v hydroxypropyl-methylcellulose 0.1% w/v polysorbate-80; n = 9), 88 mg( )kg(-1) day(-1) simvastatin (n = 8), 88 mg( )kg(-1) day(-1) simvastatin + 30 mg kg(-1) day(-1) DCA (n = 9) or 88 mg kg(-1) day(-1) simvastatin + 40 mg kg(-1) day(-1) DCA (n = 9). Compared with control, simvastatin reduced body mass gain and food intake, increased muscle fibre necrosis, plasma creatine kinase levels, muscle PDK4, muscle atrophy F-box (MAFbx) and cathepsin-L mRNA expression, increased PDK4 protein expression, and proteasome and cathepsin-L activity, and reduced muscle PDC activity. Simvastatin with DCA maintained body mass gain and food intake, abrogated the myopathy, decreased muscle PDK4 mRNA and protein, MAFbx and cathepsin-L mRNA, increased activity of PDC and reduced proteasome activity compared with simvastatin. PDC activation abolished statin myopathy in rodent skeletal muscle, which occurred at least in part via inhibition of FOXO-mediated transcription of genes regulating muscle CHO utilisation and protein breakdown.

  8. Human 17β-hydroxysteroid dehydrogenase-ligand complexes: crystals of different space groups with various cations and combined seeding and co-crystallization

    NASA Astrophysics Data System (ADS)

    Zhu, D.-W.; Han, Q.; Qiu, W.; Campbell, R. L.; Xie, B.-X.; Azzi, A.; Lin, S.-X.

    1999-01-01

    Human estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD1) is responsible for the synthesis of active estrogens that stimulate the proliferation of breast cancer cells. The enzyme has been crystallized using a Mg 2+/PEG (3500)/β-octyl glucoside system [Zhu et al., J. Mol. Biol. 234 (1993) 242]. The space group of these crystals is C2. Here we report that cations can affect 17β-HSD1 crystallization significantly. In the presence of Mn 2+ instead of Mg 2+, crystals have been obtained in the same space group with similar unit cell dimensions. In the presence of Li + and Na + instead of Mg 2+, the space group has been changed to P2 12 12 1. A whole data set for a crystal of 17ß-HSD1 complex with progesterone grown in the presence of Li + has been collected to 1.95 Å resolution with a synchrotron source. The cell dimensions are a=41.91 Å, b=108.21 Å, c=117.00 Å. The structure has been preliminarily determined by molecular replacement, yielding important information on crystal packing in the presence of different cations. In order to further understand the structure-function relationship of 17β-HSD1, enzyme complexes with several ligands have been crystallized. As the steroids have very low aqueous solubility, we used a combined method of seeding and co-crystallization to obtain crystals of 17β-HSD1 complexed with various ligands. This method provides ideal conditions for growing complex crystals, with ligands such as 20α-hydroxysteroid progesterone, testosterone and 17β-methyl-estradiol-NADP +. Several complex structures have been determined with reliable electronic density of the bound ligands.

  9. Untangling the glutamate dehydrogenase allosteric nightmare.

    PubMed

    Smith, Thomas J; Stanley, Charles A

    2008-11-01

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

  10. Higher thermostability of l-lactate dehydrogenases is a key factor in decreasing the optical purity of d-lactic acid produced from Lactobacillus coryniformis.

    PubMed

    Gu, Sol-A; Jun, Chanha; Joo, Jeong Chan; Kim, Seil; Lee, Seung Hwan; Kim, Yong Hwan

    2014-05-10

    Lactobacillus coryniformis is known to produce d-lactic acid as a dominant fermentation product at a cultivation temperature of approximately 30°C. However, the considerable production of l-lactic acid is observed when the fermentation temperature is greater than 40°C. Because optically pure lactates are synthesized from pyruvate by the catalysis of chiral-specific d- or l-lactate dehydrogenase, the higher thermostability of l-LDHs is assumed to be one of the key factors decreasing the optical purity of d-lactic acid produced from L. coryniformis at high temperature. To verify this hypothesis, two types of d-ldh genes and six types of l-ldh genes based on the genomic information of L. coryniformis were synthesized and expressed in Escherichia coli. Among the LDHs tested, five LDHs showed activity and were used to construct polyclonal antibodies. d-LDH1, l-LDH2, and l-LDH3 were found to be expressed in L. coryniformis by Western blotting analysis. The half-life values (t1/2) of the LDHs at 40°C were estimated to be 10.50, 41.76, and 2311min, and the T50(10) values were 39.50, 39.90, and 58.60°C, respectively. In addition, the Tm values were 36.0, 41.0, and 62.4°C, respectively, which indicates that l-LDH has greater thermostability than d-LDH. The higher thermostability of l-LDHs compared with that of d-LDH1 may be a major reason why the enantiopurity of d-lactic acid is decreased at high fermentation temperatures. The key enzymes characterized will suggest a direction for the design of genetically modified lactic acid bacteria to produce optically pure d-lactic acid.

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

    PubMed

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

    1975-02-28

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

  12. 2-ketogluconic acid secretion by incorporation of Pseudomonas putida KT 2440 gluconate dehydrogenase (gad) operon in Enterobacter asburiae PSI3 improves mineral phosphate solubilization.

    PubMed

    Kumar, Chanchal; Yadav, Kavita; Archana, G; Naresh Kumar, G

    2013-09-01

    Enterobacter asburiae PSI3 is known to efficiently solubilize rock phosphate by secretion of approximately 50 mM gluconic acid in Tris-buffered medium in the presence of 75 mM glucose and in a mixture of seven aldosugars each at 15 mM concentration, mimicking alkaline vertisol soils. Efficacy of this bacterium in the rhizosphere requires P release in the presence of low amount of sugars. To achieve this, E. asburiae PSI3 has been manipulated to express gluconate dehydrogenase (gad) operon of Pseudomonas putida KT 2440 to produce 2-ketogluconic acid. E. asburiae PSI3 harboring gad operon had 438 U of GAD activity, secreted 11.63 mM 2-ketogluconic and 21.65 mM gluconic acids in Tris-rock phosphate-buffered medium containing 45 mM glucose. E. asburiae PSI3 gad transformant solubilized 0.84 mM P from rock phosphate in TRP-buffered liquid medium. In the presence of a mixture of seven sugars each at 12 mM, the transformant brought about a drop in pH to 4.1 and released 0.53 mM P. PMID:23666029

  13. Pathways of Amino Acid Degradation in Nilaparvata lugens (Stål) with Special Reference to Lysine-Ketoglutarate Reductase/Saccharopine Dehydrogenase (LKR/SDH)

    PubMed Central

    Wan, Pin-Jun; Yuan, San-Yue; Tang, Yao-Hua; Li, Kai-Long; Yang, Lu; Fu, Qiang; Li, Guo-Qing

    2015-01-01

    Nilaparvata lugens harbors yeast-like symbionts (YLSs). In present paper, a genome-wide analysis found 115 genes from Ni. lugens and 90 genes from YLSs that were involved in the metabolic degradation of 20 proteinogenic amino acids. These 205 genes encoded for 77 enzymes. Accordingly, the degradation pathways for the 20 amino acids were manually constructed. It is postulated that Ni. lugens can independently degrade fourteen amino acids (threonine, alanine, glycine, serine, aspartate, asparagine, phenylalanine, tyrosine, glutamate, glutamine, proline, histidine, leucine and lysine). Ni. lugens and YLSs enzymes may work collaboratively to break down tryptophan, cysteine, arginine, isoleucine, methionine and valine. We cloned a lysine-ketoglutarate reductase/saccharopine dehydrogenase gene (Nllkr/sdh) that encoded a bifunctional enzyme catalyzing the first two steps of lysine catabolism. Nllkr/sdh is widely expressed in the first through fifth instar nymphs and adults, and is highly expressed in the fat body, ovary and gut in adults. Ingestion of dsNllkr/sdh by nymphs successfully knocked down the target gene, and caused nymphal/adult mortality, shortened nymphal development stage and reduced adult fresh weight. Moreover, Nllkr/sdh knockdown resulted in three defects: wings were shortened and thickened; cuticles were stretched and thinned; and old nymphal cuticles remained on the tips of legs and abdomen and were not completely shed. These data indicate that impaired lysine degradation negatively affects the survival and development of Ni. lugens. PMID:26000452

  14. Crystallization and preliminary X-ray analysis of the complex of NADH and 3α-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831

    SciTech Connect

    Kataoka, Sachiyo; Nakamura, Shota; Ohkubo, Tadayasu; Ueda, Shigeru; Uchiyama, Susumu; Kobayashi, Yuji; Oda, Masayuki

    2006-06-01

    The complex of NADH and 3α-HSD from Pseudomonas sp. B-0831 has been crystallized and X-ray diffraction data have been collected to 1.8 Å resolution. The NAD(P){sup +}-dependent enzyme 3α-hydroxysteroid dehydrogenase (3α-HSD) catalyzes the reversible interconversion of hydroxyl and oxo groups at position 3 of the steroid nucleus. The complex of NADH and 3α-HSD from Pseudomonas sp. B-0831 was crystallized by the hanging-drop vapour-diffusion method. Refinement of crystallization conditions with microseeding improved the quality of the X-ray diffraction data to a resolution of 1.8 Å. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 62.46, b = 82.25, c = 86.57 Å, and contained two molecules, reflecting dimer formation of 3α-HSD, in the asymmetric unit.

  15. Photodissociation spectroscopy of the Mg{sup +}-acetic acid complex

    SciTech Connect

    Abate, Yohannes; Kleiber, P. D.

    2006-11-14

    We have studied the structure and photodissociation of Mg{sup +}-acetic acid clusters. Ab initio calculations suggest four relatively strongly bound ground state isomers for the [MgC{sub 2}H{sub 4}O{sub 2}]{sup +} complex. These isomers include the cis and trans forms of the Mg{sup +}-acetic acid association complex with Mg{sup +} bonded to the carbonyl O atom of acetic acid, the Mg{sup +}-acetic acid association complex with Mg{sup +} bonded to the hydroxyl O atom of acetic acid, or to a Mg{sup +}-ethenediol association complex. Photodissociation through the Mg{sup +}-based 3p<-3s absorption bands in the near UV leads to direct (nonreactive) and reactive dissociation products: Mg{sup +}, MgOH{sup +}, Mg(H{sub 2}O){sup +}, CH{sub 3}CO{sup +}, and MgCH{sub 3}{sup +}. At low energies the dominant reactive quenching pathway is through dehydration to Mg(H{sub 2}O){sup +}, but additional reaction channels involving C-H and C-C bond activation are also open at higher energies.

  16. De novo fatty acid biosynthesis and elongation in very long-chain acyl-CoA dehydrogenase-deficient mice supplemented with odd or even medium-chain fatty acids.

    PubMed

    Tucci, Sara; Behringer, Sidney; Spiekerkoetter, Ute

    2015-11-01

    An even medium-chain triglyceride (MCT)-based diet is the mainstay of treatment in very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD). Previous studies with magnetic resonance spectroscopy have shown an impact of MCT on the average fatty acid chain length in abdominal fat. We therefore assume that medium-chain fatty acids (MCFAs) are elongated and accumulate in tissue as long-chain fatty acids. In this study, we explored the hepatic effects of long-term supplementation with MCT or triheptanoin, an odd-chain C7-based triglyceride, in wild-type and VLCAD-deficient (VLCAD(-/-) ) mice after 1 year of supplementation as compared with a control diet. The de novo biosynthesis and elongation of fatty acids, and peroxisomal β-oxidation, were quantified by RT-PCR. This was followed by a comprehensive analysis of hepatic and cardiac fatty acid profiles by GC-MS. Long-term application of even and odd MCFAs strongly induced de novo biosynthesis and elongation of fatty acids in both wild-type and VLCAD(-/-) mice, leading to an alteration of the hepatic fatty acid profiles. We detected de novo-synthesized and elongated fatty acids, such as heptadecenoic acid (C17:1n9), eicosanoic acid (C20:1n9), erucic acid (C22:1n9), and mead acid (C20:3n9), that were otherwise completely absent in mice under control conditions. In parallel, the content of monounsaturated fatty acids was massively increased. Furthermore, we observed strong upregulation of peroxisomal β-oxidation in VLCAD(-/-) mice, especially when they were fed an MCT diet. Our data raise the question of whether long-term MCFA supplementation represents the most efficient treatment in the long term. Studies on the hepatic toxicity of triheptanoin are still ongoing.

  17. De novo fatty acid biosynthesis and elongation in very long-chain acyl-CoA dehydrogenase-deficient mice supplemented with odd or even medium-chain fatty acids.

    PubMed

    Tucci, Sara; Behringer, Sidney; Spiekerkoetter, Ute

    2015-11-01

    An even medium-chain triglyceride (MCT)-based diet is the mainstay of treatment in very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD). Previous studies with magnetic resonance spectroscopy have shown an impact of MCT on the average fatty acid chain length in abdominal fat. We therefore assume that medium-chain fatty acids (MCFAs) are elongated and accumulate in tissue as long-chain fatty acids. In this study, we explored the hepatic effects of long-term supplementation with MCT or triheptanoin, an odd-chain C7-based triglyceride, in wild-type and VLCAD-deficient (VLCAD(-/-) ) mice after 1 year of supplementation as compared with a control diet. The de novo biosynthesis and elongation of fatty acids, and peroxisomal β-oxidation, were quantified by RT-PCR. This was followed by a comprehensive analysis of hepatic and cardiac fatty acid profiles by GC-MS. Long-term application of even and odd MCFAs strongly induced de novo biosynthesis and elongation of fatty acids in both wild-type and VLCAD(-/-) mice, leading to an alteration of the hepatic fatty acid profiles. We detected de novo-synthesized and elongated fatty acids, such as heptadecenoic acid (C17:1n9), eicosanoic acid (C20:1n9), erucic acid (C22:1n9), and mead acid (C20:3n9), that were otherwise completely absent in mice under control conditions. In parallel, the content of monounsaturated fatty acids was massively increased. Furthermore, we observed strong upregulation of peroxisomal β-oxidation in VLCAD(-/-) mice, especially when they were fed an MCT diet. Our data raise the question of whether long-term MCFA supplementation represents the most efficient treatment in the long term. Studies on the hepatic toxicity of triheptanoin are still ongoing. PMID:26284828

  18. Evidence in support of lysine 77 and histidine 96 as acid-base catalytic residues in saccharopine dehydrogenase from Saccharomyces cerevisiae.

    PubMed

    Kumar, Vidya Prasanna; Thomas, Leonard M; Bobyk, Kostyantyn D; Andi, Babak; Cook, Paul F; West, Ann H

    2012-01-31

    Saccharopine dehydrogenase (SDH) catalyzes the final reaction in the α-aminoadipate pathway, the conversion of l-saccharopine to l-lysine (Lys) and α-ketoglutarate (α-kg) using NAD⁺ as an oxidant. The enzyme utilizes a general acid-base mechanism to conduct its reaction with a base proposed to accept a proton from the secondary amine of saccharopine in the oxidation step and a group proposed to activate water to hydrolyze the resulting imine. Crystal structures of an open apo form and a closed form of the enzyme with saccharopine and NADH bound have been determined at 2.0 and 2.2 Å resolution, respectively. In the ternary complex, a significant movement of domain I relative to domain II that closes the active site cleft between the two domains and brings H96 and K77 into the proximity of the substrate binding site is observed. The hydride transfer distance is 3.6 Å, and the side chains of H96 and K77 are properly positioned to act as acid-base catalysts. Preparation of the K77M and H96Q single-mutant and K77M/H96Q double-mutant enzymes provides data consistent with their role as the general acid-base catalysts in the SDH reaction. The side chain of K77 initially accepts a proton from the ε-amine of the substrate Lys and eventually donates it to the imino nitrogen as it is reduced to a secondary amine in the hydride transfer step, and H96 protonates the carbonyl oxygen as the carbinolamine is formed. The K77M, H976Q, and K77M/H96Q mutant enzymes give 145-, 28-, and 700-fold decreases in V/E(t) and >10³-fold increases in V₂/K(Lys)E(t) and V₂/K(α-kg)E(t) (the double mutation gives >10⁵-fold decreases in the second-order rate constants). In addition, the K77M mutant enzyme exhibits a primary deuterium kinetic isotope effect of 2.0 and an inverse solvent deuterium isotope effect of 0.77 on V₂/K(Lys). A value of 2.0 was also observed for (D)(V₂/K(Lys))(D₂O) when the primary deuterium kinetic isotope effect was repeated in D₂O, consistent with a

  19. Evidence in support of lysine 77 and histidine 96 as acid-base catalytic residues in saccharopine dehydrogenase from Saccharomyces cerevisiae.

    PubMed

    Kumar, Vidya Prasanna; Thomas, Leonard M; Bobyk, Kostyantyn D; Andi, Babak; Cook, Paul F; West, Ann H

    2012-01-31

    Saccharopine dehydrogenase (SDH) catalyzes the final reaction in the α-aminoadipate pathway, the conversion of l-saccharopine to l-lysine (Lys) and α-ketoglutarate (α-kg) using NAD⁺ as an oxidant. The enzyme utilizes a general acid-base mechanism to conduct its reaction with a base proposed to accept a proton from the secondary amine of saccharopine in the oxidation step and a group proposed to activate water to hydrolyze the resulting imine. Crystal structures of an open apo form and a closed form of the enzyme with saccharopine and NADH bound have been determined at 2.0 and 2.2 Å resolution, respectively. In the ternary complex, a significant movement of domain I relative to domain II that closes the active site cleft between the two domains and brings H96 and K77 into the proximity of the substrate binding site is observed. The hydride transfer distance is 3.6 Å, and the side chains of H96 and K77 are properly positioned to act as acid-base catalysts. Preparation of the K77M and H96Q single-mutant and K77M/H96Q double-mutant enzymes provides data consistent with their role as the general acid-base catalysts in the SDH reaction. The side chain of K77 initially accepts a proton from the ε-amine of the substrate Lys and eventually donates it to the imino nitrogen as it is reduced to a secondary amine in the hydride transfer step, and H96 protonates the carbonyl oxygen as the carbinolamine is formed. The K77M, H976Q, and K77M/H96Q mutant enzymes give 145-, 28-, and 700-fold decreases in V/E(t) and >10³-fold increases in V₂/K(Lys)E(t) and V₂/K(α-kg)E(t) (the double mutation gives >10⁵-fold decreases in the second-order rate constants). In addition, the K77M mutant enzyme exhibits a primary deuterium kinetic isotope effect of 2.0 and an inverse solvent deuterium isotope effect of 0.77 on V₂/K(Lys). A value of 2.0 was also observed for (D)(V₂/K(Lys))(D₂O) when the primary deuterium kinetic isotope effect was repeated in D₂O, consistent with a

  20. Structures of the G81A mutant form of the active chimera of (S)-mandelate dehydrogenase and its complex with two of its substrates

    SciTech Connect

    Sukumar, Narayanasami; Dewanti, Asteriani; Merli, Angelo; Rossi, Gian Luigi; Mitra, Bharati; Mathews, F. Scott

    2009-06-01

    The crystal structure of the G81A mutant form of the chimera of (S)-mandelate dehydrogenase and of its complexes with two of its substrates reveal productive and non-productive modes of binding for the catalytic reaction. The structure also indicates the role of G81A in lowering the redox potential of the flavin co-factor leading to an ∼200-fold slower catalytic rate of substrate oxidation. (S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a membrane-associated flavoenzyme, catalyzes the oxidation of (S)-mandelate to benzoylformate. Previously, the structure of a catalytically similar chimera, MDH-GOX2, rendered soluble by the replacement of its membrane-binding segment with the corresponding segment of glycolate oxidase (GOX), was determined and found to be highly similar to that of GOX except within the substituted segments. Subsequent attempts to cocrystallize MDH-GOX2 with substrate proved unsuccessful. However, the G81A mutants of MDH and of MDH-GOX2 displayed ∼100-fold lower reactivity with substrate and a modestly higher reactivity towards molecular oxygen. In order to understand the effect of the mutation and to identify the mode of substrate binding in MDH-GOX2, a crystallographic investigation of the G81A mutant of the MDH-GOX2 enzyme was initiated. The structures of ligand-free G81A mutant MDH-GOX2 and of its complexes with the substrates 2-hydroxyoctanoate and 2-hydroxy-3-indolelactate were determined at 1.6, 2.5 and 2.2 Å resolution, respectively. In the ligand-free G81A mutant protein, a sulfate anion previously found at the active site is displaced by the alanine side chain introduced by the mutation. 2-Hydroxyoctanoate binds in an apparently productive mode for subsequent reaction, while 2-hydroxy-3-indolelactate is bound to the enzyme in an apparently unproductive mode. The results of this investigation suggest that a lowering of the polarity of the flavin environment resulting from the displacement of nearby water molecules caused by

  1. The lactate dehydrogenase--reduced nicotinamide--adenine dinucleotide--pyruvate complex. Kinetics of pyruvate binding and quenching of coeznyme fluorescence.

    PubMed

    Südi, J

    1974-04-01

    The stopped-flow kinetic studies described in this and the following paper (Südi, 1974) demonstrate that a Haldane-type description of the reversible lactate dehydrogenase reaction presents an experimentally feasible task. Combined results of these two papers yield numerical values for the six rate constants defined by the following equilibrium scheme, where E represents lactate dehydrogenase: [Formula: see text] The experiments were carried out at pH8.4 at a relatively low temperature (6.3 degrees C) with the pig heart enzyme. Identification of the above two intermediates and determination of the corresponding rate constants actually involve four series of independent observations in these studies, since (a) the reaction can be followed in both directions, and (b) both the u.v. absorption and the fluorescence of the coenzymes are altered in the reaction, and it is shown that these two spectral changes do not occur simultaneously. Kinetic observations made in the reverse direction are reported in this paper. It is demonstrated that the fluorescence of NADH can no longer be observed in the ternary complex E(NADH) (Pyr). Even though the oxidation-reduction reaction rapidly follows the formation of this complex, the numerical values of k(-4) (8.33x10(5)m(-1).s(-1)) and k(+4) (222s(-1)) are easily obtained from a directly observed second-order reaction step in which fluorescent but not u.v.-absorbing material is disappearing. U.v.-absorption measurements do not clearly resolve the subsequent oxidation-reduction step from the dissociation of lactate. It is shown that this must be due partly to the instrumental dead time, and partly to a low transient concentration of E(NAD+) (Lac) in the two-step sequential reaction in which the detectable disappearance of u.v.-absorbing material takes place. It is estimated that about one-tenth of the total change in u.v. absorption is due to a ;burst reaction' in which E(NAD+) (Lac) is produced, and this estimation yields, from k

  2. Improved production of homo-D-lactic acid via xylose fermentation by introduction of xylose assimilation genes and redirection of the phosphoketolase pathway to the pentose phosphate pathway in L-Lactate dehydrogenase gene-deficient Lactobacillus plantarum.

    PubMed

    Okano, Kenji; Yoshida, Shogo; Yamada, Ryosuke; Tanaka, Tsutomu; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

    2009-12-01

    The production of optically pure d-lactic acid via xylose fermentation was achieved by using a Lactobacillus plantarum NCIMB 8826 strain whose l-lactate dehydrogenase gene was deficient and whose phosphoketolase genes were replaced with a heterologous transketolase gene. After 60 h of fermentation, 41.2 g/liter of d-lactic acid was produced from 50 g/liter of xylose.

  3. A tightly bound quinone functions in the ubiquinone reaction sites of quinoprotein alcohol dehydrogenase of an acetic acid bacterium, Gluconobacter suboxydans.

    PubMed

    Matsushita, Kazunobu; Kobayashi, Yoshiki; Mizuguchi, Mitsuhiro; Toyama, Hirohide; Adachi, Osao; Sakamoto, Kimitoshi; Miyoshi, Hideto

    2008-10-01

    Quinoprotein alcohol dehydrogenase (ADH) of acetic acid bacteria is a membrane-bound enzyme that functions as the primary dehydrogenase in the ethanol oxidase respiratory chain. It consists of three subunits and has a pyrroloquinoline quinone (PQQ) in the active site and four heme c moieties as electron transfer mediators. Of these, three heme c sites and a further site have been found to be involved in ubiquinone (Q) reduction and ubiquinol (QH2) oxidation respectively (Matsushita et al., Biochim. Biophys. Acta, 1409, 154-164 (1999)). In this study, it was found that ADH solubilized and purified with dodecyl maltoside, but not with Triton X-100, had a tightly bound Q, and thus two different ADHs, one having the tightly bound Q (Q-bound ADH) and Q-free ADH, could be obtained. The Q-binding sites of both the ADHs were characterized using specific inhibitors, a substituted phenol PC16 (a Q analog inhibitor) and antimycin A. Based on the inhibition kinetics of Q2 reductase and ubiquinol-2 (Q2H2) oxidase activities, it was suggested that there are one and two PC16-binding sites in Q-bound ADH and Q-free ADH respectively. On the other hand, with antimycin A, only one binding site was found for Q2 reductase and Q2H2 oxidase activities, irrespective of the presence of bound Q. These results suggest that ADH has a high-affinity Q binding site (QH) besides low-affinity Q reduction and QH2 oxidation sites, and that the bound Q in the QH site is involved in the electron transfer between heme c moieties and bulk Q or QH2 in the low-affinity sites.

  4. Two D-2-hydroxy-acid dehydrogenases in Arabidopsis thaliana with catalytic capacities to participate in the last reactions of the methylglyoxal and beta-oxidation pathways.

    PubMed

    Engqvist, Martin; Drincovich, María F; Flügge, Ulf-Ingo; Maurino, Verónica G

    2009-09-11

    The Arabidopsis thaliana locus At5g06580 encodes an ortholog to Saccharomyces cerevisiae d-lactate dehydrogenase (AtD-LDH). The recombinant protein is a homodimer of 59-kDa subunits with one FAD per monomer. A substrate screen indicated that AtD-LDH catalyzes the oxidation of d- and l-lactate, d-2-hydroxybutyrate, glycerate, and glycolate using cytochrome c as an electron acceptor. AtD-LDH shows a clear preference for d-lactate, with a catalytic efficiency 200- and 2000-fold higher than that for l-lactate and glycolate, respectively, and a K(m) value for d-lactate of approximately 160 microm. Knock-out mutants showed impaired growth in the presence of d-lactate or methylglyoxal. Collectively, the data indicated that the protein is a d-LDH that participates in planta in the methylglyoxal pathway. Web-based bioinformatic tools revealed the existence of a paralogous protein encoded by locus At4g36400. The recombinant protein is a homodimer of 61-kDa subunits with one FAD per monomer. A substrate screening revealed highly specific d-2-hydroxyglutarate (d-2HG) conversion in the presence of an organic cofactor with a K(m) value of approximately 580 microm. Thus, the enzyme was characterized as a d-2HG dehydrogenase (AtD-2HGDH). Analysis of knock-out mutants demonstrated that AtD-2HGDH is responsible for the total d-2HGDH activity present in A. thaliana. Gene coexpression analysis indicated that AtD-2HGDH is in the same network as several genes involved in beta-oxidation and degradation of branched-chain amino acids and chlorophyll. It is proposed that AtD-2HGDH participates in the catabolism of d-2HG most probably during the mobilization of alternative substrates from proteolysis and/or lipid degradation.

  5. Photolysis of copper(II)-amino acid complexes in water

    SciTech Connect

    Hayase, K.; Zepp, R.G. )

    1991-07-01

    Kinetics of the photolysis of Cu{sup 2+}-amino acid complexes were investigated under sunlight or monochromatic radiation. Under sunlight (latitude 40{degree}N) in the absence of dioxygen, the mean half-lives estimated for the photoreduction of the bis-Cu{sup 2+} complexes (CuL{sub 2}) at pH 8.0 and 25C were 0.55, 1.6, 8.6, 8.8, 45. and 71 days for L = tris(hydroxymethyl)aminomethane (Tris), {beta}-alanine, L-aspartic acid, L-glutamic acid, glycine, and histidine, respectively. Under monochromatic UV irradiation (310 nm) with dioxygen absent, quantum yields for the photoproduction of Cu(I) from CuL{sub 2} were found to be 0.071, 0.098, 0.063, 0.066, 0.025, and 0.0011 for L = Tris, {beta}-alanine, L-aspartic acid, L-glutamic acid, glycine, and L-histidine, respectively. The effects of pH on the quantum yields were determined for Cu{sup 2+}-{beta}-alanine and Cu{sup 2+}-Tris complexes. Decreases in quantum yields observed with increasing acidity were attributed to changes in speciation from predominantly CuL{sub 2} to less photoreactive complexes, mainly CuL and Cu{sup 2+}. In air-saturated solutions, rates and quantum yields for Cu(I) formation were sharply reduced and hydrogen peroxide was formed. Results of the study suggest that photoreactions of Cu{sup 2+} coordination complexes may contribute to its toxicity to aquatic biota.

  6. Functional Characterization of the Subunits N, H, J, and O of the NAD(P)H Dehydrogenase Complexes in Synechocystis sp. Strain PCC 6803.

    PubMed

    He, Zhihui; Mi, Hualing

    2016-06-01

    The cyanobacterial NAD(P)H dehydrogenase (NDH-1) complexes play crucial roles in variety of bioenergetic reactions such as respiration, CO2 uptake, and cyclic electron transport around PSI. Recently, substantial progress has been made in identifying the composition of subunits of NDH-1 complexes. However, the localization and the physiological roles of several subunits in cyanobacteria are not fully understood. Here, by constructing fully segregated ndhN, ndhO, ndhH, and ndhJ null mutants in Synechocystis sp. strain PCC 6803, we found that deletion of ndhN, ndhH, or ndhJ but not ndhO severely impaired the accumulation of the hydrophilic subunits of the NDH-1 in the thylakoid membrane, resulting in disassembly of NDH-1MS, NDH-1MS', as well as NDH-1L, finally causing the severe growth suppression phenotype. In contrast, deletion of NdhO affected the growth at pH 6.5 in air. In the cytoplasm, either NdhH or NdhJ deleted mutant, but neither NdhN nor NdhO deleted mutant, failed to accumulate the NDH-1 assembly intermediate consisting of NdhH, NdhJ, NdhK, and NdhM. Based on these results, we suggest that NdhN, NdhH, and NdhJ are essential for the stability and the activities of NDH-1 complexes, while NdhO for NDH-1 functions under the condition of inorganic carbon limitation in Synechocystis sp. strain PCC 6803. We discuss the roles of these subunits and propose a new NDH-1 model. PMID:27208236

  7. Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan-1-ol production at theoretical yield in Escherichia coli.

    PubMed

    Bastian, Sabine; Liu, Xiang; Meyerowitz, Joseph T; Snow, Christopher D; Chen, Mike M Y; Arnold, Frances H

    2011-05-01

    2-methylpropan-1-ol (isobutanol) is a leading candidate biofuel for the replacement or supplementation of current fossil fuels. Recent work has demonstrated glucose to isobutanol conversion through a modified amino acid pathway in a recombinant organism. Although anaerobic conditions are required for an economically competitive process, only aerobic isobutanol production has been feasible due to an imbalance in cofactor utilization. Two of the pathway enzymes, ketol-acid reductoisomerase and alcohol dehydrogenase, require nicotinamide dinucleotide phosphate (NADPH); glycolysis, however, produces only nicotinamide dinucleotide (NADH). Here, we compare two solutions to this imbalance problem: (1) over-expression of pyridine nucleotide transhydrogenase PntAB and (2) construction of an NADH-dependent pathway, using engineered enzymes. We demonstrate that an NADH-dependent pathway enables anaerobic isobutanol production at 100% theoretical yield and at higher titer and productivity than both the NADPH-dependent pathway and transhydrogenase over-expressing strain. Our results show how engineering cofactor dependence can overcome a critical obstacle to next-generation biofuel commercialization.

  8. Electrospun polymer nanofibers reinforced by tannic acid/Fe+++ complexes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nanofibers and fibrous mats of polyvinyl alcohol (PVA) loaded with tannic acid (TA) and ferric ion (Fe+++) complexes (TA-Fe+++) were synthesized by the electrospinning technique. The spinning solutions were characterized for surface tension, electrical conductivity, and viscosity. It was found that ...

  9. Supramolecular complex formation and crystallization of isocitrate dehydrogenase from Thermus thermophilus HB8: preliminary studies with X-Ray crystallography and atomic force microscopy.

    PubMed

    Ishii, Noriyuki; Umemura, Kazuo; Miyazaki, Kentaro

    2008-09-01

    Atomic force microscopy (AFM) observation of a crystal surface of the thermostable isocitrate dehydrogenase (ICDH) from a thermophilic eubacterium, Thermus thermophilus HB8, suggested that the crystal consists of huge homo-complexed ellipsoidal bodies of the protein, with averaged long- and short-axis diameters of 18.6 nm and 10.9 nm respectively. Thick diamond-shaped crystals of about 0.4 mm on the longest axis were obtained by the vapor diffusion method from a solution of 100 mM sodium cacodylate, pH 6.6-8.4, containing 1.4 M sodium acetate as the precipitate, and diffracted X-rays at 3.7 A resolution. The crystals belonged to the monoclinic lattice type with space group C2 and had cell dimensions of a=495.5, b=189.2, c=336.2 A, and beta=126.4 degrees , indicating that an asymmetric unit contained more than 33 molecules with a molecular mass of 54.2 kDa. Calculations based on data obtained by the X-ray method showed good agreement with AFM observation. These results suggest the possibility that the residing T. thermophilus HB8 ICDH molecules are piled one on top another as a preformed supramolecular nano-architecture in the crystal lattice. The system appears suitable for further investigation using a bottom-up approach to the self-associated construction of nano-architectures with proteins.

  10. Structure of the Alpha-2 Epsilon-2 Ni-dependent CO Dehydrogenase Component of the Methanosarcina Barkeri Acetyl-CoA Decarbonylase/Synthase Complex

    SciTech Connect

    Gong, W.; Hao, B; Wei, Z; Ferguson, Jr., D; Tallant, T; Krzycki, J; Chan, M

    2008-01-01

    Ni-dependent carbon monoxide dehydrogenases (Ni-CODHs) are a diverse family of enzymes that catalyze reversible CO:CO2 oxidoreductase activity in acetogens, methanogens, and some CO-using bacteria. Crystallography of Ni-CODHs from CO-using bacteria and acetogens has revealed the overall fold of the Ni-CODH core and has suggested structures for the C cluster that mediates CO:CO2 interconversion. Despite these advances, the mechanism of CO oxidation has remained elusive. Herein, we report the structure of a distinct class of Ni-CODH from methanogenic archaea: the ?2?2 component from the ?8?8?8?8?8 CODH/acetyl-CoA decarbonylase/synthase complex, an enzyme responsible for the majority of biogenic methane production on Earth. The structure of this Ni-CODH component provides support for a hitherto unobserved state in which both CO and H2O/OH- bind to the Ni and the exogenous FCII iron of the C cluster, respectively, and offers insight into the structures and functional roles of the ?-subunit and FeS domain not present in nonmethanogenic Ni-CODHs.

  11. Structure of the Alpha(2)F(2) Ni-Dependent CO Dehydrogenase Component of the Methanosarcina Barkeri Acetyl-CoA Decarbonylase/Synthase Complex

    SciTech Connect

    Gong, W.; Hao, B.; Wei, Z.; Ferguson, D.J.; Jr.; Tallant, T.; Krzycki, J.A.; Chang, M.K.

    2009-05-18

    Ni-dependent carbon monoxide dehydrogenases (Ni-CODHs) are a diverse family of enzymes that catalyze reversible CO:CO{sub 2} oxidoreductase activity in acetogens, methanogens, and some CO-using bacteria. Crystallography of Ni-CODHs from CO-using bacteria and acetogens has revealed the overall fold of the Ni-CODH core and has suggested structures for the C cluster that mediates CO:CO{sub 2} interconversion. Despite these advances, the mechanism of CO oxidation has remained elusive. Herein, we report the structure of a distinct class of Ni-CODH from methanogenic archaea: the {alpha}{sub 2}{epsilon}{sub 2} component from the {alpha}{sub 8}{beta}{sub 8}{gamma}{sub 8}{delta}{sub 8}{epsilon}{sub 8} CODH/acetyl-CoA decarbonylase/synthase complex, an enzyme responsible for the majority of biogenic methane production on Earth. The structure of this Ni-CODH component provides support for a hitherto unobserved state in which both CO and H{sub 2}O/OH{sup -} bind to the Ni and the exogenous FCII iron of the C cluster, respectively, and offers insight into the structures and functional roles of the {epsilon}-subunit and FeS domain not present in nonmethanogenic Ni-CODHs.

  12. Photosystem I cyclic electron flow via chloroplast NADH dehydrogenase-like complex performs a physiological role for photosynthesis at low light.

    PubMed

    Yamori, Wataru; Shikanai, Toshiharu; Makino, Amane

    2015-09-11

    Cyclic electron transport around photosystem I (PS I) was discovered more than a half-century ago and two pathways have been identified in angiosperms. Although substantial progress has been made in understanding the structure of the chloroplast NADH dehydrogenase-like (NDH) complex, which mediates one route of the cyclic electron transport pathways, its physiological function is not well understood. Most studies focused on the role of the NDH-dependent PS I cyclic electron transport in alleviation of oxidative damage in strong light. In contrast, here it is shown that impairment of NDH-dependent cyclic electron flow in rice specifically causes a reduction in the electron transport rate through PS I (ETR I) at low light intensity with a concomitant reduction in CO2 assimilation rate, plant biomass and importantly, grain production. There was no effect on PS II function at low or high light intensity. We propose a significant physiological function for the chloroplast NDH at low light intensities commonly experienced during the reproductive and ripening stages of rice cultivation that have adverse effects crop yield.

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

    PubMed Central

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

    1998-01-01

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

  14. Crystal structure of homoisocitrate dehydrogenase from Schizosaccharomyces pombe

    SciTech Connect

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

    2013-09-18

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

  15. Amino acid residues involved in the catalytic mechanism of NAD-dependent glutamate dehydrogenase from Halobacterium salinarum.

    PubMed

    Pérez-Pomares, F; Ferrer, J; Camacho, M; Pire, C; LLorca, F; Bonete, M J

    1999-02-01

    The pH dependence of kinetic parameters for a competitive inhibitor (glutarate) was determined in order to obtain information on the chemical mechanism for NAD-dependent glutamate dehydrogenase from Halobacterium salinarum. The maximum velocity is pH dependent, decreasing at low pHs giving a pK value of 7.19+/-0.13, while the V/K for l-glutamate at 30 degrees C decreases at low and high pHs, yielding pK values of 7.9+/-0.2 and 9.8+/-0.2, respectively. The glutarate pKis profile decreases at high pHs, yielding a pK of 9. 59+/-0.09 at 30 degrees C. The values of ionization heat calculated from the change in pK with temperature are: 1.19 x 10(4), 5.7 x 10(3), 7 x 10(3), 6.6 x 10(3) cal mol-1, for the residues involved. All these data suggest that the groups required for catalysis and/or binding are lysine, histidine and tyrosine. The enzyme shows a time-dependent loss in glutamate oxidation activity when incubated with diethyl pyrocarbonate (DEPC). Inactivation follows pseudo-first-order kinetics with a second-order rate constant of 53 M-1min-1. The pKa of the titratable group was pK1=6.6+/-0.6. Inactivation with ethyl acetimidate also shows pseudo-first-order kinetics as well as inactivation with TNM yielding second-order constants of 1.2 M-1min-1 and 2.8 M-1min-1, and pKas of 8.36 and 9.0, respectively. The proposed mechanism involves hydrogen binding of each of the two carboxylic groups to tyrosyl residues; histidine interacts with one of the N-hydrogens of the l-glutamate amino group. We also corroborate the presence of a conservative lysine that has a remarkable ability to coordinate a water molecule that would act as general base.

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

    PubMed

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

    1994-06-01

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

  17. Maple syrup urine disease: The E1{beta} gene of human branched-chain {alpha}-ketoacid dehydrogenase complex has 11 rather than 10 exons, and the 3{prime} UTR in one of the two E1{beta} mRNAs arises from intronic sequences

    SciTech Connect

    Chuang, J.L.; Chuang, D.T.; Cox, R.P.

    1996-06-01

    Maple syrup urine disease (MSUD) or branched-chain ketoaciduria is caused by a deficiency in the mitochondrial branched-chain {alpha}-ketoacid dehydrogenase (BCKAD) complex. The clinical manifestations are characterized by accumulation of branched chain amino and {alpha}-ketoacids, which leads to severe cerebral edema with seizures, ketoacidosis, and mental retardation. The BCKAD complex comprises three catalytic components, i.e., a decarboxylase (E1) consisting of two E1{alpha} (M{sub r} = 46,000) and two E1{Beta} (M{sub r} = 37,500) subunits, a transacylase (E2) that contains 24 lipoic acid-bearing subunits, and a dehydrogenase (E3), which is a homodimeric flavoprotein. MSUD is genetically heterogeneous, since mutations in the E1{alpha} subunit (type IA MSUD), the E1{Beta} subunit (type IB), the E2 subunit (type II) and the E3 subunit (type III) have been described. The functional consequences of certain mutations in the BCKAD complex have been studied. 23 refs., 3 figs.

  18. Nickel-phendione complex covalently attached onto carbon nanotube/cross linked glucose dehydrogenase as bioanode for glucose/oxygen compartment-less biofuel cell

    NASA Astrophysics Data System (ADS)

    Korani, Aazam; Salimi, Abdollah; Hadadzadeh, Hasan

    2015-05-01

    Here, [Ni(phendion) (phen)]Cl2 complex, (phendion and phen are 1,10-phenanthroline-5,6-dione and 5-amino-1, 10-phenanthrolin) covalently attached onto carboxyl functionalized multi walls carbon nanotube modified glassy carbon electrode (GCE/MWCNTs-COOH) using solid phase interactions and combinatorial approaches.The attached [Ni(phendion) (phen)]Cl2 complex displays a surface controlled electrode process and it acts as an effective redox mediator for electrocatalytic oxidation of dihydronicotinamide adenine dinucleotide (NADH) at reduced overpotentials. With co-immobilization of glucose dehydrogenase enzyme (GDH) by crosslinking an effective biocatalyst for glucose oxidation designed. The onset potential and current density are -0.1 V versus Ag/AgCl electrode and 0.550 mA cm-2, which indicate the applicability of the proposed system as an efficient bioanode for biofuel cell (BFC) design. A GCE/MWCNTs modified with electrodeposited gold nanoparticles (AuNPs) as a platform for immobilization of bilirubin oxidase (BOD) and the prepared GCE/MWCNTs/AuNPs/BOD biocathode exhibits an onset potential of 0.56 V versus Ag/AgCl. The performance of the fabricated bioanode and biocathode in a membraneless enzyme based glucose/O2 biofuel cell is evaluated. The open circuit voltage of the cell and maximum current density are 520 mV and 0.233 mA cm-2, respectively, while maximum power density of 40 μWcm-2 achieves at voltage of 280 mV with stable output power after 24 h continues operation.

  19. Communication between Thiamin Cofactors in the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Active Centers EVIDENCE FOR A DIRECT PATHWAY BETWEEN THE 4′-AMINOPYRIMIDINE N1′ ATOMS

    SciTech Connect

    Nemeria, Natalia S; Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Mossad, Madouna; Tittmann, Kai; Furey, William; Jordan, Frank

    2010-11-03

    Kinetic, spectroscopic, and structural analysis tested the hypothesis that a chain of residues connecting the 4{prime}-aminopyrimidine N1{prime} atoms of thiamin diphosphates (ThDPs) in the two active centers of the Escherichia coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (Glu{sup 571}, Glu{sup 235}, and Glu{sup 237}) and Arg{sup 606} resulted in impaired binding of the second ThDP, once the first active center was filled, suggesting a pathway for communication between the two ThDPs. (1) Steady-state kinetic and fluorescence quenching studies revealed that upon E571A, E235A, E237A, and R606A substitutions, ThDP binding in the second active center was affected. (2) Analysis of the kinetics of thiazolium C2 hydrogen/deuterium exchange of enzyme-bound ThDP suggests half-of-the-sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for the slow exchanging site, indicating that only one of two active sites is filled with ThDP. (3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. (4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that Glu{sup 235} makes no direct contact with the cofactor. The role of the conserved Glu{sup 571} residue in both catalysis and cofactor orientation is revealed by the combined results for the first time.

  20. Production of natural antimicrobial compound D-phenyllactic acid using Leuconostoc mesenteroides ATCC 8293 whole cells involving highly active D-lactate dehydrogenase.

    PubMed

    Li, L; Shin, S-Y; Lee, K W; Han, N S

    2014-10-01

    Phenyllactic acid (PLA) is an antimicrobial compound naturally synthesized in various fermented foods and its D-form of PLA is known to be more active than the L-isomer. In this study, Leuconostoc mesenteroides ATCC 8293 cells, elaborating D-lactate dehydrogenase (D-ldh) were used to produce D-PLA from phenylpyruvic acid (PPA). When cultured in the presence of PPA (≤50 mmol l(-1)), growing cells produced a maximum yield of 35 mmol l(-1) of D-PLA, and the yields were between 75·2 and 83·3%. Higher conversion yields were obtained at pH 6·0-7·0 when growing cells were used, while the optimum pH range was broader for resting cells. The time required for the complete conversion of PPA into PLA could be shortened to 3 h using resting cells. D-ldh, an enzyme encoded by the LEUM_1756 gene of Leuc. mesenteroides ATCC 8293, was found to be responsible for the conversion of PPA into PLA. The Km and kcat values of the enzyme for PPA were found to be 15·4 mmol l(-1) and 5645 s(-1), respectively. The conditions required for the efficient production of D-PLA were optimized for both growing and resting cells of Leuc. mesenteroides, with special emphasis on achieving high stereoselectivity and conversion yield. Significance and impact of the study: This is the first study on the production of D-phenyllactic acid, which is a natural antimicrobial compound, from phenylpyruvate using Leuconostoc mesenteroides cells. The strain, ATCC 8293, that was used in the study, possesses high stereoselectivity and delivers a high yield. Therefore, it might be a promising candidate for use in large-scale production facilities and in fermented foods.

  1. Glucose-6-phosphate dehydrogenase

    MedlinePlus

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

  2. Nucleic Acid-Peptide Complex Phase Controlled by DNA Hybridization

    NASA Astrophysics Data System (ADS)

    Vieregg, Jeffrey; Lueckheide, Michael; Leon, Lorraine; Marciel, Amanda; Tirrell, Matthew

    When polyanions and polycations are mixed, counterion release drives formation of polymer-rich complexes that can either be solid (precipitates) or liquid (coacervates) depending on the properties of the polyelectrolytes. These complexes are important in many fields, from encapsulation of industrial polymers to membrane-free segregation of biomolecules such as nucleic acids and proteins. Condensation of long double-stranded DNA has been studied for several decades, but comparatively little attention has been paid to the polyelectrolyte behavior of oligonucleotides. We report here studies of DNA oligonucleotides (10 - 88 nt) complexed with polylysine (10 - 100 aa). Unexpectedly, we find that the phase of the resulting complexes is controlled by the hybridization state of the nucleic acid, with double-stranded DNA forming precipitates and single-stranded DNA forming coacervates. Stability increases with polyelectrolyte length and decreases with solution salt concentration, with complexes of the longer double-stranded polymers undergoing precipitate/coacervate/soluble transitions as ionic strength is increased. Mixing coacervates formed by complementary single-stranded oligonucleotides results in precipitate formation, raising the possibility of stimulus-responsive material design.

  3. Combinational expression of sorbose/sorbosone dehydrogenases and cofactor pyrroloquinoline quinone increases 2-keto-L-gulonic acid production in Ketogulonigenium vulgare-Bacillus cereus consortium.

    PubMed

    Du, Jin; Bai, Wei; Song, Hao; Yuan, Ying-Jin

    2013-09-01

    The expression levels of sorbose/sorbosone dehydrogenase genes (sdh and sndh) and the synthesis genes (pqqABCDEN) of the adjoint cofactor pyrroloquinoline quinone (PQQ) were genetically manipulated in Ketogulonigenium vulgare to increase the production of 2-keto-l-gulonic acid (2-KLG), the precursor of vitamin C, in the consortium of K. vulgare and Bacillus cereus. We found that overexpression of sdh-sndh alone in K. vulgare could not significantly enhance the production of 2-KLG, revealing the cofactor PQQ was required for the biosynthesis of 2-KLG. Various expression levels of PQQ were achieved by differential expression of pqqA, pqqABCDE and pqqABCDEN, respectively. The combinatorial expression of sdh/sndh and pqqABCDEN in K. vulgare enabled a 20% increase in the production of 2-KLG (79.1±0.6gl(-1)) than that of the parental K. vulgare (65.9±0.4gl(-1)) in shaking flasks. Our results demonstrated the balanced co-expression of both the key enzymes and the related cofactors was an efficient strategy to increase chemicals' biosynthesis.

  4. Cloning of a Serratia marcescens DNA fragment that induces quinoprotein glucose dehydrogenase-mediated gluconic acid production in Escherichia coli in the presence of stationary phase Serratia marcescens.

    PubMed

    Krishnaraj, P U; Goldstein, A H

    2001-12-18

    Serratia marcescens ER2 was isolated from an endorhizosphere sample based on its high level of mineral phosphate solubilizing (MPS) activity. This phenotype was correlated with expression of the direct oxidation pathway. An ER2 plasmid library constructed in Escherichia coli strain DH5alpha was screened for MPS activity. A recombinant clone DH5alpha (pKG3791) was capable of gluconic acid (GA) production and tricalcium phosphate solubilization but only in the presence of stationary phase ER2 cells. GA production in DH5alpha (pKG3791) was apparently the result of the quinoprotein glucose dehydrogenase activity because AG121 (a Tn5 knockout of gcd) carrying pKG3791 did not produce GA under the same conditions. GA production by DH5alpha (pKG3791) was not observed when ER2 was replaced by another PQQ-producing strain bacterium. These data add to a growing body of evidence that E. coli contains some type of PQQ biosynthesis pathway distinct from those previously characterized in Gram-negative bacteria and that these genes may be induced under appropriate conditions.

  5. The enhancement of tolerance to salt and cold stresses by modifying the redox state and salicylic acid content via the cytosolic malate dehydrogenase gene in transgenic apple plants.

    PubMed

    Wang, Qing-Jie; Sun, Hong; Dong, Qing-Long; Sun, Tian-Yu; Jin, Zhong-Xin; Hao, Yu-Jin; Yao, Yu-Xin

    2016-10-01

    In this study, we characterized the role of an apple cytosolic malate dehydrogenase gene (MdcyMDH) in the tolerance to salt and cold stresses and investigated its regulation mechanism in stress tolerance. The MdcyMDH transcript was induced by mild cold and salt treatments, and MdcyMDH-overexpressing apple plants possessed improved cold and salt tolerance compared to wild-type (WT) plants. A digital gene expression tag profiling analysis revealed that MdcyMDH overexpression largely altered some biological processes, including hormone signal transduction, photosynthesis, citrate cycle and oxidation-reduction. Further experiments verified that MdcyMDH overexpression modified the mitochondrial and chloroplast metabolisms and elevated the level of reducing power, primarily caused by increased ascorbate and glutathione, as well as the increased ratios of ascorbate/dehydroascorbate and glutathione/glutathione disulphide, under normal and especially stress conditions. Concurrently, the transgenic plants produced a high H2 O2 content, but a low O2·- production rate was observed compared to the WT plants. On the other hand, the transgenic plants accumulated more free and total salicylic acid (SA) than the WT plants under normal and stress conditions. Taken together, MdcyMDH conferred the transgenic apple plants a higher stress tolerance by producing more reductive redox states and increasing the SA level; MdcyMDH could serve as a target gene to genetically engineer salt- and cold-tolerant trees. PMID:26923485

  6. Oligosaccharide-based Surfactant/Citric Acid Buffer System Stabilizes Lactate Dehydrogenase during Freeze-drying and Storage without the Addition of Natural Sugar.

    PubMed

    Ogawa, Shigesaburo; Kawai, Ryuichiro; Koga, Maito; Asakura, Kouichi; Takahashi, Isao; Osanai, Shuichi

    2016-06-01

    Experiments were conducted to assess the maintenance effects of oligosaccharide-based surfactants on the enzymatic activity of a model protein, lactate dehydrogenase (LDH), during freeze-drying and room temperature storage using the citric acid buffer system. Oligosaccharide-based surfactants, which exhibit a high glass transition temperature (Tg), promoted the eminent retention of enzymatic activity during these protocols, whereas monosaccharide-based surfactants with a low Tg displayed poor performance at high concentration, albeit much better than that of Tween 80 at middle concentration. The increase in the alkyl chain length did not exert positive effects as observed for the maintenance effect during freeze-thawing, but an amphiphilic nature and a glass forming ability were crucial for the effective stabilization at a low excipient concentration during freeze-drying. Even a low oligosaccharide-based surfactant content (0.1 mg mL(-1)) could maintain LDH activity during freeze-drying, but a high surfactant content (1.0 mg mL(-1)) was required to prevent buffer precipitation and retain high LDH activity on storage. Regarding storage, glass formation restricted molecular mobility in the lyophilized matrix, and LDH activity was effectively retained. The present results describe a strategy based on the glass-forming ability of surfactant-type excipients that affords a natural sugar-free formulation or an alternative use for polysorbate-type surfactants.

  7. Pharmacology of bile acid receptors: Evolution of bile acids from simple detergents to complex signaling molecules.

    PubMed

    Copple, Bryan L; Li, Tiangang

    2016-02-01

    For many years, bile acids were thought to only function as detergents which solubilize fats and facilitate the uptake of fat-soluble vitamins in the intestine. Many early observations; however, demonstrated that bile acids regulate more complex processes, such as bile acids synthesis and immune cell function through activation of signal transduction pathways. These studies were the first to suggest that receptors may exist for bile acids. Ultimately, seminal studies by many investigators led to the discovery of several bile acid-activated receptors including the farnesoid X receptor, the vitamin D receptor, the pregnane X receptor, TGR5, α5 β1 integrin, and sphingosine-1-phosphate receptor 2. Several of these receptors are expressed outside of the gastrointestinal system, indicating that bile acids may have diverse functions throughout the body. Characterization of the functions of these receptors over the last two decades has identified many important roles for these receptors in regulation of bile acid synthesis, transport, and detoxification; regulation of glucose utilization; regulation of fatty acid synthesis and oxidation; regulation of immune cell function; regulation of energy expenditure; and regulation of neural processes such as gastric motility. Through these many functions, bile acids regulate many aspects of digestion ranging from uptake of essential vitamins to proper utilization of nutrients. Accordingly, within a short time period, bile acids moved beyond simple detergents and into the realm of complex signaling molecules. Because of the important processes that bile acids regulate through activation of receptors, drugs that target these receptors are under development for the treatment of several diseases, including cholestatic liver disease and metabolic syndrome. In this review, we will describe the various bile acid receptors, the signal transduction pathways activated by these receptors, and briefly discuss the physiological processes that

  8. Amino acid residues interacting with both the bound quinone and coenzyme, pyrroloquinoline quinone, in Escherichia coli membrane-bound glucose dehydrogenase.

    PubMed

    Mustafa, Golam; Ishikawa, Yoshinori; Kobayashi, Kazuo; Migita, Catharina T; Elias, M D; Nakamura, Satsuki; Tagawa, Seiichi; Yamada, Mamoru

    2008-08-01

    The Escherichia coli membrane-bound glucose dehydrogenase (mGDH) as the primary component of the respiratory chain possesses a tightly bound ubiquinone (UQ) flanking pyrroloquinoline quinone (PQQ) as a coenzyme. Several mutants for Asp-354, Asp-466, and Lys-493, located close to PQQ, that were constructed by site-specific mutagenesis were characterized by enzymatic, pulse radiolysis, and EPR analyses. These mutants retained almost no dehydrogenase activity or ability of PQQ reduction. CD and high pressure liquid chromatography analyses revealed that K493A, D466N, and D466E mutants showed no significant difference in molecular structure from that of the wild-type mGDH but showed remarkably reduced content of bound UQ. A radiolytically generated hydrated electron (e(aq)(-)) reacted with the bound UQ of the wild enzyme and K493R mutant to form a UQ neutral semiquinone with an absorption maximum at 420 nm. Subsequently, intramolecular electron transfer from the bound UQ semiquinone to PQQ occurred. In K493R, the rate of UQ to PQQ electron transfer is about 4-fold slower than that of the wild enzyme. With D354N and D466N mutants, on the other hand, transient species with an absorption maximum at 440 nm, a characteristic of the formation of a UQ anion radical, appeared in the reaction of e(aq)(-), although the subsequent intramolecular electron transfer was hardly affected. This indicates that D354N and D466N are prevented from protonation of the UQ semiquinone radical. Moreover, EPR spectra showed that mutations on Asp-466 or Lys-493 residues changed the semiquinone state of bound UQ. Taken together, we reported here for the first time the existence of a semiquinone radical of bound UQ in purified mGDH and the difference in protonation of ubisemiquinone radical because of mutations in two different amino acid residues, located around PQQ. Furthermore, based on the present results and the spatial arrangement around PQQ, Asp-466 and Lys-493 are suggested to interact both

  9. Quantification of acidic compounds in complex biomass-derived streams

    SciTech Connect

    Karp, Eric M.; Nimlos, Claire T.; Deutch, Steve; Salvachúa, Davinia; Cywar, Robin M.; Beckham, Gregg T.

    2016-01-01

    Biomass-derived streams that contain acidic compounds from the degradation of lignin and polysaccharides (e.g. black liquor, pyrolysis oil, pyrolytic lignin, etc.) are chemically complex solutions prone to instability and degradation during analysis, making quantification of compounds within them challenging. Here we present a robust analytical method to quantify acidic compounds in complex biomass-derived mixtures using ion exchange, sample reconstitution in pyridine and derivatization with BSTFA. The procedure is based on an earlier method originally reported for kraft black liquors and, in this work, is applied to identify and quantify a large slate of acidic compounds in corn stover derived alkaline pretreatment liquor (APL) as a function of pretreatment severity. Analysis of the samples is conducted with GCxGC-TOFMS to achieve good resolution of the components within the complex mixture. The results reveal the dominant low molecular weight components and their concentrations as a function of pretreatment severity. Application of this method is also demonstrated in the context of lignin conversion technologies by applying it to track the microbial conversion of an APL substrate. Here too excellent results are achieved, and the appearance and disappearance of compounds is observed in agreement with the known metabolic pathways of two bacteria, indicating the sample integrity was maintained throughout analysis. Finally, it is shown that this method applies more generally to lignin-rich materials by demonstrating its usefulness in analysis of pyrolysis oil and pyrolytic lignin.

  10. Structures of the G81A mutant form of the active chimera of (S)-mandelate dehydrogenase and its complex with two of its substrates

    SciTech Connect

    Sukumar, Narayanasami; Dewanti, Asteriani; Merli, Angelo; Rossi, Gian Luigi; Mitra, Bharati; Mathews, F. Scott

    2009-06-12

    (S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a membrane-associated flavoenzyme, catalyzes the oxidation of (S)-mandelate to benzoylformate. Previously, the structure of a catalytically similar chimera, MDH-GOX2, rendered soluble by the replacement of its membrane-binding segment with the corresponding segment of glycolate oxidase (GOX), was determined and found to be highly similar to that of GOX except within the substituted segments. Subsequent attempts to cocrystallize MDH-GOX2 with substrate proved unsuccessful. However, the G81A mutants of MDH and of MDH-GOX2 displayed {approx}100-fold lower reactivity with substrate and a modestly higher reactivity towards molecular oxygen. In order to understand the effect of the mutation and to identify the mode of substrate binding in MDH-GOX2, a crystallographic investigation of the G81A mutant of the MDH-GOX2 enzyme was initiated. The structures of ligand-free G81A mutant MDH-GOX2 and of its complexes with the substrates 2-hydroxyoctanoate and 2-hydroxy-3-indolelactate were determined at 1.6, 2.5 and 2.2 {angstrom} resolution, respectively. In the ligand-free G81A mutant protein, a sulfate anion previously found at the active site is displaced by the alanine side chain introduced by the mutation. 2-Hydroxyoctanoate binds in an apparently productive mode for subsequent reaction, while 2-hydroxy-3-indolelactate is bound to the enzyme in an apparently unproductive mode. The results of this investigation suggest that a lowering of the polarity of the flavin environment resulting from the displacement of nearby water molecules caused by the glycine-to-alanine mutation may account for the lowered catalytic activity of the mutant enzyme, which is consistent with the 30 mV lower flavin redox potential. Furthermore, the altered binding mode of the indolelactate substrate may account for its reduced activity compared with octanoate, as observed in the crystalline state.

  11. A comparison of potato and vertebrate lactate dehydrogenases.

    PubMed Central

    Poerio, E; Davies, D D

    1980-01-01

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

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

    PubMed Central

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

    1999-01-01

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

  13. Estimating the acidity of transition metal hydride and dihydrogen complexes by adding ligand acidity constants.

    PubMed

    Morris, Robert H

    2014-02-01

    A simple equation (pKa(THF) = ∑AL + Ccharge + Cnd + Cd6) can be used to obtain an estimate of the pKa of diamagnetic transition metal hydride and dihydrogen complexes in tetrahydrofuran, and, by use of conversion equations, in other solvents. It involves adding acidity constants AL for each of the ligands in the 5-, 6-, 7-, or 8-coordinate conjugate base complex of the hydride or dihydrogen complex along with a correction for the charge (Ccharge = -15, 0 or 30 for x = +1, 0 or -1 charge, respectively) and the periodic row of the transition metal (Cnd = 0 for 3d or 4d metal, 2 for 5d metal) as well as a correction for d(6) octahedral acids (Cd6 = 6 for d(6) metal ion in the acid, 0 for others) that are not dihydrogen complexes. Constants AL are provided for 13 commonly occurring ligand types; of these, nine neutral ligands are correlated with Lever's electrochemical ligand parameters EL. This method gives good estimates of the over 170 literature pKa values that range from less than zero to 50 with a standard deviation of 3 pKa units for complexes of the metals chromium to nickel, molybdenum, ruthenium to palladium, and tungsten to platinum in the periodic table. This approach allows a quick assessment of the acidity of hydride complexes found in nature (e.g., hydrogenases) and in industry (e.g., catalysis and hydrogen energy applications). The pKa values calculated for acids that have bulky or large bite angle chelating ligands deviate the most from this correlation. The method also provides an estimate of the base strength of the deprotonated form of the complex.

  14. Estimating the acidity of transition metal hydride and dihydrogen complexes by adding ligand acidity constants.

    PubMed

    Morris, Robert H

    2014-02-01

    A simple equation (pKa(THF) = ∑AL + Ccharge + Cnd + Cd6) can be used to obtain an estimate of the pKa of diamagnetic transition metal hydride and dihydrogen complexes in tetrahydrofuran, and, by use of conversion equations, in other solvents. It involves adding acidity constants AL for each of the ligands in the 5-, 6-, 7-, or 8-coordinate conjugate base complex of the hydride or dihydrogen complex along with a correction for the charge (Ccharge = -15, 0 or 30 for x = +1, 0 or -1 charge, respectively) and the periodic row of the transition metal (Cnd = 0 for 3d or 4d metal, 2 for 5d metal) as well as a correction for d(6) octahedral acids (Cd6 = 6 for d(6) metal ion in the acid, 0 for others) that are not dihydrogen complexes. Constants AL are provided for 13 commonly occurring ligand types; of these, nine neutral ligands are correlated with Lever's electrochemical ligand parameters EL. This method gives good estimates of the over 170 literature pKa values that range from less than zero to 50 with a standard deviation of 3 pKa units for complexes of the metals chromium to nickel, molybdenum, ruthenium to palladium, and tungsten to platinum in the periodic table. This approach allows a quick assessment of the acidity of hydride complexes found in nature (e.g., hydrogenases) and in industry (e.g., catalysis and hydrogen energy applications). The pKa values calculated for acids that have bulky or large bite angle chelating ligands deviate the most from this correlation. The method also provides an estimate of the base strength of the deprotonated form of the complex. PMID:24410025

  15. Structural Basis for "Flip-Flop" Action of Human Pyruvate Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Ciszak, Ewa; Korotchkina, Lioubov; Dominiak, Paulina; Sidhu, Sukhdeep; Patel, Mulchand

    2003-01-01

    The derivative of vitamin B1, thiamin pyrophosphate is a cofactor of pyruvate dehydrogenase, a component enzyme of the mitochondrial pyruvate dehydrogenase multienzyme complex that plays a major role in directing energy metabolism in the cell. This cofactor is used to cleave the C(sup alpha)-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. In alpha(sub 2)beta(sub 2)-tetrameric human pyruvate dehydrogenase, there are two cofactor binding sites, each of them being a center of independently conducted, although highly coordinated enzymatic reactions. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites can now be understood based on the recently determined crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95A resolution. The structure of pyruvate dehydrogenase was determined using a combination of MAD phasing and molecular replacement followed by rounds of torsion-angles molecular-dynamics simulated-annealing refinement. The final pyruvate dehydrogenase structure included coordinates for all protein amino acids two cofactor molecules, two magnesium and two potassium ions, and 742 water molecules. The structure was refined to R = 0.202 and R(sub free) = 0.244. Our structural analysis of the enzyme folding and domain assembly identified a simple mechanism of this protein motion required for the conduct of catalytic action.

  16. Alteration of substrate specificity of alanine dehydrogenase

    PubMed Central

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

    2015-01-01

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

  17. Complexes of polyadenylic acid and the methyl esters of amino acids

    NASA Technical Reports Server (NTRS)

    Khaled, M. A.; Mulins, D. W., Jr.; Swindle, M.; Lacey, J. C., Jr.

    1983-01-01

    A study of amino acid methyl esters binding to polyadenylic acid supports the theory that the genetic code originated through weak but selective affinities between amino acids and nucleotides. NMR, insoluble complex analysis, and ultraviolet spectroscopy are used to illustrate a correlation between the hydrophybicities of A amino acids and their binding constants, which, beginning with the largest, are in the order of Phe (having nominally a hydrophobic AAA anticodon), Ile, Leu, Val and Gly (having a hydrophilic anticodon with no A). In general, the binding constants are twice the values by Reuben and Polk (1980) for monomeric AMP, which suggests that polymer amino acids are interacting with only one base. No real differences are found betwen poly A binding for free Phe, Phe methyl ester or Phe amide, except that the amide value is slightly lower.

  18. Shikimate dehydrogenase from Pinu sylvestris L. needles

    SciTech Connect

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

    1986-07-10

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

  19. Biomonitoring acidic drainage impact in a complex setting using periphyton.

    PubMed

    de la Peña, Santiago; Barreiro, Rodolfo

    2009-03-01

    Acid mine drainage (AMD) often exerts various environmental pressures on nearby water courses: chemical stress from low pH and dissolved metals; physical stress from metal oxide deposits. Affected streams can thus display a spatially variable combination of stress agents that may complicate its biomonitoring using native communities such as periphyton. Here, we have measured water and periphyton variables in four streams that surround an abandoned copper mine to determine which periphyton attributes consistently detected AMD impact in a complex environmental setting. Seventeen years after the end of commercial exploitation, the abandoned mine still decreases water quality in nearby streams: moderate acidification, very high metal load (Al, Ni, Cu, Zn), and a conspicuous presence of metal oxide deposits with diverse composition. Even under the resultant complex pattern of polluted conditions, periphyton was a reliable bioindicator of AMD. Epilithic diatom taxa tolerant of acidic conditions increased in AMD sites and, at severely impacted locations, species richness decreased. Also, algal biomass may have been negatively affected in some stream reaches affected by metal oxide deposits. Other periphyton attributes (total biomass, diatom diversity) seemed mostly unrelated to AMD. Diatom assemblage composition was the most sensitive and consistent bioindicator of mine drainage; besides, it rendered a biological assessment of AMD impact that largely coincided with the physicochemical evaluation. Still, including other taxonomic (proportion of acid-tolerant diatom species, diatom richness) and non-taxonomic (algal biomass) attributes in the biomonitoring procedure rendered a more comprehensive assessment of the negative consequences generated by AMD.

  20. Complexes of carborane acids linked by strong hydrogen bonds: acidity scales.

    PubMed

    Grabowski, Sławomir J

    2016-06-28

    Carborane acids, currently known as the strongest acis, are analyzed and compared with other species classified as superacids as well as with selected mineral acids and carbocations. Calculations (B3LYP/6-311++G(d,p) level) on these moieties as well as on their conjugate bases were performed to evaluate corresponding proton affinities. In addition, the complexes of these species with the CHB11F11(-) anion and the complexes of the conjugate bases with the trimethylammonium cation were analyzed. The scales, based on spectroscopic results, DFT energies and on the topological QTAIM (Quantum Theory of Atoms in Molecules) parameters, are introduced and discussed to order the acidity of the species analyzed here. The properties of the bond critical points corresponding to the intermolecular contacts are discussed. The majority of the results show that HCHB11F11 carborane acid is the strongest in agreement with the previous experimental studies of Reed and coworkers. Very often the abovementioned acidity scales show that carborane acids are able to protonate hydrocarbons. PMID:27253195

  1. Oxygenation of Organoboronic Acids by a Nonheme Iron(II) Complex: Mimicking Boronic Acid Monooxygenase Activity.

    PubMed

    Chatterjee, Sayanti; Paine, Tapan Kanti

    2015-10-19

    Phenolic compounds are important intermediates in the bacterial biodegradation of aromatic compounds in the soil. An Arthrobacter sp. strain has been shown to exhibit boronic acid monooxygenase activity through the conversion of different substituted phenylboronic acids to the corresponding phenols using dioxygen. While a number of methods have been reported to cleave the C-B bonds of organoboronic acids, there is no report on biomimetic iron complex exhibiting this activity using dioxygen as the oxidant. In that direction, we have investigated the reactivity of a nucleophilic iron-oxygen oxidant, generated upon oxidative decarboxylation of an iron(II)-benzilate complex [(Tp(Ph2))Fe(II)(benzilate)] (Tp(Ph2) = hydrotris(3,5-diphenyl-pyrazol-1-yl)borate), toward organoboronic acids. The oxidant converts different aryl/alkylboronic acids to the corresponding oxygenated products with the incorporation of one oxygen atom from dioxygen. This method represents an efficient protocol for the oxygenation of boronic acids with dioxygen as the terminal oxidant.

  2. Complexes of carborane acids linked by strong hydrogen bonds: acidity scales.

    PubMed

    Grabowski, Sławomir J

    2016-06-28

    Carborane acids, currently known as the strongest acis, are analyzed and compared with other species classified as superacids as well as with selected mineral acids and carbocations. Calculations (B3LYP/6-311++G(d,p) level) on these moieties as well as on their conjugate bases were performed to evaluate corresponding proton affinities. In addition, the complexes of these species with the CHB11F11(-) anion and the complexes of the conjugate bases with the trimethylammonium cation were analyzed. The scales, based on spectroscopic results, DFT energies and on the topological QTAIM (Quantum Theory of Atoms in Molecules) parameters, are introduced and discussed to order the acidity of the species analyzed here. The properties of the bond critical points corresponding to the intermolecular contacts are discussed. The majority of the results show that HCHB11F11 carborane acid is the strongest in agreement with the previous experimental studies of Reed and coworkers. Very often the abovementioned acidity scales show that carborane acids are able to protonate hydrocarbons.

  3. Oxygenation of Organoboronic Acids by a Nonheme Iron(II) Complex: Mimicking Boronic Acid Monooxygenase Activity.

    PubMed

    Chatterjee, Sayanti; Paine, Tapan Kanti

    2015-10-19

    Phenolic compounds are important intermediates in the bacterial biodegradation of aromatic compounds in the soil. An Arthrobacter sp. strain has been shown to exhibit boronic acid monooxygenase activity through the conversion of different substituted phenylboronic acids to the corresponding phenols using dioxygen. While a number of methods have been reported to cleave the C-B bonds of organoboronic acids, there is no report on biomimetic iron complex exhibiting this activity using dioxygen as the oxidant. In that direction, we have investigated the reactivity of a nucleophilic iron-oxygen oxidant, generated upon oxidative decarboxylation of an iron(II)-benzilate complex [(Tp(Ph2))Fe(II)(benzilate)] (Tp(Ph2) = hydrotris(3,5-diphenyl-pyrazol-1-yl)borate), toward organoboronic acids. The oxidant converts different aryl/alkylboronic acids to the corresponding oxygenated products with the incorporation of one oxygen atom from dioxygen. This method represents an efficient protocol for the oxygenation of boronic acids with dioxygen as the terminal oxidant. PMID:26430780

  4. Plasma Amino Acids Changes in Complex Regional Pain Syndrome

    PubMed Central

    Alexander, Guillermo M.; Reichenberger, Erin; Peterlin, B. Lee; Perreault, Marielle J.; Grothusen, John R.; Schwartzman, Robert J.

    2013-01-01

    Complex regional pain syndrome (CRPS) is a severe chronic pain condition that most often develops following trauma. Blood samples were collected from 220 individuals, 160 CRPS subjects, and 60 healthy pain-free controls. Plasma amino acid levels were compared and contrasted between groups. L-Aspartate, L-glutamate, and L-ornithine were significantly increased, whereas L-tryptophan and L-arginine were significantly decreased in CRPS subjects as compared to controls. In addition, the L-kynurenine to L-tryptophan ratio demonstrated a significant increase, whereas the global arginine bioavailability ratio (GABR) was significantly decreased in the CRPS subjects. The CRPS subjects demonstrated a significant correlation between overall pain and the plasma levels of L-glutamate and the L-kynurenine to L-tryptophan ratio. CRPS subjects also showed a correlation between the decrease in plasma L-tryptophan and disease duration. This study shows that CRPS subjects exhibit significant changes in plasma levels of amino acids involved in glutamate receptor activation and in amino acids associated with immune function as compared to healthy pain-free controls. A better understanding of the role plasma amino acids play in the pathophysiology of CRPS may lead to novel treatments for this crippling condition. PMID:24303215

  5. Folic acid supplementation during pregnancy induces sex-specific changes in methylation and expression of placental 11β-hydroxysteroid dehydrogenase 2 in rats.

    PubMed

    Penailillo, Reyna; Guajardo, Angelica; Llanos, Miguel; Hirsch, Sandra; Ronco, Ana Maria

    2015-01-01

    In the placenta, 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) limits fetal glucocorticoid exposure and its inhibition has been associated to low birth weight. Its expression, encoded by the HSD11B2 gene is regulated by DNA methylation. We hypothesized that maternal diets supplemented with folic acid (FA) during pregnancy modify the expression of placental HSD11B2 through gene methylation. Wistar rats were fed with high (8 mg/kg) or normal low (1mg/kg, control) levels of FA during pregnancy. Concentrations of mRNA and protein in placentas were determined by qRT-PCR and Western blot respectively. Methylation in five CpG sites of the placental HSD11B2 promoter (-378 to -275) was analyzed by bacterial cloning and subsequent sequencing. In the FA-supplemented group, mRNA and protein levels of 11β-HSD2 decreased by 58% and increased by 89%, respectively, only in placentas attached to males. In controls, most CpG sites were not methylated except for the CpG2 site which was 80% methylated. CpG2 methylation level increased under the FA treatment; however, only in placentas attached to females was this increase significant (113%). This change was not related to HSD11B2 expression. Fetal weight of females from FA- supplemented mothers was 6% higher than females from control mothers. In conclusion, this is the first study reporting that FA over supplementation during pregnancy modifies the placental HSD11B2 gene expression and methylation in a sex-dependent manner, suggesting that maternal diets with high content of FA can induce early sex-specific responses, which may lead to long-term consequences for the offspring.

  6. An integrated bienzyme glucose oxidase-fructose dehydrogenase-tetrathiafulvalene-3-mercaptopropionic acid-gold electrode for the simultaneous determination of glucose and fructose.

    PubMed

    Campuzano, Susana; Loaiza, Oscar A; Pedrero, María; de Villena, F Javier Manuel; Pingarrón, José M

    2004-06-01

    A bienzyme biosensor for the simultaneous determination of glucose and fructose was developed by coimmobilising glucose oxidase (GOD), fructose dehydrogenase (FDH), and the mediator, tetrathiafulvalene (TTF), by cross-linking with glutaraldehyde atop a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM) on a gold disk electrode (AuE). The performance of this bienzyme electrode under batch and flow injection (FI) conditions, as well as an amperometric detection in high-performance liquid chromatography (HPLC), are reported. The order of enzyme immobilisation atop the MPA-SAM affected the biosensor amperometric response in terms of sensitivity, with the immobilisation order GOD, FDH, TTF being selected. Similar analytical characteristics to those obtained with single GOD or FDH SAM-based biosensors for glucose and fructose were achieved with the bienzyme electrode, indicating that no noticeable changes in the biosensor responses to the analytes occurred as a consequence of the coimmobilisation of both enzymes on the same MPA-AuE. The suitability of the bienzyme biosensor for the analysis of real samples under flow injection conditions was tested by determining glucose in two certified serum samples. The simultaneous determination of glucose and fructose in the same sample cannot be performed without a separation step because at the detection potential used (+0.10 V), both sugars show amperometric response. Consequently, HPLC with amperometric detection at the TTF-FDH-GOD-MPA-AuE was accomplished. Glucose and fructose were simultaneously determined in honey, cola softdrink, and commercial apple juice, and the results were compared with those obtained by using other reference methods.

  7. Co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol using resting cells of recombinant Klebsiella pneumoniae J2B strain overexpressing aldehyde dehydrogenase.

    PubMed

    Kumar, Vinod; Sankaranarayanan, Mugesh; Jae, Kyeung-Eun; Durgapal, Meetu; Ashok, Somasundar; Ko, Yeounjoo; Sarkar, Ritam; Park, Sunghoon

    2012-10-01

    The co-production of 3-hydroxypropionic acid (3HP) and 1,3-propanediol (PDO) from glycerol was studied using the resting cells of a recombinant Klebsiella pneumoniae J2B strain that overexpresses an aldehyde dehydrogenase (KGSADH). Active biomass was produced in a mineral salt medium containing yeast extract and glycerol under a range of aeration conditions, and shifted to potassium phosphate buffer containing glycerol for bioconversion. The microaerobic or anaerobic conditions were favorable for both the production of active biomass and subsequent bioconversion. At the flask level, the recombinant strain (2.0 g CDW/L) grown under microaerobic conditions produced 43.2 mM 3HP and 59.0 mM PDO from glycerol (117 mM) in 30 min with a cumulative yield of 0.87 (mol/mol). The fed-batch bioconversion, which was performed in a 1.5-L bioreactor with 1.0 g CDW/L at a constant pH 7.0 under anaerobic conditions, resulted in 125.6 mM 3HP and 209.5 mM PDO in 12 h with a cumulative overall productivity, yield, and maximum specific production rate of 27.9 mmol/L/h, 0.71 (mol/mol), and 128.5 mmol/g CDW/h, respectively. Lactate, succinate and 2,3-butanediol were the major by-products, whereas the production of acetate and ethanol was marginal. This is the first report of the simultaneous production of 3HP and PDO from glycerol using a resting cell system.

  8. Amino acid substitutions in malate dehydrogenases of piezophilic bacteria isolated from intestinal contents of deep-sea fishes retrieved from the abyssal zone.

    PubMed

    Saito, Rie; Kato, Chiaki; Nakayama, Akihiko

    2006-02-01

    To examine the occurrence in other deep-sea bacteria of two amino acid substitutions (Ala-180 and His-229) in malate dehydrogenase (MDH) found previously in the deep-sea piezophilic Moritella sp. strain 2D2, we cloned and sequenced MDH genes of deep-sea piezophilic Moritella and Shewanella strains isolated from intestinal contents of deep-sea fishes, as well as other Moritella species from deep-sea water and sediments: M. marina, M. japonica, and M. yayanosii. The piezophilic Moritella strains had a Val residue or an Ala residue at position 180 and all the Moritella strains except for one had a His residue at position 229. However, four piezophilic-strain-specific substitutions at positions 103, 111, 229, and 283 were found to be completely conserved in the MDH of the intestinal Moritella strains of deep-sea fishes, indicating the substitutions may be habitat-specific. The piezophilic Shewanella strains had a Val residue and a Gln residue at positions 180 and 229, respectively. However, the MDHs of the Shewanella strains had five piezophilic-strain-specific substitutions at positions 61, 65, 107, 161, and 202. Therefore, the enzymatic strategies for responding to deep-sea high pressure environments of the MDHs between the genera Moritella and Shewanella are potentially different. Moreover, homology modeling shows these substitutions found in the MDHs of both genera except for position 229 in the subunit interface are located on the exposed region of the MDH molecules, indicating the substitutions may be related to the hydration state of the molecules. PMID:16598154

  9. Protein-mediated assembly of succinate dehydrogenase and its cofactors.

    PubMed

    Van Vranken, Jonathan G; Na, Un; Winge, Dennis R; Rutter, Jared

    2015-01-01

    Succinate dehydrogenase (or complex II; SDH) is a heterotetrameric protein complex that links the tribarboxylic acid cycle with the electron transport chain. SDH is composed of four nuclear-encoded subunits that must translocate independently to the mitochondria and assemble into a mature protein complex embedded in the inner mitochondrial membrane. Recently, it has become clear that failure to assemble functional SDH complexes can result in cancer and neurodegenerative syndromes. The effort to thoroughly elucidate the SDH assembly pathway has resulted in the discovery of four subunit-specific assembly factors that aid in the maturation of individual subunits and support the assembly of the intact complex. This review will focus on these assembly factors and assess the contribution of each factor to the assembly of SDH. Finally, we propose a model of the SDH assembly pathway that incorporates all extant data.

  10. [PREPARATIONS OF PAMIDRONOVIC ACID IN COMPLEX TREATMENT ON OSTEOGENESIS IMPERFECTA].

    PubMed

    Zyma, A M; Guk, Yu M; Magomedov, O M; Gayko, O G; Kincha-Polishchuk, T A

    2015-07-01

    Modern view of drug therapy in the complex treatment of orthopedic manifestations of osteogenesis imperfecta (OI) was submitted. Developed and tested system of drug correction of structural and functional state of bone tissue (BT) using drugs pamidronovic acid, depending on osteoporosis severity and type of disease. Such therapy is appropriate to apply both independently and in conjunction with surgery to correct deformations of long bones of the lower extremities. Effectiveness and feasibility of the proposed methods of drug therapy was proved, most patients resume features walking and support. PMID:26591224

  11. Structure and function analysis of protein-nucleic acid complexes

    NASA Astrophysics Data System (ADS)

    Kuznetsova, S. A.; Oretskaya, T. S.

    2016-05-01

    The review summarizes published data on the results and achievements in the field of structure and function analysis of protein-nucleic acid complexes by means of main physical and biochemical methods, including X-ray diffraction, nuclear magnetic resonance spectroscopy, electron and atomic force microscopy, small-angle X-ray and neutron scattering, footprinting and cross-linking. Special attention is given to combined approaches. The advantages and limitations of each method are considered, and the prospects of their application for wide-scale structural studies in vivo are discussed. The bibliography includes 145 references.

  12. Structure and function analysis of protein–nucleic acid complexes

    NASA Astrophysics Data System (ADS)

    Kuznetsova, S. A.; Oretskaya, T. S.

    2016-05-01

    The review summarizes published data on the results and achievements in the field of structure and function analysis of protein–nucleic acid complexes by means of main physical and biochemical methods, including X-ray diffraction, nuclear magnetic resonance spectroscopy, electron and atomic force microscopy, small-angle X-ray and neutron scattering, footprinting and cross-linking. Special attention is given to combined approaches. The advantages and limitations of each method are considered, and the prospects of their application for wide-scale structural studies in vivo are discussed. The bibliography includes 145 references.

  13. Formation and characterization of thioglycolic acid-silver cluster complexes.

    PubMed

    Bellina, Bruno; Antoine, Rodolphe; Broyer, Michel; Gell, Lars; Sanader, Željka; Mitrić, Roland; Bonačić-Koutecký, Vlasta; Dugourd, Philippe

    2013-06-21

    Gas phase reactivity observed in an ion trap was used to produce silver clusters protected with thioglycolic acid. Fragmentation pathways as well as optical properties were explored experimentally and theoretically. Sequential losses of SCH2 and CO2 in the ion trap lead to redox reactions with charge transfers between the metal part and the carboxylate and thiolate groups. This allows us to control the number of electrons in the metallic subunit and thus optical properties of the complexes. The presented formation process can be used as a prototype for tuning optical and chemical properties of ligated metal clusters by varying the number of confined electrons within the metallic subunit.

  14. Human liver alcohol dehydrogenase: amino acid substitution in the beta 2 beta 2 Oriental isozyme explains functional properties, establishes an active site structure, and parallels mutational exchanges in the yeast enzyme.

    PubMed Central

    Jörnvall, H; Hempel, J; Vallee, B L; Bosron, W F; Li, T K

    1984-01-01

    The homodimeric Oriental beta 2 beta 2 isozyme of human liver alcohol dehydrogenase, corresponding to an allelic variant at the ADH2 gene locus, was studied in order to define the amino acid exchange in relation to the beta 1 beta 1 isozyme, the predominant allelic form among Caucasians. Sequence analysis reveals that the amino acid substitution occurs at position 7 of the largest CNBr fragment, corresponding to position 47 of the whole protein chain. Here, the beta 2 form has a histidine residue, while, in common with other characterized mammalian liver alcohol dehydrogenases, the beta 1 form has an arginine residue. This exchange does not affect the adjacent cysteine-46 residue, which is a protein ligand to the active-site zinc atom, thus clarifying previously inconsistent results. The histidine/arginine-47 mutational replacement corresponds to a position that binds the pyrophosphate group of the coenzyme NAD(H); this explains the functional differences between the beta 1 beta 1 and beta 2 beta 2 isozymes, including both a lower pH optimum and higher turnover number of beta 2 beta 2, which is likely to be the mutant form. The exchange demonstrates the existence of parallel but separate mutations in the evolution of alcohol dehydrogenases because these mammalian enzymes differ at exactly the same position by the same type of substitution as is found between a mutant and the wild-type constitutive forms of the corresponding yeast enzyme. PMID:6374651

  15. Platform engineering of Corynebacterium glutamicum with reduced pyruvate dehydrogenase complex activity for improved production of L-lysine, L-valine, and 2-ketoisovalerate.

    PubMed

    Buchholz, Jens; Schwentner, Andreas; Brunnenkan, Britta; Gabris, Christina; Grimm, Simon; Gerstmeir, Robert; Takors, Ralf; Eikmanns, Bernhard J; Blombach, Bastian

    2013-09-01

    Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the l-valine biosynthetic genes ilvBNCE, all strains produced l-valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 Δpqo Δppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression of ilvBNCD instead of ilvBNCE transformed the l-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a YP/S of 0.24 mol per mol of glucose and a QP of 6.9 mM per h [0.8 g/(liter × h)]. The replacement of the aceE promoter by the dapA-A16 promoter in the two C. glutamicum l-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.

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

    PubMed

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

    2004-03-01

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

  17. The complexation behavior of neptunium and plutonium with nitrilotriacetic acid

    SciTech Connect

    Nitsche, H.; Becraft, K.

    1990-08-01

    The first stability constant of NpO{sub 2}{sup +} with nitrilotriacetic acid (NTA) was determined at four ionic strengths (0.5, 1.0, 2.0, 3.0 M) using spectrophotometry. Nonlinear least-squares data fitting identified the complex as NpO{sub 2}NTA{sup 2-}. The Specific Ion Interaction Theory (S.I.T) approximation method was used to determine the stability constants at infinite dilution. First results on Pu{sup 4+} and PuO{sub 2}{sup 2+} complexation with NTA are reported. The stability constant for the Pu(NTA){sup +} complex at I = 0.1 M strength is given. From results for PuO{sub 2}{sup 2+} complexation with NTA (I = 1 M) at pH < 3, the stability constant was derived for PuO{sub 2} NTA{sup {minus}}. At pH > 3, NTA partially reduced PuO{sub 2}{sup 2+} to PuO{sub 2}{sup +}. 3 refs., 5 figs., 4 tabs.

  18. Murine branched chain alpha-ketoacid dehydrogenase kinase; cDNA cloning, tissue distribution, and temporal expression during embryonic development.

    PubMed

    Doering, C B; Coursey, C; Spangler, W; Danner, D J

    1998-06-01

    These studies were designed to demonstrate the structural and functional similarity of murine branched chain alpha-ketoacid dehydrogenase and its regulation by the complex-specific kinase. Nucleotide sequence and deduced amino acid sequence for the kinase cDNA demonstrate a highly conserved coding sequence between mouse and human. Tissue-specific expression in adult mice parallels that reported in other mammals. Kinase expression in female liver is influenced by circadian rhythm. Of special interest is the fluctuating expression of this kinase during embryonic development against the continuing increase in the catalytic subunits of this mitochondrial complex during development. The need for regulation of the branched chain alpha-ketoacid dehydrogenase complex by kinase expression during embryogenesis is not understood. However, the similarity of murine branched chain alpha-ketoacid dehydrogenase and its kinase to the human enzyme supports the use of this animal as a model for the human system. PMID:9611264

  19. Safety, efficacy and physiological actions of a lysine-free, arginine-rich formula to treat glutaryl-CoA dehydrogenase deficiency: focus on cerebral amino acid influx.

    PubMed

    Strauss, Kevin A; Brumbaugh, Joan; Duffy, Alana; Wardley, Bridget; Robinson, Donna; Hendrickson, Christine; Tortorelli, Silvia; Moser, Ann B; Puffenberger, Erik G; Rider, Nicholas L; Morton, D Holmes

    2011-01-01

    Striatal degeneration from glutaryl-CoA dehydrogenase deficiency (glutaric aciduria type 1, GA1) is associated with cerebral formation and entrapment of glutaryl-CoA and its derivatives that depend on cerebral lysine influx. In 2006 we designed a lysine-free study formula enriched with arginine to selectively block lysine transport across cerebral endothelia and thereby limit glutaryl-CoA production by brain. Between 2006 and present, we treated twelve consecutive children with study formula (LYSx group) while holding all other treatment practices constant. Clinical and biochemical outcomes were compared to 25 GA1 patients (PROx group) treated between 1995 and 2005 with natural protein restriction (dietary lysine/arginine ratio of 1.7±0.3 mg:mg). We used published kinetic parameters of the y+and LAT1 blood-brain barrier transporters to model the influx of amino acids into the brain. Arginine fortification to achieve a mean dietary lysine/arginine ratio of 0.7±0.2 mg:mg was neuroprotective. All 12 LYSx patients are physically and neurologically healthy after 28 aggregate patient-years of follow up (current ages 28±21 months) and there were no adverse events related to formula use. This represents a 36% reduction of neurological risk (95% confidence interval 14-52%, p=0.018) that we can directly attribute to altered amino acid intake. During the first year of life, 20% lower lysine intake and two-fold higher arginine intake by LYSx patients were associated with 50% lower plasma lysine, 3-fold lower plasma lysine/arginine concentration ratio, 42% lower mean calculated cerebral lysine influx, 54% higher calculated cerebral arginine influx, 15-26% higher calculated cerebral influx of several anaplerotic precursors (isoleucine, threonine, methionine, and leucine), 50% less 3-hydroxyglutarate excretion, and a 3-fold lower hospitalization rate (0.8 versus 2.3 hospitalizations per patient per year). The relationship between arginine fortification and plasma lysine

  20. Removal of acidic or basic α-amino acids in water by poorly water soluble scandium complexes.

    PubMed

    Hayashi, Nobuyuki; Jin, Shigeki; Ujihara, Tomomi

    2012-11-01

    To recognize α-amino acids with highly polar side chains in water, poorly water soluble scandium complexes with both Lewis acidic and basic portions were synthesized as artificial receptors. A suspension of some of these receptor molecules in an α-amino acid solution could remove acidic and basic α-amino acids from the solution. The compound most efficient at preferentially removing basic α-amino acids (arginine, histidine, and lysine) was the receptor with 7,7'-[1,3-phenylenebis(carbonylimino)]bis(2-naphthalenesulfonate) as the ligand. The neutral α-amino acids were barely removed by these receptors. Removal experiments using a mixed amino acid solution generally gave results similar to those obtained using solutions containing a single amino acid. The results demonstrated that the scandium complex receptors were useful for binding acidic and basic α-amino acids. PMID:23050492

  1. Removal of acidic or basic α-amino acids in water by poorly water soluble scandium complexes.

    PubMed

    Hayashi, Nobuyuki; Jin, Shigeki; Ujihara, Tomomi

    2012-11-01

    To recognize α-amino acids with highly polar side chains in water, poorly water soluble scandium complexes with both Lewis acidic and basic portions were synthesized as artificial receptors. A suspension of some of these receptor molecules in an α-amino acid solution could remove acidic and basic α-amino acids from the solution. The compound most efficient at preferentially removing basic α-amino acids (arginine, histidine, and lysine) was the receptor with 7,7'-[1,3-phenylenebis(carbonylimino)]bis(2-naphthalenesulfonate) as the ligand. The neutral α-amino acids were barely removed by these receptors. Removal experiments using a mixed amino acid solution generally gave results similar to those obtained using solutions containing a single amino acid. The results demonstrated that the scandium complex receptors were useful for binding acidic and basic α-amino acids.

  2. Dual Fatty Acid Elongase Complex Interactions in Arabidopsis.

    PubMed

    Morineau, Céline; Gissot, Lionel; Bellec, Yannick; Hematy, Kian; Tellier, Frédérique; Renne, Charlotte; Haslam, Richard; Beaudoin, Frédéric; Napier, Johnathan; Faure, Jean-Denis

    2016-01-01

    Very long chain fatty acids (VLCFAs) are involved in plant development and particularly in several cellular processes such as membrane trafficking, cell division and cell differentiation. However, the precise role of VLCFAs in these different cellular processes is still poorly understood in plants. In order to identify new factors associated with the biosynthesis or function of VLCFAs, a yeast multicopy suppressor screen was carried out in a yeast mutant strain defective for fatty acid elongation. Loss of function of the elongase 3 hydroxyacyl-CoA dehydratase PHS1 in yeast and PASTICCINO2 in plants prevents growth and induces cytokinesis defects. PROTEIN TYROSIN PHOSPHATASE-LIKE (PTPLA) previously characterized as an inactive dehydratase was able to restore yeast phs1 growth and VLCFAs elongation but not the plant pas2-1 defects. PTPLA interacted with elongase subunits in the Endoplasmic Reticulum (ER) and its absence induced the accumulation of 3-hydroxyacyl-CoA as expected from a dehydratase involved in fatty acid (FA) elongation. However, loss of PTPLA function increased VLCFA levels, an effect that was dependent on the presence of PAS2 indicating that PTPLA activity repressed FA elongation. The two dehydratases have specific expression profiles in the root with PAS2, mostly restricted to the endodermis, while PTPLA was confined in the vascular tissue and pericycle cells. Comparative ectopic expression of PTPLA and PAS2 in their respective domains confirmed the existence of two independent elongase complexes based on PAS2 or PTPLA dehydratase that are functionally interacting. PMID:27583779

  3. Dual Fatty Acid Elongase Complex Interactions in Arabidopsis

    PubMed Central

    Morineau, Céline; Gissot, Lionel; Bellec, Yannick; Hematy, Kian; Tellier, Frédérique; Renne, Charlotte; Haslam, Richard; Beaudoin, Frédéric; Napier, Johnathan; Faure, Jean-Denis

    2016-01-01

    Very long chain fatty acids (VLCFAs) are involved in plant development and particularly in several cellular processes such as membrane trafficking, cell division and cell differentiation. However, the precise role of VLCFAs in these different cellular processes is still poorly understood in plants. In order to identify new factors associated with the biosynthesis or function of VLCFAs, a yeast multicopy suppressor screen was carried out in a yeast mutant strain defective for fatty acid elongation. Loss of function of the elongase 3 hydroxyacyl-CoA dehydratase PHS1 in yeast and PASTICCINO2 in plants prevents growth and induces cytokinesis defects. PROTEIN TYROSIN PHOSPHATASE-LIKE (PTPLA) previously characterized as an inactive dehydratase was able to restore yeast phs1 growth and VLCFAs elongation but not the plant pas2-1 defects. PTPLA interacted with elongase subunits in the Endoplasmic Reticulum (ER) and its absence induced the accumulation of 3-hydroxyacyl-CoA as expected from a dehydratase involved in fatty acid (FA) elongation. However, loss of PTPLA function increased VLCFA levels, an effect that was dependent on the presence of PAS2 indicating that PTPLA activity repressed FA elongation. The two dehydratases have specific expression profiles in the root with PAS2, mostly restricted to the endodermis, while PTPLA was confined in the vascular tissue and pericycle cells. Comparative ectopic expression of PTPLA and PAS2 in their respective domains confirmed the existence of two independent elongase complexes based on PAS2 or PTPLA dehydratase that are functionally interacting. PMID:27583779

  4. α-Lactalbumin:Oleic Acid Complex Spontaneously Delivers Oleic Acid to Artificial and Erythrocyte Membranes.

    PubMed

    Wen, Hanzhen; Strømland, Øyvind; Halskau, Øyvind

    2015-09-25

    Human α-lactalbumin made lethal to tumor cells (HAMLET) is a tumoricidal complex consisting of human α-lactalbumin and multiple oleic acids (OAs). OA has been shown to play a key role in the activity of HAMLET and its related complexes, generally known as protein-fatty acid (PFA) complexes. In contrast to what is known about the fate of the protein component of such complexes, information about what happens to OA during their action is still lacking. We monitored the membrane, OA and protein components of bovine α-lactalbumin complexed with OA (BLAOA; a HAMLET-like substance) and how they associate with each other. Using ultracentrifugation, we found that the OA and lipid components follow each other closely. We then firmly identify a transfer of OA from BLAOA to both artificial and erythrocyte membranes, indicating that natural cells respond similarly to BLAOA treatment as artificial membranes. Uncomplexed OA is unable to similarly affect membranes at the conditions tested, even at elevated concentrations. Thus, BLAOA can spontaneously transfer OA to a lipid membrane. After the interaction with the membrane, the protein is likely to have lost most or all of its OA. We suggest a mechanism for passive import of mainly uncomplexed protein into cells, using existing models for OA's effect on membranes. Our results are consistent with a membrane destabilization mediated predominantly by OA insertion being a significant contribution to PFA cytotoxicity.

  5. [Adsorption Properties of Fluorine onto Fulvic Acid-Bentonite Complex].

    PubMed

    Fang, Dun; Tian, Hua-jing; Ye, Xin; He, Ci-li; Dan, You-meng; Wei, Shi-yong

    2016-03-15

    Fulvic Acid-Bentonite (FA-BENT) complex was prepared using coprecipitation method, and basic properties of the complex and sorption properties of fluorine at different environmental conditions were studied. XRD results showed that the d₀₀₁ spacing of FA- BENT complex had no obvious change compared with the raw bentonite, although the diffraction peak intensity of smectite in FA-BENT complex reduced, and indicated that FA mainly existed as a coating on the external surface of bentonite. Some functional groups (such as C==O, −OH, etc. ) of FA were observed in FA-BENT FTIR spectra, thus suggesting ligand exchange-surface complexation between FA and bentonite. Higher initial pH values of the reaction system were in favor of the adsorption of fluorine onto FA-BENT, while the equilibrium capacity decreased with the increase of pH at initial pH ≥ 4.50. The adsorption of fluorine onto FA-BENT was also affected by ionic strength, and the main reason might be the "polarity" effect. The adsorption of fluorine onto FA-BENT followed pseudo-second-order kinetic model and was controlled by chemical process ( R² = 0.999 2). Compared with the Freundlich model, Langmuir model was apparently of a higher goodness of fit (R² > 0.994 9) for absorption of fluorine onto FA-BENT. Thermodynamic parameters indicated that the adsorption process of fluorine was an spontaneously endothermic reaction, and was an entropy-driven process (ΔH 32.57 kJ · mol⁻¹, ΔS 112.31 J · (mol · K)⁻¹, ΔG −0.65- −1.76 kJ · mol⁻¹).

  6. [Adsorption Properties of Fluorine onto Fulvic Acid-Bentonite Complex].

    PubMed

    Fang, Dun; Tian, Hua-jing; Ye, Xin; He, Ci-li; Dan, You-meng; Wei, Shi-yong

    2016-03-15

    Fulvic Acid-Bentonite (FA-BENT) complex was prepared using coprecipitation method, and basic properties of the complex and sorption properties of fluorine at different environmental conditions were studied. XRD results showed that the d₀₀₁ spacing of FA- BENT complex had no obvious change compared with the raw bentonite, although the diffraction peak intensity of smectite in FA-BENT complex reduced, and indicated that FA mainly existed as a coating on the external surface of bentonite. Some functional groups (such as C==O, −OH, etc. ) of FA were observed in FA-BENT FTIR spectra, thus suggesting ligand exchange-surface complexation between FA and bentonite. Higher initial pH values of the reaction system were in favor of the adsorption of fluorine onto FA-BENT, while the equilibrium capacity decreased with the increase of pH at initial pH ≥ 4.50. The adsorption of fluorine onto FA-BENT was also affected by ionic strength, and the main reason might be the "polarity" effect. The adsorption of fluorine onto FA-BENT followed pseudo-second-order kinetic model and was controlled by chemical process ( R² = 0.999 2). Compared with the Freundlich model, Langmuir model was apparently of a higher goodness of fit (R² > 0.994 9) for absorption of fluorine onto FA-BENT. Thermodynamic parameters indicated that the adsorption process of fluorine was an spontaneously endothermic reaction, and was an entropy-driven process (ΔH 32.57 kJ · mol⁻¹, ΔS 112.31 J · (mol · K)⁻¹, ΔG −0.65- −1.76 kJ · mol⁻¹). PMID:27337896

  7. Injectable hydrogels derived from phosphorylated alginic acid calcium complexes.

    PubMed

    Kim, Han-Sem; Song, Minsoo; Lee, Eun-Jung; Shin, Ueon Sang

    2015-06-01

    Phosphorylation of sodium alginate salt (NaAlg) was carried out using H3PO4/P2O5/Et3PO4 followed by acid-base reaction with Ca(OAc)2 to give phosphorylated alginic acid calcium complexes (CaPAlg), as a water dispersible alginic acid derivative. The modified alginate derivatives including phosphorylated alginic acid (PAlg) and CaPAlg were characterized by nuclear magnetic resonance spectroscopy for (1)H, and (31)P nuclei, high resolution inductively coupled plasma optical emission spectroscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. CaPAlg hydrogels were prepared simply by mixing CaPAlg solution (2w/v%) with NaAlg solution (2w/v%) in various ratios (2:8, 4:6, 6:4, 8:2) of volume. No additional calcium salts such as CaSO4 or CaCl2 were added externally. The gelation was completed within about 3-40min indicating a high potential of hydrogel delivery by injection in vivo. Their mechanical properties were tested to be ≤6.7kPa for compressive strength at break and about 8.4kPa/mm for elastic modulus. SEM analysis of the CaPAlg hydrogels showed highly porous morphology with interconnected pores of width in the range of 100-800μm. Cell culture results showed that the injectable hydrogels exhibited comparable properties to the pure alginate hydrogel in terms of cytotoxicity and 3D encapsulation of cells for a short time period. The developed injectable hydrogels showed suitable physicochemical and mechanical properties for injection in vivo, and could therefore be beneficial for the field of soft tissue engineering. PMID:25842118

  8. Ancestral genetic complexity of arachidonic acid metabolism in Metazoa.

    PubMed

    Yuan, Dongjuan; Zou, Qiuqiong; Yu, Ting; Song, Cuikai; Huang, Shengfeng; Chen, Shangwu; Ren, Zhenghua; Xu, Anlong

    2014-09-01

    Eicosanoids play an important role in inducing complex and crucial physiological processes in animals. Eicosanoid biosynthesis in animals is widely reported; however, eicosanoid production in invertebrate tissue is remarkably different to vertebrates and in certain respects remains elusive. We, for the first time, compared the orthologs involved in arachidonic acid (AA) metabolism in 14 species of invertebrates and 3 species of vertebrates. Based on parsimony, a complex AA-metabolic system may have existed in the common ancestor of the Metazoa, and then expanded and diversified through invertebrate lineages. A primary vertebrate-like AA-metabolic system via cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) pathways was further identified in the basal chordate, amphioxus. The expression profiling of AA-metabolic enzymes and lipidomic analysis of eicosanoid production in the tissues of amphioxus supported our supposition. Thus, we proposed that the ancestral complexity of AA-metabolic network diversified with the different lineages of invertebrates, adapting with the diversity of body plans and ecological opportunity, and arriving at the vertebrate-like pattern in the basal chordate, amphioxus.

  9. Ancestral genetic complexity of arachidonic acid metabolism in Metazoa.

    PubMed

    Yuan, Dongjuan; Zou, Qiuqiong; Yu, Ting; Song, Cuikai; Huang, Shengfeng; Chen, Shangwu; Ren, Zhenghua; Xu, Anlong

    2014-09-01

    Eicosanoids play an important role in inducing complex and crucial physiological processes in animals. Eicosanoid biosynthesis in animals is widely reported; however, eicosanoid production in invertebrate tissue is remarkably different to vertebrates and in certain respects remains elusive. We, for the first time, compared the orthologs involved in arachidonic acid (AA) metabolism in 14 species of invertebrates and 3 species of vertebrates. Based on parsimony, a complex AA-metabolic system may have existed in the common ancestor of the Metazoa, and then expanded and diversified through invertebrate lineages. A primary vertebrate-like AA-metabolic system via cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) pathways was further identified in the basal chordate, amphioxus. The expression profiling of AA-metabolic enzymes and lipidomic analysis of eicosanoid production in the tissues of amphioxus supported our supposition. Thus, we proposed that the ancestral complexity of AA-metabolic network diversified with the different lineages of invertebrates, adapting with the diversity of body plans and ecological opportunity, and arriving at the vertebrate-like pattern in the basal chordate, amphioxus. PMID:24801744

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

    PubMed

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

    2015-10-01

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

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

    PubMed

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

    2015-10-01

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

  12. Ternary Complexes of Iron, Amyloid-β and Nitrilotriacetic Acid

    PubMed Central

    Jiang, Dianlu; Li, Xiangjun; Williams, Renee; Patel, Sveti; Men, Lijie; Wang, Yinsheng; Zhou, Feimeng

    2009-01-01

    The interaction of amyloid-β (Aβ) and redox-active metals, two important biomarkers present in the senile plaques of AD brain, has been suggested to either enhance the Aβ aggregation or facilitate the generation of reactive oxygen species (ROS). The present study investigates the nature of the interaction between the metal-binding domain of Aβ, viz, Aβ(1-16), and the Fe(III) or Fe(II) complex with nitrilotriacetic acid (NTA). Using electrospray ionization mass spectrometry (ESI-MS), the formation of a ternary complex of Aβ(1-16), Fe(III), and NTA with a stoichiometry of 1:1:1 was identified. MS also revealed that the NTA moiety can be detached via collision-induced dissociation. The cumulative dissociation constants of both Aβ-Fe(III)-NTA and Aβ-Fe(II)-NTA were deduced to be 6.3 × 10-21 M2 and 5.0 × 10-12 M2, respectively, via measuring the fluorescence quenching of the sole tyrosine residue on Aβ upon the complex formation. The redox properties of these two complexes were investigated by cyclic voltammetry. The redox potential of the Aβ-Fe(III)-NTA complex was found to be 0.03 V vs. Ag/AgCl, which is negatively shifted by 0.54 V when compared to the redox potential of free Fe(III)/Fe(II). Despite such a large potential modulation, the redox potential of the Aβ-Fe(III)-NTA complex is still sufficiently high for occurrence of a range of redox reactions with cellular species. Aβ-Fe(II)-NTA electrogenerated from Aβ-Fe(III)-NTA was also found to catalyze the reduction of oxygen to produce H2O2. These findings provide significant insight into the role of iron and Aβ in the development of AD. The binding of iron by Aβ modulates the redox potential to a level where its redox cycling occurs. In the presence of a biological reductant (antioxidant), redox cycling of iron could disrupt the redox balance within the cellular milieu. As a consequence, not only ROS is continuously produced, but also oxygen and biological reductants can be depleted. A cascade of

  13. Effects of humic acid-metal complexes on hepatic carnitine palmitoyltransferase, carnitine acetyltransferase and catalase activities

    SciTech Connect

    Fungjou Lu; Youngshin Chen . Dept. of Biochemistry); Tienshang Huang . Dept. of Medicine)

    1994-03-01

    A significant increase in activities of hepatic carnitine palmitoyltransferase and carnitine acetyltransferase was observed in male Balb/c mice intraperitoneally injected for 40 d with 0.125 mg/0.1 ml/d humic acid-metal complexes. Among these complexes, the humic acid-As complex was relatively effective, whereas humic acid-25 metal complex was more effective, and humic acid-26 metal complex was most effective. However, humic acid or metal mixtures, or metal such as As alone, was not effective. Humic acid-metal complexes also significantly decreased hepatic catalase activity. A marked decrease of 60-kDa polypeptide in liver cytoplasm was also observed on SDS-polyacrylamide gel electrophoresis after the mice had been injected with the complexes. Morphological analysis of a histopathological biopsy of such treated mice revealed several changes in hepatocytes, including focal necrosis and cell infiltration, mild fatty changes, reactive nuclei, and hypertrophy. Humic acid-metal complexes affect activities of metabolic enzymes of fatty acids, and this results in accumulation of hydrogen peroxide and increase of the lipid peroxidation. The products of lipid peroxidation may be responsible for liver damage and possible carcinogenesis. Previous studies in this laboratory had shown that humic acid-metal complex altered the coagulation system and that humic acid, per se, caused vasculopathy. Therefore, humic acid-metal complexes may be main causal factors of not only so-called blackfoot disease, but also the liver cancer prevailing on the southwestern coast of Taiwan.

  14. A microwave study of the ammonia-nitric acid complex

    SciTech Connect

    Ott, M.E.; Leopold, K.R.

    1999-03-11

    Microwave spectra of H{sub 3}{sup 14}N-H{sup 14}NO{sub 3}, H{sub 3}{sup 15}N-H{sup 14}NO{sub 3}, H{sub 3}{sup 14}N-H{sup 15}NO{sub 3}, and H{sub 3}{sup 14}N-D{sup 14}NO{sub 3} have been obtained by Fourier transform spectroscopy in a supersonic jet. The spectra are consistent with a hydrogen-bonded structure having a planar arrangement of the heavy atom frame and a linear or near-linear hydrogen bond involving the HNO{sub 3} proton and the lone pair on the ammonia. The hydrogen bond length obtained from the rotational constants is 1.736(63) {angstrom} and the nitrogen-nitrogen separation is 3.344(12) {angstrom}. The NO single bond of the nitric acid and the C{sub 3} axis of the NH{sub 3} form angles of 54(4){degree} and 21(3){degree}, respectively, with the line joining the centers of mass of the monomers. The hydrogen bond length is significantly shorter than that observed in related complexes such as H{sub 3}N-HCl and H{sub 3}N-HBr, indicating a particularly strong interaction between ammonia and nitric acid.

  15. Centrosymmetric dimer of quinuclidine betaine and squaric acid complex

    NASA Astrophysics Data System (ADS)

    Dega-Szafran, Z.; Katrusiak, A.; Szafran, M.

    2012-12-01

    The complex of squaric acid (3,4-dihydroxy-3-cyclobuten-1,2-dion, H2SQ) with quinuclidine betaine (1-carboxymethyl-1-azabicyclo[2.2.2]octane inner salt, QNB), 1, has been characterized by single-crystal X-ray analysis, FTIR and NMR spectroscopies and by DFT calculations. In the crystal of 1, monoclinic space group P21/n, one proton from H2SQ is transferred to QNB. QNBH+ and HSQ- are linked together by a Osbnd H⋯O hydrogen bond of 2.553(2) Å. Two such QNBH+·HSQ- complexes form a centrosymmetric dimer bridged by two Osbnd H⋯O bonds of 2.536(2) Å. The FTIR spectrum is consistent with the X-ray results. The structures of monomer QNBH+·HSQ- (1a) and dimer [QNB·H2SQ]2 (2) have been optimized at the B3LYP/6-311++G(d,p) level of theory. Isolated dimer 2 optimized back to a molecular aggregate of H2SQ and QNB. The calculated frequencies for the optimized structure of dimer 2 have been used to explain the frequencies of the experimental FTIR spectrum. The interpretation of 1H and 13C NMR spectra has been based on the calculated GIAO/B3LYP/6-311++G(d,p) magnetic isotropic shielding constants for monomer 1a.

  16. Solution structures of europium(III) complexes of ethylenediaminetetraacetic acid

    SciTech Connect

    Latva, M.; Kankara, J.; Haapakka, K.

    1996-04-01

    Coordination of ethylenediaminetetraacetic acid (EDTA) with europium(III) has been studied at different concentrations in solution using {sup 7}F{sub 0}{yields}{sup 5}D{sub 0} excitation spectroscopy and excited-state lifetime measurements. EDTA forms with Eu(III) ion three different species in equimolar solutions at room temperature. At low pH values EuEDTAH is formed and at higher pH values than 1.5 two EuEDTA{sup -} complexes, which differ from each other with one water molecule in the first coordination sphere of the Eu(III) ion, total coordination number and coordination geometry, are also formed. When the concentration of EDTA is higher than the concentration of Eu(III), an EuEDTA(EDTAH){sup 4-} species where the second EDTA is weakly coordinated to EuEDTA{sup -}, is formed. If the concentration of Eu(III) ion is higher than EDTA, the extra Eu(III) ions associate with EuEDTA{sup -} and link to one of the carboxylate groups of EDTA thus causing a shortening of the excited-state lifetime of the EuEDTA{sup -} complex.

  17. Polymer complexes. LX. Supramolecular coordination and structures of N(4-(acrylamido)-2-hydroxybenzoic acid) polymer complexes

    NASA Astrophysics Data System (ADS)

    Ghoneim, M. M.; El-Sonbati, A. Z.; El-Bindary, A. A.; Diab, M. A.; Serag, L. S.

    2015-04-01

    A number of novel polymer complexes of various anions of copper(II), cobalt(II), nickel(II) and uranyl(II) with N(4-(acrylamido)-2-hydroxy benzoic acid) (ABH) have been synthesized and characterized by elemental analysis, IR, 1H NMR, magnetic susceptibility measurements, electronic spin resonance, vibrational spectra and thermal analysis. The molecular structures of the ligand are optimized theoretically and the quantum chemical parameters are calculated. Tentative structures for the polymeric metal complexes due to their potential application are also suggested. The IR data exhibit the coordination of ONO2/OAc/SO4 with the metal ions in the polymeric metal complex. Vibrational spectra indicate coordination of carboxylate oxygen and phenolic OH of the ligand giving a MO4 square planar chromophore. Ligand field ESR spectra support square planar geometry around Cu(II). The thermal decomposition of the polymer complexes were discussed in relation to structure, and the thermodynamic parameters of the decomposition stages were evaluated applying Coast-Redfern and Horowitz-Metzger methods.

  18. Fluorescent complexes of nucleic acids/8-hydroxyquinoline/lanthanum(III) and the fluorometry of nucleic acids

    SciTech Connect

    Cheng Zhi Huang; Ke An Li; Shen Yang Tong

    1996-07-01

    The ternary fluorescent complexes of nucleic acids/8-hydroxyquinoline/lanthanum (III) were studied. Nucleic acids in the study involve natured and thermally denatured calf thymus DNA, fish sperm DNA and yeast RNA. In the range of PH 8.0-8.4 (controlled by NH{sub 3}-NH{sub 4}Cl buffer) ternary fluorescent complexes are formed which emit at 485.0 nm for calf thymus DNA and at 480.0 nm for fish sperm DNA when excited at 265.0 nm. Based on the fluorescence reactions sensitive fluorometric methods for nucleic acids were proposed. Using optimal conditions, the calibration curves were linear in the range of 0.4 --3.6 {mu}g{sup .}ml{sup -1} for calf thymus DNA, 0.4 -- 4.0 {mu}g{sup .}ml{sup -1} for fish sperm DNA and 0.4 --4.0{mu}g{sup .}ml{sup -1} for yeast RNA, respectively. Five synthetic samples were determined with satisfaction.

  19. 40 CFR 721.10089 - Modified salicylic acid, zirconium complex (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Modified salicylic acid, zirconium... Specific Chemical Substances § 721.10089 Modified salicylic acid, zirconium complex (generic). (a) Chemical... as modified salicylic acid, zirconium complex (PMN P-00-552) is subject to reporting under...

  20. 40 CFR 721.10089 - Modified salicylic acid, zirconium complex (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Modified salicylic acid, zirconium... Specific Chemical Substances § 721.10089 Modified salicylic acid, zirconium complex (generic). (a) Chemical... as modified salicylic acid, zirconium complex (PMN P-00-552) is subject to reporting under...

  1. 40 CFR 721.10089 - Modified salicylic acid, zirconium complex (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Modified salicylic acid, zirconium... Specific Chemical Substances § 721.10089 Modified salicylic acid, zirconium complex (generic). (a) Chemical... as modified salicylic acid, zirconium complex (PMN P-00-552) is subject to reporting under...

  2. 40 CFR 721.10089 - Modified salicylic acid, zirconium complex (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Modified salicylic acid, zirconium... Specific Chemical Substances § 721.10089 Modified salicylic acid, zirconium complex (generic). (a) Chemical... as modified salicylic acid, zirconium complex (PMN P-00-552) is subject to reporting under...

  3. 40 CFR 721.10089 - Modified salicylic acid, zirconium complex (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Modified salicylic acid, zirconium... Specific Chemical Substances § 721.10089 Modified salicylic acid, zirconium complex (generic). (a) Chemical... as modified salicylic acid, zirconium complex (PMN P-00-552) is subject to reporting under...

  4. Identification and characterization of kinetically competent carbinolamine and alpha-iminoglutarate complexes in the glutamate dehydrogenase-catalyzed oxidation of L-glutamate using a multiwavelength transient state approach.

    PubMed

    Maniscalco, S J; Saha, S K; Fisher, H F

    1998-10-13

    A highly constrained and heavily overdetermined multiwavelength transient state kinetic approach has been used to study the oxidative deamination of L-glutamate catalyzed by beef liver glutamate dehydrogenase. Spectra generated using the known enzyme-reduced coenzyme-substrate spectrum served as models for deconvolution of kinetic scan data. Deconvolution of the multiwavelength time course array shows formation of three distinguishable intermediates in the reaction sequence, an ultrablue-shifted complex, an ultrared-shifted complex, and a blue-shifted complex. The ultrablue-shifted entity is identified as the enzyme-NADPH-alpha-iminoglutarate complex (ERI) and the ultrared as the enzyme-NADPH-alpha-carbinolamine complex (ERC). The blue-shifted complex is characterized as the E-NADPH-ketoglutarate species (ERK). The location of these species along the reaction coordinate has been determined and their kinetic competency in the reaction sequence has been established by fitting the concentration time courses of the components for both the alpha-deuterio- and the alpha-protio-L-glutamate reactions to the now highly constrained differential equations derived from a kinetic scheme involving the sequential formation of alpha-iminoglutarate, alpha-carbinolamine, and alpha-ketoglutarate-reduced coenzyme complexes, following the formation of two prehydride transfer complexes. PMID:9772187

  5. Molecular complexes of alprazolam with carboxylic acids, boric acid, boronic acids, and phenols. Evaluation of supramolecular heterosynthons mediated by a triazole ring.

    PubMed

    Varughese, Sunil; Azim, Yasser; Desiraju, Gautam R

    2010-09-01

    A series of molecular complexes, both co-crystals and salts, of a triazole drug-alprazolam-with carboxylic acids, boric acid, boronic acids, and phenols have been analyzed with respect to heterosynthons present in the crystal structures. In all cases, the triazole ring behaves as an efficient hydrogen bond acceptor with the acidic coformers. The hydrogen bond patterns exhibited with aromatic carboxylic acids were found to depend on the nature and position of the substituents. Being a strong acid, 2,6-dihydroxybenzoic acid forms a salt with alprazolam. With aliphatic dicarboxylic acids alprazolam forms hydrates and the water molecules play a central role in synthon formation and crystal packing. The triazole ring makes two distinct heterosynthons in the molecular complex with boric acid. Boronic acids and phenols form consistent hydrogen bond patterns, and these are seemingly independent of the substitutional effects. Boronic acids form noncentrosymmetric cyclic synthons, while phenols form O--H...N hydrogen bonds with the triazole ring.

  6. Crystallization and preliminary X-ray analysis of L-serine 3-dehydrogenase complexed with NADP+ from the hyperthermophilic archaeon Pyrobaculum calidifontis.

    PubMed

    Yoneda, Kazunari; Sakuraba, Haruhiko; Araki, Tomohiro; Shibata, Takeshi; Nikki, Takahiro; Ohshima, Toshihisa

    2013-02-01

    An NAD(P)(+)-dependent L-serine 3-dehydrogenase from the hyperthermophilic archaeon Pyrobaculum calidifontis was crystallized using the sitting-drop vapour-diffusion method with ammonium sulfate as the precipitant. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 120.81, b = 57.40, c = 56.37 Å, β = 106.88°. Diffraction data were collected to 1.57 Å resolution on beamline NE3A at the Photon Factory. The overall R(merge) was 4.2% and the data completeness was 90.1%. PMID:23385753

  7. Molybdopterin cofactor from Methanobacterium formicicum formate dehydrogenase.

    PubMed Central

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

    1986-01-01

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

  8. Partial Similarities Between Yeast and Liver Alcohol Dehydrogenases

    PubMed Central

    Jörnvall, Hans

    1973-01-01

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

  9. Acute Carnosine Administration Increases Respiratory Chain Complexes and Citric Acid Cycle Enzyme Activities in Cerebral Cortex of Young Rats.

    PubMed

    Macedo, Levy W; Cararo, José H; Maravai, Soliany G; Gonçalves, Cinara L; Oliveira, Giovanna M T; Kist, Luiza W; Guerra Martinez, Camila; Kurtenbach, Eleonora; Bogo, Maurício R; Hipkiss, Alan R; Streck, Emilio L; Schuck, Patrícia F; Ferreira, Gustavo C

    2016-10-01

    Carnosine (β-alanyl-L-histidine) is an imidazole dipeptide synthesized in excitable tissues of many animals, whose biochemical properties include carbonyl scavenger, anti-oxidant, bivalent metal ion chelator, proton buffer, and immunomodulating agent, although its precise physiological role(s) in skeletal muscle and brain tissues in vivo remain unclear. The aim of the present study was to investigate the in vivo effects of acute carnosine administration on various aspects of brain bioenergetics of young Wistar rats. The activity of mitochondrial enzymes in cerebral cortex was assessed using a spectrophotometer, and it was found that there was an increase in the activities of complexes I-III and II-III and succinate dehydrogenase in carnosine-treated rats, as compared to vehicle-treated animals. However, quantitative real-time RT-PCR (RT-qPCR) data on mRNA levels of mitochondrial biogenesis-related proteins (nuclear respiratory factor 1 (Nrf1), peroxisome proliferator-activated receptor-γ coactivator 1-α (Ppargc1α), and mitochondrial transcription factor A (Tfam)) were not altered significantly and therefore suggest that short-term carnosine administration does not affect mitochondrial biogenesis. It was in agreement with the finding that immunocontent of respiratory chain complexes was not altered in animals receiving carnosine. These observations indicate that acute carnosine administration increases the respiratory chain and citric acid cycle enzyme activities in cerebral cortex of young rats, substantiating, at least in part, a neuroprotector effect assigned to carnosine against oxidative-driven disorders. PMID:26476839

  10. Acute Carnosine Administration Increases Respiratory Chain Complexes and Citric Acid Cycle Enzyme Activities in Cerebral Cortex of Young Rats.

    PubMed

    Macedo, Levy W; Cararo, José H; Maravai, Soliany G; Gonçalves, Cinara L; Oliveira, Giovanna M T; Kist, Luiza W; Guerra Martinez, Camila; Kurtenbach, Eleonora; Bogo, Maurício R; Hipkiss, Alan R; Streck, Emilio L; Schuck, Patrícia F; Ferreira, Gustavo C

    2016-10-01

    Carnosine (β-alanyl-L-histidine) is an imidazole dipeptide synthesized in excitable tissues of many animals, whose biochemical properties include carbonyl scavenger, anti-oxidant, bivalent metal ion chelator, proton buffer, and immunomodulating agent, although its precise physiological role(s) in skeletal muscle and brain tissues in vivo remain unclear. The aim of the present study was to investigate the in vivo effects of acute carnosine administration on various aspects of brain bioenergetics of young Wistar rats. The activity of mitochondrial enzymes in cerebral cortex was assessed using a spectrophotometer, and it was found that there was an increase in the activities of complexes I-III and II-III and succinate dehydrogenase in carnosine-treated rats, as compared to vehicle-treated animals. However, quantitative real-time RT-PCR (RT-qPCR) data on mRNA levels of mitochondrial biogenesis-related proteins (nuclear respiratory factor 1 (Nrf1), peroxisome proliferator-activated receptor-γ coactivator 1-α (Ppargc1α), and mitochondrial transcription factor A (Tfam)) were not altered significantly and therefore suggest that short-term carnosine administration does not affect mitochondrial biogenesis. It was in agreement with the finding that immunocontent of respiratory chain complexes was not altered in animals receiving carnosine. These observations indicate that acute carnosine administration increases the respiratory chain and citric acid cycle enzyme activities in cerebral cortex of young rats, substantiating, at least in part, a neuroprotector effect assigned to carnosine against oxidative-driven disorders.

  11. Morphological characteristics, oxidative stability and enzymic hydrolysis of amylose-fatty acid complexes.

    PubMed

    Marinopoulou, Anna; Papastergiadis, Efthimios; Raphaelides, Stylianos N; Kontominas, Michael G

    2016-05-01

    Complexes of amylose with fatty acids varying in carbon chain length and degree of unsaturation were prepared at 30, 50 or 70°C by dissolving amylose in 0.1N KOH and mixing with fatty acid potassium soap solution. The complexes were obtained in solid form as precipitates after neutralization. SEM microscopy revealed that the morphology of the complexes was that of ordered lamellae separated from amorphous regions whereas confocal laser scanning microscopy showed images of the topography of the guest molecules in the complex matrix. FTIR spectroscopy revealed that the absorption peak attributed to carbonyl group of free fatty acid was shifted when the fatty acid was in the form of amylose complex. Thermo-gravimetry showed that the unsaturated fatty acids were effectively protected from oxidation when they were complexed with amylose whereas enzymic hydrolysis experiments showed that the guest molecules were quantitatively released from the amylose complexes. PMID:26877002

  12. Morphological characteristics, oxidative stability and enzymic hydrolysis of amylose-fatty acid complexes.

    PubMed

    Marinopoulou, Anna; Papastergiadis, Efthimios; Raphaelides, Stylianos N; Kontominas, Michael G

    2016-05-01

    Complexes of amylose with fatty acids varying in carbon chain length and degree of unsaturation were prepared at 30, 50 or 70°C by dissolving amylose in 0.1N KOH and mixing with fatty acid potassium soap solution. The complexes were obtained in solid form as precipitates after neutralization. SEM microscopy revealed that the morphology of the complexes was that of ordered lamellae separated from amorphous regions whereas confocal laser scanning microscopy showed images of the topography of the guest molecules in the complex matrix. FTIR spectroscopy revealed that the absorption peak attributed to carbonyl group of free fatty acid was shifted when the fatty acid was in the form of amylose complex. Thermo-gravimetry showed that the unsaturated fatty acids were effectively protected from oxidation when they were complexed with amylose whereas enzymic hydrolysis experiments showed that the guest molecules were quantitatively released from the amylose complexes.

  13. Functional Replacement of the Escherichia coli d-(−)-Lactate Dehydrogenase Gene (ldhA) with the l-(+)-Lactate Dehydrogenase Gene (ldhL) from Pediococcus acidilactici†

    PubMed Central

    Zhou, Shengde; Shanmugam, K. T.; Ingram, L. O.

    2003-01-01

    The microbial production of l-(+)-lactic acid is rapidly expanding to allow increased production of polylactic acid (PLA), a renewable, biodegradable plastic. The physical properties of PLA can be tailored for specific applications by controlling the ratio of l-(+) and d-(−) isomers. For most uses of PLA, the l-(+) isomer is more abundant. As an approach to reduce costs associated with biocatalysis (complex nutrients, antibiotics, aeration, product purification, and waste disposal), a recombinant derivative of Escherichia coli W3110 was developed that contains five chromosomal deletions (focA-pflB frdBC adhE ackA ldhA). This strain was constructed from a d-(−)-lactic acid-producing strain, SZ63 (focA-pflB frdBC adhE ackA), by replacing part of the chromosomal ldhA coding region with Pediococcus acidilactici ldhL encoding an l-lactate dehydrogenase. Although the initial strain (SZ79) grew and fermented poorly, a mutant (SZ85) was readily isolated by selecting for improved growth. SZ85 exhibited a 30-fold increase in l-lactate dehydrogenase activity in comparison to SZ79, functionally replacing the native d-lactate dehydrogenase activity. Sequencing revealed mutations in the upstream, coding, and terminator regions of ldhL in SZ85, which are presumed to be responsible for increased l-lactate dehydrogenase activity. SZ85 produced l-lactic acid in M9 mineral salts medium containing glucose or xylose with a yield of 93 to 95%, a purity of 98% (based on total fermentation products), and an optical purity greater than 99%. Unlike other recombinant biocatalysts for l-lactic acid, SZ85 remained prototrophic and is devoid of plasmids and antibiotic resistance genes. PMID:12676706

  14. Synthesis of conjugated linoleic acid by the linoleate isomerase complex in food-derived lactobacilli

    PubMed Central

    Yang, B.; Chen, H.; Gu, Z.; Tian, F.; Ross, R. P.; Stanton, C.; Chen, Y. Q.; Chen, W.; Zhang, H.

    2015-01-01

    Aims To assess strains of lactobacilli for their capacity to produce functional fatty acid-conjugated linoleic acid. To assess the linoleate isomerase for CLA production in the most efficient CLA producer. Methods and Results In this study, strains of food-derived lactobacilli were cultured in media with linoleic acid and CLA production was assessed. Most of the selected strains produced CLA at different levels, with Lactobacillus plantarum ZS2058 being the most efficient CLA producer converting over 50% of linoleic acid to c9, t11-CLA and t9, t11-CLA. Some intermediates 10-hydroxy-cis-12-octadecenoic acid, 10-oxo-cis-12-octadecenoic acid and 10-oxo-trans-11-octadecenoic acid were determined via GC-MS. The genes coding the multicomponent linoleate isomerase containing myosin-cross-reactive antigen, short-chain dehydrogenase/oxidoreductase and acetoacetate decarboxylase for CLA production in Lact. plantarum ZS2058 were cloned and expressed in Escherichia coli. With the mixture of recombinant E. coli, c9, t11-CLA and three kinds of intermediates were produced from linoleic acid, which were in line with those in the lactobacilli. Conclusions The ability for CLA production by lactobacilli exhibited variation. Lactobacillus plantarum and Lact. bulgaricus were the most efficient producers in the selected strains. Lact. plantarum ZS2058 converted linoleic acid to CLAs with 10-hydroxy-cis-12-octadecenoic acid, 10-oxo-cis-12-octadecenoic acid and 10-oxo-trans-11-octadecenoic acid as intermediates. The multiple-step reactions for CLA production catalysed by multicomponent linoleate isomerase in Lact. plantarum ZS2058 were confirmed successfully. Significance and Impact of the study Multicomponent linoleate isomerase provides important results for the illustration of the mechanism for CLA production in lactic acid bacteria. Food-derived lactobacilli with CLA production ability offers novel opportunities for functional foods development. PMID:24750362

  15. Plant Formate Dehydrogenase

    SciTech Connect

    John Markwell

    2005-01-10

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

  16. Complexation of NpO2+ with N-methyl-iminodiacetic Acid: in Comparison with Iminodiacetic and Dipicolinic Acids

    SciTech Connect

    Tian, Guoxin; Rao, Linfeng

    2010-10-01

    Complexation of Np(V) with N-methyl-iminodiacetic acid (MIDA) in 1 M NaClO{sub 4} solution was studied with multiple techniques including potentiometry, spectrophotometry, and microcalorimetry. The 1:2 complex, NpO{sub 2}(MIDA){sub 2}{sup 3-} was identified for the first time in aqueous solution. The correlation between its optical absorption properties and symmetry was discussed, in comparison with Np(V) complexes with two structurally related nitrilo-dicarboxylic acids, iminodiacetic acid (IDA) and dipicolinic acid (DPA). The order of the binding strength (DPA > MIDA > IDA) is explained by the difference in the structural and electronic properties of the ligands. In general, the nitrilo-dicarboxylates form stronger complexes with Np(V) than oxy-dicarboxylates due to a much more favorable enthalpy of complexation.

  17. Thermodynamics of the complexation of arabinogalactan with salicylic and p-aminobenzoic acids in aqueous solutions

    NASA Astrophysics Data System (ADS)

    Mudarisova, R. Kh.; Badykova, L. A.

    2016-03-01

    The thermodynamics of complexation of arabinogalactan with salicylic and p-aminobenzoic acids in aqueous solutions is studied by means spectroscopy. The standard thermodynamic characteristics (Δ H°; Δ G°; Δ S°) of complexation are calculated.

  18. Lipid complex effect on fatty acid profile and chemical composition of cow milk and cheese.

    PubMed

    Bodkowski, R; Czyż, K; Kupczyński, R; Patkowska-Sokoła, B; Nowakowski, P; Wiliczkiewicz, A

    2016-01-01

    The effect of administration of lipid complex (LC) on cow milk and cheese characteristics was studied. Lipid complex was elaborated based on grapeseed oil with synthesized conjugated linoleic acid (CLA) and Atlantic mackerel oil enriched in n-3 fatty acids. The 4-wk experiment was conducted on 30 Polish Holstein Friesian cows. The experimental group cow diet was supplemented with 400 g/d of LC (containing 38% CLA, and eicosapentaenoic acid + docosahexaenoic acid in a relative amount of 36.5%) on a humic-mineral carrier. The chemical composition and fatty acid profile of milk and rennet cheese from raw fresh milk were analyzed. Lipid complex supplementation of the total mixed ration had no effect on milk yield and milk composition, except fat content, which decreased from 4.6 to 4.1%, a 10.9% decrease. Milk from cows treated with LC had greater relative amounts of unsaturated fatty acids, particularly polyunsaturated fatty acids, and lesser relative amounts of saturated fatty acids. Lipid complex addition changed milk fat fatty acid profile: C18:2 cis-9,trans-11 and trans-10,cis-12 isomer (CLA) contents increased by 278 and 233%, respectively, as did eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6) contents. Milk fat fatty acid profile changes were correlated with the modifications in rennet cheese fatty acid profile. Lipid complex supplementation of dairy cows produced considerable changes in the biological value of milk and cheese fat.

  19. Lipid complex effect on fatty acid profile and chemical composition of cow milk and cheese.

    PubMed

    Bodkowski, R; Czyż, K; Kupczyński, R; Patkowska-Sokoła, B; Nowakowski, P; Wiliczkiewicz, A

    2016-01-01

    The effect of administration of lipid complex (LC) on cow milk and cheese characteristics was studied. Lipid complex was elaborated based on grapeseed oil with synthesized conjugated linoleic acid (CLA) and Atlantic mackerel oil enriched in n-3 fatty acids. The 4-wk experiment was conducted on 30 Polish Holstein Friesian cows. The experimental group cow diet was supplemented with 400 g/d of LC (containing 38% CLA, and eicosapentaenoic acid + docosahexaenoic acid in a relative amount of 36.5%) on a humic-mineral carrier. The chemical composition and fatty acid profile of milk and rennet cheese from raw fresh milk were analyzed. Lipid complex supplementation of the total mixed ration had no effect on milk yield and milk composition, except fat content, which decreased from 4.6 to 4.1%, a 10.9% decrease. Milk from cows treated with LC had greater relative amounts of unsaturated fatty acids, particularly polyunsaturated fatty acids, and lesser relative amounts of saturated fatty acids. Lipid complex addition changed milk fat fatty acid profile: C18:2 cis-9,trans-11 and trans-10,cis-12 isomer (CLA) contents increased by 278 and 233%, respectively, as did eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6) contents. Milk fat fatty acid profile changes were correlated with the modifications in rennet cheese fatty acid profile. Lipid complex supplementation of dairy cows produced considerable changes in the biological value of milk and cheese fat. PMID:26506539

  20. Effects of chemical and enzymatic modifications on starch-linoleic acid complex formation.

    PubMed

    Arijaje, Emily Oluwaseun; Wang, Ya-Jane

    2017-02-15

    This study investigated the complexation yield and physicochemical properties of soluble and insoluble starch complexes with linoleic acid when a β-amylase treatment was applied to acetylated and debranched potato starch. The degree of acetylation was generally higher in the soluble complexes than in the insoluble ones. The insoluble complexes from the acetylated starch displayed the V-type pattern, whereas, the soluble complexes displayed a mixture of either the A-/V-type or the B-/V-type pattern. Acetylation decreased onset and peak melting temperatures for the insoluble complexes, whereas no melting endotherm was observed in the soluble complexes. Acetylation substantially increased the amount of complexed linoleic acid in the insoluble complexes, but had little positive effect on the formation of the soluble complexes. The β-amylase treatment significantly increased the complexed linoleic content in both soluble and insoluble complexes for the low acetylated starch, but not for the high acetylated starch. PMID:27664602

  1. Amylose inclusion complexation of ferulic acid via lipophilization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ferulic acid is an interesting phytochemical that exhibits antioxidant, anti-inflammatory, antimicrobial, UV-absorber, and anticarcinogenic activities. These properties make it of interest in food formulations, cosmetics, polymer, and pharmaceutical applications. However, delivery of ferulic acid in...

  2. The behavior and importance of lactic acid complexation in Talspeak extraction systems

    SciTech Connect

    Grimes, Travis S.; Nilsson, Mikael; Nash, Kenneth L.

    2008-07-01

    Advanced partitioning of spent nuclear fuel in the UREX +la process relies on the TALSPEAK process for separation of fission-product lanthanides from trivalent actinides. The classic TALSPEAK utilizes an aqueous medium of both lactic acid and diethylenetriaminepentaacetic acid and the extraction reagent di(2-ethylhexyl)phosphoric acid in an aromatic diluent. In this study, the specific role of lactic acid and the complexes involved in the extraction of the trivalent actinides and lanthanides have been investigated using {sup 14}C-labeled lactic acid. Our results show that lactic acid partitions between the phases in a complex fashion. (authors)

  3. Efficient production of optically pure D-lactic acid from raw corn starch by using a genetically modified L-lactate dehydrogenase gene-deficient and alpha-amylase-secreting Lactobacillus plantarum strain.

    PubMed

    Okano, Kenji; Zhang, Qiao; Shinkawa, Satoru; Yoshida, Shogo; Tanaka, Tsutomu; Fukuda, Hideki; Kondo, Akihiko

    2009-01-01

    In order to achieve direct and efficient fermentation of optically pure D-lactic acid from raw corn starch, we constructed L-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 alpha-amylase (AmyA). The resulting strain produced only D-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct D-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct D-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct D-lactic acid fermentation from raw starch.

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

    PubMed

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

    1995-08-01

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

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

    PubMed Central

    Koenig, K; Andreesen, J R

    1990-01-01

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

  6. Diverging regulation of pyruvate dehydrogenase kinase isoform gene expression in cultured human muscle cells.

    PubMed

    Abbot, Emily L; McCormack, James G; Reynet, Christine; Hassall, David G; Buchan, Kevin W; Yeaman, Stephen J

    2005-06-01

    The pyruvate dehydrogenase complex occupies a central and strategic position in muscle intermediary metabolism and is primarily regulated by phosphorylation/dephosphorylation. The identification of multiple isoforms of pyruvate dehydrogenase kinase (PDK1-4) and pyruvate dehydrogenase phosphatase (PDP1-2) has raised intriguing new possibilities for chronic pyruvate dehydrogenase complex control. Experiments to date suggest that PDK4 is the major isoenzyme responsible for changes in pyruvate dehydrogenase complex activity in response to various different metabolic conditions. Using a cultured human skeletal muscle cell model system, we found that expression of both PDK2 and PDK4 mRNA is upregulated in response to glucose deprivation and fatty acid supplementation, the effects of which are reversed by insulin treatment. In addition, insulin directly downregulates PDK2 and PDK4 mRNA transcript abundance via a phosphatidylinositol 3-kinase-dependent pathway, which may involve glycogen synthase kinase-3 but does not utilize the mammalian target of rapamycin or mitogen-activated protein kinase signalling pathways. In order to further elucidate the regulation of PDK, the role of the peroxisome proliferators-activated receptors (PPAR) was investigated using highly potent subtype selective agonists. PPARalpha and PPARdelta agonists were found to specifically upregulate PDK4 mRNA expression, whereas PPARgamma activation selectively decreased PDK2 mRNA transcript abundance. PDP1 mRNA expression was unaffected by all conditions analysed. These results suggest that in human muscle, hormonal and nutritional conditions may control PDK2 and PDK4 mRNA expression via a common signalling mechanism. In addition, PPARs appear to independently regulate specific PDK isoform transcipt levels, which are likely to impart important metabolic mediation of fuel utilization by the muscle. PMID:15955060

  7. Preparation and investigation of acetyl salicylic acid-caffeine complex for rectal administration.

    PubMed

    Fouad, Ehab A; El-Badry, Mahmoud; Alanazi, Fars K; Arafah, Maha M; Al-Ashban, Riyadh; Alsarra, Ibrahim A

    2010-06-01

    An acetyl salicylic acid-caffeine complex was prepared and evaluated for the potential use in rectal administration. The results revealed the formation of a complex between acetyl salicylic acid and caffeine in a 1:1 molar ratio by a charge transfer mechanism. The effects of acetyl salicylic acid and complex on the rectal tissues showed destruction in the mucosal epithelium in case of acetyl salicylic acid; however, no change in the rectal tissues was noticed upon the administration of the complex. The effect of suppository bases on the release of the complex was studied using Witepsol H15 as fatty base and polyethylene glycols (PEG) 1000 and 4000 as a water soluble suppository base. The release profiles of acetyl salicylic acid and the complex were faster from PEG than from that of Witepsol H15. The percent release for the complex and acetyl salicylic acid from PEG base were 45.8, and 34.9%, respectively. However, it was 8.7 and 7.8%, respectively, from Witepsol H15 fatty base. The release kinetic was found to follow the non-Fickian diffusion model for complex from the suppository bases. It was concluded that acetyl salicylic acid caffeine complex can be used safely for rectal administration.

  8. Effects of chemical and enzymatic modifications on starch-oleic acid complex formation.

    PubMed

    Arijaje, Emily Oluwaseun; Wang, Ya-Jane

    2015-04-29

    The solubility of starch-inclusion complexes affects the digestibility and bioavailability of the included molecules. Acetylation with two degrees of substitution, 0.041 (low) and 0.091 (high), combined without or with a β-amylase treatment was employed to improve the yield and solubility of the inclusion complex between debranched potato starch and oleic acid. Both soluble and insoluble complexes were recovered and analyzed for their degree of acetylation, complexation yields, molecular size distributions, X-ray diffraction patterns, and thermal properties. Acetylation significantly increased the amount of recovered soluble complexes as well as the complexed oleic acid in both soluble and insoluble complexes. High-acetylated debranched-only starch complexed the highest amount of oleic acid (38.0 mg/g) in the soluble complexes; low-acetylated starch with or without the β-amylase treatment resulted in the highest complexed oleic acid in the insoluble complexes (37.6-42.9 mg/g). All acetylated starches displayed the V-type X-ray pattern, and the melting temperature generally decreased with acetylation. The results indicate that starch acetylation with or without the β-amylase treatment can improve the formation and solubility of the starch-oleic acid complex. PMID:25877005

  9. Acetic acid treatment in S. cerevisiae creates significant energy deficiency and nutrient starvation that is dependent on the activity of the mitochondrial transcriptional complex Hap2-3-4-5.

    PubMed

    Kitanovic, Ana; Bonowski, Felix; Heigwer, Florian; Ruoff, Peter; Kitanovic, Igor; Ungewiss, Christin; Wölfl, Stefan

    2012-01-01

    Metabolic pathways play an indispensable role in supplying cellular systems with energy and molecular building blocks for growth, maintenance and repair and are tightly linked with lifespan and systems stability of cells. For optimal growth and survival cells rapidly adopt to environmental changes. Accumulation of acetic acid in stationary phase budding yeast cultures is considered to be a primary mechanism of chronological aging and induction of apoptosis in yeast, which has prompted us to investigate the dependence of acetic acid toxicity on extracellular conditions in a systematic manner. Using an automated computer controlled assay system, we investigated and model the dynamic interconnection of biomass yield- and growth rate-dependence on extracellular glucose concentration, pH conditions and acetic acid concentration. Our results show that toxic concentrations of acetic acid inhibit glucose consumption and reduce ethanol production. In absence of carbohydrates uptake, cells initiate synthesis of storage carbohydrates, trehalose and glycogen, and upregulate gluconeogenesis. Accumulation of trehalose and glycogen, and induction of gluconeogenesis depends on mitochondrial activity, investigated by depletion of the Hap2-3-4-5 complex. Analyzing the activity of glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PYK), and glucose-6-phosphate dehydrogenase (G6PDH) we found that while high acetic acid concentration increased their activity, lower acetic acids concentrations significantly inhibited these enzymes. With this study we determined growth and functional adjustment of metabolism to acetic acid accumulation in a complex range of extracellular conditions. Our results show that substantial acidification of the intracellular environment, resulting from accumulation of dissociated acetic acid in the cytosol, is required for acetic acid toxicity, which creates a state of energy deficiency and nutrient starvation.

  10. Acetic acid treatment in S. cerevisiae creates significant energy deficiency and nutrient starvation that is dependent on the activity of the mitochondrial transcriptional complex Hap2-3-4-5

    PubMed Central

    Kitanovic, Ana; Bonowski, Felix; Heigwer, Florian; Ruoff, Peter; Kitanovic, Igor; Ungewiss, Christin; Wölfl, Stefan

    2012-01-01

    Metabolic pathways play an indispensable role in supplying cellular systems with energy and molecular building blocks for growth, maintenance and repair and are tightly linked with lifespan and systems stability of cells. For optimal growth and survival cells rapidly adopt to environmental changes. Accumulation of acetic acid in stationary phase budding yeast cultures is considered to be a primary mechanism of chronological aging and induction of apoptosis in yeast, which has prompted us to investigate the dependence of acetic acid toxicity on extracellular conditions in a systematic manner. Using an automated computer controlled assay system, we investigated and model the dynamic interconnection of biomass yield- and growth rate-dependence on extracellular glucose concentration, pH conditions and acetic acid concentration. Our results show that toxic concentrations of acetic acid inhibit glucose consumption and reduce ethanol production. In absence of carbohydrates uptake, cells initiate synthesis of storage carbohydrates, trehalose and glycogen, and upregulate gluconeogenesis. Accumulation of trehalose and glycogen, and induction of gluconeogenesis depends on mitochondrial activity, investigated by depletion of the Hap2-3-4-5 complex. Analyzing the activity of glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PYK), and glucose-6-phosphate dehydrogenase (G6PDH) we found that while high acetic acid concentration increased their activity, lower acetic acids concentrations significantly inhibited these enzymes. With this study we determined growth and functional adjustment of metabolism to acetic acid accumulation in a complex range of extracellular conditions. Our results show that substantial acidification of the intracellular environment, resulting from accumulation of dissociated acetic acid in the cytosol, is required for acetic acid toxicity, which creates a state of energy deficiency and nutrient starvation. PMID:23050242

  11. Complex Regulation of cyp26a1 Creates a Robust Retinoic Acid Gradient in the Zebrafish Embryo

    PubMed Central

    White, Richard J; Nie, Qing; Lander, Arthur D; Schilling, Thomas F

    2007-01-01

    Positional identities along the anterior–posterior axis of the vertebrate nervous system are assigned during gastrulation by multiple posteriorizing signals, including retinoic acid (RA), fibroblast growth factors (Fgfs), and Wnts. Experimental evidence has suggested that RA, which is produced in paraxial mesoderm posterior to the hindbrain by aldehyde dehydrogenase 1a2 (aldh1a2/raldh2), forms a posterior-to-anterior gradient across the hindbrain field, and provides the positional information that specifies the locations and fates of rhombomeres. Recently, alternative models have been proposed in which RA plays only a permissive role, signaling wherever it is not degraded. Here we use a combination of experimental and modeling tools to address the role of RA in providing long-range positional cues in the zebrafish hindbrain. Using cell transplantation and implantation of RA-coated beads into RA-deficient zebrafish embryos, we demonstrate that RA can directly convey graded positional information over long distances. We also show that expression of Cyp26a1, the major RA-degrading enzyme during gastrulation, is under complex feedback and feedforward control by RA and Fgf signaling. The predicted consequence of such control is that RA gradients will be both robust to fluctuations in RA synthesis and adaptive to changes in embryo length during gastrulation. Such control also provides an explanation for the fact that loss of an endogenous RA gradient can be compensated for by RA that is provided in a spatially uniform manner. PMID:18031199

  12. Salicylic Acid Inhibits the Replication of Tomato bushy stunt virus by Directly Targeting a Host Component in the Replication Complex.

    PubMed

    Tian, Miaoying; Sasvari, Zsuzsanna; Gonzalez, Paulina Alatriste; Friso, Giulia; Rowland, Elden; Liu, Xiao-Min; van Wijk, Klaas J; Nagy, Peter D; Klessig, Daniel F

    2015-04-01

    Although the plant hormone salicylic acid (SA) plays a central role in signaling resistance to viral infection, the underlying mechanisms are only partially understood. Identification and characterization of SA's direct targets have been shown to be an effective strategy for dissecting the complex SA-mediated defense signaling network. In search of additional SA targets, we previously developed two sensitive approaches that utilize SA analogs in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology to identify and evaluate candidate SA-binding proteins (SABPs) from Arabidopsis. Using these approaches, we have now identified several members of the Arabidopsis glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein family, including two chloroplast-localized and two cytosolic isoforms, as SABPs. Cytosolic GAPDH is a well-known glycolytic enzyme; it also is an important host factor involved in the replication of Tomato bushy stunt virus (TBSV), a single-stranded RNA virus. Using a yeast cell-free extract, an in vivo yeast replication system, and plant protoplasts, we demonstrate that SA inhibits TBSV replication. SA does so by inhibiting the binding of cytosolic GAPDH to the negative (-)RNA strand of TBSV. Thus, this study reveals a novel molecular mechanism through which SA regulates virus replication. PMID:25584724

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

    PubMed

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

    2014-01-01

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

  14. Mitochondrial complex II participates in normoxic and hypoxic regulation of α-keto acids in the murine heart.

    PubMed

    Mühling, Jörg; Tiefenbach, Martina; López-Barneo, José; Piruat, José I; García-Flores, Paula; Pfeil, Uwe; Gries, Barbara; Mühlfeld, Christian; Weigand, Markus A; Kummer, Wolfgang; Weissmann, Norbert; Paddenberg, Renate

    2010-12-01

    α-Keto acids (α-KAs) are not just metabolic intermediates but are also powerful modulators of different cellular pathways. Here, we tested the hypothesis that α-KA concentrations are regulated by complex II (succinate dehydrogenase=SDH), which represents an intersection between the mitochondrial respiratory chain for which an important function in cardiopulmonary oxygen sensing has been demonstrated, and the Krebs cycle, a central element of α-KA metabolism. SDH subunit D heterozygous (SDHD(+/-)) and wild-type (WT) mice were housed at normoxia or hypoxia (10% O(2)) for 4 days or 3 weeks, and right ventricular pressure, right ventricle/(left ventricle+septum) ratio, cardiomyocyte ultrastructure, pulmonary vascular remodelling, ventricular complex II subunit expression, SDH activity and α-KA concentrations were analysed. In both strains, hypoxia induced increases in right ventricular pressure and enhanced muscularization of distal pulmonary arteries. Right ventricular hypertrophy was less severe in SDHD(+/-) mice although the cardiomyocyte ultrastructure and mitochondrial morphometric parameters were unchanged. Protein amounts of SDHA, SDHB and SDHC, and SDH activity were distinctly reduced in SDHD(+/-) mice. In normoxic SDHD(+/-) mice, α-ketoisocaproate concentration was lowered to 50% as compared to WT animals. Right/left ventricular concentration differences and the hypoxia-induced decline in individual α-KAs were less pronounced in SDHD(+/-) animals indicating that mitochondrial complex II participates in the adjustment of cardiac α-KA concentrations both under normoxic and hypoxic conditions. These characteristics are not related to the hemodynamic consequences of hypoxia-induced pulmonary vascular remodelling, since its extent and right ventricular pressure were not affected in SDHD(+/-) mice albeit right ventricular hypertrophy was attenuated.

  15. New insights into the binding mode of coenzymes: structure of Thermus thermophilus Delta1-pyrroline-5-carboxylate dehydrogenase complexed with NADP+.

    PubMed

    Inagaki, Eiji; Ohshima, Noriyasu; Sakamoto, Keiko; Babayeva, Nigar D; Kato, Hiroaki; Yokoyama, Shigeyuki; Tahirov, Tahir H

    2007-06-01

    Delta(1)-Pyrroline-5-carboxylate dehydrogenase (P5CDh) is known to preferentially use NAD(+) as a coenzyme. The k(cat) value of Thermus thermophilus P5CDh (TtP5CDh) is four times lower for NADP(+) than for NAD(+). The crystal structure of NADP(+)-bound TtP5CDh was solved in order to study the structure-activity relationships for the coenzymes. The binding mode of NADP(+) is essentially identical to that in the previously solved NAD(+)-bound form, except for the regions around the additional 2'-phosphate group of NADP(+). The coenzyme-binding site can only accommodate this group by the rotation of a glutamate residue and subtle shifts in the main chain. The 2'-phosphate of NADP(+) increases the number of hydrogen bonds between TtP5CDh and NADP(+) compared with that between TtP5CDh and NAD(+). Furthermore, the phosphate of the bound NADP(+) would restrict the ;bending' of the coenzyme because of steric hindrance. Such bending is important for dissociation of the coenzymes. These results provide a plausible explanation of the lower turnover rate of NADP(+) compared with NAD(+).

  16. Characterization of testis-specific isoenzyme of human pyruvate dehydrogenase.

    PubMed

    Korotchkina, Lioubov G; Sidhu, Sukhdeep; Patel, Mulchand S

    2006-04-01

    Pyruvate dehydrogenase (PDH), the first component of the human pyruvate dehydrogenase complex, has two isoenzymes, somatic cell-specific PDH1 and testis-specific PDH2 with 87% sequence identity in the alpha subunit of alpha(2) beta(2) PDH. The presence of functional testis-specific PDH2 is important for sperm cells generating nearly all their energy from carbohydrates via pyruvate oxidation. Kinetic and regulatory properties of recombinant human PDH2 and PDH1 were compared in this study. Site-specific phosphorylation/dephosphorylation of the three phosphorylation sites by four PDH kinases (PDK1-4) and two PDH phosphatases (PDP1-2) were investigated by substituting serines with alanine or glutamate in PDHs. PDH2 was found to be very similar to PDH1 as follows: (i) in specific activities and kinetic parameters as determined by the pyruvate dehydrogenase complex assay; (ii) in thermostability at 37 degrees C; (iii) in the mechanism of inactivation by phosphorylation of three sites; and (iv) in the phosphorylation of sites 1 and 2 by PDK3. In contrast, the differences for PDH2 were indicated as follows: (i) by a 2.4-fold increase in binding affinity for the PDH-binding domain of dihydrolipoamide acetyltransferase as measured by surface plasmon resonance; (ii) by possible involvement of Ser-264 (site 1) of PDH2 in catalysis as evident by its kinetic behavior; and (iii) by the lower activities of PDK1, PDK2, and PDK4 as well as PDP1 and PDP2 toward PDH2. These differences between PDH2 and PDH1 are less than expected from substitution of 47 amino acids in each PDH2 alpha subunit. The multiple substitutions may have compensated for any drastic alterations in PDH2 structure thereby preserving its kinetic and regulatory characteristics largely similar to that of PDH1. PMID:16436377

  17. Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Hong, Y. S.; Ciszak, Ewa; Patel, Mulchand

    2000-01-01

    Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For

  18. Stability Constants of Mixed Ligand Complexes of Nickel(II) with Adenine and Some Amino Acids

    PubMed Central

    Türkel, Naciye

    2015-01-01

    Nickel is one of the essential trace elements found in biological systems. It is mostly found in nickel-based enzymes as an essential cofactor. It forms coordination complexes with amino acids within enzymes. Nickel is also present in nucleic acids, though its function in DNA or RNA is still not clearly understood. In this study, complex formation tendencies of Ni(II) with adenine and certain L-amino acids such as aspartic acid, glutamic acid, asparagine, leucine, phenylalanine, and tryptophan were investigated in an aqueous medium. Potentiometric equilibrium measurements showed that both binary and ternary complexes of Ni(II) form with adenine and the above-mentioned L-amino acids. Ternary complexes of Ni(II)-adenine-L-amino acids are formed by stepwise mechanisms. Relative stabilities of the ternary complexes are compared with those of the corresponding binary complexes in terms of Δlog10⁡K, log10⁡X, and % RS values. It was shown that the most stable ternary complex is Ni(II):Ade:L-Asn while the weakest one is Ni(II):Ade:L-Phe in aqueous solution used in this research. In addition, results of this research clearly show that various binary and ternary type Ni(II) complexes are formed in different concentrations as a function of pH in aqueous solution. PMID:26843852

  19. Recovery of nickel and cobalt from organic acid complexes: adsorption mechanisms of metal-organic complexes onto aminophosphonate chelating resin.

    PubMed

    Deepatana, A; Valix, M

    2006-09-21

    This study examined the recovery of nickel and cobalt from organic acid complexes using a chelating aminophosphonate Purolite S950 resin. These metal complexes are generated by bioleaching nickel laterite ores, a commercial nickel and cobalt mineral oxide, with heterotrophic organism and their metabolites or organic acid products. Equilibrium adsorption tests were conducted as a function of Ni and Co concentrations (15-2000 mg/L), solution pH (0.01 and 0.1 M acids) and three metabolic complexing agents (citrate, malate and lactate). It was shown that the adsorption of the various Ni- and Co-complexes on Purolite were quite low, 16-18 and 5.4-9 mg/g of resin, respectively, in comparison to the smaller nickel ions and nickel sulfate. This was attributed to the bulky organic ligands which promoted crowding effect or steric hindrance. The adsorption of these complexes was further hampered by the strong affinity of the resin to H+ ions under acidic conditions. Mechanisms of adsorption, as inferred from the fitted empirical Langmuir and Freundlich models, were correlated to the proposed steric hindrance and competitive adsorption effects. Nickel and cobalt elution from the resin were found be effective and were independent of the type of metal complexes and metal concentrations. This study demonstrated the relative challenges involved in recovering nickel and cobalt from bioleaching solutions. PMID:16698178

  20. Recovery of nickel and cobalt from organic acid complexes: adsorption mechanisms of metal-organic complexes onto aminophosphonate chelating resin.

    PubMed

    Deepatana, A; Valix, M

    2006-09-21

    This study examined the recovery of nickel and cobalt from organic acid complexes using a chelating aminophosphonate Purolite S950 resin. These metal complexes are generated by bioleaching nickel laterite ores, a commercial nickel and cobalt mineral oxide, with heterotrophic organism and their metabolites or organic acid products. Equilibrium adsorption tests were conducted as a function of Ni and Co concentrations (15-2000 mg/L), solution pH (0.01 and 0.1 M acids) and three metabolic complexing agents (citrate, malate and lactate). It was shown that the adsorption of the various Ni- and Co-complexes on Purolite were quite low, 16-18 and 5.4-9 mg/g of resin, respectively, in comparison to the smaller nickel ions and nickel sulfate. This was attributed to the bulky organic ligands which promoted crowding effect or steric hindrance. The adsorption of these complexes was further hampered by the strong affinity of the resin to H+ ions under acidic conditions. Mechanisms of adsorption, as inferred from the fitted empirical Langmuir and Freundlich models, were correlated to the proposed steric hindrance and competitive adsorption effects. Nickel and cobalt elution from the resin were found be effective and were independent of the type of metal complexes and metal concentrations. This study demonstrated the relative challenges involved in recovering nickel and cobalt from bioleaching solutions.

  1. Cloning of the rat pyruvate dehydrogenase kinase 4 gene promoter: activation of pyruvate dehydrogenase kinase 4 by the peroxisome proliferator-activated receptor gamma coactivator.

    PubMed

    Ma, Ke; Zhang, Yi; Elam, Marshall B; Cook, George A; Park, Edwards A

    2005-08-19

    The pyruvate dehydrogenase complex catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the metabolism of glucose to acetyl-CoA. Phosphorylation of pyruvate dehydrogenase by the pyruvate dehydrogenase kinases (PDK) inhibits pyruvate dehydrogenase complex activity. There are four PDK isoforms, and expression of PDK4 and PDK2 genes is elevated in starvation and diabetes, allowing glucose to be conserved while fatty acid oxidation is increased. In these studies we have investigated the transcriptional mechanisms by which the expression of the PDK4 gene is increased. The peroxisome proliferator-activated receptor gamma coactivator (PGC-1alpha) stimulates the expression of genes involved in hepatic gluconeogenesis and mitochondrial fatty acid oxidation. We have found that PGC-1alpha will induce the expression of both the PDK2 and PDK4 genes in primary rat hepatocytes and ventricular myocytes. We cloned the promoter for the rat PDK4 gene. Hepatic nuclear factor 4 (HNF4), which activates many genes in the liver, will induce PDK4 expression. Although HNF4 and PGC-1alpha interact to stimulate several genes encoding gluconeogenic enzymes, the induction of PDK4 does not involve interactions of PGC-1alpha with HNF4. Using the chromatin immunoprecipitation assay, we have demonstrated that HNF4 and PGC-1alpha are associated with the PDK4 gene in vivo. Our data suggest that by inducing PDK genes PGC-1alpha will direct pyruvate away from metabolism into acetyl-CoA and toward the formation of oxaloacetate and into the gluconeogenic pathway. PMID:15967803

  2. Synthesis and spectroscopic characterization of gallic acid and some of its azo complexes

    NASA Astrophysics Data System (ADS)

    Masoud, Mamdouh S.; Hagagg, Sawsan S.; Ali, Alaa E.; Nasr, Nessma M.

    2012-04-01

    A series of gallic acid and azo gallic acid complexes were prepared and characterized by elemental analysis, IR, electronic spectra and magnetic susceptibility. The complexes were of different geometries: Octahedral, Tetrahedral and Square Planar. ESR was studied for copper complexes. All of the prepared complexes were of isotropic nature. The thermal analyses of the complexes were studied by DTA and DSC techniques. The thermodynamic parameters and the thermal transitions, such as glass transitions, crystallization and melting temperatures for some ligands and their complexes were evaluated and discussed. The entropy change values, ΔS#, showed that the transition states are more ordered than the reacting complexes. The biological activities of some ligands and their complexes are tested against Gram positive and Gram negative bacteria. The results showed that some complexes have a well considerable activity against different organisms.

  3. Effects of chemical and enzymatic modifications on starch-stearic acid complex formation.

    PubMed

    Arijaje, Emily Oluwaseun; Wang, Ya-Jane; Shinn, Sara; Shah, Utkarsh; Proctor, Andrew

    2014-04-01

    Debranched unacetylated and acetylated potato starches with two degrees of substitution, 0.041 (low) and 0.078 (high), combined with or without β-amylase hydrolysis were prepared to form soluble and insoluble complexes with stearic acid. The effects of modifications on the complexation, thermal properties, and X-ray patterns of soluble and insoluble complexes were investigated. Acetylation decreased the recovery of insoluble complexes but increased that of soluble complexes. Low acetylated, β-amylase-treated starch had a significantly increased amount of complexed stearic acid (123.1 mg/g) for insoluble complexes; high acetylated, β-amylase-treated starch had the highest complexed stearic acid (61.2 mg/g) for the soluble complexes. The melting temperature of the complexes decreased with acetylation. All β-amylase-treated acetylated complexes displayed the V-type diffraction pattern with peaks at 2θ = 7.4°, 12.9°, and 20°. These results suggest that starch can be modified by acetylation, debranching, and/or β-amylase to produce significant quantities of soluble starch-stearic acid complexes.

  4. Nickel(II) and copper(II) complexes with humic acid anions and their derivatives

    SciTech Connect

    Ryabova, I.N.

    2008-01-15

    Complexation of Ni(II) and Cu(II) in aqueous solutions with anions of humic acids, extracted from naturally oxidized coal, and with their hydroxymethyl derivatives is studied spectrophotometrically and potentiometrically. The complexation stoichiometry and the stability constants of the complexes are determined.

  5. Butachlor impact on protein, free amino acid and glutamine contents, and on activity levels of aminotransferases, glutamate dehydrogenase and glutamine synthetase in the fresh water snail, Pila globosa (Swainson).

    PubMed

    Rajyalakshmi, T; Srinivas, T; Swamy, K V; Mohan, P M

    1996-08-01

    Biochemical changes followed in the freshwater snail Pila globosa (Swainson) during exposure to sublethal concentrations of the herbicide butachlor (26.6 ppm) in the ambient medium, at 3,6,12,24 and 48 h intervals, were marked by a significant decrease in total and soluble proteins, and an increase in free amino acids in foot and hepatopancreas up to 12 h before gradually recovering. Aminotransferase activities and glutamine content decreased during the early periods of exposure, while glutamate dehydrogenase activity increased. After an initial elevation, glutamate synthetase activity decreased at later intervals. Maximum effect of butachlor on the enzymes was seen after 12 h exposure. The extent of increase or decrease in different parameters examined varied between the two tissues studied. These changes are discussed in relation to the toxic stress of butachlor.

  6. Telomerization of amino acids with butadiene, catalyzed by palladium complexes

    SciTech Connect

    Dzhemilev, U.M.; Fakhretdinov, R.N.; Telin, A.G.

    1987-01-10

    The telomerization of ..cap alpha..-, ..beta..-, ..gamma..-, and epsilon-amino acids having various structures with butadiene under the influence of the three-component palladium catalyst Pd(acac)/sub 2/-PPh/sub 3/-AlEt/sub 3/ was investigated in DMSO-toluene solution. The ..cap alpha..- and epsilon-aliphatic and also the ..cap alpha..-, ..beta..-, and ..gamma..-aromatic amino acids react with butadiene, giving the products from octadienylation at the amino group exclusively. Under the conditions of telomerization aliphatic ..beta..-amino acids are cleaved with the formation of unsaturated tertiary amines. In the case of aliphatic ..gamma..-amino acids it is possible to obtain telomers alkylated at the carbonyl group.

  7. An Experiment on Isomerism in Metal-Amino Acid Complexes.

    ERIC Educational Resources Information Center

    Harrison, R. Graeme; Nolan, Kevin B.

    1982-01-01

    Background information, laboratory procedures, and discussion of results are provided for syntheses of cobalt (III) complexes, I-III, illustrating three possible bonding modes of glycine to a metal ion (the complex cations II and III being linkage/geometric isomers). Includes spectrophotometric and potentiometric methods to distinguish among the…

  8. Complexing of amino acids to DNA by chromate in intact cells.

    PubMed Central

    Voitkun, V; Zhitkovich, A; Costa, M

    1994-01-01

    Using o-pthaldialdehyde (OPT) fluorescence, the amino acids associated with DNA were studied following exposure of intact Chinese hamster ovary cells to chromate. Rigorous extraction with EDTA, acid, or base was required to release the amino acids cross-linked to the DNA isolated from control or chromate-treated cells by standard procedures (i.e., proteinase K, phenol, etc.). Amino acids resisting extraction from DNA were not studied since analysis was limited to those that could be released by these procedures. There was a chromate dose-dependent increase in amino acids complexed with the DNA that could be released by EDTA, acid, and base, and these amino acids were separated by HPLC and identified. Substantial increases in cysteine, glutamine, glutamic acid, histidine, threonine, and tyrosine were found as a function of increasing concentrations of chromate. There was also a time-dependent increase in complexing of these amino acids to the DNA by chromate. The amino acids found complexed to DNA in intact cells by chromate were thought to originate from reactions of free amino acids or small peptides with the DNA rather than being proteolytic products derived from larger proteins that were cross-linked to the DNA. This was supported by a number of experiments: a) free amino acids or bovine serum albumin (BSA) were cross-linked by chromium to DNA in vitro and the DNA was isolated by standard procedures.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7843108

  9. The formic acid-nitric acid complex: microwave spectrum, structure, and proton transfer.

    PubMed

    Mackenzie, Rebecca B; Dewberry, Christopher T; Leopold, Kenneth R

    2014-09-11

    Rotational spectra are reported for seven isotopologues of the complex HCOOH-HNO3 in a supersonic jet. The system is planar and bound by a pair of hydrogen bonds, much like the more widely studied carboxylic acid dimers. Double proton exchange interconverts the system between a pair of equivalent structures, as revealed by a splitting of the a-type spectrum that disappears when one of the hydrogen bonding protons is replaced by deuterium. The observation of relative intensities that are consistent with nuclear spin statistics in a symmetric and antisymmetric pair of tunneling states provides additional evidence for such a motion. The observed splittings in the pure rotational spectrum are 1-2 orders of magnitude smaller than those recently reported in the pure rotational spectra of several related carboxylic acid dimers. This is a curious difference, although we note that because the observed spectra do not cross the tunneling doublet, the splittings are a measure of the difference in effective rotational constants for the two states, not the tunneling frequency itself. The observed rotational constants have been used to determine an accurate vibrationally averaged structure for the complex. The two hydrogen bond lengths, 1.686(17) Å and 1.813(10) Å for the hydrogen bonds involving the HNO3 and HCOOH protons, respectively, differ by 0.127(27) Å. Likewise, the associated oxygen-oxygen distances determined for the parent species, 2.631 and 2.794 Å, differ by 0.163 Å. These results suggest that the double proton transfer is necessarily accompanied by substantial motion of the heavy atom frame, and thus this system, in principle, provides an excellent prototype for multidimensional tunneling processes. Ab initio calculations of the binding energy and the barrier height are presented. Excellent agreement between the calculated equilibrium structure and the experimental, vibrationally averaged structure suggests that the vibrational wave function is not highly

  10. Structural models of the bimetallic subunit at the A-cluster of acetyl coenzyme a synthase/CO dehydrogenase: binuclear sulfur-bridged Ni-Cu and Ni-Ni complexes and their reactions with CO.

    PubMed

    Harrop, Todd C; Olmstead, Marilyn M; Mascharak, Pradip K

    2004-11-17

    The Ni(II)-dicarboxamido-dithiolato complexes (Et4N)2[Ni(NpPepS)] (1) and (Et4N)2[Ni(PhPepS)] (2) were used as Nid metallosynthons in the construction of higher nuclearity dinuclear Ni-Cu and Ni-Ni species to model the bimetallic Mp-Nid site of the A-cluster of acetyl coenzyme A synthase/CO dehydrogenase (ACS/CODH). Reaction of 1 with [Cu(neo)Cl] and [Ni(terpy)Cl2] in MeCN affords the dinuclear complexes (Et4N)[Cu(neo)Ni(NpPepS)] (3) and [Ni(terpy)Ni(NpPepS)] (4), respectively. Reaction of 2 with [Ni(dppe)Cl2] in MeCN yields [Ni(dppe)Ni(PhPepS)] (6). The Ni-Cu complex 3 exhibits no redox chemistry at the Nid site and no reaction with CO. In contrast, the Nip sites in 4 and 6 are readily reduced (characterized by their Ni(I) EPR spectra) and bind CO, exhibiting nuco bands at 2044 and 1997 cm-1, respectively, indicating terminal CO binding. The present Ni-Ni systems replicate the structural and chemical properties of the A-cluster site in ACS/CODH and support the presence of Ni at Mp in the catalytically active enzyme.

  11. Identification of Novel Immunogenic Proteins from Mycoplasma bovis and Establishment of an Indirect ELISA Based on Recombinant E1 Beta Subunit of the Pyruvate Dehydrogenase Complex

    PubMed Central

    Wei, Kai; Zhang, Haiyan; Zhang, Yuewei; Xu, Jian; Jiang, Fei; Liu, Xu; Xu, Wei; Wu, Wenxue

    2014-01-01

    The pathogen Mycoplasma bovis (M. bovis) is a major cause of respiratory disease, mastitis, and arthritis in cattle. Screening the key immunogenic proteins and updating rapid diagnostic techniques are necessary to the prevention and control of M. bovis infection. In this study, 19 highly immunogenic proteins from M. bovis strain PD were identified using 2-dimensional gel electrophoresis, immunoblotting and MALDI-TOF/TOF MS. Of these 19 proteins, pyruvate dehydrogenase E1 component beta subunit (PDHB) showed excellent immune reactivity and repeatability. PDHB was found to be conserved in different M. bovis isolates, as indicated by Western blot analysis. On the basis of these results, a rPDHB-based indirect ELISA (iELISA) was established for the detection of serum antibodies using prokaryotically expressed recombinant PDHB protein as the coating antigen. The specificity analysis result showed that rPDHB-based iELISA did not react with other pathogens assessed in our study except M. agalactiae (which infects sheep and goats). Moreover, 358 serum samples from several disease-affected cattle feedlots were tested using this iELISA system and a commercial kit, which gave positive rates of 50.8% and 39.9%, respectively. The estimated Kappa agreement coefficient between the two methods was 0.783. Notably, 39 positive serum samples that had been missed by the commercial kit were all found to be positive by Western blot analysis. The detection rate of rPDHB-based iELISA was significantly higher than that of the commercial kit at a serum dilution ratio of 1∶5120 to 1∶10,240 (P<0.05). Taken together, these results provide important information regarding the novel immunogenic proteins of M. bovis. The established rPDHB-based iELISA may be suitable for use as a new method of antibody detection in M. bovis. PMID:24520369

  12. Evidence for a Complex Between Thf and Acetic Acid from Broadband Rotational Spectroscopy

    NASA Astrophysics Data System (ADS)

    Zaleski, Daniel P.; Bittner, Dror M.; Mullaney, John Connor; Stephens, Susanna L.; King, Adrian; Habgood, Matthew; Walker, Nick

    2015-06-01

    Evidence for a complex between tetrahydrofuran (THF) and acetic acid from broadband rotational spectroscopy will be presented. Transitions believed to belong to the complex were first identified in a gas mixture containing small amounts of THF, triethyl borane, and acetic acid balanced in argon. Ab initio calculations suggest a complex between THF and acetic acid is more likely to form compared to the analogous acetic acid complex with triethyl borane, the initial target. The observed rotational constants are also more similar to those predicted for a complex formed between THF and acetic acid, than for those of a complex formed between triethyl borane and acetic acid. Subsequently, multiple isotopologues of acetic acid have been measured, confirming its presence in the structure. No information has yet been obtained through isotopic substitution within the THF sub-unit. Ab initio calculations predict the most likely structure is one where the acetic acid subunit coordinates over the ring creating a "bridge" between the THF oxygen, the carboxylic O-H, and the carbonyl oxygen to a hydrogen atom on the back of the ring.

  13. Design of stereoelectronically promoted super lewis acids and unprecedented chemistry of their complexes.

    PubMed

    Foroutan-Nejad, Cina; Vicha, Jan; Marek, Radek

    2014-09-01

    A new family of stereoelectronically promoted aluminum and scandium super Lewis acids is introduced on the basis of state-of-the-art computations. Structures of these molecules are designed to minimize resonance electron donation to central metal atoms in the Lewis acids. Acidity of these species is evaluated on the basis of their fluoride-ion affinities relative to the antimony pentafluoride reference system. It is demonstrated that introduced changes in the stereochemistry of the designed ligands increase acidity considerably relative to Al and Sc complexes with analogous monodentate ligands. The high stability of fluoride complexes of these species makes them ideal candidates to be used as weakly coordinating anions in combination with highly reactive cations instead of conventional Lewis acid-fluoride complexes. Further, the interaction of all designed molecules with methane is investigated. All studied acids form stable pentavalent-carbon complexes with methane. In addition, interactions of the strongest acid of this family with very weak bases, namely, H2, N2, carbon oxides, and noble gases were investigated; it is demonstrated that this compound can form considerably stable complexes with the aforementioned molecules. To the best of our knowledge, carbonyl and nitrogen complexes of this species are the first hypothetical four-coordinated carbonyl and nitrogen complexes of aluminum. The nature of bonding in these systems is studied in detail by various bonding analysis approaches.

  14. Complexation of U(VI) with 1-Hydroxyethane-1,1-diphosphonicAcid (HEDPA) in Acidic to Basic Solutions

    SciTech Connect

    Reed, W A; Rao, L; Zanonato, P; Garnov, A; Powell, B A; Nash, K L

    2007-01-24

    Complexation of U(VI) with 1-hydroxyethane-1,1-diphosphonic acid (HEDPA) in acidic to basic solutions has been studied with multiple techniques. A number of 1:1 (UO{sub 2}H{sub 3}L), 1:2 (UO{sub 2}H{sub j}L{sub 2} where j = 4, 3, 2, 1, 0 and -1) and 2:2 ((UO{sub 2}){sub 2}H{sub j}L{sub 2} where j = 1, 0 and -1) complexes form, but the 1:2 complexes are the major species in a wide pH range. Thermodynamic parameters (formation constants, enthalpy and entropy of complexation) were determined by potentiometry and calorimetry. Data indicate that the complexation of U(VI) with HEDPA is exothermic, favored by the enthalpy of complexation. This is in contrast to the complexation of U(VI) with dicarboxylic acids in which the enthalpy term usually is unfavorable. Results from electrospray ionization mass spectrometry (ESI-MS) and {sup 31}P NMR have confirmed the presence of 1:1, 1:2 and 2:2 U(VI)-HEDPA complexes.

  15. [Thermodynamic characteristics of nucleic acid complexes with silver ions].

    PubMed

    Minasian, K A; Poletaev, A I; Borob'ev, A F

    1981-01-01

    By means of mixing reaction calorimetry the enthalpy of the complexes formation between Ag+ ions and DNA and dsRNA was measured. It was shown that Ag+ ions are able to form two types of complexes (I and II) with dsRNA. Using the method of the competitive reaction with chloride ions the stability constants of complex formation were obtained for dsRNA-Ag+ complexes for different temperatures. These measurements gave the delta H and delta S values for both complexes: delta HI = -74,9 +/- 7,1 kjouls/mol, delta SI = -100.0 +/- 25.0 jouls/mol deg; delta HII = -39,8 +/- 4,2 kjouls/mol, delta SII = +2 +/- 14 jouls/mol deg. The calorimetric results of delta H determination are the same within the limits of experimental errors. The enthalpy term of dsRNA-Ag+ complexes proved to bring the main contribution into the free energy of complex formation.

  16. Sorbitol dehydrogenase. Full-length cDNA sequencing reveals a mRNA coding for a protein containing an additional 42 amino acids at the N-terminal end.

    PubMed

    Wen, Y; Bekhor, I

    1993-10-01

    A cDNA clone encoding rat sorbitol dehydrogenase (SDH) was isolated from a rat testis lambda ZAP II cDNA library. The full-length cDNA insert contained 2277 base pairs (bp), starting 182 bp upstream from an ATG codon where translation to the active enzyme SDH is presumed to be initiated. A second ATG codon, however, was found 126 bp upstream, aligned in the same reading frame as that of the active enzyme. Therefore, the coding sequence for SDH can be translated into an additional 42-amino-acid polypeptide linked to the N-terminal amino acid of the enzyme, generating a pre-sorbitol dehydrogenase. The sequence data indicate that the nucleotide environment around this ATG codon is more favorable towards it being the actual open reading frame (ORF) for a pre-SDH than the ATG codon preceding the nucleotide sequence for SDH. Since no known SDH starts with the additional 42 amino acids, it may be that post-translational removal of this polypeptide accompanies the release of the active enzyme. Next, the 3' untranslated region of the cDNA contained a non-coding 1021 bp downstream from the TAA stop codon. The latter sequence included three putative poly(A) signals: one at nucleotides 1362-1367, the second at nucleotides 1465-1470, and the third at nucleotides 2212-2217 [17 bp away from the poly(A) tail]. In addition to the above findings we also report a variance in one of the amino acids in the SDH cDNA sequence. This variance occurs at position 957-960, where threonine is coded for instead of aspartic acid; in the rat testis SDH cDNA, we find the sequence is ACG instead of GAC, as was reported for the rat liver SDH cDNA. Northern-blot hybridization analysis showed that SDH mRNA is a doublet, one band of 4 kb and the other of 2.3-2.4 kb, in both the rat liver and the rat lens, further confirming that the isolated SDH cDNA constituted a full-length cDNA.

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

    MedlinePlus

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

  18. Polymers with complexing properties. Simple poly(amino acids)

    NASA Technical Reports Server (NTRS)

    Roque, J. M.

    1978-01-01

    The free amino (0.3 equiv/residue) and carboxyl (0.5 equiv/residue) groups of thermal polylysine increased dramatically on treatment with distilled water. The total hydrolysis of such a polymer was abnormal in that only about 50% of the expected amino acids were recovered. Poly (lysine-co-alanine-co-glycine) under usual conditions hydrolyzed completely in 8 hours; whereas, when it was pretreated with diazomethane, a normal period of 24 hours was required to give (nearly) the same amounts of each free amino acid as compared with those obtained from the untreated polymer. The amino groups of the basic thermal poly(amino acids) were sterically hindered. The existence of nitrogen atoms linking two or three chains and reactive groups (anhydride, imine) were proposed.

  19. NdhM Subunit Is Required for the Stability and the Function of NAD(P)H Dehydrogenase Complexes Involved in CO2 Uptake in Synechocystis sp. Strain PCC 6803.

    PubMed

    He, Zhihui; Xu, Min; Wu, Yaozong; Lv, Jing; Fu, Pengcheng; Mi, Hualing

    2016-03-11

    The cyanobacterial type I NAD(P)H dehydrogenase (NDH-1) complexes play a crucial role in a variety of bioenergetic reactions such as respiration, CO2 uptake, and cyclic electron transport around photosystem I. Two types of NDH-1 complexes, NDH-1MS and NDH-1MS', are involved in the CO2 uptake system. However, the composition and function of the complexes still remain largely unknown. Here, we found that deletion of ndhM caused inactivation of NDH-1-dependent cyclic electron transport around photosystem I and abolishment of CO2 uptake, resulting in a lethal phenotype under air CO2 condition. The mutation of NdhM abolished the accumulation of the hydrophilic subunits of the NDH-1, such as NdhH, NdhI, NdhJ, and NdhK, in the thylakoid membrane, resulting in disassembly of NDH-1MS and NDH-1MS' as well as NDH-1L. In contrast, the accumulation of the hydrophobic subunits was not affected in the absence of NdhM. In the cytoplasm, the NDH-1 subcomplex assembly intermediates including NdhH and NdhK were seriously affected in the ΔndhM mutant but not in the NdhI-deleted mutant ΔndhI. In vitro protein interaction analysis demonstrated that NdhM interacts with NdhK, NdhH, NdhI, and NdhJ but not with other hydrophilic subunits of the NDH-1 complex. These results suggest that NdhM localizes in the hydrophilic subcomplex of NDH-1 complexes as a core subunit and is essential for the function of NDH-1MS and NDH-1MS' involved in CO2 uptake in Synechocystis sp. strain PCC 6803. PMID:26703473

  20. NdhM Subunit Is Required for the Stability and the Function of NAD(P)H Dehydrogenase Complexes Involved in CO2 Uptake in Synechocystis sp. Strain PCC 6803*

    PubMed Central

    He, Zhihui; Xu, Min; Wu, Yaozong; Lv, Jing; Fu, Pengcheng; Mi, Hualing

    2016-01-01

    The cyanobacterial type I NAD(P)H dehydrogenase (NDH-1) complexes play a crucial role in a variety of bioenergetic reactions such as respiration, CO2 uptake, and cyclic electron transport around photosystem I. Two types of NDH-1 complexes, NDH-1MS and NDH-1MS′, are involved in the CO2 uptake system. However, the composition and function of the complexes still remain largely unknown. Here, we found that deletion of ndhM caused inactivation of NDH-1-dependent cyclic electron transport around photosystem I and abolishment of CO2 uptake, resulting in a lethal phenotype under air CO2 condition. The mutation of NdhM abolished the accumulation of the hydrophilic subunits of the NDH-1, such as NdhH, NdhI, NdhJ, and NdhK, in the thylakoid membrane, resulting in disassembly of NDH-1MS and NDH-1MS′ as well as NDH-1L. In contrast, the accumulation of the hydrophobic subunits was not affected in the absence of NdhM. In the cytoplasm, the NDH-1 subcomplex assembly intermediates including NdhH and NdhK were seriously affected in the ΔndhM mutant but not in the NdhI-deleted mutant ΔndhI. In vitro protein interaction analysis demonstrated that NdhM interacts with NdhK, NdhH, NdhI, and NdhJ but not with other hydrophilic subunits of the NDH-1 complex. These results suggest that NdhM localizes in the hydrophilic subcomplex of NDH-1 complexes as a core subunit and is essential for the function of NDH-1MS and NDH-1MS′ involved in CO2 uptake in Synechocystis sp. strain PCC 6803. PMID:26703473

  1. Meat and ascorbic acid can promote Fe availability from Fe-phytate but not from Fe-tannic acid complexes.

    PubMed

    Engle-Stone, Reina; Yeung, Andrew; Welch, Ross; Glahn, Raymond

    2005-12-28

    This study utilized an in vitro digestion/Caco-2 cell model to determine the levels of ascorbic acid (AA) and "meat factor" needed to promote Fe absorption from Fe complexed with phytic acid (PA) or tannic acid (TA). AA reversed the inhibition of Fe absorption by PA beginning at a molar ratio of 1:20:1 (Fe:PA:AA) but essentially had no effect on the Fe complexed with TA. Fish also reversed the inhibition of Fe uptake by PA but not by TA. TA and fish decreased total Fe solubility. Iron in the presence of PA was highly soluble. AA, but not fish, increased the percentage of soluble Fe as Fe2+ in the presence of both inhibitors. The results indicate that monoferric phytate is a form of Fe that can be available for absorption in the presence of uptake promoters. In contrast, a TA-Fe complex is much less soluble and unavailable in the presence of promoters.

  2. Isolation of a cDNA coding for L-galactono-gamma-lactone dehydrogenase, an enzyme involved in the biosynthesis of ascorbic acid in plants. Purification, characterization, cDNA cloning, and expression in yeast.

    PubMed

    Ostergaard, J; Persiau, G; Davey, M W; Bauw, G; Van Montagu, M

    1997-11-28

    L-Galactono-gamma-lactone dehydrogenase (EC 1.3.2.3; GLDase), an enzyme that catalyzes the final step in the biosynthesis of L-ascorbic acid was purified 1693-fold from a mitochondrial extract of cauliflower (Brassica oleracea, var. botrytis) to apparent homogeneity with an overall yield of 1.1%. The purification procedure consisted of anion exchange, hydrophobic interaction, gel filtration, and fast protein liquid chromatography. The enzyme had a molecular mass of 56 kDa estimated by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis and showed a pH optimum for activity between pH 8.0 and 8.5, with an apparent Km of 3.3 mM for L-galactono-gamma-lactone. Based on partial peptide sequence information, polymerase chain reaction fragments were isolated and used to screen a cauliflower cDNA library from which a cDNA encoding GLDase was isolated. The deduced mature GLDase contained 509 amino acid residues with a predicted molecular mass of 57,837 Da. Expression of the cDNA in yeast produced a biologically active protein displaying GLDase activity. Furthermore, we identified a substrate for the enzyme in cauliflower extract, which co-eluted with L-galactono-gamma-lactone by high-performance liquid chromatography, suggesting that this compound is a naturally occurring precursor of L-ascorbic acid biosynthesis in vivo.

  3. Salicylic acid binding of mitochondrial alpha-ketoglutarate dehydrogenase E2 affects mitochondrial oxidative phosphorylation and electron transport chain components and plays a role in basal defense against tobacco mosaic virus in tomato.

    PubMed

    Liao, Yangwenke; Tian, Miaoying; Zhang, Huan; Li, Xin; Wang, Yu; Xia, Xiaojian; Zhou, Jie; Zhou, Yanhong; Yu, Jingquan; Shi, Kai; Klessig, Daniel F

    2015-02-01

    Salicylic acid (SA) plays a critical role in plant defense against pathogen invasion. SA-induced viral defense in plants is distinct from the pathways mediating bacterial and fungal defense and involves a specific pathway mediated by mitochondria; however, the underlying mechanisms remain largely unknown. The SA-binding activity of the recombinant tomato (Solanum lycopersicum) alpha-ketoglutarate dehydrogenase (Slα-kGDH) E2 subunit of the tricarboxylic acid (TCA) cycle was characterized. The biological role of this binding in plant defenses against tobacco mosaic virus (TMV) was further investigated via Slα-kGDH E2 silencing and transient overexpression in plants. Slα-kGDH E2 was found to bind SA in two independent assays. SA treatment, as well as Slα-kGDH E2 silencing, increased resistance to TMV. SA did not further enhance TMV defense in Slα-kGDH E2-silenced tomato plants but did reduce TMV susceptibility in Nicotiana benthamiana plants transiently overexpressing Slα-kGDH E2. Furthermore, Slα-kGDH E2-silencing-induced TMV resistance was fully blocked by bongkrekic acid application and alternative oxidase 1a silencing. These results indicated that binding by Slα-kGDH E2 of SA acts upstream of and affects the mitochondrial electron transport chain, which plays an important role in basal defense against TMV. The findings of this study help to elucidate the mechanisms of SA-induced viral defense.

  4. Transition Metal Complexes of Phosphinous Acids Featuring a Quasichelating Unit: Synthesis, Characterization, and Hetero-bimetallic Complexes.

    PubMed

    Allefeld, Nadine; Bader, Julia; Neumann, Beate; Stammler, Hans-Georg; Ignat'ev, Nikolai; Hoge, Berthold

    2015-08-17

    Diorganophosphane oxides were employed as preligands for the synthesis of catalytically active transition metal complexes of the phosphinous acids (CF3)2POH and (C2F5)2POH. Their reactions with solid PtCl2 and PdCl2 led to the formation of mononuclear phosphinous acid complexes [Cl2M{P(R(f))2OH}2] (M = Pd, Pt; R(f) = C2F5, CF3), which can be crystallized, for example, as its pyridinium salts, 2[HPy](+)[Cl2Pd{P(CF3)2O}2](2-). In vacuo HCl is liberated from the neutral palladium complexes affording mixtures of di- and polynuclear complexes. Moreover, (C2F5)2POH was reacted with several β-diketonato complexes of palladium, platinum, and nickel yielding air- and moisture-stable complexes [(acac)M{[P(R(f))2O]2H}], featuring a quasichelating phosphinous acid phosphinito unit {P(R(f))2O···H···O(R(f))2P}(-). Treatment of [Ni(Cp)2] (Cp = cyclopentadienyl) and [(cod)RhCl]2 (cod = 1,5-cyclooctadiene) with (C2F5)2POH leads to the substitution of one Cp or chloro ligand by a quasichelating unit. The novel coordination compounds were characterized by NMR and IR spectroscopies, mass spectrometry, and X-ray diffraction analysis. The platinum complex [(acac)Pt{[P(C2F5)2O]2H}] (acac = acetylacetonato) was used for the construction of hetero-bimetallic complexes by the treatment with [(cod)RhCl]2 and [Ni(Cp)2]. The trinuclear bimetallic complex [{(acac)Pt[P(C2F5)2O]2}2Ni] is the first structurally characterized hetero-bimetallic species containing a bis(perfluoroalkyl)phosphinito bridge.

  5. Supramolecular adducts based on weak interactions between the trimeric Lewis acid complex (perfluoro-ortho-phenylene)mercury and polypnictogen complexes.

    PubMed

    Fleischmann, Martin; Jones, James S; Balázs, Gábor; Gabbaï, François P; Scheer, Manfred

    2016-09-21

    Reactions of the trinuclear Lewis acid perfluoro-ortho-phenylene)mercury [(o-HgC6F4)3] (1) with the polypnictogen complex [CpMo(CO)2(η(3)-P3)] (2) containing a cyclo-P3 ligand and the series of E2 complexes [{CpMo(CO)2}2(μ,η(2):η(2)-E2)] (E = P(3a), As(3b), Sb(3c), Bi(3d)) are reported. In all cases, the reaction products show very weak interactions between the En ligand complexes and the Lewis acid 1, as evidenced by their highly dynamic behaviour in solution and the formation of adducts in the solid state showing HgE contacts below the respective sum of the van der Waals radii. The complexes 2 (P3), 3a (P2) and 3b (As2) show interactions of only one pnictogen atom with all three Hg atoms of 1. The complex 3c (Sb2) forms two adducts with 1 showing either a side-on coordination of the Sb2 dumbbell towards Hg or an end-on coordination of both Sb atoms towards two independent molecules of 1. The Bi2 complex 3d shows an almost parallel alignment of the Bi2 dumbbell situated above the center of the planar Lewis acid 1. The arrangements of the E2 complex series towards 1 are rationalized with the help of electrostatic potential maps obtained by DFT calculations. Finally the structural characterizations of a new modification of the free Sb2 complex 3c, the Bi2 complex 3d, the starting material of its preparation [Bi{CpMo(CO)3}3] (4) and an unprecedented 'Cr4As5' cluster 5 are presented. PMID:27461890

  6. Analysis of complexes pairing hydroperoxyl radical with peroxyformic acid.

    PubMed

    Solimannejad, Mohammad; Shirazi, Sanaz G; Scheiner, Steve

    2007-10-25

    Ab initio quantum calculations are used to analyze the binding of complexes pairing OOH with HOOCHO. Six minima are located on the potential energy surface, all of cyclic geometry. Of particular interest are the OH...O and CH...O H-bonds that arise in the complexes and the manner in which these interactions influence the internal properties of the subunits. The analysis is complicated by the presence of an intramolecular H-bond in the unperturbed HOOCHO molecule, which must be broken in order to form the pair of intermolecular H-bonds that are responsible for the binding in the most stable complex. The CH bond of HOOCHO is contracted, and its stretching frequency undergoes a blue shift, when this group participates in a H-bond. PMID:17914777

  7. Hydrogen bonds in 1:1 complex of piperidine-3-carboxylic acid with salicylic acid

    NASA Astrophysics Data System (ADS)

    Bartoszak-Adamska, Elżbieta; Dega-Szafran, Zofia; Krociak, Magdalena; Jaskolski, Mariusz; Szafran, Mirosław

    2009-02-01

    The 1:1 complex between the zwitterionic piperidinium-3-carboxylate (P3C) and salicylic acid (SAL), P3C·SAL, has been characterized by single crystal X-ray analysis, FTIR and NMR spectroscopy, and by DFT calculations. The crystals are orthorhombic, space group Pbca, with a = 11.6477(7), b = 9.1754(6), c = 23.5833(12) Å. An O sbnd H⋯O bridge (2.537(1) Å) links the SAL and P3C moieties. The proton in this H bond is located closer to the salicylic carboxylate group. In the P3C moiety, the piperidine ring adopts the chair conformation, and the carboxylate group is in the axial orientation and is stabilized by an intramolecular N +sbnd H⋯O hydrogen bond of 2.847(1) Å. In the crystal packing, two P3C·SAL units form a centrosymmetric dimer through a pair of intermolecular N +sbnd H⋯O bonds of 2.801(1) Å. The dimers form a zigzag chain linked via another N +sbnd H⋯O bond (2.799(1) Å). In the structures of the monomeric [P3C·SAL] and dimeric [(P3C·SAL) 2] species optimized by B3LYP/6-31G(d,p) calculations, both the inter- and intra-molecular hydrogen bonds are shorter than in the crystal. The FTIR spectrum shows a broad absorption in the 3100-2400 cm -1 region attributed to νNH and νOH vibrations. The broad absorption in the 1500-600 cm -1 region is attributed to the O sbnd H·O hydrogen bonds. The 1H and 13C NMR spectra have been analyzed to elucidate the structure of the P3C·SAL complex in solution. The GIAO magnetic isotropic shielding tensors have been used to predict the 1H and 13C chemical shifts in DMSO solution.

  8. Regulation of pyruvate dehydrogenase kinase expression by the farnesoid X receptor

    SciTech Connect

    Savkur, Rajesh S.; Bramlett, Kelli S.; Michael, Laura F.; Burris, Thomas P. . E-mail: burris_thomas_p@lilly.com

    2005-04-01

    The pyruvate dehydrogenase complex (PDC) functions as an important junction in intermediary metabolism by influencing the utilization of fat versus carbohydrate as a source of fuel. Activation of PDC is achieved by phosphatases, whereas, inactivation is catalyzed by pyruvate dehydrogenase kinases (PDKs). The expression of PDK4 is highly regulated by the glucocorticoid and peroxisome proliferator-activated receptors. We demonstrate that the farnesoid X receptor (FXR; NR1H4), which regulates a variety of genes involved in lipoprotein metabolism, also regulates the expression of PDK4. Treatment of rat hepatoma cells as well as human primary hepatocytes with FXR agonists stimulates the expression of PDK4 to levels comparable to those obtained with glucocorticoids. In addition, treatment of mice with an FXR agonist significantly increased hepatic PDK4 expression, while concomitantly decreasing plasma triglyceride levels. Thus, activation of FXR may suppress glycolysis and enhance oxidation of fatty acids via inactivation of the PDC by increasing PDK4 expression.

  9. 9-O-acetylated sialic acids differentiating normal haematopoietic precursors from leukemic stem cells with high aldehyde dehydrogenase activity in children with acute lymphoblastic leukaemia.

    PubMed

    Chowdhury, Suchandra; Chandra, Sarmila; Mandal, Chitra

    2014-10-01

    Childhood acute lymphoblastic leukaemia (ALL) originates from mutations in haematopoietic progenitor cells (HPCs). For high-risk patients, treated with intensified post-remission chemotherapy, haematopoietic stem cell (HSC) transplantation is considered. Autologous HSC transplantation needs improvisation till date. Previous studies established enhanced disease-associated expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs) on lymphoblasts of these patients at diagnosis, followed by its decrease with clinical remission and reappearance with relapse. Based on this differential expression of Neu5,9Ac2-GPs, identification of a normal HPC population was targeted from patients at diagnosis. This study identifies two distinct haematopoietic progenitor populations from bone marrow of diagnostic ALL patients, exploring the differential expression of Neu5,9Ac2-GPs with stem cell (CD34, CD90, CD117, CD133), haematopoietic (CD45), lineage-commitment (CD38) antigens and cytosolic aldehyde dehydrogenase (ALDH). Normal haematopoietic progenitor cells (ALDH(+)SSC(lo)CD45(hi)Neu5,9Ac2 -GPs(lo)CD34(+)CD38(-)CD90(+)CD117(+)CD133(+)) differentiated into morphologically different, lineage-specific colonies, being crucial for autologous HSC transplantation while leukemic stem cells (ALDH(+)SSC(lo)CD45(lo)Neu5,9Ac2 -GPs(hi)CD34(+)CD38(+)CD90(-)CD117(-)CD133(-)) lacking this ability can be potential targets for minimal residual disease detection and drug-targeted immunotherapy.

  10. Superiority of zinc complex of acetylsalicylic acid to acetylsalicylic acid in preventing postischemic myocardial dysfunction.

    PubMed

    Korkmaz, Sevil; Atmanli, Ayhan; Li, Shiliang; Radovits, Tamás; Hegedűs, Peter; Barnucz, Enikő; Hirschberg, Kristóf; Loganathan, Sivakkanan; Yoshikawa, Yutaka; Yasui, Hiroyuki; Karck, Matthias; Szabó, Gábor

    2015-09-01

    The pathophysiology of ischemic myocardial injury involves cellular events, reactive oxygen species, and an inflammatory reaction cascade. The zinc complex of acetylsalicylic acid (Zn(ASA)2) has been found to possess higher anti-inflammatory and lower ulcerogenic activities than acetylsalicylic acid (ASA). Herein, we studied the effects of both ASA and Zn(ASA)2 against acute myocardial ischemia. Rats were pretreated with ASA (75 mg/kg) or Zn(ASA)2 (100 mg/kg) orally for five consecutive days. Isoproterenol (85 mg/kg, subcutaneously [s.c.]) was applied to produce myocardial infarction. After 17-22 h, animals were anesthetized with sodium pentobarbital (60 mg/kg, intraperitoneally [i.p.]) and both electrical and mechanical parameters of cardiac function were evaluated in vivo. Myocardial histological and gene expression analyses were performed. In isoproterenol-treated rats, Zn(ASA)2 treatment normalized significantly impaired left-ventricular contractility index (Emax 2.6 ± 0.7 mmHg/µL vs. 4.6 ± 0.5 mmHg/µL, P < 0.05), increased stroke volume (30 ± 3 µL vs. 50 ± 6 µL, P < 0.05), decreased systemic vascular resistance (7.2 ± 0.7 mmHg/min/mL vs. 4.2 ± 0.5 mmHg/min/mL, P < 0.05) and reduced inflammatory infiltrate into the myocardial tissues. ECG revealed a restoration of elevated ST-segment (0.21 ± 0.03 mV vs. 0.09 ± 0.02 mV, P < 0.05) and prolonged QT-interval (79.2 ± 3.2 ms vs. 69.5 ± 2.5 ms, P < 0.05) by Zn(ASA)2. ASA treatment did not result in an improvement of these parameters. Additionally, Zn(ASA)2 significantly increased the mRNA-expression of superoxide dismutase 1 (+73 ± 15%), glutathione peroxidase 4 (+44 ± 12%), and transforming growth factor (TGF)-β1 (+102 ± 22%). In conclusion, our data demonstrate that oral administration of zinc and ASA in the form of bis(aspirinato)zinc(II) complex is superior to ASA in preventing electrical

  11. Superiority of zinc complex of acetylsalicylic acid to acetylsalicylic acid in preventing postischemic myocardial dysfunction

    PubMed Central

    Korkmaz, Sevil; Atmanli, Ayhan; Li, Shiliang; Radovits, Tamás; Hegedűs, Peter; Hirschberg, Kristóf; Loganathan, Sivakkanan; Yoshikawa, Yutaka; Yasui, Hiroyuki; Karck, Matthias; Szabó, Gábor

    2015-01-01

    The pathophysiology of ischemic myocardial injury involves cellular events, reactive oxygen species, and an inflammatory reaction cascade. The zinc complex of acetylsalicylic acid (Zn(ASA)2) has been found to possess higher anti-inflammatory and lower ulcerogenic activities than acetylsalicylic acid (ASA). Herein, we studied the effects of both ASA and Zn(ASA)2 against acute myocardial ischemia. Rats were pretreated with ASA (75 mg/kg) or Zn(ASA)2 (100 mg/kg) orally for five consecutive days. Isoproterenol (85 mg/kg, subcutaneously [s.c.]) was applied to produce myocardial infarction. After 17–22 h, animals were anesthetized with sodium pentobarbital (60 mg/kg, intraperitoneally [i.p.]) and both electrical and mechanical parameters of cardiac function were evaluated in vivo. Myocardial histological and gene expression analyses were performed. In isoproterenol-treated rats, Zn(ASA)2 treatment normalized significantly impaired left-ventricular contractility index (Emax 2.6 ± 0.7 mmHg/µL vs. 4.6 ± 0.5 mmHg/µL, P < 0.05), increased stroke volume (30 ± 3 µL vs. 50 ± 6 µL, P < 0.05), decreased systemic vascular resistance (7.2 ± 0.7 mmHg/min/mL vs. 4.2 ± 0.5 mmHg/min/mL, P < 0.05) and reduced inflammatory infiltrate into the myocardial tissues. ECG revealed a restoration of elevated ST-segment (0.21 ± 0.03 mV vs. 0.09 ± 0.02 mV, P < 0.05) and prolonged QT-interval (79.2 ± 3.2 ms vs. 69.5 ± 2.5 ms, P < 0.05) by Zn(ASA)2. ASA treatment did not result in an improvement of these parameters. Additionally, Zn(ASA)2 significantly increased the mRNA-expression of superoxide dismutase 1 (+73 ± 15%), glutathione peroxidase 4 (+44 ± 12%), and transforming growth factor (TGF)-β1 (+102 ± 22%). In conclusion, our data demonstrate that oral administration of zinc and ASA in the form of bis(aspirinato)zinc(II) complex is superior to ASA in preventing electrical

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

    PubMed Central

    Cafruny, W A; Plagemann, P G

    1982-01-01

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

  13. Spectroscopic studies on gallic acid and its azo derivatives and their iron(III) complexes.

    PubMed

    Masoud, Mamdouh S; Ali, Alaa E; Haggag, Sawsan S; Nasr, Nessma M

    2014-01-01

    Azo gallic derivatives and their iron(III) complexes were synthesized and characterized. The stereochemistry and the mode of bonding of the complexes were achieved based on elemental analysis, UV-Vis and IR. The thermal behaviors of the complexes were studied. The effect of pH on the electronic absorption spectra of gallic acid and its azo derivatives are discussed. Different spectroscopic methods (molar ratio, straight line method, continuous variation, slope ratio and successive method) are applied for determination of stoichiometry and pK values for the complex formation of gallic acid with iron(III) in aqueous media. Iron(III) complexes of gallic acid is formed with different ratio: 1:1, 1:2, 1:3 and 1:4 (M:L).

  14. The properties of solid Zn(II)-amino acid complexes in the form of suspensions.

    PubMed

    Dolińska, B

    2001-10-01

    An investigation was made into the experimental conditions for the formation of poorly soluble complexes of the divalent Zinc(II) combined with the following selected amino acids: tyrosine, tryptophan, cysteine, histidine, and alanine, in the form of suspensions for parenteral administration. The number of Zn(II)-binding sites in the amino acid (n) as well as the amino acid affinity to Zn(II) (Ka), were determined. Cysteine was found to have the highest number of Zn(II)-binding sites--3, whereas alanine the lowest--1. In the conditions described herein, Zn(II) amino acid complexes of diverse stability (durability) were obtained. The analysis of the kinetics of the binding revealed that the most stable complexes were those formed by Zn(II) in combination with tryptophan (Ka = 405.78 microM(-1) +/- 12.17), and with tyrosine (Ka = 343.88 microM +/- 22.35); whereas the least stable complexes were those formed by Zn(II) in combination with histidine (Ka = 29.90 microM +/- 4.78), and with alanine (Ka = 13.0 microM(-1) +/- 1.04). Cysteine formed complexes of intermediate stability (Ka = 168.53 microM(-1) +/- 12.36). The stability ofthe Zn(II) amino acid complexes obtained was conditioned by both the molecular weight (P = 0.033) of the amino acid and its isoelectric point (P < 0.001). PMID:11718265

  15. Stable association complex electrospray mass spectrometry for the determination of cyanuric acid.

    PubMed

    Magnuson, M L; Kelty, C A; Cantú, R

    2001-10-01

    Cyanuric acid, a suspected gastrointestinal or liver toxicant, has gained interest as a potential degradation product of triazine herbicides, such as simazine and atrazine. This paper investigates the determination of cyanuric acid by stable association complex electrospray mass spectrometry (cESI-MS). The cyanuric acid is extracted from the water through a microscale liquid-liquid extraction. The extract is evaporated to dryness, and an aqueous solution of quaternary ammonium cationic surfactant is added. When injected into the electrospray mass spectrometer, the surfactant and the cyanuric acid form a mass-selective stable association complex, which may be used for confident quantification of cyanuric acid. Several extraction solvents and surfactants were investigated. These studies provide insight into the mechanism of electrospray for the formation of these complexes, specifically with regard to the surface activity of the different surfactants and the chemistry of the surfactant-cyanuric acid complexes. From an analytical standpoint, the cESI-MS method detection limit for extraction of a 1 mL aqueous solution of cyanuric acid was 130 microg/L based on 3.14sigma(n-1) of seven replicate injections. Standard additions were used for quantification of eight aqueous samples. The cyanuric acid concentrations determined with cESI-MS were not significantly different at the 95% confidence level to those determined by conventional high-performance liquid chromatography (HPLC). A recovery of 100% from a fortified urine sample illustrates the robustness of the technique.

  16. Identification of 9α-Hydroxy-17-Oxo-1,2,3,4,10,19-Hexanorandrostan-5-Oic Acid in Steroid Degradation by Comamonas testosteroni TA441 and Its Conversion to the Corresponding 6-En-5-Oyl Coenzyme A (CoA) Involving Open Reading Frame 28 (ORF28)- and ORF30-Encoded Acyl-CoA Dehydrogenases

    PubMed Central

    Hayashi, Toshiaki; Koshino, Hiroyuki; Malon, Michal; Hirota, Hiroshi; Kudo, Toshiaki

    2014-01-01

    Comamonas testosteroni TA441 degrades steroids via aromatization and meta-cleavage of the A ring, followed by hydrolysis, and produces 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid as an intermediate compound. Herein, we identify a new intermediate compound, 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid. Open reading frame 28 (ORF28)- and ORF30-encoded acyl coenzyme A (acyl-CoA) dehydrogenase was shown to convert the CoA ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid to the CoA ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid. A homology search of the deduced amino acid sequences suggested that the ORF30-encoded protein is a member of the acyl-CoA dehydrogenase_fadE6_17_26 family, whereas the deduced amino acid sequence of ORF28 showed no significant similarity to specific acyl-CoA dehydrogenase family proteins. Possible steroid degradation gene clusters similar to the cluster of TA441 appear in bacterial genome analysis data. In these clusters, ORFs similar to ORFs 28 and 30 are often found side by side and ordered in the same manner as ORFs 28 and 30. PMID:25092028

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

    PubMed Central

    Her, Yeng F.; Maher, L. James

    2015-01-01

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

  18. Complexation of di-amides of dipicolinic acid with neodymium

    SciTech Connect

    Lapka, J.L.; Paulenova, A.

    2013-07-01

    Di-amides have undergone significant studies as possible ligands for use in the partitioning of trivalent minor actinides and lanthanides. The binding affinities of three isomeric ligands with neodymium in acetonitrile solution have been investigated. The stability constants of the metal-ligand complexes formed between different isomers of N,N'-diethyl-N,N'- ditolyl-di-picolinamide (EtTDPA) and trivalent neodymium in acetonitrile have been determined by spectrophotometric and calorimetric methods. Each isomer of EtTDPA has been found to be capable of forming three complexes with trivalent neodymium, Nd(EtTDPA), Nd(EtTDPA){sub 2}, and Nd(EtTDPA){sub 3}. Values from spectrophotometric and calorimetric titrations are within reasonable agreement with each other. The order of stability constants for each metal:ligand complex decreases in the order Et(m)TDPA > Et(p)TDPA > Et(o)TDPA. The obtained values are comparable to other di-amidic ligands obtained under similar system conditions and mirror previously obtained solvent extraction data for EtTDPA at low ionic strengths. (authors.

  19. Metal complexes of salicylhydroxamic acid (H2Sha), anthranilic hydroxamic acid and benzohydroxamic acid. Crystal and molecular structure of [Cu(phen)2(Cl)]Cl x H2Sha, a model for a peroxidase-inhibitor complex.

    PubMed

    O'Brien, E C; Farkas, E; Gil, M J; Fitzgerald, D; Castineras, A; Nolan, K B

    2000-04-01

    Stability constants of iron(III), copper(II), nickel(II) and zinc(II) complexes of salicylhydroxamic acid (H2Sha), anthranilic hydroxamic acid (HAha) and benzohydroxamic acid (HBha) have been determined at 25.0 degrees C, I=0.2 mol dm(-3) KCl in aqueous solution. The complex stability order, iron(III) > copper(II) > nickel(II) approximately = zinc(II) was observed whilst complexes of H2Sha were found to be more stable than those of the other two ligands. In the preparation of ternary metal ion complexes of these ligands and 1,10-phenanthroline (phen) the crystalline complex [Cu(phen)2(Cl)]Cl x H2Sha was obtained and its crystal structure determined. This complex is a model for hydroxamate-peroxidase inhibitor interactions.

  20. 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.

  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. PMID:25542170

  2. Glutamates 78 and 122 in the active site of saccharopine dehydrogenase contribute to reactant binding and modulate the basicity of the acid-base catalysts.

    PubMed

    Ekanayake, Devi K; Andi, Babak; Bobyk, Kostyantyn D; West, Ann H; Cook, Paul F

    2010-07-01

    Saccharopine dehydrogenase catalyzes the NAD-dependent oxidative deamination of saccharopine to give l-lysine and alpha-ketoglutarate. There are a number of conserved hydrophilic, ionizable residues in the active site, all of which must be important to the overall reaction. In an attempt to determine the contribution to binding and rate enhancement of each of the residues in the active site, mutations at each residue are being made, and double mutants are being made to estimate the interrelationship between residues. Here, we report the effects of mutations of active site glutamate residues, Glu(78) and Glu(122), on reactant binding and catalysis. Site-directed mutagenesis was used to generate E78Q, E122Q, E78Q/E122Q, E78A, E122A, and E78A/E122A mutant enzymes. Mutation of these residues increases the positive charge of the active site and is expected to affect the pK(a) values of the catalytic groups. Each mutant enzyme was completely characterized with respect to its kinetic and chemical mechanism. The kinetic mechanism remains the same as that of wild type enzymes for all of the mutant enzymes, with the exception of E78A, which exhibits binding of alpha-ketoglutarate to E and E.NADH. Large changes in V/K(Lys), but not V, suggest that Glu(78) and Glu(122) contribute binding energy for lysine. Shifts of more than a pH unit to higher and lower pH of the pK(a) values observed in the V/K(Lys) pH-rate profile of the mutant enzymes suggests that the presence of Glu(78) and Glu(122) modulates the basicity of the catalytic groups.

  3. Production of optically pure L-lactic acid from lignocellulosic hydrolysate by using a newly isolated and D-lactate dehydrogenase gene-deficient Lactobacillus paracasei strain.

    PubMed

    Kuo, Yang-Cheng; Yuan, Shuo-Fu; Wang, Chun-An; Huang, Yin-Jung; Guo, Gia-Luen; Hwang, Wen-Song

    2015-12-01

    The use of lignocellulosic feedstock for lactic acid production with a difficulty is that the release of inhibitory compounds during the pretreatment process which inhibit the growth of microorganism. Thus we report a novel lactic acid bacterium, Lactobacillus paracasei 7 BL, that has a high tolerance to inhibitors and produced optically pure l-lactic acid after the interruption of ldhD gene. The strain 7 BL fermented glucose efficiently and showed high titer of l-lactic acid (215 g/l) by fed-batch strategy. In addition, 99 g/l of l-lactic acid with high yield (0.96 g/g) and productivity (2.25-3.23 g/l/h) was obtained by using non-detoxified wood hydrolysate. Rice straw hydrolysate without detoxification was also tested and yielded a productivity rate as high as 5.27 g/l/h. Therefore, L. paracasei 7 BL represents a potential method of l-lactic acid production from lignocellulosic biomass and has attractive application for industries. PMID:26433790

  4. Kinetic study of the complexation of gallic acid with Fe(II)

    NASA Astrophysics Data System (ADS)

    Lu, Li-li; Li, Ying-hua; Lu, Xiu-yang

    2009-10-01

    Kinetic study on the complexation of gallic acid with ferrous sulfate was performed using UV-Vis absorption spectroscopy. Under the experimental conditions, the stoichiometric composition of the formed complex is 1:1. The complexation reaction was found to be a second-order one. The influences of temperature, ionic strength and solvents on the complexation reaction were investigated. According to the Arrhenius equation, the apparent activation energy of the complexation reaction was evaluated to be 71.64 kJ × mol -1. A three-step reaction mechanism was proposed, which can well explain the kinetic results obtained.

  5. Crystallization of bFGF-DNA Aptamer Complexes Using a Sparse Matrix Designed for Protein-Nucleic Acid Complexes

    NASA Technical Reports Server (NTRS)

    Cannone, Jaime J.; Barnes, Cindy L.; Achari, Aniruddha; Kundrot, Craig E.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    The Sparse Matrix approach for obtaining lead crystallization conditions has proven to be very fruitful for the crystallization of proteins and nucleic acids. Here we report a Sparse Matrix developed specifically for the crystallization of protein-DNA complexes. This method is rapid and economical, typically requiring 2.5 mg of complex to test 48 conditions. The method was originally developed to crystallize basic fibroblast growth factor (bFGF) complexed with DNA sequences identified through in vitro selection, or SELEX, methods. Two DNA aptamers that bind with approximately nanomolar affinity and inhibit the angiogenic properties of bFGF were selected for co-crystallization. The Sparse Matrix produced lead crystallization conditions for both bFGF-DNA complexes.

  6. Horseradish peroxidase. Complex formation with anions and hydrocyanic acid.

    PubMed

    Araiso, T; Dunford, H B

    1981-10-10

    Equilibrium binding experiments have been performed with perchlorate, chloride, and acetate in the presence of horseradish peroxidase. The binding of perchlorate and acetate appears to be like that of nitrate, at a site other than the sixth coordination position of the heme iron. Competitive experiments using both nitrate and cyanide demonstrate that two different binding sites are present on the enzyme. Chloride appears to bind at the sixth coordination position as do both fluoride and cyanide. Temperature jump experiments indicate that it is likely the nitrate anion and not undissociated nitric acid which is the binding species. Competitive stopped flow experiments indicate that the bound nitrate slows both the association rate and dissociation rate of cyanide, indicating that nitrate binds close to the sixth coordination position.

  7. Spectra, energy levels, and energy transition of lanthanide complexes with cinnamic acid and its derivatives

    NASA Astrophysics Data System (ADS)

    Zhou, Kaining; Feng, Zhongshan; Shen, Jun; Wu, Bing; Luo, Xiaobing; Jiang, Sha; Li, Li; Zhou, Xianju

    2016-04-01

    High resolution spectra and luminescent lifetimes of 6 europium(III)-cinnamic acid complex {[Eu2L6(DMF)(H2O)]·nDMF·H2O}m (L = cinnamic acid I, 4-methyl-cinnamic acid II, 4-chloro-cinnamic acid III, 4-methoxy-cinnamic acid IV, 4-hydroxy-cinnamic acid V, 4-nitro-cinnamic acid VI; DMF = N, N-dimethylformamide, C3H7NO) were recorded from 8 K to room temperature. The energy levels of Eu3 + in these 6 complexes are obtained from the spectra analysis. It is found that the energy levels of the central Eu3 + ions are influenced by the nephelauxetic effect, while the triplet state of ligand is lowered by the p-π conjugation effect of the para-substituted functional groups. The best energy matching between the ligand triplet state and the central ion excited state is found in complex I. While the other complexes show poorer matching because the gap of 5D0 and triplet state contracts.

  8. Spectra, energy levels, and energy transition of lanthanide complexes with cinnamic acid and its derivatives.

    PubMed

    Zhou, Kaining; Feng, Zhongshan; Shen, Jun; Wu, Bing; Luo, Xiaobing; Jiang, Sha; Li, Li; Zhou, Xianju

    2016-04-01

    High resolution spectra and luminescent lifetimes of 6 europium(III)-cinnamic acid complex {[Eu2L6(DMF)(H2O)]·nDMF·H2O}m (L=cinnamic acid I, 4-methyl-cinnamic acid II, 4-chloro-cinnamic acid III, 4-methoxy-cinnamic acid IV, 4-hydroxy-cinnamic acid V, 4-nitro-cinnamic acid VI; DMF=N, N-dimethylformamide, C3H7NO) were recorded from 8 K to room temperature. The energy levels of Eu(3+) in these 6 complexes are obtained from the spectra analysis. It is found that the energy levels of the central Eu(3+) ions are influenced by the nephelauxetic effect, while the triplet state of ligand is lowered by the p-π conjugation effect of the para-substituted functional groups. The best energy matching between the ligand triplet state and the central ion excited state is found in complex I. While the other complexes show poorer matching because the gap of (5)D0 and triplet state contracts.

  9. Spectra, energy levels, and energy transition of lanthanide complexes with cinnamic acid and its derivatives.

    PubMed

    Zhou, Kaining; Feng, Zhongshan; Shen, Jun; Wu, Bing; Luo, Xiaobing; Jiang, Sha; Li, Li; Zhou, Xianju

    2016-04-01

    High resolution spectra and luminescent lifetimes of 6 europium(III)-cinnamic acid complex {[Eu2L6(DMF)(H2O)]·nDMF·H2O}m (L=cinnamic acid I, 4-methyl-cinnamic acid II, 4-chloro-cinnamic acid III, 4-methoxy-cinnamic acid IV, 4-hydroxy-cinnamic acid V, 4-nitro-cinnamic acid VI; DMF=N, N-dimethylformamide, C3H7NO) were recorded from 8 K to room temperature. The energy levels of Eu(3+) in these 6 complexes are obtained from the spectra analysis. It is found that the energy levels of the central Eu(3+) ions are influenced by the nephelauxetic effect, while the triplet state of ligand is lowered by the p-π conjugation effect of the para-substituted functional groups. The best energy matching between the ligand triplet state and the central ion excited state is found in complex I. While the other complexes show poorer matching because the gap of (5)D0 and triplet state contracts. PMID:26802538

  10. Zinc complexes as fluorescent chemosensors for nucleic acids: new perspectives for a "boring" element.

    PubMed

    Terenzi, Alessio; Lauria, Antonino; Almerico, Anna Maria; Barone, Giampaolo

    2015-02-28

    Zinc(II) complexes are effective and selective nucleic acid-binders and strongly fluorescent molecules in the low energy range, from the visible to the near infrared. These two properties have often been exploited to quantitatively detect nucleic acids in biological samples, in both in vitro and in vivo models. In particular, the fluorescent emission of several zinc(II) complexes is drastically enhanced or quenched by the binding to nucleic acids and/or upon visible light exposure, in a different fashion in bulk solution and when bound to DNA. The twofold objective of this perspective is (1) to review recent utilisations of zinc(II) complexes as selective fluorescent probes for nucleic acids and (2) to highlight their novel potential applications as diagnostic tools based on their photophysical properties.

  11. Detailed kinetics and regulation of mammalian 2-oxoglutarate dehydrogenase

    PubMed Central

    2011-01-01

    Background Mitochondrial 2-oxoglutarate (α-ketoglutarate) dehydrogenase complex (OGDHC), a key regulatory point of tricarboxylic acid (TCA) cycle, plays vital roles in multiple pathways of energy metabolism and biosynthesis. The catalytic mechanism and allosteric regulation of this large enzyme complex are not fully understood. Here computer simulation is used to test possible catalytic mechanisms and mechanisms of allosteric regulation of the enzyme by nucleotides (ATP, ADP), pH, and metal ion cofactors (Ca2+ and Mg2+). Results A model was developed